Cross-interference of two model peroxisome proliferators in peroxisomal and estrogenic pathways in brown trout hepatocytes

Energy Technology Data Exchange (ETDEWEB)

Madureira, Tânia Vieira, E-mail: tvmadureira@icbas.up.pt [Interdisciplinary Centre of Marine and Environmental Research (CIIMAR/CIMAR), University of Porto (U. Porto), Terminal de Cruzeiros do Porto de Leixões, Av. General Norton de Matos s/n, 4450-208 Matosinhos (Portugal); Institute of Biomedical Sciences Abel Salazar (ICBAS), University of Porto (U. Porto), Laboratory of Histology and Embryology, Department of Microscopy, Rua Jorge Viterbo Ferreira 228, P 4050-313 Porto (Portugal); Pinheiro, Ivone; Malhão, Fernanda; Lopes, Célia [Interdisciplinary Centre of Marine and Environmental Research (CIIMAR/CIMAR), University of Porto (U. Porto), Terminal de Cruzeiros do Porto de Leixões, Av. General Norton de Matos s/n, 4450-208 Matosinhos (Portugal); Institute of Biomedical Sciences Abel Salazar (ICBAS), University of Porto (U. Porto), Laboratory of Histology and Embryology, Department of Microscopy, Rua Jorge Viterbo Ferreira 228, P 4050-313 Porto (Portugal); Urbatzka, Ralph [Interdisciplinary Centre of Marine and Environmental Research (CIIMAR/CIMAR), University of Porto (U. Porto), Terminal de Cruzeiros do Porto de Leixões, Av. General Norton de Matos s/n, 4450-208 Matosinhos (Portugal); Castro, L. Filipe C. [Interdisciplinary Centre of Marine and Environmental Research (CIIMAR/CIMAR), University of Porto (U. Porto), Terminal de Cruzeiros do Porto de Leixões, Av. General Norton de Matos s/n, 4450-208 Matosinhos (Portugal); Faculty of Sciences (FCUP), University of Porto (U. Porto), Department of Biology, Rua do Campo Alegre, P 4169-007 Porto (Portugal); and others

2017-06-15

Highlights: • Brown trout hepatocytes seem to be a low responder to model peroxisome proliferators. • Most peroxisomal targets were not affected by Wy-14,643 and clofibrate exposures. • Some estrogenic-related genes were up-regulated after 150 μM of Wy-14,643. • Wy-14,643 increase VtgA and ERα mRNA levels, while ICI 182,780 revert the effect. • Cross-interference in peroxisomal and estrogenic pathways should be more explored. - Abstract: Peroxisome proliferators cause species-specific effects, which seem to be primarily transduced by peroxisome proliferator-activated receptor alpha (PPARα). Interestingly, PPARα has a close interrelationship with estrogenic signaling, and this latter has already been promptly activated in brown trout primary hepatocytes. Thus, and further exploring this model, we assess here the reactivity of two PPARα agonists in direct peroxisomal routes and, in parallel the cross-interferences in estrogen receptor (ER) mediated paths. To achieve these goals, three independent in vitro studies were performed using single exposures to clofibrate – CLF (50, 500 and 1000 μM), Wy-14,643 – Wy (50 and 150 μM), GW6471 – GW (1 and 10 μM), and mixtures, including PPARα agonist or antagonist plus an ER agonist or antagonist. Endpoints included gene expression analysis of peroxisome/lipidic related genes (encoding apolipoprotein AI – ApoAI, fatty acid binding protein 1 – Fabp1, catalase – Cat, 17 beta-hydroxysteroid dehydrogenase 4 – 17β-HSD4, peroxin 11 alpha – Pex11α, PPARαBb, PPARαBa and urate oxidase – Uox) and those encoding estrogenic targets (ERα, ERβ-1 and vitellogenin A – VtgA). A quantitative morphological approach by using a pre-validated catalase immunofluorescence technique allowed checking possible changes in peroxisomes. Our results show a low responsiveness of trout hepatocytes to model PPARα agonists in direct target receptor pathways. Additionally, we unveiled interferences in estrogenic

Developmental changes in concentrations of vitellin, vitellogenin, and lipids in hemolymph, hepatopancreas, and ovaries from different ovarian stages of Indian white prawn Fenneropenaeus indicus

DEFF Research Database (Denmark)

Boucard, C.G.V.; Levy, P.; Ceccaldi, H.J.

2002-01-01

The objective of the present study was to characterize the relationship between vitellogenin (Vtg) and vitellin (Vt) concentration profiles during the reproductive cycle of the penaeid prawn Fenneropenaeus indicus. Vt was purified from ovaries of vitellogenic females by gradient ultracentrifugati...

Effects of adrenergic agents on the expression of zebrafish (Danio rerio) vitellogenin Ao1

International Nuclear Information System (INIS)

Yin Naida; Jin Xia; He Jiangyan; Yin Zhan

2009-01-01

Teleost vitellogenins (VTGs) are large multidomain apolipoproteins, traditionally considered to be estrogen-responsive precursors of the major egg yolk proteins, expressed and synthesized mainly in hepatic tissue. The inducibility of VTGs has made them one of the most frequently used in vivo and in vitro biomarkers of exposure to estrogen-active substances. A significant level of zebrafish vtgAo1, a major estrogen responsive form, has been unexpectedly found in heart tissue in our present studies. Our studies on zebrafish cardiomyopathy, caused by adrenergic agonist treatment, suggest a similar protective function of the cardiac expressed vtgAo1. We hypothesize that its function is to unload surplus intracellular lipids in cardiomyocytes for 'reverse triglyceride transportation' similar to that found in lipid transport proteins in mammals. Our results also demonstrated that zebrafish vtgAo1 mRNA expression in heart can be suppressed by both α-adrenergic agonist, phenylephrine (PE) and β-adrenergic agonist, isoproterenol (ISO). Furthermore, the strong stimulation of zebrafish vtgAo1 expression in plasma induced by the β-adrenergic antagonist, MOXIsylyl, was detected by Enzyme-Linked ImmunoSorbent Assay (ELISA). Such stimulation cannot be suppressed by taMOXIfen, an antagonist to estrogen receptors. Thus, our present data indicate that the production of teleost VTG in vivo can be regulated not only by estrogenic agents, but by adrenergic signals as well.

A latex agglutination test for the field determination of abnormal vitellogenin production in male fishes contaminated by estrogen mimics

International Nuclear Information System (INIS)

Magalhaes, Ilizabete; Pihan, Jean-Claude; Falla, Jairo

2004-01-01

Estrogen mimics are pollutants present in the aquatic environment. These compounds induce abnormalities in the reproductive system of male fishes, which lead to a total or partial male feminization, or to their demasculinization. Ultimately, these alterations could lead to a disappearance of the total contaminated fish population. Moreover, these toxic substances possess the capacity to mimic endogenous estrogens and to induce the abnormal production of vitellogenin (VTG) in male and immature fishes. The purpose of this research was to develop an easy, specific, cheap and fast method for diagnosing the contamination of male fishes by estrogen mimics, using VTG as biomarker. The selected method is based on a reverse latex agglutination test (rLAT), developed with monoclonal antibodies specific of this biomarker. The development of this VTG-rLAT has involved, firstly, the purification of carp VTG to produce monoclonal antibodies, specifics of this protein. One of these antibodies was selected to recover latex particles (diameter: 1 μm). Finally, the immunoreactivity of the VTG-rLAT was verified with different fish plasma samples from males treated with 17β-estradiol and non-treated males or females in vitellogenesis

Estrogenic and anti-estrogenic influences in cultured brown trout hepatocytes: Focus on the expression of some estrogen and peroxisomal related genes and linked phenotypic anchors

Energy Technology Data Exchange (ETDEWEB)

Madureira, Tânia Vieira, E-mail: tvmadureira@icbas.up.pt [Interdisciplinary Centre of Marine and Environmental Research (CIIMAR/CIMAR), U.Porto—University of Porto, Rua dos Bragas 289, P 4050-123 Porto (Portugal); Institute of Biomedical Sciences Abel Salazar, U.Porto (ICBAS)—University of Porto, Laboratory of Histology and Embryology, Department of Microscopy, Rua Jorge Viterbo Ferreira 228, P 4050-313 Porto (Portugal); Malhão, Fernanda; Pinheiro, Ivone; Lopes, Célia; Ferreira, Nádia [Interdisciplinary Centre of Marine and Environmental Research (CIIMAR/CIMAR), U.Porto—University of Porto, Rua dos Bragas 289, P 4050-123 Porto (Portugal); Institute of Biomedical Sciences Abel Salazar, U.Porto (ICBAS)—University of Porto, Laboratory of Histology and Embryology, Department of Microscopy, Rua Jorge Viterbo Ferreira 228, P 4050-313 Porto (Portugal); Urbatzka, Ralph [Interdisciplinary Centre of Marine and Environmental Research (CIIMAR/CIMAR), U.Porto—University of Porto, Rua dos Bragas 289, P 4050-123 Porto (Portugal); Castro, L. Filipe C. [Interdisciplinary Centre of Marine and Environmental Research (CIIMAR/CIMAR), U.Porto—University of Porto, Rua dos Bragas 289, P 4050-123 Porto (Portugal); Faculty of Sciences (FCUP), U.Porto—University of Porto, Department of Biology, Rua do Campo Alegre, P 4169-007 Porto (Portugal); Rocha, Eduardo [Interdisciplinary Centre of Marine and Environmental Research (CIIMAR/CIMAR), U.Porto—University of Porto, Rua dos Bragas 289, P 4050-123 Porto (Portugal); Institute of Biomedical Sciences Abel Salazar, U.Porto (ICBAS)—University of Porto, Laboratory of Histology and Embryology, Department of Microscopy, Rua Jorge Viterbo Ferreira 228, P 4050-313 Porto (Portugal)

2015-12-15

Highlights: • Evidence of crosstalk between estrogens and peroxisomal pathways in brown trout. • VtgA and ERα mRNA levels increased after 1, 10 and 50 μM of ethinylestradiol (EE2). • ERβ-1, catalase and urate oxidase mRNA levels decreased after estrogenic stimuli. • Estrogenic effects in VtgA, ERα and Uox mRNA levels were reverted by ICI 182,780. • Immunofluorescence/electron microscopy shows smaller peroxisomes after 50 μM of EE2. - Abstract: Estrogens, estrogenic mimics and anti-estrogenic compounds are known to target estrogen receptors (ER) that can modulate other nuclear receptor signaling pathways, such as those controlled by the peroxisome proliferator-activated receptor (PPAR), and alter organelle (inc. peroxisome) morphodynamics. By using primary isolated brown trout (Salmo trutta f. fario) hepatocytes after 72 and 96 h of exposure we evaluated some effects in selected molecular targets and in peroxisomal morphological features caused by: (1) an ER agonist (ethinylestradiol—EE2) at 1, 10 and 50 μM; (2) an ER antagonist (ICI 182,780) at 10 and 50 μM; and (3) mixtures of both (Mix I—10 μM EE2 and 50 μM ICI; Mix II—1 μM EE2 and 10 μM ICI and Mix III—1 μM EE2 and 50 μM ICI). The mRNA levels of the estrogenic targets (ERα, ERβ-1 and vitellogenin A—VtgA) and the peroxisome structure/function related genes (catalase, urate oxidase—Uox, 17β-hydroxysteroid dehydrogenase 4—17β-HSD4, peroxin 11α—Pex11α and PPARα) were analyzed by real-time polymerase chain reaction (RT-PCR). Stereology combined with catalase immunofluorescence revealed a significant reduction in peroxisome volume densities at 50 μM of EE2 exposure. Concomitantly, at the same concentration, electron microscopy showed smaller peroxisome profiles, exacerbated proliferation of rough endoplasmic reticulum, and a generalized cytoplasmic vacuolization of hepatocytes. Catalase and Uox mRNA levels decreased in all estrogenic stimuli conditions. VtgA and ERα m

Estrogenic and anti-estrogenic influences in cultured brown trout hepatocytes: Focus on the expression of some estrogen and peroxisomal related genes and linked phenotypic anchors

International Nuclear Information System (INIS)

Madureira, Tânia Vieira; Malhão, Fernanda; Pinheiro, Ivone; Lopes, Célia; Ferreira, Nádia; Urbatzka, Ralph; Castro, L. Filipe C.; Rocha, Eduardo

2015-01-01

Highlights: • Evidence of crosstalk between estrogens and peroxisomal pathways in brown trout. • VtgA and ERα mRNA levels increased after 1, 10 and 50 μM of ethinylestradiol (EE2). • ERβ-1, catalase and urate oxidase mRNA levels decreased after estrogenic stimuli. • Estrogenic effects in VtgA, ERα and Uox mRNA levels were reverted by ICI 182,780. • Immunofluorescence/electron microscopy shows smaller peroxisomes after 50 μM of EE2. - Abstract: Estrogens, estrogenic mimics and anti-estrogenic compounds are known to target estrogen receptors (ER) that can modulate other nuclear receptor signaling pathways, such as those controlled by the peroxisome proliferator-activated receptor (PPAR), and alter organelle (inc. peroxisome) morphodynamics. By using primary isolated brown trout (Salmo trutta f. fario) hepatocytes after 72 and 96 h of exposure we evaluated some effects in selected molecular targets and in peroxisomal morphological features caused by: (1) an ER agonist (ethinylestradiol—EE2) at 1, 10 and 50 μM; (2) an ER antagonist (ICI 182,780) at 10 and 50 μM; and (3) mixtures of both (Mix I—10 μM EE2 and 50 μM ICI; Mix II—1 μM EE2 and 10 μM ICI and Mix III—1 μM EE2 and 50 μM ICI). The mRNA levels of the estrogenic targets (ERα, ERβ-1 and vitellogenin A—VtgA) and the peroxisome structure/function related genes (catalase, urate oxidase—Uox, 17β-hydroxysteroid dehydrogenase 4—17β-HSD4, peroxin 11α—Pex11α and PPARα) were analyzed by real-time polymerase chain reaction (RT-PCR). Stereology combined with catalase immunofluorescence revealed a significant reduction in peroxisome volume densities at 50 μM of EE2 exposure. Concomitantly, at the same concentration, electron microscopy showed smaller peroxisome profiles, exacerbated proliferation of rough endoplasmic reticulum, and a generalized cytoplasmic vacuolization of hepatocytes. Catalase and Uox mRNA levels decreased in all estrogenic stimuli conditions. VtgA and ERα m

Standardization of plasma vitellogenin level for surveying environmental estrogen pollution using the Japanese common goby Acanthogobius flavimanus.

Science.gov (United States)

Song, Jing; Nagae, Masaki; Soyano, Kiyoshi

2018-07-01

Field surveys of the impact of environmental estrogen (EE) pollution in aquatic wildlife have been conducted using vitellogenin (VTG) as a biomarker to evaluate the influence of EE. However, a standard baseline of VTG level that can be used to evaluate EE pollution has not been fully determined. To the best of our knowledge, the present study is the first to determine the standard baseline VTG level for evaluating the biological effects of EE pollution using the Japanese common goby (Acanthogobius flavimanus) as the target model fish. Plasma VTG and estradiol-17β (E 2 ) levels associated with the reproductive cycle of wild goby inhabiting an unpolluted environment were measured. Mean plasma VTG and E 2 levels exhibited similar changes, increasing in the yolk vesicle stage and peaking in the tertiary yolk stage in females. However, plasma VTG and E 2 levels showed no significant changes in males, remaining at low levels throughout the reproductive cycle. The highest VTG levels in females and males were 1.6 mg ml -1 and 124.87 ng ml -1 , respectively. These results indicate that the baseline level (normal level) in males was approximately 130 ng ml -1 at most. We concluded that the threshold between normal and abnormal levels with a 10% risk rate was 150 ng ml -1 in the wild male goby. Plasma VTG levels in males captured from Nagasaki Harbor were higher than the threshold in each reproductive developmental stage, indicating the possibility of EE pollution at this site. The biological standard baseline for VTG established in this study is useful for assessing EE pollution in natural waters. Copyright © 2018 John Wiley & Sons, Ltd.

Assessing the potential for trace organic contaminants commonly found in Australian rivers to induce vitellogenin in the native rainbowfish (Melanotaenia fluviatilis) and the introduced mosquitofish (Gambusia holbrooki)

Energy Technology Data Exchange (ETDEWEB)

Scott, Philip D. [Australian Rivers Institute, Griffith School of Environment, Griffith University, Southport, Queensland, 4222 (Australia); Coleman, Heather M. [School of Pharmacy and Pharmaceutical Science, Ulster University, Coleraine, BT52 1SA, Northern Ireland (United Kingdom); Colville, Anne; Lim, Richard [School of Life Sciences, University of Technology Sydney, PO Box 123, Broadway, New South Wales, 2007 (Australia); Matthews, Benjamin [Australian Rivers Institute, Griffith School of Environment, Griffith University, Southport, Queensland, 4222 (Australia); McDonald, James A. [School of Civil & Environmental Engineering, University of New South Wales, New South Wales, 2052 (Australia); Miranda, Ana [School of Applied Sciences, Royal Melbourne Institute of Technology, PO Box 71, Bundoora, Victoria, 3083 (Australia); Neale, Peta A. [Australian Rivers Institute, Griffith School of Environment, Griffith University, Southport, Queensland, 4222 (Australia); Nugegoda, Dayanthi [School of Applied Sciences, Royal Melbourne Institute of Technology, PO Box 71, Bundoora, Victoria, 3083 (Australia); Tremblay, Louis A. [Cawthron Institute, 98 Halifax St. East, Nelson 7042 (New Zealand); School of Biological Sciences, University of Auckland, PO Box 92019, Auckland, 1142 (New Zealand); Leusch, Frederic D.L., E-mail: f.leusch@griffith.edu.au [Australian Rivers Institute, Griffith School of Environment, Griffith University, Southport, Queensland, 4222 (Australia)

2017-04-15

Highlights: • Mosquitofish and rainbowfish exposed in the lab to trace organic compounds. • Vitellogenin protein and mRNA monitored as biomarker of estrogenicity. • Minimal response in both species to environmentally relevant concentrations. • Native rainbowfish was slightly more sensitive than the invasive mosquitofish. • Results suggest that risk of estrogenic disruption in Australian rivers is low. - Abstract: In Australia, trace organic contaminants (TrOCs) and endocrine active compounds (EACs) have been detected in rivers impacted by sewage effluent, urban stormwater, agricultural and industrial inputs. It is unclear whether these chemicals are at concentrations that can elicit endocrine disruption in Australian fish species. In this study, native rainbowfish (Melanotaenia fluviatilis) and introduced invasive (but prevalent) mosquitofish (Gambusia holbrooki) were exposed to the individual compounds atrazine, estrone, bisphenol A, propylparaben and pyrimethanil, and mixtures of compounds including hormones and personal care products, industrial compounds, and pesticides at environmentally relevant concentrations. Vitellogenin (Vtg) protein and liver Vtg mRNA induction were used to assess the estrogenic potential of these compounds. Vtg expression was significantly affected in both species exposed to estrone at concentrations that leave little margin for safety (p < 0.001). Propylparaben caused a small but statistically significant 3× increase in Vtg protein levels (p = 0.035) in rainbowfish but at a concentration 40× higher than that measured in the environment, therefore propylparaben poses a low risk of inducing endocrine disruption in fish. Mixtures of pesticides and a mixture of hormones, pharmaceuticals, industrial compounds and pesticides induced a small but statistically significant increase in plasma Vtg in rainbowfish, but did not affect mosquitofish Vtg protein or mRNA expression. These results suggest that estrogenic activity represents a

Assessment of potential biomarkers, metallothionein and vitellogenin mRNA expressions in various chemically exposed benthic Chironomus riparius larvae

Science.gov (United States)

Park, Kiyun; Kwak, Inn-Sil

2012-12-01

The objective of this study was conducted to identify the possibility of using Chironomus metallothionein (MT) and vitellogenin (VTG) as biomarkers of stress caused by endocrinedisrupting chemicals (EDCs), heavy metals, herbicides and veterinary antibiotics. We characterized the MT and VTG cDNA in Chironomus riparius and evaluated their mRNA expression profiles following exposure to different environmental pollutants. The gene expression analysis showed that the MT mRNA levels increased significantly after long-term exposure to cadmium (Cd), copper (Cu), Lead (Pb), di(2-ethylhexyl) phthalate (DEHP), and 2,4-dichlorophenoxyacetic acid (2,4-D). Moreover, the VTG mRNA expression increased significantly in C. riparius larvae exposed to BPA, NP, DEHP, Cd, 2,4-D and fenbendazole. Evaluation of the long-term effects of environmental pollutants revealed up regulation of Chironomus MT mRNA in response to DEHP exposure among EDCs, and the level of the VTG mRNA was increased significantly following treatment with Cd and herbicide 2,4-D at all concentrations in a dose-dependent manner. These results indicate that VTG could be used as a potential biomarker of herbicide and Cd as well as EDCs, while MT was a potential biomarker of heavy metals such as Cd, Cu, and Pb in aquatic environments.

Cross-interference of two model peroxisome proliferators in peroxisomal and estrogenic pathways in brown trout hepatocytes.

Science.gov (United States)

Madureira, Tânia Vieira; Pinheiro, Ivone; Malhão, Fernanda; Lopes, Célia; Urbatzka, Ralph; Castro, L Filipe C; Rocha, Eduardo

2017-06-01

Peroxisome proliferators cause species-specific effects, which seem to be primarily transduced by peroxisome proliferator-activated receptor alpha (PPARα). Interestingly, PPARα has a close interrelationship with estrogenic signaling, and this latter has already been promptly activated in brown trout primary hepatocytes. Thus, and further exploring this model, we assess here the reactivity of two PPARα agonists in direct peroxisomal routes and, in parallel the cross-interferences in estrogen receptor (ER) mediated paths. To achieve these goals, three independent in vitro studies were performed using single exposures to clofibrate - CLF (50, 500 and 1000μM), Wy-14,643 - Wy (50 and 150μM), GW6471 - GW (1 and 10μM), and mixtures, including PPARα agonist or antagonist plus an ER agonist or antagonist. Endpoints included gene expression analysis of peroxisome/lipidic related genes (encoding apolipoprotein AI - ApoAI, fatty acid binding protein 1 - Fabp1, catalase - Cat, 17 beta-hydroxysteroid dehydrogenase 4 - 17β-HSD4, peroxin 11 alpha - Pex11α, PPARαBb, PPARαBa and urate oxidase - Uox) and those encoding estrogenic targets (ERα, ERβ-1 and vitellogenin A - VtgA). A quantitative morphological approach by using a pre-validated catalase immunofluorescence technique allowed checking possible changes in peroxisomes. Our results show a low responsiveness of trout hepatocytes to model PPARα agonists in direct target receptor pathways. Additionally, we unveiled interferences in estrogenic signaling caused by Wy, leading to an up-regulation VtgA and ERα at 150μM; these effects seem counteracted with a co-exposure to an ER antagonist. The present data stress the potential of this in vitro model for further exploring the physiological/toxicological implications related with this nuclear receptor cross-regulation. Copyright © 2017 Elsevier B.V. All rights reserved.

Estrogenic and anti-estrogenic influences in cultured brown trout hepatocytes: Focus on the expression of some estrogen and peroxisomal related genes and linked phenotypic anchors.

Science.gov (United States)

Madureira, Tânia Vieira; Malhão, Fernanda; Pinheiro, Ivone; Lopes, Célia; Ferreira, Nádia; Urbatzka, Ralph; Castro, L Filipe C; Rocha, Eduardo

2015-12-01

Estrogens, estrogenic mimics and anti-estrogenic compounds are known to target estrogen receptors (ER) that can modulate other nuclear receptor signaling pathways, such as those controlled by the peroxisome proliferator-activated receptor (PPAR), and alter organelle (inc. peroxisome) morphodynamics. By using primary isolated brown trout (Salmo trutta f. fario) hepatocytes after 72 and 96h of exposure we evaluated some effects in selected molecular targets and in peroxisomal morphological features caused by: (1) an ER agonist (ethinylestradiol-EE2) at 1, 10 and 50μM; (2) an ER antagonist (ICI 182,780) at 10 and 50μM; and (3) mixtures of both (Mix I-10μM EE2 and 50μM ICI; Mix II-1μM EE2 and 10μM ICI and Mix III-1μM EE2 and 50μM ICI). The mRNA levels of the estrogenic targets (ERα, ERβ-1 and vitellogenin A-VtgA) and the peroxisome structure/function related genes (catalase, urate oxidase-Uox, 17β-hydroxysteroid dehydrogenase 4-17β-HSD4, peroxin 11α-Pex11α and PPARα) were analyzed by real-time polymerase chain reaction (RT-PCR). Stereology combined with catalase immunofluorescence revealed a significant reduction in peroxisome volume densities at 50μM of EE2 exposure. Concomitantly, at the same concentration, electron microscopy showed smaller peroxisome profiles, exacerbated proliferation of rough endoplasmic reticulum, and a generalized cytoplasmic vacuolization of hepatocytes. Catalase and Uox mRNA levels decreased in all estrogenic stimuli conditions. VtgA and ERα mRNA increased after all EE2 treatments, while ERβ-1 had an inverse pattern. The EE2 action was reversed by ICI 182,780 in a concentration-dependent manner, for VtgA, ERα and Uox. Overall, our data show the great value of primary brown trout hepatocytes to study the effects of estrogenic/anti-estrogenic inputs in peroxisome kinetics and in ER and PPARα signaling, backing the still open hypothesis of crosstalk interactions between these pathways and calling for more mechanistic

Natural mixtures of persistent organic pollutants (POPs) suppress ovarian follicle development, liver vitellogenin immunostaining and hepatocyte proliferation in female zebrafish (Danio rerio)

International Nuclear Information System (INIS)

Kraugerud, Marianne; Doughty, Richard William; Lyche, Jan L.; Berg, Vidar; Tremoen, Nina H.; Alestrøm, Peter; Aleksandersen, Mona; Ropstad, Erik

2012-01-01

Persistent organic pollutants such as polychlorinated biphenyls (PCBs), polybrominated diphenyl ethers (PBDEs) and dichlorodiphenyltrichloroethane (DDT) are present in high concentrations in livers of burbot (Lota lota) in Lake Mjøsa, Norway. In order to assess effects of such pollutants on fish gonadal morphology, female zebrafish were exposed in two generations by food to mixtures of pollutants extracted from livers of burbot from Lake Mjøsa (high and low dose) and Lake Losna, which represents background pollution, and compared to a control group. Ovarian follicle counts detected a significant decrease in late vitellogenic follicle stages in fish exposed to the Losna and the high concentrations of Mjøsa mixtures in fish from the first generation. In addition, proliferation of granulosa cells, visualized by immunohistochemistry against proliferating cell nuclear antigen (PCNA), was decreased in all exposure groups in either early or late vitellogenic follicle stages compared to control. This was accompanied by increased apoptosis of granulosa cells. There was a decrease in proliferation of liver hepatocytes with exposure to both Mjøsa mixtures. In addition, immunopositivity for vitellogenin in the liver was significantly lower in the Mjøsa high group than in the control group. When analysing effects of parental exposure, fish with parents exposed to Mjøsa high mixture had significantly higher numbers of perinucleolar follicles than fish with control parents. We conclude that long-term exposure of a real-life mixture of pollutants containing high- and background levels of chemicals supress ovarian follicle development, liver vitellogenin immunostaining intensity and hepatocyte proliferation in the zebrafish model.

Natural mixtures of persistent organic pollutants (POPs) suppress ovarian follicle development, liver vitellogenin immunostaining and hepatocyte proliferation in female zebrafish (Danio rerio)

Energy Technology Data Exchange (ETDEWEB)

Kraugerud, Marianne, E-mail: Marianne.Kraugerud@nvh.no [Dept. of Basic Sciences and Aquatic Medicine, Norwegian School of Veterinary Science, POB 8146 Dep., 0033 Oslo (Norway); Doughty, Richard William, E-mail: vetrwdoughty@yahoo.co.uk [Sundveien 22, 2015 Leirsund (Norway); Lyche, Jan L., E-mail: Jan.Lyche@nvh.no [Dept. of Food Safety and Infection Biology, Norwegian School of Veterinary Science, POB 8146 Dep., 0033 Oslo (Norway); Berg, Vidar, E-mail: Vidar.Berg@nvh.no [Dept. of Food Safety and Infection Biology, Norwegian School of Veterinary Science, POB 8146 Dep., 0033 Oslo (Norway); Tremoen, Nina H., E-mail: Nina.Hardnes@nvh.no [Dept. of Production Animal Clinical Sciences, Norwegian School of Veterinary Science, POB 8146 Dep., 0033 Oslo (Norway); Alestrom, Peter, E-mail: Peter.Alestrom@nvh.no [Dept. of Basic Sciences and Aquatic Medicine, Norwegian School of Veterinary Science, POB 8146 Dep., 0033 Oslo (Norway); Aleksandersen, Mona, E-mail: Mona.Aleksandersen@nvh.no [Dept. of Basic Sciences and Aquatic Medicine, Norwegian School of Veterinary Science, POB 8146 Dep., 0033 Oslo (Norway); Ropstad, Erik, E-mail: Erik.Ropstad@nvh.no [Dept. of Production Animal Clinical Sciences, Norwegian School of Veterinary Science, POB 8146 Dep., 0033 Oslo (Norway)

2012-07-15

Persistent organic pollutants such as polychlorinated biphenyls (PCBs), polybrominated diphenyl ethers (PBDEs) and dichlorodiphenyltrichloroethane (DDT) are present in high concentrations in livers of burbot (Lota lota) in Lake Mjosa, Norway. In order to assess effects of such pollutants on fish gonadal morphology, female zebrafish were exposed in two generations by food to mixtures of pollutants extracted from livers of burbot from Lake Mjosa (high and low dose) and Lake Losna, which represents background pollution, and compared to a control group. Ovarian follicle counts detected a significant decrease in late vitellogenic follicle stages in fish exposed to the Losna and the high concentrations of Mjosa mixtures in fish from the first generation. In addition, proliferation of granulosa cells, visualized by immunohistochemistry against proliferating cell nuclear antigen (PCNA), was decreased in all exposure groups in either early or late vitellogenic follicle stages compared to control. This was accompanied by increased apoptosis of granulosa cells. There was a decrease in proliferation of liver hepatocytes with exposure to both Mjosa mixtures. In addition, immunopositivity for vitellogenin in the liver was significantly lower in the Mjosa high group than in the control group. When analysing effects of parental exposure, fish with parents exposed to Mjosa high mixture had significantly higher numbers of perinucleolar follicles than fish with control parents. We conclude that long-term exposure of a real-life mixture of pollutants containing high- and background levels of chemicals supress ovarian follicle development, liver vitellogenin immunostaining intensity and hepatocyte proliferation in the zebrafish model.

Mechanistic insights into induction of vitellogenin gene expression by estrogens in Sydney rock oysters, Saccostrea glomerata.

Science.gov (United States)

Tran, Thi Kim Anh; MacFarlane, Geoff R; Kong, Richard Yuen Chong; O'Connor, Wayne A; Yu, Richard Man Kit

2016-05-01

Marine molluscs, such as oysters, respond to estrogenic compounds with the induction of the egg yolk protein precursor, vitellogenin (Vtg), availing a biomarker for estrogenic pollution. Despite this application, the precise molecular mechanism through which estrogens exert their action to induce molluscan vitellogenesis is unknown. As a first step to address this question, we cloned a gene encoding Vtg from the Sydney rock oyster Saccostrea glomerata (sgVtg). Using primers designed from a partial sgVtg cDNA sequence available in Genbank, a full-length sgVtg cDNA of 8498bp was obtained by 5'- and 3'-RACE. The open reading frame (ORF) of sgVtg was determined to be 7980bp, which is substantially longer than the orthologs of other oyster species. Its deduced protein sequence shares the highest homology at the N- and C-terminal regions with other molluscan Vtgs. The full-length genomic DNA sequence of sgVtg was obtained by genomic PCR and genome walking targeting the gene body and flanking regions, respectively. The genomic sequence spans 20kb and consists of 30 exons and 29 introns. Computer analysis identified three closely spaced half-estrogen responsive elements (EREs) in the promoter region and a 210-bp CpG island 62bp downstream of the transcription start site. Upregulation of sgVtg mRNA expression was observed in the ovaries following in vitro (explants) and in vivo (tank) exposure to 17β-estradiol (E2). Notably, treatment with an estrogen receptor (ER) antagonist in vitro abolished the upregulation, suggesting a requirement for an estrogen-dependent receptor for transcriptional activation. DNA methylation of the 5' CpG island was analysed using bisulfite genomic sequencing of the in vivo exposed ovaries. The CpG island was found to be hypomethylated (with 0-3% methylcytosines) in both control and E2-exposed oysters. However, no significant differential methylation or any correlation between methylation and sgVtg expression levels was observed. Overall, the

Gender determination in the Paiche or Pirarucu (Arapaima gigas) using plasma vitellogenin, 17beta-estradiol, and 11-ketotestosterone levels.

Science.gov (United States)

Chu-Koo, F; Dugué, R; Alván Aguilar, M; Casanova Daza, A; Alcántara Bocanegra, F; Chávez Veintemilla, C; Duponchelle, F; Renno, J-F; Tello, Salvador; Nuñez, J

2009-03-01

Arapaima gigas is an air-breathing giant fish of Amazonian rivers. Given its great economic and cultural importance, the aquaculture development of this species represents an evident solution to face the decline of wild populations. In captivity, reproduction occurs generally in large earthen ponds where stocks of a few tens of brooders are maintained together at the beginning of the rainy season (December-March in the Peruvian Amazon). Fry production relies on the spontaneous formation of male and female pairs, which build a nest, delimit a territory and guard the offspring for at least 20 days from other congeners and predators. However, as sex determination of A. gigas is not possible by morphological criteria, it is very difficult to optimize reproduction conditions and fry production in each pond, which seriously hampers the culture of this species. This situation prompted us to develop sexing methodologies based on (1) the detection of female specific plasma Vitellogenin (Vtg) using an enzyme immuno assay (EIA), and (2) the determination of plasma 17beta-estradiol and 11-ketotestosterone levels for immature specimens. The Vtg purification was performed by electro-elution after polyacrilamide gel electrophoresis (PAGE) from plasma of 17beta-estradiol treated A. gigas juveniles. Two different Vtg molecules were isolated, (Vtg(1) and Vtg(2)) with 184 and 112 kDa apparent molecular masses, respectively, and two antibodies were raised in rabbits for each Vtg molecule. Adult fish were 100% accurately sexed by Vtg EIA, while 100% of immature fish and 95% of adults were accurately sexed by 17beta-Estradiol and 11-Ketestosterone ratios. We also observed different color pattern development in male and female adult fish (6-year-olds) around the reproductive period.

Stress-related gene expression changes in rainbow trout hepatocytes exposed to various municipal wastewater treatment influents and effluents.

Science.gov (United States)

Gagné, F; Smyth, S A; André, C; Douville, M; Gélinas, M; Barclay, K

2013-03-01

The present study sought to examine the performance of six different wastewater treatment processes from 12 wastewater treatment plants using a toxicogenomic approach in rainbow trout hepatocytes. Freshly prepared rainbow trout hepatocytes were exposed to increasing concentrations of influent (untreated wastewaters) and effluent (C(18)) extracts for 48 h at 15 °C. A test battery of eight genes was selected to track changes in xenobiotic biotransformation, estrogenicity, heavy metal detoxification, and oxidative stress. The wastewaters were processed by six different treatment systems: facultative and aerated lagoons, activated sludge, biological aerated filter, biological nutrient removal, chemically assisted primary treated, and trickling filter/solids contact. Based on the chemical characteristics of the effluents, the treatment plants were generally effective in removing total suspended solids and chemical oxygen demand, but less so for ammonia and alkalinity. The 12 influents differed markedly with each other, which makes the comparison among treatment processes difficult. For the influents, both population size and flow rate influenced the increase in the following mRNA levels in exposed hepatocytes: metallothionein (MT), cytochrome P4503A4 (CYP3A4), and vitellogenin (VTG). Gene expression of glutathione S-transferase (GST) and the estrogen receptor (ER), were influenced only by population size in exposed cells to the influent extracts. The remaining genes-superoxide dismutase (SOD) and multidrug resistance transporter (MDR)-were not influenced by either population size or flow rate in exposed cells. It is noteworthy that the changes in MT, ER, and VTG in cells exposed to the effluents were significantly affected by the influents across the 12 cities examined. However, SOD, CYP1A1, CYP3A4, GST, and MDR gene expression were the least influenced by the incoming influents. The data also suggest that wastewater treatments involving biological or aeration

Induction of vitellogenin synthesis by estrogen in avian liver: relationship between level of vitellogenin mRNA and vitellogenin synthesis.

Science.gov (United States)

Mullinix, K P; Wetekam, W; Deeley, R G; Gordon, J I; Meyers, M; Kent, K A; Goldberger, R F

1976-01-01

We have investigated the estrogen-mediated induction of vitellogenin synthesis in rooster liver. We compared the concentrations of vitellogenin messenger RNA (mRNA) in the liver with the concentrations of vitellogenin in the sera of roosters that had recieved various treatments with estrogen. We found no vitellogenin mRNA in the livers of the unstimulated roosters. An initial injection of estrogen was attended by de novo synthesis of vitellogenin mRNA in the liver and accumulation of vitellogenin in the serum. When vitellogenin was no longer present in the serum or liver (the "post-estrogen-serum-negative" state), the liver was found to contain appreciable amounts of vitellogenin mRNA. This mRNA was of the same size as that found in the liver of the rooster actively synthesizing vitellogenin in response to estrogen. Whereas vitellogenin mRNA was in large polysomes in the livers of the roosters actively synthesizing vitellogenin, the vitellogenin mRNA in the liver of the post-estrogen-serum-negative rooster was not associated with polysomes. The possible relevance of these findings to the fact that the rooster responds differently to a primary stimulation with estrogen than to subsequent stimulations is discussed. PMID:1064017

The Expression of Leptin, Estrogen Receptors, and Vitellogenin mRNAs in Migrating Female Chum Salmon, : The Effects of Hypo-osmotic Environmental Changes

Directory of Open Access Journals (Sweden)

Young Jae Choi

2014-04-01

Full Text Available Leptin plays an important role in energy homeostasis and reproductive function in fish, especially in reproduction. Migrating fish, such as salmonoids, are affected by external environmental factors, and salinity changes are a particularly important influence on spawning migrations. The aim of this study was to test whether changes in salinity affect the expression of leptin, estrogen receptors (ERs, and vitellogenin (VTG in chum salmon (Oncorhynchus keta. The expression and activity of leptin, the expression of ERs and VTG, and the levels of estradiol-17β and cortisol increased after the fish were transferred to FW, demonstrating that changes in salinity stimulate the HPG axis in migrating female chum salmon. These findings reveal details about the role of elevated leptin levels and sex steroid hormones in stimulating sexual maturation and reproduction in response to salinity changes in chum salmon.

Functional characterization of estrogen receptor subtypes, ERα and ERβ, mediating vitellogenin production in the liver of rainbow trout

International Nuclear Information System (INIS)

Leanos-Castaneda, Olga; Kraak, Glen van der

2007-01-01

The estrogen-dependent process of vitellogenesis is a key function on oviparous fish reproduction and it has been widely used as an indicator of xenoestrogen exposure. The two estrogen receptor (ER) subtypes, ERα and ERβ, are often co-expressed in the liver of fish. The relative contribution of each ER subtype to modulate vitellogenin production by hepatocytes was studied using selected compounds known to preferentially interact with specific ER subtypes: propyl-pyrazole-triol (PPT) an ERα selective agonist, methyl-piperidino-pyrazole (MPP) an ERα selective antagonist, and diarylpropionitrile (DPN) an ERβ selective agonist. First, the relative binding affinity of the test compounds to estradiol for rainbow trout hepatic nuclear ER was determined using a competitive ligand binding assay. All the test ligands achieved complete displacement of specific [ 3 H]-estradiol binding from the nuclear ER extract. This indicates that the test ligands have the potential to modify the ER function in the rainbow trout liver. Secondly, the ability of the test compounds to induce or inhibit vitellogenin production by primary cultures of rainbow trout hepatocytes was studied. Estradiol and DPN were the only compounds that induced a dose-dependent increase on vitellogenin synthesis. The lack of vitellogenin induction by PPT indicates that ERα could not have a role on this reproductive process whereas the ability of DPN to induce vitellogenin production supports the participation of ERβ. In addition, this hypothesis is reinforced by the results obtained from MPP plus estradiol. On one hand, the absence of suppressive activity of MPP in the estradiol-induced vitellogenin production does not support the participation of ERα. On the other hand, once blocked ERα with MPP, the only manifestation of agonist activity of estradiol would be achieved via ERβ. In conclusion, the present results indicate that vitellogenin production is mainly mediated through ERβ, implying, furthermore

Fecundity, 17ß-estradiol concentrations and expression of vitellogenin and estrogen receptor genes throughout the ovarian cycle in female Eastern mosquitofish from three lakes in Florida

DEFF Research Database (Denmark)

Kristensen, T.; Edwards, T. M.; Kohno, S.

2007-01-01

Previous studies of Eastern mosquitofish in contaminated Lake Apopka, Florida, have documented reduced sperm count and sexual behaviour in males but increased fecundity and liver weight in females, compared to nearby reference lakes. Liver weight can be an indicator of vitellogenin (Vtg) synthesis...... in fish, such as the mosquitofish. It was therefore hypothesized that estrogenic organochlorine pesticides, present at elevated concentrations in animals from Lake Apopka, could cause the reproductive disorders in males, as well as increase female fecundity. We initiated a test of this hypothesis...... by examining the relationship between 17β-estradiol (E2) tissue concentrations, hepatic estrogen receptor α (ERα) and Vtg A, B and C gene expression and fecundity in sexually mature female Eastern mosquitofish from Lake Apopka and two reference lakes, Lake Woodruff and Lake Orange. We observed that female...

Potential use of Vitellogenin and Zona radiata proteins as biomarkers of endocrine disruption in Peregrine falcon exposed to organochlorine compounds (DDTs, PCBs, PCDDs and PCDFs)

Energy Technology Data Exchange (ETDEWEB)

Jimenez, B. [CSIC, Inst. of Organic Chemistry, Madrid (Spain); Mori, G.; Concejero, M.A.; Casini, S.; Fossi, M.C. [Siena Univ. (Italy)

2004-09-15

Many different classes of environmental contaminants such as industrial chemicals (e.g. alkylphenols, polychlorinated biphenyls, pesticides, PAHs, polychlorinated dibenzo-p-dioxins, and dibenzofurans), ''can cause adverse effects in the reproductive functions of intact organisms or their progenies, consequent to changes in endocrine functions'' showing a so-called Endocrine disruptor activity. Avian raptor species, such as peregrine falcon (Falco peregrinus) for their peculiar position in the food web are potentially at risk in relation to the accumulation of Persistent Organic Pollutants (POPs) and toxic metals. Recent studies carried out with Peregrine falcon (Falco peregrinus) in Spain reveal a contamination with organochlorine compounds (PCDDs, PCDFs, PCBs and DDTs) which could be responsible of the decrease of successful pairs observed during the last ten years. Thus there is a need to develop sensitive diagnostic monitoring tools for the evaluation of toxicological risk and potential effects on the reproductive function and population dynamic of avian top predator species. Two markers for the detection of EDs effects in oviparous vertebrates are induction of Vitellogenin (Vtg) and Zona Radiata Proteins (ZR). Vtg, a complex phospholipoglycoprotein, is the major egg-yolk protein precursor and is normally synthesized by females in response to estradiol. ZR together with Zona Pellucida (ZP) constitutes in birds part of the eggshell. These proteins (Vtg, ZR and ZP) are normally synthesised in the liver as a response to an estrogen signal given by Estradiol. Males and sexually undifferentiated specimens also have the Vtg and ZR genes but do not express them unless exposed to estrogenic compounds. The main aim of this preliminary study was to develop methods for the detection of Vtg and ZR in plasma obtained from peregrine falcon as a specific biomarker for the evaluation of the effects of EDCs.

Comparing the effects of tetrabromobisphenol-A, bisphenol A, and their potential replacement alternatives, TBBPA-bis(2,3-dibromopropyl ether) and bisphenol S, on cell viability and messenger ribonucleic acid expression in chicken embryonic hepatocytes.

Science.gov (United States)

Ma, Melissa; Crump, Doug; Farmahin, Reza; Kennedy, Sean W

2015-02-01

A market for alternative brominated flame retardants (BFRs) has emerged recently due to the phase out of persistent and inherently toxic BFRs. Several of these replacement compounds have been detected in environmental matrices, including wild birds. A chicken embryonic hepatocyte (CEH) assay was utilized to assess the effects of the BFR, tetrabromobisphenol-A (TBBPA), and its replacement alternative, tetrabromobisphenol A bis(2,3-dibromopropyl ether [TBBPA-DBPE]) on cell viability and messenger ribonucleic acid (mRNA) expression. Bisphenol A (BPA) and 1 of its replacement alternatives, bisphenol S (BPS), were also screened for effects. Both TBBPA and BPA decreased CEH viability with calculated median lethal concentration (LC50) values of 40.6 μM and 61.7 μM, respectively. However, the replacement alternatives, TBBPA-DBPE and BPS, did not affect cell viability (up to 300 μM). Effects on mRNA expression were determined using an Avian ToxChip polymerse chain reaction (PCR) array and a real-time (RT)-PCR assay for the estrogen-responsive genes, apolipoproteinII (ApoII) and vitellogenin (Vtg). A luciferase reporter gene assay was used to assess dioxin-like effects. Tetrabromobisphenol-A altered mRNA levels of 4 genes from multiple toxicity pathways and increased luciferase activity in the luciferase reporter gene assay, whereas its alternative, TBBPA-DBPE, only altered 1 gene on the array, Cyp1a4, and increased luciferase activity. At 300 μM, a concentration that decreased cell viability for TBBPA and BPA, the BPA replacement, BPS, altered the greatest number of transcripts, including both ApoII and Vtg. Bisphenol A exposure did not alter any genes on the array but did up-regulate Vtg at 10 μM. Characterization of the potential toxicological and molecular-level effects of these compounds will ideally be useful to chemical regulators tasked with assessing the risk of new and existing chemicals. © 2014 SETAC.

Mechanisms of Egg Yolk Formation and Implications on Early Life History of White Perch (Morone americana.

Directory of Open Access Journals (Sweden)

Justin Schilling

Full Text Available The three white perch (Morone americana vitellogenins (VtgAa, VtgAb, VtgC were quantified accurately and precisely in the liver, plasma, and ovary during pre-, early-, mid-, and post-vitellogenic oocyte growth using protein cleavage-isotope dilution mass spectrometry (PC-IDMS. Western blotting generally mirrored the PC-IDMS results. By PC-IDMS, VtgC was quantifiable in pre-vitellogenic ovary tissues and VtgAb was quantifiable in pre-vitellogenic liver tissues however, neither protein was detected by western blotting in these respective tissues at this time point. Immunohistochemistry indicated that VtgC was present within pre-vitellogenic oocytes and localized to lipid droplets within vitellogenic oocytes. Affinity purification coupled to tandem mass spectrometry using highly purified VtgC as a bait protein revealed a single specific interacting protein (Y-box binding protein 2a-like [Ybx2a-like] that eluted with suramin buffer and confirmed that VtgC does not bind the ovary vitellogenin receptors (LR8 and Lrp13. Western blotting for LR8 and Lrp13 showed that both receptors were expressed during vitellogenesis with LR8 and Lrp13 expression highest in early- and mid-vitellogenesis, respectively. The VtgAa within the ovary peaked during post-vitellogenesis, while VtgAb peaked during early-vitellogenesis in both white perch and the closely related striped bass (M. saxatilis. The VtgC was steadily accumulated by oocytes beginning during pre-vitellogenesis and continued until post-vitellogenesis and its composition varies widely between striped bass and white perch. In striped bass, the VtgC accounted for 26% of the vitellogenin-derived egg yolk, however in the white perch it comprised only 4%. Striped bass larvae have an extended developmental window and these larvae have yolk stores that may enable them to survive in the absence of food for twice as long as white perch after hatch. Thus, the VtgC may play an integral role in providing nutrients to

Characterization of plasma vitellogenin and sex hormone concentrations during the annual reproductive cycle of the endangered razorback sucker

Science.gov (United States)

Hinck, Jo Ellen; Papoulias, Diana M.; Annis, Mandy L.; Tillitt, Donald E.; Marr, Carrie; Denslow, Nancy D.; Kroll, Kevin J.; Nachtmann, Jason

2011-01-01

Population declines of the endangered razorback sucker Xyrauchen texanus in the Colorado River basin have been attributed to predation by and competition with nonnative fishes, habitat alteration, and dam construction. The reproductive health and seasonal variation of the reproductive end points of razorback sucker populations are currently unknown. Using nonlethal methods, we characterized the plasma hormonal fluctuations of reproductively mature female and male razorback suckers over a 12-month period in a hatchery by measuring their vitellogenin (VTG) and three sex hormones: 17β-estradiol (E2), testosterone (T), and 11-ketotestosterone (KT). Fish were identified as reproductive or nonreproductive based on their body weight, VTG, and sex hormone profiles. In reproductive females, the E2 concentration increased in the fall and winter, and increases in T and VTG concentrations were generally associated with the spawning period. Mean T concentrations were consistently greater in reproductive females than in nonreproductive females, but this pattern was even more pronounced during the spawning period (spring). Consistently low T concentrations (the spawning period may indicate reproductive impairment. In reproductive males, spring increases in KT and T concentrations were associated with spawning; concentrations of E2 (the study. In addition, the E2 : KT ratio and T were the best metrics by which to distinguish female from male adult razorback suckers throughout the year. These metrics of reproductive health and condition may be particularly important to recovery efforts of razorback suckers given that the few remaining wild populations are located in a river where water quality and quantity issues are well documented. In addition to the size, age, and recruitment information currently considered in the recovery goals of this endangered species, reproductive end points could be included as recovery metrics with which to monitor seasonal trends and determine whether

Effects of estrogen exposure in mussels, Mytilus edulis, at different stages of gametogenesis

Energy Technology Data Exchange (ETDEWEB)

Ciocan, Corina M.; Cubero-Leon, Elena; Puinean, Alin M.; Hill, Elizabeth M. [Department of Biology and Environmental Science, School of Life Sciences, University of Sussex, Falmer, Brighton BN1 9QJ (United Kingdom); Minier, Christophe [Laboratoire d' Ecotoxicologie, Universite du Havre, 25 Rue Philippe Lebon, BP540, 766058 Le Havre (France); Osada, Makoto [Laboratory of Aquacultural Biology, Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori-amamiyamachi, Sendai 981-8555 (Japan); Fenlon, Kate [Department of Biology and Environmental Science, School of Life Sciences, University of Sussex, Falmer, Brighton BN1 9QJ (United Kingdom); Rotchell, Jeanette M., E-mail: j.rotchell@sussex.ac.u [Department of Biology and Environmental Science, School of Life Sciences, University of Sussex, Falmer, Brighton BN1 9QJ (United Kingdom)

2010-09-15

Mytilus edulis were exposed to 17{beta}-estradiol (E2) and the synthetic estrogens ethinyl estradiol (EE2) and estradiol benzoate (EB) for 10 days. Two exposures were performed to determine their effect on vitellogenin (VTG) and estrogen receptor 2 (ER2) mRNA expression at different stages of the reproductive cycle. Significant natural variation was not observed in VTG mRNA expression, though ER2 mRNA expression displayed significantly lower values during January, February and July compared with other times of the year. A significant increase in VTG and ER2 mRNA expression was observed in mussels exposed to estrogens at the early stage of gametogenesis. In contrast, mature mussels displayed no statistically significant change in the VTG or ER2 mRNA expression. The data presented suggests that the reproductive physiology of molluscs, in terms of VTG and ER2 mRNA expression, may be susceptible to damage by environmental estrogens at certain points in their gametogenesis process. - This study concerns vitellogenin and estrogen receptor mRNA expression in a mollusc and is relevant to those studying endocrine disruption in invertebrate species.

Sex assignment of lake sturgeon (Acipenser fluvescens) based on plasma sex hormone and vitellogenin levels

Science.gov (United States)

Craig, J.M.; Papoulias, D.M.; Thomas, M.V.; Annis, M.L.; Boase, J.

2009-01-01

This study focused on identifying the sex of lake sturgeon by measuring the sex hormones estradiol and testosterone, and the phosphoprotein vitellogenin (Vtg) in blood plasma by radioimmunoassay and enzyme-linked immunosorbent assay, respectively, and evaluating these techniques as tools in lake sturgeon population management. Surveys of the St Clair River (SCR) lake sturgeon population have characterized it as rebounding by having steady or increasing recruitment since 1997. However, researchers have not been able to effectively determine the sex for most of the sturgeon they capture because few fish caught during surveys are releasing gametes. A total of 115 fish were sampled from May through June in 2004 and 2005 from the SCR, Michigan, USA. Of these, only four females and eight males were verified (i.e. they were releasing gametes at time of capture), resulting in very few fish with which to validate blood hormone and Vtg biomarkers of sex. Fifty-six percent of the fish were assigned a sex designation based on biomarker criteria. Correspondence between actual gonadal sex and biomarker-directed classification was good for the small subset of fish for which gonadal sex was definitively determined. Moreover, application of the steroid values in a predictive sex assignment model developed for white sturgeon misclassified only the same two fish that were misclassified with the steroid and Vtg biomarkers. The experimental results suggest a sex ratio of 1 : 2.7 (F:M), however more conclusive methods are needed to confirm this ratio because so few fish were available for sex validation. Of the 43 males, 14 were within the legal slot limit, 11 were smaller than 1067 mm total length (TL), and 18 were larger than 1270 mm TL. All 15 females were larger than 1270 mm TL, and thus protected by the slot limit criteria. Considering that lake sturgeon are threatened in Michigan, an advantage to using blood plasma assays was that fish were not harmed, and sample collection was

ESTRADIOL-INDUCED SYNTHESIS OF VITELLOGENIN .3. ISOLATION AND CHARACTERIZATION OF VITELLOGENIN MESSENGER-RNA FROM AVIAN LIVER

NARCIS (Netherlands)

AB, G.; Roskam, W. G.; Dijkstra, J.; Mulder, J.; Willems, M.; van der Ende, A.; Gruber, M.

1976-01-01

The messenger RNA of the hormone-induced protein vitellogenin was isolated from the liver of estrogen-treated roosters. Starting from total polysomal RNA, the vitellogenin messenger was purified 67-fold by oligo (dT)-cellulose chromatography and sizing on a sucrose gradient. The messenger was

The induction of vitellogenin mRNA in avian liver

NARCIS (Netherlands)

Boogaart, Paul van den

1980-01-01

Vitellogenine is het precursor eiwit van de belangrijkste dooiereiwitten lipovitelline en fosvitine; vitellogenine wordt gemaakt in de lever van leggende hennen onder invloed van oestrogenen. In hanen kan de synthese van vitellogenine worden geïnduceerd door inspuiting van 17B-oestradiol. Na

Protein incorporation by isolated amphibian oocytes. VI. Comparison of autologous and xenogeneic vitellogenins

Energy Technology Data Exchange (ETDEWEB)

Wallace, R A; Deufel, R A; Misulovin, Z

1980-01-01

1. Labeled vitellogenins were isolated from the sera of several amphibians, a turtle, and a pigeon, and were incubated in vitro with oocytes from Xenopus laevis and Rana pipiens. 2. Oocytes from X. laevis sequestered vitellogenin from salamanders, turtle, and pigeon at rates comparable to that for autologous vitellogenin, while anuran vitellogenins were sequestered at somewhat lower rates. 3. Oocytes from R. pipiens sequestered X. laevis vitellogenin at a rate comparable to autologous vitellogenin, while salamander, turtle, and pigeon vitellogenins were sequestered at faster rates. 4. All vitellogenins examined appear to be recognized and incorporated specifically by X. laevis and R. pipiens oocytes.

Efficiency of advanced wastewater treatment technologies for the reduction of hormonal activity in effluents and connected surface water bodies by means of vitellogenin analyses in rainbow trout (Oncorhynchus mykiss) and brown trout (Salmo trutta f. fario).

Science.gov (United States)

Henneberg, Anja; Triebskorn, Rita

2015-01-01

Endocrine effects in the aquatic environment are in the focus of scientists and media along with debates on the necessity of further steps in wastewater treatment. In the present study VTG responses were compared to evaluate upgrades at wastewater treatment plants (WWTPs). We investigated several advanced sewage treatment technologies at two WWTPs connected to the Schussen, a tributary of Lake Constance, for the reduction of hormonal activity: (1) a powdered activated charcoal filter at the WWTP Langwiese; and (2) a combination of ozonation, sand filter, and granulated activated carbon filter at the WWTP Eriskirch. Rainbow trout and brown trout were either directly exposed to the effluents in aquaria or cages, or in a bypass system flown through by surface water of the Schussen. As a reference, trout were kept in bypass aquaria at the Argen River, which is less influenced by micropollutants. As a biomarker for estrogenicity, we analyzed the yolk precursor protein vitellogenin in immature rainbow trout and brown trout and in trout larvae (100 days post-fertilization) prior to and after the upgrade with the new technologies. Trout of different ages and species were used to detect differences in their sensitivity. At both bypass stations, larvae of brown trout showed significantly higher vitellogenin levels prior to the upgrade compared to negative control levels. Female brown trout exposed at the bypass station downstream of the WWTP showed decreased vitellogenin levels after the upgrade. In 1-year-old immature trout directly exposed to the respective effluents, no significant effects of the upgrades on vitellogenin levels were found. In general, larger effects were observed in brown trout than in rainbow trout, indicating that they are more sensitive test organisms.

Effect of TBT and PAHs on CYP1A, AhR and Vitellogenin Gene Expression in the Japanese Eel, Anguilla japonica.

Science.gov (United States)

Choi, Min Seop; Kwon, Se Ryun; Choi, Seong Hee; Kwon, Hyuk Chu

2012-12-01

Gene expressions of cytochrome P4501A (CYP1A), aryl hydrocarbon receptor (AhR) and vitellogenin (Vg) by endocrine disruptors, benzo[α]pyrene (B[a]P) and tributyltin (TBT) were examined in cultured eel hepatocytes which were isolated from eels treated previously with B[a]P (10 mg/kg) or estradiol-17β (20 mg/kg) in vivo, and the relationship between CYP1A, AhR and Vg genes were studied. When the cultured eel hepatocytes were treated with B[a]P (10(-6)-10(-5) M) the gene expressions of CYP1A and AhR were enhanced in a concentration-dependent manner. However, when treated with TBT (10(-9)-10(-5) M) the gene expressions of CYP1A and AhR were suppressed at high concentrations (10(-6)-10(-5) M), while having no effects at low concentrations (10(-9)-10(-7) M). Gene expression of Vg was also suppressed by TBT in a concentration-dependent manner in cultured eel hepatocytes which was previously treated in vivo with estradiol-17β.

Plasma vitellogenin in Morelet's crocodiles from contaminated habitats in northern Belize

International Nuclear Information System (INIS)

Rainwater, Thomas R.; Selcer, Kyle W.; Nespoli, Lisa M.; Finger, Adam G.; Ray, David A.; Platt, Steven G.; Smith, Philip N.; Densmore, Llewellyn D.; Anderson, Todd A.; McMurry, Scott T.

2008-01-01

Vitellogenin induction has been widely used as a biomarker of endocrine disruption in wildlife, but few studies have investigated its use in wild reptiles living in contaminated habitats. This study examined vitellogenin induction in Morelet's crocodiles (Crocodylus moreletii) from wetlands in northern Belize contaminated with organochlorine (OC) pesticides. Vitellogenin was measured in 381 crocodile plasma samples using a vitellogenin ELISA previously developed for this species. Vitellogenin was detected in nine samples, all from adult females sampled during the breeding season. Males and juvenile females did not contain detectable levels of vitellogenin; however, many of these animals contained OC pesticides in their caudal scutes, confirming contaminant exposure. The lack of a vitellogenic response in these animals may be attributable to several factors related to the timing and magnitude of exposure to endocrine-disrupting chemicals and should not be interpreted as an absence of other contaminant-induced biological responses. - Wild crocodiles living in habitats polluted with organochlorine pesticides did not exhibit contaminant-induced vitellogenin induction in blood plasma

Effects of the UV-filter 2-ethyl-hexyl-4-trimethoxycinnamate (EHMC) on expression of genes involved in hormonal pathways in fathead minnows (Pimephales promelas) and link to vitellogenin induction and histology

International Nuclear Information System (INIS)

Christen, Verena; Zucchi, Sara; Fent, Karl

2011-01-01

UV-filters are increasingly used in cosmetics and in the protection of materials against UV-irradiation, and ultimately they reach aquatic systems. The lipophilic UV-filter 2-ethyl-hexyl-4-trimethoxycinnamate (EHMC) belongs to one of the most frequently used UV-filters and accumulates in aquatic animals. Despite its ubiquitous presence in water and biota, very little is known about its potential hormonal effects on aquatic organisms. In our study, we evaluated the effects of measured water concentration of 5.4, 37.5, 244.5 and 394 μg/L EHMC on the expression of genes involved in hormonal pathways in the liver, testis and brain of male and female fathead minnows (Pimephales promelas). We compare the transcription profile with the plasma vitellogenin (VTG) content, secondary sex characteristics, and gonad histology. Transcripts of the androgen receptor (ar) were significantly down-regulated in the liver of females at 37.5, 244.5 μg/L and 394 μg/L EHMC. Additionally, the 3β-hydroxysteroid dehydrogenase (3β-HSD) transcript was significantly decreased in the liver of males at 37.5, 244.5 and 394 μg/L EHMC, and at 244.5 and 394 μg/L EHMC in females. The expressional changes were tissue-specific in most cases, being most significant in the liver. Vitellogenin plasma concentration was significantly increased at 244.5 μg/L EHMC in males. EHMC induced significant histological changes in testes and ovaries at 394 μg/L. Testes displayed a decrease in spermatocytes, and ovaries a decrease in previtellogenic oocytes. The induction of VTG plasma concentration and the histological changes in gonads suggest an estrogenic and/or antiandrogenic activity of EHMC. On the other hand, the gene expression profile shows an antiestrogenic (e.g.: down-regulation of esr1) activity of EHMC. In conclusion, our data demonstrate that EHMC displays low but multiple hormonal activities in fish.

Effects of the UV-filter 2-ethyl-hexyl-4-trimethoxycinnamate (EHMC) on expression of genes involved in hormonal pathways in fathead minnows (Pimephales promelas) and link to vitellogenin induction and histology

Energy Technology Data Exchange (ETDEWEB)

Christen, Verena; Zucchi, Sara [University of Applied Sciences Northwestern Switzerland, School of Life Sciences, Gruendenstrasse 40, CH-4132 Muttenz (Switzerland); Fent, Karl, E-mail: karl.fent@fhnw.ch [University of Applied Sciences Northwestern Switzerland, School of Life Sciences, Gruendenstrasse 40, CH-4132 Muttenz (Switzerland); Swiss Federal Institute of Technology Zuerich (ETH Zuerich), Department of Environmental Sciences, 8092 Zuerich (Switzerland)

2011-04-15

UV-filters are increasingly used in cosmetics and in the protection of materials against UV-irradiation, and ultimately they reach aquatic systems. The lipophilic UV-filter 2-ethyl-hexyl-4-trimethoxycinnamate (EHMC) belongs to one of the most frequently used UV-filters and accumulates in aquatic animals. Despite its ubiquitous presence in water and biota, very little is known about its potential hormonal effects on aquatic organisms. In our study, we evaluated the effects of measured water concentration of 5.4, 37.5, 244.5 and 394 {mu}g/L EHMC on the expression of genes involved in hormonal pathways in the liver, testis and brain of male and female fathead minnows (Pimephales promelas). We compare the transcription profile with the plasma vitellogenin (VTG) content, secondary sex characteristics, and gonad histology. Transcripts of the androgen receptor (ar) were significantly down-regulated in the liver of females at 37.5, 244.5 {mu}g/L and 394 {mu}g/L EHMC. Additionally, the 3{beta}-hydroxysteroid dehydrogenase (3{beta}-HSD) transcript was significantly decreased in the liver of males at 37.5, 244.5 and 394 {mu}g/L EHMC, and at 244.5 and 394 {mu}g/L EHMC in females. The expressional changes were tissue-specific in most cases, being most significant in the liver. Vitellogenin plasma concentration was significantly increased at 244.5 {mu}g/L EHMC in males. EHMC induced significant histological changes in testes and ovaries at 394 {mu}g/L. Testes displayed a decrease in spermatocytes, and ovaries a decrease in previtellogenic oocytes. The induction of VTG plasma concentration and the histological changes in gonads suggest an estrogenic and/or antiandrogenic activity of EHMC. On the other hand, the gene expression profile shows an antiestrogenic (e.g.: down-regulation of esr1) activity of EHMC. In conclusion, our data demonstrate that EHMC displays low but multiple hormonal activities in fish.

Effects of the UV-filter 2-ethyl-hexyl-4-trimethoxycinnamate (EHMC) on expression of genes involved in hormonal pathways in fathead minnows (Pimephales promelas) and link to vitellogenin induction and histology.

Science.gov (United States)

Christen, Verena; Zucchi, Sara; Fent, Karl

2011-04-01

UV-filters are increasingly used in cosmetics and in the protection of materials against UV-irradiation, and ultimately they reach aquatic systems. The lipophilic UV-filter 2-ethyl-hexyl-4-trimethoxycinnamate (EHMC) belongs to one of the most frequently used UV-filters and accumulates in aquatic animals. Despite its ubiquitous presence in water and biota, very little is known about its potential hormonal effects on aquatic organisms. In our study, we evaluated the effects of measured water concentration of 5.4, 37.5, 244.5 and 394 μg/L EHMC on the expression of genes involved in hormonal pathways in the liver, testis and brain of male and female fathead minnows (Pimephales promelas). We compare the transcription profile with the plasma vitellogenin (VTG) content, secondary sex characteristics, and gonad histology. Transcripts of the androgen receptor (ar) were significantly down-regulated in the liver of females at 37.5, 244.5 μg/L and 394 μg/L EHMC. Additionally, the 3β-hydroxysteroid dehydrogenase (3β-HSD) transcript was significantly decreased in the liver of males at 37.5, 244.5 and 394 μg/L EHMC, and at 244.5 and 394 μg/L EHMC in females. The expressional changes were tissue-specific in most cases, being most significant in the liver. Vitellogenin plasma concentration was significantly increased at 244.5 μg/L EHMC in males. EHMC induced significant histological changes in testes and ovaries at 394 μg/L. Testes displayed a decrease in spermatocytes, and ovaries a decrease in previtellogenic oocytes. The induction of VTG plasma concentration and the histological changes in gonads suggest an estrogenic and/or antiandrogenic activity of EHMC. On the other hand, the gene expression profile shows an antiestrogenic (e.g.: down-regulation of esr1) activity of EHMC. In conclusion, our data demonstrate that EHMC displays low but multiple hormonal activities in fish. Copyright © 2011 Elsevier B.V. All rights reserved.

DNA methylation induced changes in chromatin conformation of the promoter of the vitellogenin II gene of Japanese quail during aging.

Science.gov (United States)

Gupta, Sanjay; Pathak, Rashmi U; Kanungo, Madhu S

2006-08-01

One approach to the understanding of the molecular basis of aging in higher organisms may be to use genes whose timing and rate of expression during the life span run parallel with specific functions that can be monitored. The genes for egg proteins, such as vitellogenin (VTG), which is expressed in the liver, and ovalbumin, lysozyme etc. that are expressed in the oviduct of birds, meet these requirements. Egg laying function is dependent on the production of these proteins, which, in turn, depends on the expression of their genes. In this communication we present the age-related studies on the VTG II gene of the bird, Japanese quail. The gene is expressed only in the liver and its expression is considerably lower in old birds that do not lay eggs. Comparison of the promoter region of the gene carrying the two important cis-acting elements, estrogen responsive element (ERE) and progesterone responsive element (PRE), shows it to be 100% homologous to the corresponding region of the chicken VTG II gene. Methylation of DNA and conformation of chromatin of this region were studied, as they are known to be important for regulation of expression of genes. Our studies show that in the liver of adult female quails which lay eggs, a -CCGG- sequence located in this region is hypomethylated, and the chromatin encompassing this region of the gene is relaxed. In the old, the -CCGG- sequence is hypermethylated and the chromatin is compact. This is correlated with a decrease in the expression of the gene and decrease in egg production. Further, electrophoretic mobility shift assay (EMSA) shows that the levels/affinity of specific trans-acting factors that bind to ERE and PRE present in the region, are not different in adult and old birds. Hence the methylation status of the -CCGG- sequence that is located in-between the ERE and the PRE may be crucial for the conformation of chromatin and availability of these two important cis-acting elements for the binding of the trans

Control of transcriptional repression of the vitellogenin receptor gene in largemouth bass (Micropterus salmoides) by select estrogen receptors isotypes.

Science.gov (United States)

Dominguez, Gustavo A; Bisesi, Joseph H; Kroll, Kevin J; Denslow, Nancy D; Sabo-Attwood, Tara

2014-10-01

The vitellogenin receptor (Vtgr) plays an important role in fish reproduction. This receptor functions to incorporate vitellogenin (Vtg), a macromolecule synthesized and released from the liver in the bloodstream, into oocytes where it is processed into yolk. Although studies have focused on the functional role of Vtgr in fish, the mechanistic control of this gene is still unexplored. Here we report the identification and analysis of the first piscine 5' regulatory region of the vtgr gene which was cloned from largemouth bass (Micropterus salmoides). Using this putative promoter sequence, we investigated a role for hormones, including insulin and 17β-estradiol (E2), in transcriptional regulation through cell-based reporter assays. No effect of insulin was observed, however, E2 was able to repress transcriptional activity of the vtgr promoter through select estrogen receptor subtypes, Esr1 and Esr2a but not Esr2b. Electrophoretic mobility shift assay demonstrated that Esr1 likely interacts with the vtgr promoter region through half ERE and/or SP1 sites, in part. Finally we also show that ethinylestradiol (EE2), but not bisphenol-A (BPA), represses promoter activity similarly to E2. These results reveal for the first time that the Esr1 isoform may play an inhibitory role in the expression of LMB vtgr mRNA under the influence of E2, and potent estrogens such as EE2. In addition, this new evidence suggests that vtgr may be a target of select endocrine disrupting compounds through environmental exposures. © The Author 2014. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Primary induction of vitellogenin mRNA in the rooster by 17beta-estradiol.

Science.gov (United States)

Burns, A T; Deeley, R G; Gordon, J I; Udell, D S; Mullinix, K P; Goldberger, R F

1978-01-01

We have studied the kinetics of vitellogenin mRNA accumulation in rooster liver after a primary injection of 17beta-estradiol. The levels of vitellogenin mRNA have been determined both by hybridization of total cellular RNA to vitellogenin cDNA and by translation of vitellogenin mRNA in a wheat germ cell-free system. The results obtained by both methods of analysis are in good agreement and indicate that vitellogenin mRNA is present in the liver of normal roosters at a level of 0-5 molecules per liver cell and increases in amount during the 3 days following injection of estrogen, reaching a level of almost 6000 molecules per cell at the peak of the response. The level of vitellogenin mRNA declined exponentially during the next 14 days with a half-life of 29 hr, reaching a level of less than 10 molecules per cell at 17 days after injection of the hormone. The levels of vitellogenin mRNA after stimulation with estrogen have been correlated with the in vivo rate of synthesis of the vitellogenin polypeptide. The results indicate that the rate of vitellogenin synthesis is closely correlated with the level of vitellogenin mRNA. On the basis of these findings, we conclude that vitellogenin mRNA does not exist in the liver in an untranslated form after withdrawal from estrogen. PMID:273910

In Vivo Anti-estrogenic Effects of Menadione on Hepatic Estrogen-responsive Gene Expression in Male Medaka (Oryzias latipes)

OpenAIRE

Yamaguchi, Akemi; Kohra, Shinya; Ishibashi, Hiroshi; Arizono, Koji; Tominaga, Nobuaki

2008-01-01

Menadione, a synthetic vitamin K3, exhibits anti-estrogenic activity on in vitro assay. However, the in vivo anti-estrogenic effects of menadione have not been determined, while correlations between biological effects and structural changes are unclear. Thus, we investigated the in vivo anti-estrogenic activity of menadione under fluorescent light and dark conditions. Suppression of the hepatic estrogen response genes vitellogenin1 (VTG1), VTG2 and estrogen receptor-α (ER-α) was used as an in...

Excision of foreign gene product with cathepsin D in chicken hepatoma cell line

International Nuclear Information System (INIS)

Sato, Masaharu; Kawashima, Tsuyoshi; Aosasa, Masayoshi; Horiuchi, Hiroyuki; Furusawa, Shuichi; Matsuda, Haruo

2005-01-01

To easily and rapidly recover exogenous gene products from chicken egg yolk, we constructed pVTG-catD (VTG, vitellogenin; catD, cathepsin D), a vector cassette carrying two catD-recognition signal peptides (catD-RSPs) in addition to the cloning site. An enhanced green fluorescence protein (EGFP)-encoding DNA fragment was ligated into the pVTG-catD. When the resultant construct pVTG-EGFP-catD containing histidine- and myc-tags was transfected into the chicken hepatoma cell line LMH, EGFP-expression at 24 h post-cultivation was confirmed by fluorescence microscopy. Because a signal peptide (NTVLAEF) encoded in pVTG-EGFP-catD is recognized by catD, the VTG-EGFP fusion protein digested with catD was detectable by Western blotting. Digested exogenous gene product was recovered with nickel resin. These results indicate that catD-recognition sites bearing pVTG-catD and His-tags are functional in chicken LMH cells. Therefore, the system described here may be of use in making excision exogenous gene products in the chicken and in creating homozygous knock-in chickens

Vitellogenin RNAi halts ovarian growth and diverts reproductive proteins and lipids in young grasshoppers.

Science.gov (United States)

Tokar, Derek R; Veleta, Katherine A; Canzano, Joseph; Hahn, Daniel A; Hatle, John D

2014-11-01

Reduced reproduction extends lifespan of females in many animals. To test the effects of reproduction on storage of macronutrients, we block reproductive output in the lubber grasshopper by injecting RNAi against the precursor to egg-yolk protein, vitellogenin, in early adulthood. Controls were injected with either buffer or RNAi against the major storage protein in the hemolymph, hexamerin-90. Vitellogenin RNAi greatly reduced both levels of mRNA for vitellogenin and ovarian growth, in comparison to both controls. Fat body mass was increased upon vitellogenin RNAi, but concentrations of the three hexameric storage proteins from the hemolymph were not. Surprisingly, hemolymph vitellogenin levels were increased upon vitellogenin RNAi. Total reproductive protein (hemolymph vitellogenin plus ovarian vitellin) was unchanged by vitellogenin RNAi, as reproductive protein was diverted to the hemolymph. Similarly, the increased lipid storage upon vitellogenin RNAi was largely attributable to the reduction in lipid in the ovary, due to decreased ovarian growth. A BLAST search revealed that the 515 bp sequence of vitellogenin used for RNAi had three 11 bp regions identical to the vitellogenin receptor of the cockroach Leucophaea maderae. This suggests that our treatment, in addition to reducing levels of vitellogenin transcript, may have also blocked transport of vitellogenin from the hemolymph to the ovary. This would be consistent with halted ovarian growth simultaneous with high levels of vitellogenin in the hemolymph. Nonetheless, the accumulation of vitellogenin, instead of hexameric storage proteins, is inconsistent with a simple model of the trade-off between reproduction and storage. This was observed in young females; future studies will address whether investment of proteins may shift to the soma as individuals age. Overall, our results suggest that blockage of reproduction in young grasshoppers redirects lipids to storage and reproductive proteins to the hemolymph

Testosterone-induced modulation of peroxisomal morphology and peroxisome-related gene expression in brown trout (Salmo trutta f. fario) primary hepatocytes.

Science.gov (United States)

Lopes, Célia; Malhão, Fernanda; Guimarães, Cláudia; Pinheiro, Ivone; Gonçalves, José F; Castro, L Filipe C; Rocha, Eduardo; Madureira, Tânia V

2017-12-01

Disruption of androgenic signaling has been linked to possible cross-modulation with other hormone-mediated pathways. Therefore, our objective was to explore effects caused by testosterone - T (1, 10 and 50μM) in peroxisomal signaling of brown trout hepatocytes. To study the underlying paths involved, several co-exposure conditions were tested, with flutamide - F (anti-androgen) and ICI 182,780 - ICI (anti-estrogen). Molecular and morphological approaches were both evaluated. Peroxisome proliferator-activated receptor alpha (PPARα), catalase and urate oxidase were the selected targets for gene expression analysis. The vitellogenin A gene was also included as a biomarker of estrogenicity. Peroxisome relative volumes were estimated by immunofluorescence, and transmission electron microscopy was used for qualitative morphological control. The single exposures of T caused a significant down-regulation of urate oxidase (10 and 50μM) and a general up-regulation of vitellogenin. A significant reduction of peroxisome relative volumes and smaller peroxisome profiles were observed at 50μM. Co-administration of T and ICI reversed the morphological modifications and vitellogenin levels. The simultaneous exposure of T and F caused a significant and concentration-dependent diminishing in vitellogenin expression. Together, the findings suggest that in the tested model, T acted via both androgen and estrogen receptors to shape the peroxisomal related targets. Copyright © 2017 Elsevier B.V. All rights reserved.

Plasma vitellogenin in Morelet's crocodiles from contaminated habitats in northern Belize

Energy Technology Data Exchange (ETDEWEB)

Rainwater, Thomas R. [Institute of Environmental and Human Health and Department of Environmental Toxicology, Texas Tech University, Box 41163, Lubbock, TX 79409-1163 (United States)], E-mail: thomas.rainwater@gmail.com; Selcer, Kyle W. [Department of Biological Sciences, Bayer School of Natural and Environmental Sciences, Duquesne University, Pittsburgh, PA 15282 (United States)], E-mail: selcer@duq.edu; Nespoli, Lisa M. [Department of Biological Sciences, Bayer School of Natural and Environmental Sciences, Duquesne University, Pittsburgh, PA 15282 (United States)], E-mail: nespoli345@duq.edu; Finger, Adam G. [Institute of Environmental and Human Health and Department of Environmental Toxicology, Texas Tech University, Box 41163, Lubbock, TX 79409-1163 (United States)], E-mail: agfinger@tiehh.ttu.edu; Ray, David A. [Department of Biological Sciences, Texas Tech University, Lubbock, TX 79409 (United States)], E-mail: david.ray@mail.wvu.edu; Platt, Steven G. [Department of Biology, P.O. Box C-64, Sul Ross State University, Alpine, TX 79832 (United States)], E-mail: splatt@sulross.edu; Smith, Philip N. [Institute of Environmental and Human Health and Department of Environmental Toxicology, Texas Tech University, Box 41163, Lubbock, TX 79409-1163 (United States)], E-mail: philip.smith@tiehh.ttu.edu; Densmore, Llewellyn D. [Department of Biological Sciences, Texas Tech University, Lubbock, TX 79409 (United States)], E-mail: lou.densmore@ttu.edu; Anderson, Todd A. [Institute of Environmental and Human Health and Department of Environmental Toxicology, Texas Tech University, Box 41163, Lubbock, TX 79409-1163 (United States)], E-mail: todd.anderson@tiehh.ttu.edu; McMurry, Scott T. [Institute of Environmental and Human Health and Department of Environmental Toxicology, Texas Tech University, Box 41163, Lubbock, TX 79409-1163 (United States)], E-mail: scott.mcmurry@tiehh.ttu.edu

2008-05-15

Vitellogenin induction has been widely used as a biomarker of endocrine disruption in wildlife, but few studies have investigated its use in wild reptiles living in contaminated habitats. This study examined vitellogenin induction in Morelet's crocodiles (Crocodylus moreletii) from wetlands in northern Belize contaminated with organochlorine (OC) pesticides. Vitellogenin was measured in 381 crocodile plasma samples using a vitellogenin ELISA previously developed for this species. Vitellogenin was detected in nine samples, all from adult females sampled during the breeding season. Males and juvenile females did not contain detectable levels of vitellogenin; however, many of these animals contained OC pesticides in their caudal scutes, confirming contaminant exposure. The lack of a vitellogenic response in these animals may be attributable to several factors related to the timing and magnitude of exposure to endocrine-disrupting chemicals and should not be interpreted as an absence of other contaminant-induced biological responses. - Wild crocodiles living in habitats polluted with organochlorine pesticides did not exhibit contaminant-induced vitellogenin induction in blood plasma.

Estrogenic response of bisphenol A in rainbow trout (Oncorhynchus mykiss)

DEFF Research Database (Denmark)

Lindholst, Christian; Pedersen, Knud Ladegaard; Pedersen, Søren Nørby

2000-01-01

Bisphenol A (BPA) previously shown to possess xenoestrogenic activities was administered to rainbow trout (Oncorhynchus mykiss) through a continuos flow system. The estrogenic response expressed as the induction of vitellogenin (VTG) synthesis was measured during 12 days of exposure, using a direct...

17beta-estradiol induced vitellogenesis is inhibited by cortisol at the post-transcriptional level in Arctic char (Salvelinus alpinus

Directory of Open Access Journals (Sweden)

Modig Carina

2004-09-01

Full Text Available Abstract This study was performed to investigate stress effects on the synthesis of egg yolk precursor, vitellogenin (Vtg in Arctic char (Salvelinus alpinus. In particular the effect of cortisol (F was determined since this stress hormone has been suggested to interfere with vitellogenesis and is upregulated during sexual maturation in teleosts. Arctic char Vtg was purified and polyclonal antibodies were produced in order to develop tools to study regulation of vitellogenesis. The Vtg antibodies were used to develop an enzyme-linked immunosorbent assay. The corresponding Vtg cDNA was cloned from a hepatic cDNA library in order to obtain DNA probes to measure Vtg mRNA expression. Analysis of plasma from juvenile Arctic char, of both sexes, exposed to different steroids showed that production of Vtg was induced in a dose dependent fashion by 17β-estradiol (E2, estrone and estriol. Apart from estrogens a high dose of F also upregulated Vtg. In addition, F, progesterone (P and tamoxifen were tested to determine these compounds ability to modulate E2 induced Vtg synthesis at both the mRNA and protein level. Tamoxifen was found to inhibit E2 induced Vtg mRNA and protein upregulation. P did not alter the Vtg induction while F reduced the Vtg protein levels without affecting the Vtg mRNA levels. Furthermore the inhibition of Vtg protein was found to be dose dependent. Thus, the inhibitory effect of F on Vtg appears to be mediated at the post-transcriptional level.

Effects of exposure to the xenoestrogen octylphenol and subsequent transfer to clean water on liver and gonad ultrastructure during early development of Zoarces viviparus embryos

DEFF Research Database (Denmark)

Jespersen, Åse; Rasmussen, Tina H.; Hirche, Majken

2010-01-01

) during exposure and after transfer to clean water (depuration). In the mother fish, significantly higher concentrations of plasma vitellogenin (vtg) and calcium were found in the two exposed groups, when compared with the C group after exposure and depuration. When compared, however, with the respective...

Plasma Vitellogenin in Free-Ranging Loggerhead Sea Turtles (Caretta caretta of the Northwest Atlantic Ocean

Directory of Open Access Journals (Sweden)

Kimberly Smelker

2014-01-01

Full Text Available Vitellogenin is the egg yolk precursor protein produced by oviparous vertebrates. As endogenous estrogen increases during early reproductive activity, hepatic production of vitellogenin is induced and is assumed to be complete in female sea turtles before the first nesting event. Until the present study, innate production of vitellogenin has not been described in free-ranging sea turtles. Our study describes circulating concentrations of vitellogenin in loggerhead sea turtles (Caretta caretta from the Northwest Atlantic Ocean. We collected blood samples from juveniles and adults via in-water captures off the coast of the Southeast USA from May to August, and from nesting females in June and July at Hutchinson Island, Florida. All samples were analyzed using an in-house ELISA developed specifically to measure Caretta caretta vitellogenin concentration. As expected, plasma vitellogenin declined in nesting turtles as the nesting season progressed, although it still remained relatively elevated at the end of the season. In addition, mean vitellogenin concentration in nesting turtles was 1,000 times greater than that measured in samples from in-water captures. Our results suggest that vitellogenesis may continue throughout the nesting season, albeit at a decreasing rate. Further, vitellogenin detected in turtles captured in-water may have resulted from exposure to endocrine disrupting chemicals.

Transfer of Immunity from Mother to Offspring Is Mediated via Egg-Yolk Protein Vitellogenin.

Directory of Open Access Journals (Sweden)

Heli Salmela

2015-07-01

Full Text Available Insect immune systems can recognize specific pathogens and prime offspring immunity. High specificity of immune priming can be achieved when insect females transfer immune elicitors into developing oocytes. The molecular mechanism behind this transfer has been a mystery. Here, we establish that the egg-yolk protein vitellogenin is the carrier of immune elicitors. Using the honey bee, Apis mellifera, model system, we demonstrate with microscopy and western blotting that vitellogenin binds to bacteria, both Paenibacillus larvae--the gram-positive bacterium causing American foulbrood disease--and to Escherichia coli that represents gram-negative bacteria. Next, we verify that vitellogenin binds to pathogen-associated molecular patterns; lipopolysaccharide, peptidoglycan and zymosan, using surface plasmon resonance. We document that vitellogenin is required for transport of cell-wall pieces of E. coli into eggs by imaging tissue sections. These experiments identify vitellogenin, which is distributed widely in oviparous species, as the carrier of immune-priming signals. This work reveals a molecular explanation for trans-generational immunity in insects and a previously undescribed role for vitellogenin.

Application of Mass Spectrometry for the Analysis of Vitellogenin, a Unique Biomarker for Xenobiotic Compounds

Science.gov (United States)

Cohen, Alejandro M.; Banoub, Joseph H.

Vitellogenin is a complex phosphoglycolipoprotein that is secreted into the bloodstream of sexually mature, female, oviparous animals in response to circulating estrogens. It is then incorporated into the ovaries by receptor mediated endocytosis, where it is further cleaved to form the major constituents of the egg yolk proteins. It is generally accepted that these protein and peptide products serve as the main nutritional reserve for the developing embryo. Quantification of vitellogenin in blood is useful for different purposes. The reproductive status and degree of sexual maturation of oviparous animals can be assessed according to the levels of vitellogenin in plasma. The expression of this protein can also be induced in males under the effect of estrogenic compounds. Relying on this observation, vitellogenin has been used as a unique biomarker of environmental endocrine disruption in many species. In this respect, vitellogenin levels could potentially be used to assess the use of chemical warefare compounds with estrogenic activity. In this paper we review a technique developed for measuring vitellogenin plasma levels of different fish species using high performance liquid chromatography coupled to tandem mass spectrometry.

The temporal pattern of vitellogenin synthesis in Drosophila grimshawi

International Nuclear Information System (INIS)

Kambysellis, M.P.; Hatzopoulos, P.; Craddock, E.M.

1989-01-01

The temporal pattern of protein production and, in particular, vitellogenin protein synthesis during the sexual maturation of Drosophila grimshawi females has been studied in vivo by briefly feeding the flies with 35S-methionine and 3H-amino acids. The overall level of incorporation was very low in young flies; it then progressively increased to reach a maximum with the onset of sexual maturity at 13-15 days. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses revealed three classes of proteins: those synthesized throughout the age spectrum, which constitute the majority of protein species; proteins synthesized primarily or only in young flies; and proteins synthesized only by the older flies. In this Drosophila species, the three vitellogenins (V1, V2, and V3) appeared to be synthesized in a two-phase pattern. In the first phase, small quantities of V1 and V2 were detected immunologically in the fat body and hemolymph of newly emerged and 1 day-old flies. These proteins did not accumulate in the hemolymph or the ovaries, apparently being unstable proteins. The second phase commenced in early vitellogenesis (7-9 days of age) with synthesis in the fat body of small quantities of V1 and V2, followed by V3 proteins. These proteins were secreted and accumulated in the hemolymph and 24 h later were found in the ovaries. Their quantities increased rapidly and a steady state of synthesis, release into the hemolymph, and uptake by the ovaries was reached by days 13-15. We have estimated that during the steady state of vitellogenin synthesis, a fly can synthesize in 24 h at least 152 micrograms of vitellogenins, which is more than 2% of its body weight, at an average rate of about 6.3 micrograms vitellogenins/h. About 2 micrograms of this are synthesized in the fat body, and about 4 micrograms in the ovaries

Vitellogenin messenger rna in rooster liver

NARCIS (Netherlands)

Bos, Ebo Sijbren

1975-01-01

The investigations described in this thesis were carried out as a part of the studies in our laboratory on the control of gene expression in animal cells. They represent an example of the hormonal regulation of protein synthesis, viz. the induction of vitellogenin synthesis in rooster liver by the

Plasma levels of vitellogenin in Chrysemys picta during the annual gonadal cycle: Measurement by specific radioimmunoassay

International Nuclear Information System (INIS)

Gapp, D.A.; Ho, S.M.; Callard, I.P.

1979-01-01

A RIA for turtle (Chrysemys picta) vitellogenin is described. After dimethylformamide precipitation of vitellogenin from the plasma of estrogen-treated female turtles, antibodies were developed in rabbits. The dimethylformamide precipitate was further purified by o-triethylaminoethyl cellulose column chromatography; the vitellogenin component eluted as a single peak. This material was used for iodination by a mild chloramine method. Antibodies to turtle vitellogenin did not cross-react with plasma from male turtles or vitellogenic females of other vertebrate groups, including lizards and snakes. Limited cross-reactivity exists among the chelonians, however. Using a 1:5000 dilution of antiserum, the limit of detection was 15 ng, and the midrange was 320 +- 45 ng. For an antiserum dilution of 1:1000, these figures were 30 and 600 +- 37 ng, respectively. Using this assay, the seasonal pattern of plasma vitellogenin in the turtle has been described, and preliminary studies on in vitro hepatic vitellogenesis have been performed

A characterization of the ZFL cell line and primary hepatocytes as in vitro liver cell models for the zebrafish (Danio rerio)

International Nuclear Information System (INIS)

Eide, Marta; Rusten, Marte; Male, Rune; Jensen, Knut Helge Midtbø; Goksøyr, Anders

2014-01-01

Highlights: •The ZFL cell line and primary hepatocytes were characterized. •Basic and induced expression of nuclear receptors and target genes were found. •The ZFL cell line expresses very low basic levels of most genes. •The ZFL cells have low induction of gene expression following exposures. •Primary hepatocytes show large sex-dependent differences in gene expression. -- Abstract: The zebrafish (Danio rerio) is a widely used model species in biomedical research. The ZFL cell line, established from zebrafish liver, and freshly isolated primary hepatocytes from zebrafish have been used in several toxicological studies. However, no previous report has compared and characterized these two systems at the level of gene expression. The aim of this study was to evaluate the ZFL cell line in comparison to primary hepatocytes as in vitro models for studying effects of environmental contaminants in zebrafish liver. Using quantitative real-time PCR, the basal level and transcriptional induction potential of key genes involved in toxic responses in the ZFL cell line, primary hepatocytes and whole liver from zebrafish were compared. The study showed that the ZFL cells have lower levels of mRNA of most selected genes compared to zebrafish liver. The induced gene transcription following exposure to ligand was much lower in ZFL cells compared to zebrafish primary hepatocytes at the doses tested. Importantly, oestrogen receptor and vitellogenin genes showed low basal transcription and no induction response in the ZFL cell line. In conclusion, it appears that primary hepatocytes are well suited for studying environmental contaminants including xenoestrogens, but may show large sex-dependent differences in gene transcription. The ZFL cell line shows potential in toxicological studies involving the aryl hydrocarbon receptor pathway. However, low potential for transcriptional induction of genes in general should be expected, especially notable when studying estrogenic

A characterization of the ZFL cell line and primary hepatocytes as in vitro liver cell models for the zebrafish (Danio rerio)

Energy Technology Data Exchange (ETDEWEB)

Eide, Marta, E-mail: marta.eide@bio.uib.no [Department of Biology, University of Bergen, Bergen (Norway); Rusten, Marte; Male, Rune [Department of Molecular Biology, University of Bergen, Bergen (Norway); Jensen, Knut Helge Midtbø; Goksøyr, Anders [Department of Biology, University of Bergen, Bergen (Norway)

2014-02-15

Highlights: •The ZFL cell line and primary hepatocytes were characterized. •Basic and induced expression of nuclear receptors and target genes were found. •The ZFL cell line expresses very low basic levels of most genes. •The ZFL cells have low induction of gene expression following exposures. •Primary hepatocytes show large sex-dependent differences in gene expression. -- Abstract: The zebrafish (Danio rerio) is a widely used model species in biomedical research. The ZFL cell line, established from zebrafish liver, and freshly isolated primary hepatocytes from zebrafish have been used in several toxicological studies. However, no previous report has compared and characterized these two systems at the level of gene expression. The aim of this study was to evaluate the ZFL cell line in comparison to primary hepatocytes as in vitro models for studying effects of environmental contaminants in zebrafish liver. Using quantitative real-time PCR, the basal level and transcriptional induction potential of key genes involved in toxic responses in the ZFL cell line, primary hepatocytes and whole liver from zebrafish were compared. The study showed that the ZFL cells have lower levels of mRNA of most selected genes compared to zebrafish liver. The induced gene transcription following exposure to ligand was much lower in ZFL cells compared to zebrafish primary hepatocytes at the doses tested. Importantly, oestrogen receptor and vitellogenin genes showed low basal transcription and no induction response in the ZFL cell line. In conclusion, it appears that primary hepatocytes are well suited for studying environmental contaminants including xenoestrogens, but may show large sex-dependent differences in gene transcription. The ZFL cell line shows potential in toxicological studies involving the aryl hydrocarbon receptor pathway. However, low potential for transcriptional induction of genes in general should be expected, especially notable when studying estrogenic

Comparative analysis of distinctive transcriptome profiles with biochemical evidence in bisphenol S- and benzo[a]pyrene-exposed liver tissues of the olive flounder Paralichthys olivaceus.

Directory of Open Access Journals (Sweden)

Jee-Hyun Jung

Full Text Available Flounder is a promising model species for environmental monitoring of coastal regions. To assess the usefulness of liver transcriptome profiling, juvenile olive flounder Paralichthys olivaceus were exposed to two pollutants, bisphenol S (BPS and benzo[a]pyrene (BaP, which have different chemical characteristics and have distinct modes of metabolic action in teleost. Six hours after intraperitoneal injection with BPS (50 mg/kg bw or BaP (20 mg/kg bw, liver transcriptomes were analyzed using the Illumina Hiseq 3000 platform. Interestingly, the transcriptome was highly sensitive and was distinctively expressed in response to each chemical. The primary effect of BPS was significantly increased transcription of egg process and vitellogenesis related genes, including vitellogenins (vtg1, vtg2, zona pellucida sperm-binding proteins (zp3, zp4, and estrogen receptors (erα, erβ, with increases in plasma 17β-estradiol (E2 and vitellogenin (VTG concentrations. Following BaP treatment, detoxification- and biotransformation-related genes such as cyp1a1 and UDP-glucuronosyltransferase (ugt1a1 were significantly increased, with an increase in EROD activity. In both transcriptomes, mRNA expression of genes involved in antioxidant defense systems was increased, while genes involved in innate immunity were decreased upon BPS or BaP exposure with a decrease in complement activity. This study provides useful insight into the chemical-specific hepatic transcriptional response of P. olivaceus and suggests a basis for further studies examining biomarker application of liver transcriptomes for environmental pollution.

RNAi-mediated silencing of vitellogenin gene function turns honeybee ( Apis mellifera) workers into extremely precocious foragers

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Marco Antonio, David Santos; Guidugli-Lazzarini, Karina Rosa; Do Nascimento, Adriana Mendes; Simões, Zilá Luz Paulino; Hartfelder, Klaus

2008-10-01

The switch from within-hive activities to foraging behavior is a major transition in the life cycle of a honeybee ( Apis mellifera) worker. A prominent regulatory role in this switch has long been attributed to juvenile hormone (JH), but recent evidence also points to the yolk precursor protein vitellogenin as a major player in behavioral development. In the present study, we injected vitellogenin double-stranded RNA (dsVg) into newly emerged worker bees of Africanized genetic origin and introduced them together with controls into observation hives to record flight behavior. RNA interference-mediated silencing of vitellogenin gene function shifted the onset of long-duration flights (>10 min) to earlier in life (by 3 4 days) when compared with sham and untreated control bees. In fact, dsVg bees were observed conducting such flights extremely precociously, when only 3 days old. Short-duration flights (<10 min), which bees usually perform for orientation and cleaning, were not affected. Additionally, we found that the JH titer in dsVg bees collected after 7 days was not significantly different from the controls. The finding that depletion of the vitellogenin titer can drive young bees to become extremely precocious foragers could imply that vitellogenin is the primary switch signal. At this young age, downregulation of vitellogenin gene activity apparently had little effect on the JH titer. As this unexpected finding stands in contrast with previous results on the vitellogenin/JH interaction at a later age, when bees normally become foragers, we propose a three-step sequence in the constellation of physiological parameters underlying behavioral development.

Estradiol-induced gene expression in largemouth bass (Micropterus salmoides)

Science.gov (United States)

Bowman, C.J.; Kroll, K.J.; Gross, T.G.; Denslow, N.D.

2002-01-01

Vitellogenin (Vtg) and estrogen receptor (ER) gene expression levels were measured in largemouth bass to evaluate the activation of the ER-mediated pathway by estradiol (E2). Single injections of E2 ranging from 0.0005 to 5 mg/kg up-regulated plasma Vtg in a dose-dependent manner. Vtg and ER mRNAs were measured using partial cDNA sequences corresponding to the C-terminal domain for Vtg and the ligand-binding domain of ER?? sequences. After acute E2-exposures (2 mg/kg), Vtg and ER mRNAs and plasma Vtg levels peaked after 2 days. The rate of ER mRNA accumulation peaked 36-42 h earlier than Vtg mRNA. The expression window for ER defines the primary response to E2 in largemouth bass and that for Vtg a delayed primary response. The specific effect of E2 on other estrogen-regulated genes was tested during these same time windows using differential display RT-PCR. Specific up-regulated genes that are expressed in the same time window as Vtg were ERp72 (a membrane-bound disulfide isomerase) and a gene with homology to an expressed gene identified in zebrafish. Genes that were expressed in a pattern that mimics the ER include the gene for zona radiata protein ZP2, and a gene with homology to an expressed gene found in winter flounder. One gene for fibrinogen ?? was down-regulated and an unidentified gene was transiently up-regulated after 12 h of exposure and returned to basal levels by 48 h. Taken together these studies indicate that the acute molecular response to E2 involves a complex network of responses over time. ?? 2002 Elsevier Science Ireland Ltd. All rights reserved.

Vitellogenin and vitellogenin receptor gene expression and 20-hydroxyecdysone concentration in Macrobrachium rosenbergii exposed to chlordecone.

Science.gov (United States)

Lafontaine, Anne; Hanikenne, Marc; Boulangé-Lecomte, Céline; Forget-Leray, Joëlle; Thomé, Jean-Pierre; Gismondi, Eric

2016-10-01

Chlordecone is a persistent organochlorine pesticide widely used in Guadeloupe (French West Indies) to control the banana weevil Cosmopolites sordidus. Although it was previously highlighted that chlordecone may affect the reproduction and growth of vertebrate species, little information is available on the chlordecone effects in invertebrates. The present study investigated the effects of chlordecone on a hormone and a protein having key roles in reproduction and growth of the decapod crustacean Macrobrachium rosenbergii, by measuring the 20-hydroxyecdysone concentration, vitellogenin, and vitellogenin receptor gene expression, as well as the bioconcentration of chlordecone in exposed prawns. First, the results revealed that chlordecone was accumulated in M. rosenbergii. Then, it was found that Vg and VgR gene expression were increased in male and female M. rosenbergii exposed to chlordecone for 90 and 240 days, while the 20-hydroxyecdysone concentrations were decreased. This work suggests that chlordecone accumulates in prawn tissues and could affect key molecules involved in the reproduction and the growth of the invertebrate M. rosenbergii. However, many questions remain unresolved regarding the impacts of chlordecone on growth and reproduction and the signaling pathways responsible for these effects, as well as the potential role of confounding factors present in in situ studies.

Effect of methomyl on sex steroid hormone and vitellogenin levels in serum of male tilapia (Oreochromis niloticus) and recovery pattern.

Science.gov (United States)

Meng, Shun-Long; Qiu, Li-Ping; Hu, Geng-Dong; Fan, Li-Min; Song, Chao; Zheng, Yao; Wu, Wei; Qu, Jian-Hong; Li, Dan-Dan; Chen, Jia-Zhang; Xu, Pao

2017-07-01

Tilapia were exposed to sub-lethal concentrations of 0, 0.2, 2, 20 or 200 μg/L for 30 days, then transferred to methomyl-free water for 18 days. E 2 , T, 11-KTand VTG in serum were examined. There were no significant changes in all the parameters in serum of tilapia exposed to 0.2 μg/L and 2 μg/L methomyl compared to the control. However, 20 μg/L and 200 μg/L have the potential to disrupt the endocrine system of male tilapia, as shown by its ability to increase VTG and E 2 and decrease T and 11-KT in serum. Thus it would appear the no observed adverse effect level for sexual steroid hormones of methomyl is lower than 2 μg/L. Recovery data showed that the effects produced by 20μg/L were reversible but not at 200μg/L. Furthermore, the sensitivity of above parameters to methomyl followed the order of VTG>E 2 >11-KT>T>GSI, suggesting VTG being the better biomarkers. © 2017 Wiley Periodicals, Inc.

Quantification of vitellogenin in Atlantic salmon (Salmo salar) plasma by radioimmunoassay

International Nuclear Information System (INIS)

Idler, D.R.; Hwang, S.J.; Crim, L.W.

1979-01-01

An antibody prepared against salmon egg yolk proteins has been used to quantify Atlantic salmon (Salmo salar) plasma vitellogenin using radioimmunoassay. A low molecular weight fraction isolated from salmon egg yolk was used for radioiodination and as standard solution because plasma vitellogenin could not be iodinated successfully. Parallelism of the egg yolk standard to displacement given by a fraction isolated from vitellogenic salmon plasma and dilutions of plasma samples allowed the assay to be used to evaluate the state of gonadal development of migrating females several months in advance of spawning and for sexing relatively immature salmon. (author)

DNA sequence and prokaryotic expression analysis of vitellogenin ...

African Journals Online (AJOL)

In this study, the DNA sequence of vitellogenin from Antheraea pernyi (Ap-Vg) was identified and its functional domain (30-740 aa, Ap-Vg-1) was expressed in Escherichia coli BL21 (DE3) cells. The recombinant Ap-Vg-1 proteins were purified and used for antibody preparation. The results showed that the intact DNA ...

FATHEAD MINNOW VITELLOGENIN: CDNA SEQUENCE AND MRNA AND PROTEIN EXPRESSION AFTER 17 BETA-ESTRADIOL TREATMENT

Science.gov (United States)

In the present study, a sensitive ribonuclease protection assay (RPA) for VTG mRNA was developed for the fathead minnow (Pimephales promelas), a species proposed for routine endocrine-disrupting chemical (EDC) screening.

Vitellogenin and vitellogenin receptor gene expression profiles in Spodoptera exigua are related to host plant suitability.

Science.gov (United States)

Zhao, Jing; Sun, Yang; Xiao, Liubin; Tan, Yongan; Jiang, Yiping; Bai, Lixin

2018-04-01

The beet armyworm Spodoptera exigua, a worldwide phytophagous pest, causes considerable economic agricultural losses. Understanding the relationship between its fecundity and the host plant is a basic and important component of early forecasting of beet armyworm outbreaks. However, little is known about the molecular mechanism by which distinct hosts affect S. exigua fecundity. In this study, key life-history parameters of S. exigua reared on distinct hosts were investigated; the host plants could be ranked as lettuce > shallot > tomato > celery in their order of suitability. Full-length S. exigua vitellogenin receptor (SeVgR) cDNA was cloned, and sex-, stage- and tissue-specific expression characteristics were assessed. Spodoptera exigua vitellogenin (SeVg) and SeVgR expression levels were markedly modulated by host nutrients (P lettuce, the most preferred and most nutritive host, than in those reared on tomato and celery. Interestingly, significant linear regression correlations were found between SeVg and SeVgR expression levels and key S. exigua life-history parameters, especially life span, pupa weight, and female fecundity (P < 0.01). Host plant type and suitability could affect the expression pattern of SeVg and SeVgR, which influenced S. exigua fecundity. Vg and VgR have the potential to be used as molecular markers of S. exigua fecundity and for forecasting outbreaks of S. exigua on different hosts. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

Effect of Bisphenol A on the Levels of Vitellogenin and ...

African Journals Online (AJOL)

Effect of Bisphenol A on the Levels of Vitellogenin and Metallothionein in Adult ... Purpose: To investigate the potential role of bisphenol A (BPA) in inducing endocrine disruption in fish. ... EMAIL FREE FULL TEXT EMAIL FREE FULL TEXT

Plasma biomarkers in juvenile marine fish provide evidence for endocrine modulation potential of organotin compounds.

Science.gov (United States)

Min, Byung Hwa; Kim, Bo-Mi; Kim, Moonkoo; Kang, Jung-Hoon; Jung, Jee-Hyun; Rhee, Jae-Sung

2018-08-01

Organotin compounds, such as tributyltin (TBT) and triphenyltin (TPT), have been widely used to control marine fouling. Here, we show that organotin stimulation reduces the hormone levels in the plasma of two economically important aquaculture fish. Blood plasma samples were collected from juvenile red seabream and black rockfish exposed to environmentally realistic concentrations of TBT and TPT for 14 days. The levels of two plasma biomarkers, namely the yolk protein precursor vitellogenin (VTG) and the sex steroid 17β-estradiol (E2), were measured to determine the endocrine disrupting potential of the organotin compounds. Both organotin compounds were dose-dependently accumulated in the blood of two fish. Exposure to waterborne TBT and TBT significantly decreased the plasma VTG levels in both the juvenile fish in a dose-dependent manner. In contrast, the treatment with E2, a well-known VTG inducer, significantly increased the plasma VTG levels in both the fish. In addition, the mRNA levels of vtg were also downregulated in the liver tissues of both the fish at 100 and/or 1000 ng L -1 of TBT or TPT exposure. The plasma E2 titers were significantly suppressed at 100 and/or 1000 ng L -1 of TBT or TPT exposure for 14 days compared to their titer in the control. Since estrogen directly regulates vtg gene expression and VTG synthesis, our results reveal the endocrine disrupting potential of organotin compounds, and subsequently the endocrine modulation at early stage of fish can trigger further fluctuations in sexual differentiation, maturation, sex ration or egg production. In addition, the results demonstrate their effects on non-target organisms, particularly on animals reared in aquaculture and fisheries. Copyright © 2018 Elsevier Inc. All rights reserved.

Generation of human hepatocytes by stem cell technology: definition of the hepatocyte.

Science.gov (United States)

Hengstler, Jan G; Brulport, Marc; Schormann, Wiebke; Bauer, Alexander; Hermes, Matthias; Nussler, Andreas K; Fandrich, Fred; Ruhnke, Maren; Ungefroren, Hendrik; Griffin, Louise; Bockamp, Ernesto; Oesch, Franz; von Mach, Marc-Alexander

2005-06-01

Since 1999, numerous articles have reported the generation of hepatocytes from different types of extrahepatic stem or precursor cells. This opens exciting new possibilities for pharmacology and toxicology, as well as for cell therapy. Hepatocyte marker expression, including albumin, cytokeratin 18, c-met, alpha-fetoprotein and cytochrome P450 3A4 and -2B6, has been observed after transplantation of different types of human stem cells into the liver of laboratory animals or in vitro after incubation with cytokines. These intriguing observations have prompted scientists to classify stem cell-derived cell populations as hepatocytes. However, this conclusion may be premature. It has been shown that factors of the liver microenvironment can induce expression of a limited number of hepatocyte marker genes in nonhepatic cell types. To conclude on the grounds of a limited number of markers that these cells are true hepatocytes is not indicated. In this case one should carefully evaluate crucial hepatocyte-defining enzymatic properties. The present article: i) reviews studies describing the fate of extrahepatic human stem and precursor cells in livers of laboratory animals, including the possibility of cell fusion; and ii) critically discusses the phenotype of stem cells after application of various differentiation protocols aimed at generating human hepatocytes. In addition, the necessary criteria needed for defining a true hepatocyte are suggested. Establishing the necessary properties for stem cell-derived hepatocytes is timely and reasonable, and thus avoids further misleading semantic confusion. Finally, it is essential to understand that the definition of a bona fide hepatocyte should not be limited to qualitative assays, such as reverse transcriptase polymerase chain reaction and immunohistochemistry, but has to include a quantitative analysis of enzymatic activities, which allows direct comparison with primary hepatocytes. Although the stem cell-derived-hepatocyte

Effects of triclosan on hormones and reproductive axis in female Yellow River carp (Cyprinus carpio): Potential mechanisms underlying estrogen effect.

Science.gov (United States)

Wang, Fan; Guo, Xiangmeng; Chen, Wanguang; Sun, Yaowen; Fan, Chaojie

2017-12-01

Triclosan (TCS), a member of the class of compounds called pharmaceutical and personal care products (PPCPs), is a broad antibacterial and antifungal agent found in a lot of consumer products. However, TCS hormone effect mechanism in teleost female fish is not clear. Female Yellow River carp (Cyprinus carpio) were exposed to 1/20, 1/10 and 1/5 LC 50 TCS (96h LC 50 of TCS to carp) under semi-static conditions for 42days. Vitellogenin (Vtg), 17β-estradiol (E 2 ), testosterone(T), estrogen receptor (Er), gonadotropin (GtH), and gonadotropin-releasing hormone (GnRH) levels were measured by enzyme-linked immunosorbent assay (ELISA). Meanwhile, we also examined the mRNA expressions of aromatase, GtHs-β, GnRH, and Er by quantitative real-time PCR (qRT-PCR). The results indicated that 1/5 LC 50 TCS induced Vtg in hepatopancreas of female carps by interference with the hypothalamic-pituitary-gonadal (HPG) axis at multiple potential loci through three mechanisms: (a) TCS exposure enhanced the mRNA expression of hypothalamus and gonadal aromatase which converts androgens into estrogens, subsequently increasing serum concentrations of E 2 to induce Vtg in hepatopancreas; (b) TCS treatment increased GnRH and GtH-β mRNA expression and secretion, causing the disturbance of reproductive endocrine and the increase of E 2 to induce Vtg in hepatopancreas; (c) TCS exposure enhanced synthesis and secretion of Er, then it bound to Er to active Vtg synthesis. These mechanisms showed that TCS may induce Vtg production in female Yellow River carp by Er-mediated and non-Er-mediated pathways. Copyright © 2017 Elsevier Inc. All rights reserved.

The fraction haemolymph vitellogenin of a honey bee colony, derived from a pooled haemolymph sample, a colony vitality parameter

NARCIS (Netherlands)

Steen, van der J.J.M.; Martel, Anne Claire; Hendrickx, Patrick

2015-01-01

The number of bees, amount of brood and haemolymph vitellogenin titre are parameters to establish the vitality of a honey bee colony. Increasing numbers of bees during summer until autumn; increasing amounts of brood in spring towards summer followed by a decrease; and low haemolymph vitellogenin

Chronic exposure of killifish to a highly polluted environment desensitizes estrogen-responsive reproductive and biomarker genes

International Nuclear Information System (INIS)

Bugel, Sean M.; Bonventre, Josephine A.; White, Lori A.; Tanguay, Robert L.; Cooper, Keith R.

2014-01-01

Highlights: • Reproductive biomarker genes in Newark Bay killifish are desensitized to estrogen. • Gene desensitization indicates pre-transcriptional effects on estrogen signaling. • Desensitization does not have a metabolic or epigenetic basis (gene methylation). • Modulation of vitellogenin and choriogenin genes correlates with reproductive impacts. • Choriogenin L appears less prone to false negatives and may be a sensitive biomarker. - Abstract: Reproductive and endocrine disruption is commonly reported in aquatic species exposed to complex contaminant mixtures. We previously reported that Atlantic killifish (Fundulus heteroclitus) from the chronically contaminated Newark Bay, NJ, exhibit multiple endocrine disrupting effects, including inhibition of vitellogenesis (yolk protein synthesis) in females and false negative vitellogenin biomarker responses in males. Here, we characterized the effects on estrogen signaling and the transcriptional regulation of estrogen-responsive genes in this model population. First, a dose–response study tested the hypothesis that reproductive biomarkers (vtg1, vtg2, chg H, chg Hm, chg L) in Newark Bay killifish are relatively less sensitive to 17β-estradiol at the transcriptional level, relative to a reference (Tuckerton, NJ) population. The second study assessed expression for various metabolism (cyp1a, cyp3a30, mdr) and estrogen receptor (ER α, ER βa, ER βb) genes under basal and estrogen treatment conditions in both populations. Hepatic metabolism of 17β-estradiol was also evaluated in vitro as an integrated endpoint for adverse effects on metabolism. In the third study, gene methylation was evaluated for promoters of vtg1 (8 CpGs) and vtg2 (10 CpGs) in both populations, and vtg1 promoter sequences were examined for single nucleotide polymorphism (SNPs). Overall, these studies show that multi-chemical exposures at Newark Bay have desensitized all reproductive biomarkers tested to estrogen. For example, at 10 ng

Chronic exposure of killifish to a highly polluted environment desensitizes estrogen-responsive reproductive and biomarker genes

Energy Technology Data Exchange (ETDEWEB)

Bugel, Sean M., E-mail: Sean.Bugel@oregonstate.edu [Department of Environmental and Molecular Toxicology, Environmental Health Sciences Center, Oregon State University, Corvallis, OR 97331 (United States); Bonventre, Josephine A. [Department of Environmental and Molecular Toxicology, Environmental Health Sciences Center, Oregon State University, Corvallis, OR 97331 (United States); White, Lori A. [Department of Biochemistry and Microbiology, Rutgers, The State University of New Jersey, New Brunswick, NJ 08901 (United States); Tanguay, Robert L. [Department of Environmental and Molecular Toxicology, Environmental Health Sciences Center, Oregon State University, Corvallis, OR 97331 (United States); Cooper, Keith R. [Department of Biochemistry and Microbiology, Rutgers, The State University of New Jersey, New Brunswick, NJ 08901 (United States)

2014-07-01

Highlights: • Reproductive biomarker genes in Newark Bay killifish are desensitized to estrogen. • Gene desensitization indicates pre-transcriptional effects on estrogen signaling. • Desensitization does not have a metabolic or epigenetic basis (gene methylation). • Modulation of vitellogenin and choriogenin genes correlates with reproductive impacts. • Choriogenin L appears less prone to false negatives and may be a sensitive biomarker. - Abstract: Reproductive and endocrine disruption is commonly reported in aquatic species exposed to complex contaminant mixtures. We previously reported that Atlantic killifish (Fundulus heteroclitus) from the chronically contaminated Newark Bay, NJ, exhibit multiple endocrine disrupting effects, including inhibition of vitellogenesis (yolk protein synthesis) in females and false negative vitellogenin biomarker responses in males. Here, we characterized the effects on estrogen signaling and the transcriptional regulation of estrogen-responsive genes in this model population. First, a dose–response study tested the hypothesis that reproductive biomarkers (vtg1, vtg2, chg H, chg Hm, chg L) in Newark Bay killifish are relatively less sensitive to 17β-estradiol at the transcriptional level, relative to a reference (Tuckerton, NJ) population. The second study assessed expression for various metabolism (cyp1a, cyp3a30, mdr) and estrogen receptor (ER α, ER βa, ER βb) genes under basal and estrogen treatment conditions in both populations. Hepatic metabolism of 17β-estradiol was also evaluated in vitro as an integrated endpoint for adverse effects on metabolism. In the third study, gene methylation was evaluated for promoters of vtg1 (8 CpGs) and vtg2 (10 CpGs) in both populations, and vtg1 promoter sequences were examined for single nucleotide polymorphism (SNPs). Overall, these studies show that multi-chemical exposures at Newark Bay have desensitized all reproductive biomarkers tested to estrogen. For example, at 10 ng

cis- and trans-acting elements of the estrogen-regulated vitellogenin gene B1 of Xenopus laevis.

Science.gov (United States)

Wahli, W; Martinez, E; Corthésy, B; Cardinaux, J R

1989-01-01

Vitellogenin genes are expressed under strict estrogen control in the liver of female oviparous vertebrates. Gene transfer experiments using estrogen-responsive cells have shown that the 13 bp perfect palindromic element GGTCACTGTGACC found upstream of the Xenopus laevis vitellogenin gene A2 promoter mediates hormonal stimulation and thus, was called the estrogen-responsive element (ERE). In the Xenopus vitellogenin genes B1 and B2 there are two closely adjacent EREs with one or more base substitutions when compared to the consensus ERE GGTCANNNTGACC. On their own, these degenerated elements have only a low or no regulatory capacity at all but act together synergistically to form an estrogen-responsive unit (ERU) with the same strength as the perfect palindromic 13 bp element. Analysis of estrogen receptor binding to the gene B1 ERU revealed a cooperative interaction of receptor dimers to the two adjacent imperfect EREs which most likely explains the synergistic stimulation observed in vivo. Furthermore, a promoter activator element located between positions --113 and --42 of the gene B1 and functional in the human MCF-7 and the Xenopus B3.2 cells has been identified and shown to be involved in the high level of induced transcription activity when the ERE is placed at a distance from the promoter. Finally, a hormone-controlled in vitro transcription system derived from Xenopus liver nuclear extracts was exploited to characterize two additional novel cis-acting elements within the vitellogenin gene B1 promoter. One of them, a negative regulatory element (NRE), is responsible for repression of promoter activity in the absence of hormone. The second is related to the NF-I binding site and is required, together with the ERE, to mediate hormonal induction. Moreover, we detected three trans-acting activities in Xenopus liver nuclear extracts that interact with these regions and demonstrated that they participate in the regulation of the expression of the vitellogenin

Alterations of tissue metallothionein and vitellogenin concentrations in tropical cup oysters (Saccostrea sp.) following short-term (96 h) exposure to cadmium

International Nuclear Information System (INIS)

Moncaleano-Niño, Angela M.; Barrios-Latorre, Sergio A.; Poloche-Hernández, Javier F.; Becquet, Vanessa; Huet, Valérie; Villamil, Luisa; Thomas-Guyon, Hélène; Ahrens, Michael J.; Luna-Acosta, Andrea

2017-01-01

Highlights: • The cup oyster Saccostrea sp. is present in Santa Marta, Colombian Caribbean. • 96 h exposure of oysters to Cd increased metallothionein concentrations in digestive glands up to 2-fold. • 96 h exposure of oysters to Cd decreased vitellogenin concentrations in gonads up to 6-fold. • Metallothionein and vitellogenin tissue concentrations correlated with whole tissue Cd concentrations. • Significant changes in metallothionein and vitellogenin levels were only evident at Cd concentrations above 100 μg/L. - Abstract: Metallothioneins and vitellogenins are low molecular weight proteins that have been used widely in environmental monitoring as biomarkers of exposure and damage to metals and estrogenic compounds, respectively. In the present study, the responses of metallothionein and vitellogenin tissue concentrations were measured following acute (96 h) aqueous exposures to cadmium in Saccostrea sp., a tropical cup oyster native to the Western Pacific Ocean that has recently established itself in the Caribbean Sea. Adult oysters (1.5–5.0 cm shell length) collected from the municipal marina of Santa Marta, Colombia (Caribbean Sea) and acclimated for 5 days in the laboratory, were exposed to Cd at five concentrations (0, 1, 10, 100 and 1000 μg/L) and their tissues (gills, digestive gland and adductor muscle) were analyzed in pools of 5 individuals (3 replicates per concentration). Metallothioneins in digestive glands of oysters exposed to Cd concentrations ≥ 100 μg/L showed a significant increase, from 8.0 to 14.8 μg MT/mg total protein, whereas metallothionein concentrations in gills increased to lesser extent, and no differences were observed in adductor muscle. Metallothionein concentrations in digestive gland and gills correlated directly with whole soft tissue Cd concentrations (ranging from 2 to 297 μg/g dw Cd). Vitellogenin in homogenates of oyster gonad tissue, after 96 h of exposure to 1000 μg/L Cd, were significantly lower (0

Alterations of tissue metallothionein and vitellogenin concentrations in tropical cup oysters (Saccostrea sp.) following short-term (96 h) exposure to cadmium

Energy Technology Data Exchange (ETDEWEB)

Moncaleano-Niño, Angela M.; Barrios-Latorre, Sergio A.; Poloche-Hernández, Javier F. [Department of Biological Sciences, Universidad de Bogota Jorge Tadeo Lozano, Carrera 4 No. 22-61, Bogota (Colombia); Becquet, Vanessa; Huet, Valérie [Littoral Environnement et Sociétés (LIENSs) – UMR 7266, CNRS-Université de La Rochelle, Bâtiment ILE 2, rue Olympe de Gouges, 17 000 La Rochelle (France); Villamil, Luisa [Department of Biological Sciences, Universidad de Bogota Jorge Tadeo Lozano, Carrera 4 No. 22-61, Bogota (Colombia); Thomas-Guyon, Hélène [Littoral Environnement et Sociétés (LIENSs) – UMR 7266, CNRS-Université de La Rochelle, Bâtiment ILE 2, rue Olympe de Gouges, 17 000 La Rochelle (France); Ahrens, Michael J., E-mail: michael.ahrens@utadeo.edu.co [Department of Biological Sciences, Universidad de Bogota Jorge Tadeo Lozano, Carrera 4 No. 22-61, Bogota (Colombia); Luna-Acosta, Andrea [Department of Biological Sciences, Universidad de Bogota Jorge Tadeo Lozano, Carrera 4 No. 22-61, Bogota (Colombia)

2017-04-15

Highlights: • The cup oyster Saccostrea sp. is present in Santa Marta, Colombian Caribbean. • 96 h exposure of oysters to Cd increased metallothionein concentrations in digestive glands up to 2-fold. • 96 h exposure of oysters to Cd decreased vitellogenin concentrations in gonads up to 6-fold. • Metallothionein and vitellogenin tissue concentrations correlated with whole tissue Cd concentrations. • Significant changes in metallothionein and vitellogenin levels were only evident at Cd concentrations above 100 μg/L. - Abstract: Metallothioneins and vitellogenins are low molecular weight proteins that have been used widely in environmental monitoring as biomarkers of exposure and damage to metals and estrogenic compounds, respectively. In the present study, the responses of metallothionein and vitellogenin tissue concentrations were measured following acute (96 h) aqueous exposures to cadmium in Saccostrea sp., a tropical cup oyster native to the Western Pacific Ocean that has recently established itself in the Caribbean Sea. Adult oysters (1.5–5.0 cm shell length) collected from the municipal marina of Santa Marta, Colombia (Caribbean Sea) and acclimated for 5 days in the laboratory, were exposed to Cd at five concentrations (0, 1, 10, 100 and 1000 μg/L) and their tissues (gills, digestive gland and adductor muscle) were analyzed in pools of 5 individuals (3 replicates per concentration). Metallothioneins in digestive glands of oysters exposed to Cd concentrations ≥ 100 μg/L showed a significant increase, from 8.0 to 14.8 μg MT/mg total protein, whereas metallothionein concentrations in gills increased to lesser extent, and no differences were observed in adductor muscle. Metallothionein concentrations in digestive gland and gills correlated directly with whole soft tissue Cd concentrations (ranging from 2 to 297 μg/g dw Cd). Vitellogenin in homogenates of oyster gonad tissue, after 96 h of exposure to 1000 μg/L Cd, were significantly lower (0

17α-Ethinylestradiol (EE2) effect on global gene expression in primary rainbow trout (Oncorhynchus mykiss) hepatocytes

International Nuclear Information System (INIS)

Hultman, Maria T.; Song, You; Tollefsen, Knut Erik

2015-01-01

Highlights: • EE2 induced large scale transcriptional changes in primary hepatocytes. • Classical estrogen biomarkers were altered in a concentration-dependent manner. • EE2 altered biological processes related to lipid transport and reproduction. • EE2 interfered with lipid metabolism, biotransformation, and multidrug transport. • In vitro transcriptional changes were fairly similar to that observed in vivo. - Abstract: The potential impact of endocrine disrupting chemicals (EDCs) in the aquatic environment has driven the development of screening assays to evaluate the estrogenic properties of chemicals and their effects on aquatic organisms such as fish. However, obtaining full concentration–response relationships in animal (in vivo) exposure studies are laborious, costly and unethical, hence a need for developing feasible alternative (non-animal) methods. Use of in vitro bioassays such as primary fish hepatocytes, which retain many of the native properties of the liver, has been proposed for in vitro screening of estrogen receptor (ER) agonists and antagonists. The aim of present study was to characterize the molecular mode of action (MoA) of the ER agonist 17α-ethinylestradiol (EE2) in primary rainbow trout (Oncorhynchus mykiss) hepatocytes. A custom designed salmonid 60,000-feature (60k) oligonucleotide microarray was used to characterize the potential MoAs after 48 h exposure to EE2. The microarray analysis revealed several concentration-dependent gene expression alterations including classical estrogen sensitive biomarker gene expression (e.g. estrogen receptor α, vitellogenin, zona radiata). Gene Ontology (GO) analysis displayed transcriptional changes suggesting interference of cellular growth, fatty acid and lipid metabolism potentially mediated through the estrogen receptor (ER), which were proposed to be associated with modulation of genes involved in endocrine function and reproduction. Pathway analysis supported the identified GOs and

17α-Ethinylestradiol (EE2) effect on global gene expression in primary rainbow trout (Oncorhynchus mykiss) hepatocytes

Energy Technology Data Exchange (ETDEWEB)

Hultman, Maria T., E-mail: mhu@niva.no [Norwegian Institute for Water Research (NIVA), Section of Ecotoxicology and Risk Assessment, Gaustadalléen 21, N-0349 Oslo (Norway); Faculty of Environmental Science & Technology, Department for Environmental Sciences, Norwegian University of Life Sciences (NMBU), Post box 5003, N-1432 Ås (Norway); Song, You [Norwegian Institute for Water Research (NIVA), Section of Ecotoxicology and Risk Assessment, Gaustadalléen 21, N-0349 Oslo (Norway); Tollefsen, Knut Erik [Norwegian Institute for Water Research (NIVA), Section of Ecotoxicology and Risk Assessment, Gaustadalléen 21, N-0349 Oslo (Norway); Faculty of Environmental Science & Technology, Department for Environmental Sciences, Norwegian University of Life Sciences (NMBU), Post box 5003, N-1432 Ås (Norway)

2015-12-15

Highlights: • EE2 induced large scale transcriptional changes in primary hepatocytes. • Classical estrogen biomarkers were altered in a concentration-dependent manner. • EE2 altered biological processes related to lipid transport and reproduction. • EE2 interfered with lipid metabolism, biotransformation, and multidrug transport. • In vitro transcriptional changes were fairly similar to that observed in vivo. - Abstract: The potential impact of endocrine disrupting chemicals (EDCs) in the aquatic environment has driven the development of screening assays to evaluate the estrogenic properties of chemicals and their effects on aquatic organisms such as fish. However, obtaining full concentration–response relationships in animal (in vivo) exposure studies are laborious, costly and unethical, hence a need for developing feasible alternative (non-animal) methods. Use of in vitro bioassays such as primary fish hepatocytes, which retain many of the native properties of the liver, has been proposed for in vitro screening of estrogen receptor (ER) agonists and antagonists. The aim of present study was to characterize the molecular mode of action (MoA) of the ER agonist 17α-ethinylestradiol (EE2) in primary rainbow trout (Oncorhynchus mykiss) hepatocytes. A custom designed salmonid 60,000-feature (60k) oligonucleotide microarray was used to characterize the potential MoAs after 48 h exposure to EE2. The microarray analysis revealed several concentration-dependent gene expression alterations including classical estrogen sensitive biomarker gene expression (e.g. estrogen receptor α, vitellogenin, zona radiata). Gene Ontology (GO) analysis displayed transcriptional changes suggesting interference of cellular growth, fatty acid and lipid metabolism potentially mediated through the estrogen receptor (ER), which were proposed to be associated with modulation of genes involved in endocrine function and reproduction. Pathway analysis supported the identified GOs and

Vitellogenins Are New High Molecular Weight Components and Allergens (Api m 12 and Ves v 6) of Apis mellifera and Vespula vulgaris Venom

Science.gov (United States)

Blank, Simon; Seismann, Henning; McIntyre, Mareike; Ollert, Markus; Wolf, Sara; Bantleon, Frank I.; Spillner, Edzard

2013-01-01

Background/Objectives Anaphylaxis due to hymenoptera stings is one of the most severe clinical outcomes of IgE-mediated hypersensitivity reactions. Although allergic reactions to hymenoptera stings are often considered as a general model for the underlying principles of allergic disease, venom immunotherapy is still hampered by severe systemic side effects and incomplete protection. The identification and detailed characterization of all allergens of hymenoptera venoms might result in an improvement in this field and promote the detailed understanding of the allergological mechanism. Our aim was the identification and detailed immunochemical and allergological characterization of the low abundant IgE-reactive 200 kDa proteins of Apis mellifera and Vespula vulgaris venom. Methods/Principal Findings Tandem mass spectrometry-based sequencing of a 200 kDa venom protein yielded peptides that could be assigned to honeybee vitellogenin. The coding regions of the honeybee protein as well as of the homologue from yellow jacket venom were cloned from venom gland cDNA. The newly identified 200 kDa proteins share a sequence identity on protein level of 40% and belong to the family of vitellogenins, present in all oviparous animals, and are the first vitellogenins identified as components of venom. Both vitellogenins could be recombinantly produced as soluble proteins in insect cells and assessed for their specific IgE reactivity. The particular vitellogenins were recognized by approximately 40% of sera of venom-allergic patients even in the absence of cross-reactive carbohydrate determinants. Conclusion With the vitellogenins of Apis mellifera and Vespula vulgaris venom a new homologous pair of venom allergens was identified and becomes available for future applications. Due to their allergenic properties the honeybee and the yellow jacket venom vitellogenin were designated as allergens Api m 12 and Ves v 6, respectively. PMID:23626765

Vitellogenin Production by Fishmeal-Containing Diets in Male Tilapia (Oreochromis Mossambicus)

Science.gov (United States)

In a number of fish species, male plasma has been shown to contain substantial levels of l7~estradiol (E z) and in certain cases, vitellogenin (Vg), an Ez-induced precursor of egg yolk protein that is specific to females. The presence ofEz and Vg in males raises questions about ...

17α-Ethinylestradiol (EE2) effect on global gene expression in primary rainbow trout (Oncorhynchus mykiss) hepatocytes.

Science.gov (United States)

Hultman, Maria T; Song, You; Tollefsen, Knut Erik

2015-12-01

The potential impact of endocrine disrupting chemicals (EDCs) in the aquatic environment has driven the development of screening assays to evaluate the estrogenic properties of chemicals and their effects on aquatic organisms such as fish. However, obtaining full concentration-response relationships in animal (in vivo) exposure studies are laborious, costly and unethical, hence a need for developing feasible alternative (non-animal) methods. Use of in vitro bioassays such as primary fish hepatocytes, which retain many of the native properties of the liver, has been proposed for in vitro screening of estrogen receptor (ER) agonists and antagonists. The aim of present study was to characterize the molecular mode of action (MoA) of the ER agonist 17α-ethinylestradiol (EE2) in primary rainbow trout (Oncorhynchus mykiss) hepatocytes. A custom designed salmonid 60,000-feature (60k) oligonucleotide microarray was used to characterize the potential MoAs after 48h exposure to EE2. The microarray analysis revealed several concentration-dependent gene expression alterations including classical estrogen sensitive biomarker gene expression (e.g. estrogen receptor α, vitellogenin, zona radiata). Gene Ontology (GO) analysis displayed transcriptional changes suggesting interference of cellular growth, fatty acid and lipid metabolism potentially mediated through the estrogen receptor (ER), which were proposed to be associated with modulation of genes involved in endocrine function and reproduction. Pathway analysis supported the identified GOs and revealed modulation of additional genes associated with apoptosis and cholesterol biosynthesis. Differentially expressed genes (DEGs) related to impaired lipid metabolism (e.g. peroxisome proliferator-activated receptor α and γ), growth (e.g. insulin growth factor protein 1), phase I and II biotransformation (e.g. cytochrome P450 1A, sulfotransferase, UDP-glucuronosyltransferase and glutathione S-transferase) provided additional

Comparison of the biological features between human fetal hepatocyte and immortalized L-02 hepatocyte in vitro

International Nuclear Information System (INIS)

Kong Weiwei; Teng Gaojun

2004-01-01

Objective: To evaluate the feasibilities of the potential donors in liver cell transplantation using the human fetal hepatocytes and immortalized L-02 hepatocytes by comparing their biological features. Methods: Human fetal hepatocytes were isolated from aborted fetal livers (gestational ages from 14 w to 24 w) by an improved two-stage perfusion method and cultured in a conditioned medium without any growth factors. α-fetal protein (AFP) and albumin (ALB) were detected by radioimmunoassay (RIA) and cytokeratin-19 (CK-19 ) was identified by cellular immunochemistry study. Immortalized L-02 hepatocytes were cultured in the same condition and the characteristic proteins were detected by the same methods. Results: The viability of human fetal hepatocytes was approximately 95% using the perfusion method, and the maximum survival time of the cultured hepatocytes was 3 weeks. The expression of AFP, ALB, and CK19 was detected at the same time, especially during Day 3 to Day 7 in the culture. By comparison, the proliferation ability of L-02 hepatocyte was greater, although with a lower level of ALB secretion. The expression of AFP and CK19 was not detected. Furthermore, during the long culture, L-02 hepatocytes may undergo a morphologic change and fail to express ALB. Conclusion: Human fetal hepatocyte may be a practical donor for hepatocyte transplantation with its high-level protein expression and potential bi-differentiation ability. In view of the absent expression of ALB and the morphologic change in culture, although with better proliferation, L-02 hepatocyte seems not useful for hepatocyte transplantation

Use of vitellogenin as a biomarker for estrogenic effect: What is the baseline?

DEFF Research Database (Denmark)

Bjerregaard, Poul; Holbech, Henrik; Pedersen, Knud Ladegaard

investigated in 2004 were revisited in the 2010 study and vitellogenin concentrations had decreased. Between 2004 and 2010 some discharges (via septic tanks) from scattered houses in the open land known to discharge estrogenic activity had been removed and this may have reduced the addition of estrogenicity...

Comparative responses to endocrine disrupting compounds in early life stages of Atlantic salmon, Salmo salar.

Science.gov (United States)

Duffy, T A; Iwanowicz, L R; McCormick, S D

2014-07-01

Atlantic salmon (Salmo salar) are endangered anadromous fish that may be exposed to feminizing endocrine disrupting compounds (EDCs) during early development, potentially altering physiological capacities, survival and fitness. To assess differential life stage sensitivity to common EDCs, we carried out short-term (4 day) exposures using three doses each of 17 α-ethinylestradiol (EE2), 17 β-estradiol (E2), and nonylphenol (NP) on four early life stages; embryos, yolk-sac larvae, feeding fry and 1 year old smolts. Differential response was compared using vitellogenin (Vtg, a precursor egg protein) gene transcription. Smolts were also examined for impacts on plasma Vtg, cortisol, thyroid hormones (T4/T3) and hepatosomatic index (HSI). Compound-related mortality was not observed in any life stage, but Vtg mRNA was elevated in a dose-dependent manner in yolk-sac larvae, fry and smolts but not in embryos. The estrogens EE2 and E2 were consistently stronger inducers of Vtg than NP. Embryos responded significantly to the highest concentration of EE2 only, while older life stages responded to the highest doses of all three compounds, as well as intermediate doses of EE2 and E2. Maximal transcription was greater for fry among the three earliest life stages, suggesting fry may be the most responsive life stage in early development. Smolt plasma Vtg was also significantly increased, and this response was observed at lower doses of each compound than was detected by gene transcription suggesting plasma Vtg is a more sensitive indicator at this life stage. HSI was increased at the highest doses of EE2 and E2, and plasma T3 was decreased at the highest dose of EE2. Our results indicate that all life stages are potentially sensitive to endocrine disruption by estrogenic compounds and that physiological responses were altered over a short window of exposure, indicating the potential for these compounds to impact fish in the wild. Copyright © 2014 Elsevier B.V. All rights

In vitro and in vivo studies of the endocrine disrupting potency of cadmium in roach (Rutilus rutilus) liver

International Nuclear Information System (INIS)

Gerbron, M.; Geraudie, P.; Xuereb, B.; Marie, S.; Minier, C.

2015-01-01

Highlights: • Ex vivo and in vivo experiments suggest estrogen receptor-driven effects of cadmium. • CdCl 2 is strongly anti-estrogenic when co-exposed to E2. • CdCl 2 + E2 significantly inhibit vtg and erα mRNA expressions. • CdCl 2 compromises the E2-mediated induction of the ar mRNA expression in vivo. - Abstract: Cadmium has been reported to exert estrogenic, antiestrogenic or both effects in vertebrate species. To elucidate the endocrine disrupting action of CdCl 2 , ex vivo and in vivo experiments were performed in roach (Rutilus rutilus). Roach liver explants were exposed to a range of CdCl 2 concentrations alone (0.1–50 μM) or with an effective concentration (100 nM) of 17β-estradiol (E2). In addition, juvenile roach were intraperitoneally injected with CdCl 2 (0.1–2.5 mg/kg) with or without 1 mg E2/kg. Subsequent analysis evaluated the effect of CdCl 2 on vitellogenin (VTG) synthesis both at the mRNA and protein level, on estrogen receptors (erα and erβ1) and on androgen receptor (ar) mRNA expression. Ex vivo and in vivo experiments indicated that CdCl 2 is strongly anti-estrogenic as, when co-exposed to E2, CdCl 2 significantly inhibited VTG production as well as vtg and erα mRNA expressions. Moreover, CdCl 2 compromised the E2-mediated induction of the ar mRNA expression in vivo

Repolarization of hepatocytes in culture.

Science.gov (United States)

Talamini, M A; Kappus, B; Hubbard, A

1997-01-01

We have evaluated the biochemical, morphological, and functional redevelopment of polarity in freshly isolated hepatocytes cultured using a double layer collagen gel sandwich technique. Western blot analysis showed increased cellular levels of the cell adhesion protein uvomorulin as cultured hepatocytes repolarized. Immunofluorescence studies using antibodies against domain-specific membrane proteins showed polarity as early as 48 hours, although the pattern of the polymeric Immunoglobulin-A receptor (pIgA-R) differed from in vivo liver. Electron microscopy showed developing bile canaliculi at 1 day. However, the functional presence of tight junctions was absent at 1 day, but present at 5 days. We further showed functional polarity to be present at 4 days by documenting the ability of cultured hepatocytes to metabolize and excrete fluorescein diacetate into visible bile canaliculi. We conclude that hepatocytes cultured appropriately develop morphological and functional polarity. Hepatocyte culture is therefore a useful tool for the study of mechanisms responsible for the development of polarized function.

Identification and Transcriptional Modulation of the Largemouth Bass, Micropterus salmoides, Vitellogenin Receptor During Oocyte Development by Insulin and Sex Steroids1

OpenAIRE

Dominguez, Gustavo A.; Quattro, Joseph M.; Denslow, Nancy D.; Kroll, Kevin J.; Prucha, Melinda S.; Porak, Wesley F.; Grier, Harry J.; Sabo-Attwood, Tara L.

2012-01-01

Fish vitellogenin synthesized and released from the liver of oviparous animals is taken up into oocytes by the vitellogenin receptor. This is an essential process in providing nutrient yolk to developing embryos to ensure successful reproduction. Here we disclose the full length vtgr cDNA sequence for largemouth bass (LMB) that reveals greater than 90% sequence homology with other fish vtgr sequences. We classify LMB Vtgr as a member of the low density lipoprotein receptor superfamily based o...

Induction of ovarian maturation in Penaeus monodon by molecular signal interventional approach.

Science.gov (United States)

Devaraj, Halagowder; Saravanakumar, Marimuthu; Thiyagu, Mani

2012-11-01

Vitellogenin (VTG) synthesis in the hepatopancreas and ovary is negatively regulated by vitellogenesis-inhibiting hormone (VIH) produced in the neurosecretory cell of X-organ/sinus gland complex of the eyestalks of penaeid shrimp. Eyestalk ablation is used commercially to induce ovarian maturation in shrimps which leads to an eventual loss of the spawner. The aim of the present study was to understand the molecular mechanism of VIH regulation in ovarian development and its inhibition of VTG gene expression by using a MEK-specific inhibitor (U0126). The real-time quantitative PCR results showed VTG mRNA level was progressively increased in the ovary and hepatopancreas of unilateral eyestalk-ablated and inhibitor-treated shrimps. Western blot analysis also showed that phosphoMEK was detected only in the unilateral eyestalk-ablated and control shrimp, whereas phospho-MEK was not detected in inhibitor-treated shrimp. DAX-1, SF-1, and StAR expression correlated with changes in VIH mRNA and altered phospho-ERK levels. This is consistent with the hypothesis that suppression of DAX-1 results in SF-1-mediated StAR protein upregulation of estradiol that is implicated in vitellogenesis. This is the first report that demonstrates the molecular mechanism of VIH suppression via MEK pathway to induce ovarian maturation in female Penaeus monodon by molecular signal intervention, a less-invasive method than traditional eyestalk ablation. Copyright © 2012 Wiley Periodicals, Inc.

Hepatocyte nuclear factor 4A improves hepatic differentiation of immortalized adult human hepatocytes and improves liver function and survival.

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Hang, Hua-Lian; Liu, Xin-Yu; Wang, Hai-Tian; Xu, Ning; Bian, Jian-Min; Zhang, Jian-Jun; Xia, Lei; Xia, Qiang

2017-11-15

Immortalized human hepatocytes (IHH) could provide an unlimited supply of hepatocytes, but insufficient differentiation and phenotypic instability restrict their clinical application. This study aimed to determine the role of hepatocyte nuclear factor 4A (HNF4A) in hepatic differentiation of IHH, and whether encapsulation of IHH overexpressing HNF4A could improve liver function and survival in rats with acute liver failure (ALF). Primary human hepatocytes were transduced with lentivirus-mediated catalytic subunit of human telomerase reverse transcriptase (hTERT) to establish IHH. Cells were analyzed for telomerase activity, proliferative capacity, hepatocyte markers, and tumorigenicity (c-myc) expression. Hepatocyte markers, hepatocellular functions, and morphology were studied in the HNF4A-overexpressing IHH. Hepatocyte markers and karyotype analysis were completed in the primary hepatocytes using shRNA knockdown of HNF4A. Nuclear translocation of β-catenin was assessed. Rat models of ALF were treated with encapsulated IHH or HNF4A-overexpressing IHH. A HNF4A-positive IHH line was established, which was non-tumorigenic and conserved properties of primary hepatocytes. HNF4A overexpression significantly enhanced mRNA levels of genes related to hepatic differentiation in IHH. Urea levels were increased by the overexpression of HNF4A, as measured 24h after ammonium chloride addition, similar to that of primary hepatocytes. Chromosomal abnormalities were observed in primary hepatocytes transfected with HNF4A shRNA. HNF4α overexpression could significantly promote β-catenin activation. Transplantation of HNF4A overexpressing IHH resulted in better liver function and survival of rats with ALF compared with IHH. HNF4A improved hepatic differentiation of IHH. Transplantation of HNF4A-overexpressing IHH could improve the liver function and survival in a rat model of ALF. Copyright © 2017 Elsevier Inc. All rights reserved.

Methylation of the chicken vitellogenin gene: influence of estradiol administration.

Science.gov (United States)

Meijlink, F C; Philipsen, J N; Gruber, M; Ab, G

1983-01-01

The degree of methylation of the chicken vitellogenin gene has been investigated. Upon induction by administration of estradiol to a rooster, methyl groups at specific sites near the 5'-end of the gene are eliminated. The process of demethylation is slower than the activation of the gene. Demethylation is therefore probably not a prerequisite to gene transcription. At least two other sites in the coding region of the gene are methylated in the liver of estrogenized roosters, but not in the liver of a laying hen, where the gene is naturally active. Images PMID:6298743

Phenotypic and functional analyses show stem cell-derived hepatocyte-like cells better mimic fetal rather than adult hepatocytes.

Science.gov (United States)

Baxter, Melissa; Withey, Sarah; Harrison, Sean; Segeritz, Charis-Patricia; Zhang, Fang; Atkinson-Dell, Rebecca; Rowe, Cliff; Gerrard, Dave T; Sison-Young, Rowena; Jenkins, Roz; Henry, Joanne; Berry, Andrew A; Mohamet, Lisa; Best, Marie; Fenwick, Stephen W; Malik, Hassan; Kitteringham, Neil R; Goldring, Chris E; Piper Hanley, Karen; Vallier, Ludovic; Hanley, Neil A

2015-03-01

Hepatocyte-like cells (HLCs), differentiated from pluripotent stem cells by the use of soluble factors, can model human liver function and toxicity. However, at present HLC maturity and whether any deficit represents a true fetal state or aberrant differentiation is unclear and compounded by comparison to potentially deteriorated adult hepatocytes. Therefore, we generated HLCs from multiple lineages, using two different protocols, for direct comparison with fresh fetal and adult hepatocytes. Protocols were developed for robust differentiation. Multiple transcript, protein and functional analyses compared HLCs to fresh human fetal and adult hepatocytes. HLCs were comparable to those of other laboratories by multiple parameters. Transcriptional changes during differentiation mimicked human embryogenesis and showed more similarity to pericentral than periportal hepatocytes. Unbiased proteomics demonstrated greater proximity to liver than 30 other human organs or tissues. However, by comparison to fresh material, HLC maturity was proven by transcript, protein and function to be fetal-like and short of the adult phenotype. The expression of 81% phase 1 enzymes in HLCs was significantly upregulated and half were statistically not different from fetal hepatocytes. HLCs secreted albumin and metabolized testosterone (CYP3A) and dextrorphan (CYP2D6) like fetal hepatocytes. In seven bespoke tests, devised by principal components analysis to distinguish fetal from adult hepatocytes, HLCs from two different source laboratories consistently demonstrated fetal characteristics. HLCs from different sources are broadly comparable with unbiased proteomic evidence for faithful differentiation down the liver lineage. This current phenotype mimics human fetal rather than adult hepatocytes. Copyright © 2014 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Magnetic cell labeling of primary and stem cell-derived pig hepatocytes for MRI-based cell tracking of hepatocyte transplantation.

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Dwayne R Roach

Full Text Available Pig hepatocytes are an important investigational tool for optimizing hepatocyte transplantation schemes in both allogeneic and xenogeneic transplant scenarios. MRI can be used to serially monitor the transplanted cells, but only if the hepatocytes can be labeled with a magnetic particle. In this work, we describe culture conditions for magnetic cell labeling of cells from two different pig hepatocyte cell sources; primary pig hepatocytes (ppHEP and stem cell-derived hepatocytes (PICM-19FF. The magnetic particle is a micron-sized iron oxide particle (MPIO that has been extensively studied for magnetic cell labeling for MRI-based cell tracking. ppHEP could endocytose MPIO with labeling percentages as high as 70%, achieving iron content as high as ~55 pg/cell, with >75% viability. PICM-19FF had labeling >97%, achieving iron content ~38 pg/cell, with viability >99%. Extensive morphological and functional assays indicated that magnetic cell labeling was benign to the cells. The results encourage the use of MRI-based cell tracking for the development and clinical use of hepatocyte transplantation methodologies. Further, these results generally highlight the importance of functional cell assays in the evaluation of contrast agent biocompatibility.

Mutational studies reveal a complex set of positive and negative control elements within the chicken vitellogenin II promoter.

Science.gov (United States)

Seal, S N; Davis, D L; Burch, J B

1991-05-01

The endogenous chicken vitellogenin II (VTGII) gene is transcribed exclusively in hepatocytes in response to estrogen. We previously identified two estrogen response elements (EREs) upstream of this gene. We now present an analysis of the VTGII promoter activated by these EREs in response to estrogen. Chimeric VTGII-CAT genes were cotransfected into LMH chicken hepatoma cells along with an estrogen receptor expression vector, and transient CAT expression was assayed after culturing the cells in the absence or presence of estrogen. An analysis of constructs bearing deletions downstream of the more proximal ERE indicated that promoter elements relevant to transcription in LMH cells extend to between -113 and -96. The relative importance of sequences within the VTGII promoter was examined by using 10 contiguous linker scanner mutations spanning the region from -117 to -24. Although most of these mutations compromised VTGII promoter function, one dramatically increased expression in LMH cells and also rendered the VTGII promoter capable of being activated by cis-linked EREs in fibroblasts cotransfected with an estrogen receptor expression vector. Gel retardation and DNase I footprinting assays revealed four factor-binding sites within this promoter. We demonstrate that three of these sites bind C/EBP, SP1, and USF (or related factors), respectively; the fourth site binds a factor that we denote TF-V beta. The biological relevance of these findings is suggested by the fact that three of these binding sites map to sites previously shown to be occupied in vivo in response to estrogen.

Reproductive biomarkers responses induced by xenoestrogens in the characid fish Astyanax fasciatus inhabiting a South American reservoir: An integrated field and laboratory approach

Energy Technology Data Exchange (ETDEWEB)

Prado, Paula S.; Pinheiro, Ana Paula B. [Departamento de Morfologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, UFMG, Belo Horizonte, C.P. 486, 30161-970, Minas Gerais (Brazil); Bazzoli, Nilo [Programa de Pós-Graduação em Zoologia de Vertebrados, Pontifícia Universidade Católica de Minas Gerais, PUC Minas, Belo Horizonte 30535-610, Minas Gerais (Brazil); Rizzo, Elizete, E-mail: ictio@icb.ufmg.br [Departamento de Morfologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, UFMG, Belo Horizonte, C.P. 486, 30161-970, Minas Gerais (Brazil)

2014-05-01

Field studies evaluating the effects of endocrine disruption chemicals (EDCs) on the fish reproduction are scarce worldwide. The goal of this study was to assess hepatic levels of vitellogenin (Vtg), zona radiata proteins (Zrp) and insulin-like growth factors (IGF-I and IGF-II), and relating them to reproductive endpoints in a wild fish population habiting a reservoir that receive domestic sewage, agricultural and industrial residues. Adult fish Astyanax fasciatus were sampled during the reproductive season in five sites from the Furnas Reservoir, Grande River, and Paraguay–Paraná basin. As a control to field data, fish were experimentally exposed via dietary intake, to oestradiol benzoate (OB) for 7 days. Fish from site with little anthropogenic interference showed hepatic levels of Vtg, Zrp and IGF-I and IGF-II similar to those from the non-treated experimental group. In sites located immediately downstream from the municipal wastewater discharges, the water total oestrogen was >120 ng/l, and male fish displayed increased Vtg and Zrp and decreased IGF-I levels similar to OB treated fish. In females, levels of Vtg, Zrp, IGF-I and IGF-II suggest an impairment of final oocyte maturation and spawning, as also detected by frequency of over-ripening, follicular atresia and fecundity. At the sites that receive agricultural and industrial residues, the water total oestrogen was <50 ng/l and females showed decreased Zrp and increased IGF-II levels associated to reduced diameter of vitellogenic follicles, indicating an inhibition of oocyte growth. Overall, the current study reports oestrogenic contamination impairing the reproduction of a wild fish from a hydroeletric reservoir and, the data contribute to improving the current knowledge on relationship between hepatic Vtg, Zrp and IGF-I and IGF-II, and reproductive endpoints in a teleost fish. In addition, our data point out novel reproductive biomarkers (IGF-I, IGF-II and over-ripening) to assessing xenoestrogenic

Hepatocyte Growth Factor Reduces Free Cholesterol-Mediated Lipotoxicity in Primary Hepatocytes by Countering Oxidative Stress

Directory of Open Access Journals (Sweden)

Mayra Domínguez-Pérez

2016-01-01

Full Text Available Cholesterol overload in the liver has shown toxic effects by inducing the aggravation of nonalcoholic fatty liver disease to steatohepatitis and sensitizing to damage. Although the mechanism of damage is complex, it has been demonstrated that oxidative stress plays a prominent role in the process. In addition, we have proved that hepatocyte growth factor induces an antioxidant response in hepatic cells; in the present work we aimed to figure out the protective effect of this growth factor in hepatocytes overloaded with free cholesterol. Hepatocytes from mice fed with a high-cholesterol diet were treated or not with HGF, reactive oxygen species present in cholesterol overloaded hepatocytes significantly decreased, and this effect was particularly associated with the increase in glutathione and related enzymes, such as γ-gamma glutamyl cysteine synthetase, GSH peroxidase, and GSH-S-transferase. Our data clearly indicate that HGF displays an antioxidant response by inducing the glutathione-related protection system.

Assessing reproductive and endocrine parameters in male largescale suckers (Catostomus macrocheilus) along a contaminant gradient in the lower Columbia River, USA

Science.gov (United States)

Jenkins, Jill A.; Olivier, H.M.; Draugelis-Dale, R. O.; Eilts, B.E.; Torres, L.; Patiño, R.; Nilsen, Elena B.; Goodbred, Steven L.

2014-01-01

Persistent organochlorine pollutants such as polychlorinated biphenyls (PCBs), dichlorodiphenyldichloroethylene (p,p′-DDE), and polybrominated diphenyl ethers (PBDEs) are stable, bioaccumulative, and widely found in the environment, wildlife, and the human population. To explore the hypothesis that reproduction in male fish is associated with environmental exposures in the lower Columbia River (LCR), reproductive and endocrine parameters were studied in male resident, non-anadromous largescale sucker (Catostomus macrocheilus) (LSS) in the same habitats as anadromous salmonids having conservation status. Testes, thyroid tissue and plasma collected in 2010 from Longview (LV), Columbia City (CC), and Skamania (SK; reference) were studied. Sperm morphologies and thyrocyte heights were measured by light microscopy, sperm motilities by computer-assisted sperm motion analysis, sperm adenosine triphosphate (ATP) with luciferase, and plasma vitellogenin (VTG), thyroxine (T4), and triiodothyronine (T3) by immunoassay. Sperm apoptosis, viability, mitochondrial membrane potential, nuclear DNA fragmentation, and reproductive stage were measured by flow cytometry. Sperm quality parameters (except counts) and VTG were significantly different among sites, with correlations between VTG and 7 sperm parameters. Thyrocyte heights, T4, T3, gonadosomatic index and Fulton's condition factor differed among sites, but not significantly. Sperm quality was significantly lower and VTG higher where liver contaminants and water estrogen equivalents were highest (LV site). Total PCBs (specifically PCB-138, -146, -151, -170, -174, -177, -180, -183, -187, -194, and -206) and total PBDEs (specifically BDE-47, -100, -153, and -154) were negatively correlated with sperm motility. PCB-206 and BDE-154 were positively correlated with DNA fragmentation, and pentachloroanisole and VTG were positively correlated with sperm apoptosis and negatively correlated with ATP. BDE-99 was positively correlated

Linking genomic responses of gonads with reproductive impairment in marine medaka (Oryzias melastigma) exposed chronically to the chemopreventive and antifouling agent, 3,3′-diindolylmethane (DIM)

Energy Technology Data Exchange (ETDEWEB)

Chen, Lianguo [Division of Life Science, Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong SAR (China); Au, Doris W.T. [State Key Laboratory in Marine Pollution, Department of Biology and Chemistry, City University of Hong Kong, Kowloon, Hong Kong SAR (China); Hu, Chenyan [School of Chemistry and Environmental Engineering, Wuhan Institute of Technology, Wuhan 430072 (China); Zhang, Weipeng [Division of Life Science, Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong SAR (China); Zhou, Bingsheng [State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072 (China); Cai, Lin [Division of Life Science, Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong SAR (China); Giesy, John P. [Department of Veterinary Biomedical Sciences and Toxicology Centre, University of Saskatchewan, 44 Campus Drive, Saskatoon, SK S7N 5B3 (Canada); Department of Zoology, and Center for Integrative Toxicology, Michigan State University, East Lansing, MI (United States); Qian, Pei-Yuan, E-mail: boqianpy@ust.hk [Division of Life Science, Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong SAR (China)

2017-02-15

Highlights: • Marine medaka were exposed chronically to low-doses of DIM. • Toxicogenomic responses of gonads were profiled at transcript and protein levels. • Molecular initiating events were linked to adverse apical outcomes. • VTG mobilization was blocked by lower abundance of cathepsin enzyme in ovary. • Eggshell proteins were more indicative of reproductive failure than VTG. - Abstract: 3,3′-Diindolylmethane (DIM) has been promoted as an effective chemopreventive and antifouling additive. However, the concurrent risks or side effects of DIM are not fully understood, especially on tissues responsive to estrogen. Therefore, this study employed marine medaka (Oryzias melastigma) as a test model to evaluate relative safety and explore mechanisms of toxic action of DIM on development and function of gonad after chronic (28 days) aqueous exposure to relatively low doses (0 μg/L or 8.5 μg/L). Integration of comprehensive toxicogenomic analysis at the transcriptome and proteome levels with apical endpoints, such as production of eggs and swimming performance of larvae, elucidated the molecular linkage in gonad from bottom up along the reproductive adverse outcome pathway. A series of sequential changes at the transcript and protein levels were linked to lesser fecundity and viability of larvae exposed to DIM. Anomalous production of vitellogenin (VTG) and eggshell proteins in testis confirmed the estrogenic potency of DIM. In the ovary, although storage of VTG was greater, lesser expressions of cathepsin enzymes blocked cleavage and incorporation of VTG into oocytes as yolk, which acted together with lower eggshell proteins to inhibit maturation of primary oocyte and thus contributed to impairment of fecundity. Overall, this study demonstrated that exposure to DIM impaired reproductive fitness. Diverse molecular initiating changes in gonads were linked to apical endpoints that could be used in assessment of risks posed by DIM on gametogenesis. In

Linking genomic responses of gonads with reproductive impairment in marine medaka (Oryzias melastigma) exposed chronically to the chemopreventive and antifouling agent, 3,3′-diindolylmethane (DIM)

International Nuclear Information System (INIS)

Chen, Lianguo; Au, Doris W.T.; Hu, Chenyan; Zhang, Weipeng; Zhou, Bingsheng; Cai, Lin; Giesy, John P.; Qian, Pei-Yuan

2017-01-01

Highlights: • Marine medaka were exposed chronically to low-doses of DIM. • Toxicogenomic responses of gonads were profiled at transcript and protein levels. • Molecular initiating events were linked to adverse apical outcomes. • VTG mobilization was blocked by lower abundance of cathepsin enzyme in ovary. • Eggshell proteins were more indicative of reproductive failure than VTG. - Abstract: 3,3′-Diindolylmethane (DIM) has been promoted as an effective chemopreventive and antifouling additive. However, the concurrent risks or side effects of DIM are not fully understood, especially on tissues responsive to estrogen. Therefore, this study employed marine medaka (Oryzias melastigma) as a test model to evaluate relative safety and explore mechanisms of toxic action of DIM on development and function of gonad after chronic (28 days) aqueous exposure to relatively low doses (0 μg/L or 8.5 μg/L). Integration of comprehensive toxicogenomic analysis at the transcriptome and proteome levels with apical endpoints, such as production of eggs and swimming performance of larvae, elucidated the molecular linkage in gonad from bottom up along the reproductive adverse outcome pathway. A series of sequential changes at the transcript and protein levels were linked to lesser fecundity and viability of larvae exposed to DIM. Anomalous production of vitellogenin (VTG) and eggshell proteins in testis confirmed the estrogenic potency of DIM. In the ovary, although storage of VTG was greater, lesser expressions of cathepsin enzymes blocked cleavage and incorporation of VTG into oocytes as yolk, which acted together with lower eggshell proteins to inhibit maturation of primary oocyte and thus contributed to impairment of fecundity. Overall, this study demonstrated that exposure to DIM impaired reproductive fitness. Diverse molecular initiating changes in gonads were linked to apical endpoints that could be used in assessment of risks posed by DIM on gametogenesis. In

Comparative responses to endocrine disrupting compounds in early life stages of Atlantic salmon, Salmo salar

Science.gov (United States)

Duffy, Tara A.; Iwanowicz, Luke R.; McCormick, Stephen D.

2014-01-01

Atlantic salmon (Salmo salar) are endangered anadromous fish that may be exposed to feminizing endocrine disrupting compounds (EDCs) during early development, potentially altering physiological capacities, survival and fitness. To assess differential life stage sensitivity to common EDCs, we carried out short-term (four day) exposures using three doses each of 17α-ethinylestradiol (EE2), 17β-estradiol (E2), and nonylphenol (NP) on four early life stages; embryos, yolk-sac larvae, feeding fry and one year old smolts. Differential response was compared using vitellogenin (Vtg, a precursor egg protein) gene transcription. Smolts were also examined for impacts on plasma Vtg, cortisol, thyroid hormones (T4/T3) and hepatosomatic index (HSI). Compound-related mortality was not observed in any life stage, but Vtg mRNA was elevated in a dose-dependent manner in yolk-sac larvae, fry and smolts but not in embyos. The estrogens EE2 and E2 were consistently stronger inducers of Vtg than NP. Embryos responded significantly to the highest concentration of EE2 only, while older life stages responded to the highest doses of all three compounds, as well as intermediate doses of EE2 and E2. Maximal transcription was greater for fry among the three earliest life stages, suggesting fry may be the most responsive life stage in early development. Smolt plasma Vtg was also significantly increased, and this response was observed at lower doses of each compound than was detected by gene transcription suggesting this is a more sensitive indicator at this life stage. HSI was increased at the highest doses of EE2 and E2 and plasma T3 decreased at the highest dose of EE2. Our results indicate that all life stages after hatching are potentially sensitive to endocrine disruption by estrogenic compounds and that physiological responses were altered over a short window of exposure, indicating the potential for these compounds to impact fish in the wild.

Factor VIIa binding and internalization in hepatocytes

DEFF Research Database (Denmark)

Hjortoe, G; Sorensen, B B; Petersen, L C

2005-01-01

The liver is believed to be the primary clearance organ for coagulation proteases, including factor VIIa (FVIIa). However, at present, clearance mechanisms for FVIIa in liver are unknown. To obtain information on the FVIIa clearance mechanism, we investigated the binding and internalization...... no effect. HEPG2 cells internalized FVIIa with a rate of 10 fmol 10(-5) cells h(-1). In contrast to HEPG2 cells, FVIIa binding to primary rat hepatocytes was completely independent of TF, and excess unlabeled FVIIa partly reduced the binding of 125I-FVIIa to rat hepatocytes. Further, compared with HEPG2...... cells, three- to fourfold more FVIIa bound to rat primary hepatocytes, and the bound FVIIa was internalized at a faster rate. Similar FVIIa binding and internalization profiles were observed in primary human hepatocytes. Plasma inhibitors had no effect on FVIIa binding and internalization in hepatocytes...

Selective insulin resistance in hepatocyte senescence

International Nuclear Information System (INIS)

Aravinthan, Aloysious; Challis, Benjamin; Shannon, Nicholas; Hoare, Matthew; Heaney, Judith; Alexander, Graeme J.M.

2015-01-01

Insulin resistance has been described in association with chronic liver disease for decades. Hepatocyte senescence has been demonstrated in chronic liver disease and as many as 80% of hepatocytes show a senescent phenotype in advanced liver disease. The aim of this study was to understand the role of hepatocyte senescence in the development of insulin resistance. Senescence was induced in HepG2 cells via oxidative stress. The insulin metabolic pathway was studied in control and senescent cells following insulin stimulation. GLUT2 and GLUT4 expressions were studied in HepG2 cells and human liver tissue. Further, GLUT2 and GLUT4 expressions were studied in three independent chronic liver disease cohorts. Signalling impairment distal to Akt in phosphorylation of AS160 and FoxO1 was evident in senescent HepG2 cells. Persistent nuclear localisation of FoxO1 was demonstrated in senescent cells despite insulin stimulation. Increased GLUT4 and decreased GLUT2 expressions were evident in senescent cells, human cirrhotic liver tissue and publically available liver disease datasets. Changes in GLUT expressions were associated with a poor clinical prognosis. In conclusion, selective insulin resistance is evident in senescent HepG2 cells and changes in GLUT expressions can be used as surrogate markers of hepatocyte senescence. - Highlights: • Senescent hepatocytes demonstrate selective insulin resistance. • GLUT changes act as markers of hepatocyte senescence and have prognostic value. • Study offers insight into long noticed intimacy of cirrhosis and insulin resistance

Selective insulin resistance in hepatocyte senescence

Energy Technology Data Exchange (ETDEWEB)

Aravinthan, Aloysious [Division of Gastroenterology and Hepatology, Department of Medicine, University of Cambridge, Cambridge (United Kingdom); Challis, Benjamin [Institute of Metabolic Sciences, University of Cambridge, Cambridge (United Kingdom); Shannon, Nicholas [Cancer Research UK Cambridge Institute, Cambridge (United Kingdom); Hoare, Matthew [Division of Gastroenterology and Hepatology, Department of Medicine, University of Cambridge, Cambridge (United Kingdom); Cancer Research UK Cambridge Institute, Cambridge (United Kingdom); Heaney, Judith [Division of Gastroenterology and Hepatology, Department of Medicine, University of Cambridge, Cambridge (United Kingdom); Foundation for Liver Research, Institute of Hepatology, London (United Kingdom); Alexander, Graeme J.M., E-mail: gja1000@doctors.org.uk [Division of Gastroenterology and Hepatology, Department of Medicine, University of Cambridge, Cambridge (United Kingdom)

2015-02-01

Insulin resistance has been described in association with chronic liver disease for decades. Hepatocyte senescence has been demonstrated in chronic liver disease and as many as 80% of hepatocytes show a senescent phenotype in advanced liver disease. The aim of this study was to understand the role of hepatocyte senescence in the development of insulin resistance. Senescence was induced in HepG2 cells via oxidative stress. The insulin metabolic pathway was studied in control and senescent cells following insulin stimulation. GLUT2 and GLUT4 expressions were studied in HepG2 cells and human liver tissue. Further, GLUT2 and GLUT4 expressions were studied in three independent chronic liver disease cohorts. Signalling impairment distal to Akt in phosphorylation of AS160 and FoxO1 was evident in senescent HepG2 cells. Persistent nuclear localisation of FoxO1 was demonstrated in senescent cells despite insulin stimulation. Increased GLUT4 and decreased GLUT2 expressions were evident in senescent cells, human cirrhotic liver tissue and publically available liver disease datasets. Changes in GLUT expressions were associated with a poor clinical prognosis. In conclusion, selective insulin resistance is evident in senescent HepG2 cells and changes in GLUT expressions can be used as surrogate markers of hepatocyte senescence. - Highlights: • Senescent hepatocytes demonstrate selective insulin resistance. • GLUT changes act as markers of hepatocyte senescence and have prognostic value. • Study offers insight into long noticed intimacy of cirrhosis and insulin resistance.

Study the impact of gamma irradiation on the vitellogenin gene in Galleria mellonella females by using the comparative CT method

Directory of Open Access Journals (Sweden)

Y.S. Zaghloul

2017-07-01

Full Text Available The eggs deposited by oviparous organisms contain large amounts of vitellus, or yolk, which are utilized by the growing embryo. Vitellogenesis is the process of vitellus accumulation involves massive heterosynthetic synthesis of the protein vitellogenin (Vg and its deposition in the oocyte. Vg levels are indicative for the efficiency of the oogenesis process. This work summarizes data on Vg levels in Galleria mellonella females exposed to various doses of gamma radiation. The results stated that vitellogenin was affected by irradiation. The decreased levels of Vg was dose dependent reaching an undetermined level at 160Gy which is the sterileing dose for G. mellonella female that suppress the egg production by them.

Targeted deletion of hepatocyte Ikkβ confers growth advantages

International Nuclear Information System (INIS)

Koch, Katherine S.; Maeda, Shin; He, Guobin; Karin, Michael; Leffert, Hyam L.

2009-01-01

Mice lacking hepatocyte IKKβ (Ikkβ Δhep ) are defective in TNFα-activation of hepatocellular transcription factor NF-κB, and highly susceptible to hepatotoxicity. Following diethylnitrosamine (DEN) exposure, Ikkβ Δhep mice develop more hepatocellular carcinoma (HCC) than control mice due partly to enhanced DEN-induced hepatocyte death. Here we show that Ikkβ Δhep hepatocytes display growth advantages over normal hepatocytes consisting of precocious PCNA and cyclin D1 expression during liver regeneration (shortened hepatocyte G 0 → G 1 transitions), and enhanced recovery efficiency, cyclin D1 expression and cell proliferation after plating. Ex vivo deletion of Ikkβ also accelerates hepatocyte growth. Ikkβ Δhep hepatocyte proliferative responses show heightened sensitivity to TGFα and TNFα, and heightened expression of fibronectin, collagens I/III, nidogen, β-actin and integrin β1 mRNAs. These findings suggest that altered mitogen signaling and expression of extracellular matrix and its associated components underlie growth advantages. Increased HCC development in Ikkβ Δhep mice may also be caused by growth advantages of surviving Ikkβ-deleted hepatocytes.

Brain gene expression changes elicited by peripheral vitellogenin knockdown in the honey bee.

Science.gov (United States)

Wheeler, M M; Ament, S A; Rodriguez-Zas, S L; Robinson, G E

2013-10-01

Vitellogenin (Vg) is best known as a yolk protein precursor. Vg also functions to regulate behavioural maturation in adult honey bee workers, but the underlying molecular mechanisms by which it exerts this novel effect are largely unknown. We used abdominal vitellogenin (vg) knockdown with RNA interference (RNAi) and brain transcriptomic profiling to gain insights into how Vg influences honey bee behavioural maturation. We found that vg knockdown caused extensive gene expression changes in the bee brain, with much of this transcriptional response involving changes in central biological functions such as energy metabolism. vg knockdown targeted many of the same genes that show natural, maturation-related differences, but the direction of change for the genes in these two contrasts was not correlated. By contrast, vg knockdown targeted many of the same genes that are regulated by juvenile hormone (JH) and there was a significant correlation for the direction of change for the genes in these two contrasts. These results indicate that the tight coregulatory relationship that exists between JH and Vg in the regulation of honey bee behavioural maturation is manifest at the genomic level and suggest that these two physiological factors act through common pathways to regulate brain gene expression and behaviour. © 2013 Royal Entomological Society.

Microarray analysis in the zebrafish (Danio rerio) liver and telencephalon after exposure to low concentration of 17alpha-ethinylestradiol

Energy Technology Data Exchange (ETDEWEB)

Martyniuk, Christopher J. [Centre for Advanced Research in Environmental Genomics, 30 Marie Curie, Department of Biology, University of Ottawa, Ottawa, Ontario, K1N 6N5 (Canada); Gerrie, Emily R. [Centre for Advanced Research in Environmental Genomics, 30 Marie Curie, Department of Biology, University of Ottawa, Ottawa, Ontario, K1N 6N5 (Canada); Popesku, Jason T. [Centre for Advanced Research in Environmental Genomics, 30 Marie Curie, Department of Biology, University of Ottawa, Ottawa, Ontario, K1N 6N5 (Canada); Ekker, Marc [Centre for Advanced Research in Environmental Genomics, 30 Marie Curie, Department of Biology, University of Ottawa, Ottawa, Ontario, K1N 6N5 (Canada); Trudeau, Vance L. [Centre for Advanced Research in Environmental Genomics, 30 Marie Curie, Department of Biology, University of Ottawa, Ottawa, Ontario, K1N 6N5 (Canada)]. E-mail: trudeauv@uottawa.ca

2007-08-15

17alpha-ethinylestradiol (EE2) is detected in sewage effluent at concentrations that can disrupt normal reproductive function in fish. The objectives of this study were to identify novel genomic responses to EE2 exposure using microarray and real-time RT-PCR analysis in the liver and telencephalon of male zebrafish. Zebrafish were exposed to an environmentally relevant nominal concentration of 10 ng/L EE2 for a 21-day period. In the liver, common biomarkers for estrogenic exposure such as vitellogenin 1 and 3 (vtg1; vtg3), estrogen receptor alpha (esr1), and apolipoprotein A1 (apoA1) mRNA were identified by microarray analysis as being differentially regulated. Real-time RT-PCR confirmed that vtg1 was induced {approx}700-fold, vtg3 was induced {approx}100-fold and esr1 was induced {approx}20-fold. As determined by microarray analysis, ATPase Na+/K+ alpha 1a.4 (atp1a1a.4) and ATPase Na+/K+ beta 1a (atp1b1a) mRNA were down-regulated in the liver. Gene ontology (GO) analysis revealed that there were common biological processes and molecular functions regulated by EE2 in both tissues (e.g. electron transport and cell communication) but there were tissue specific changes in gene categories. For example, genes involved in protein metabolism, carbohydrate metabolism were down-regulated in the liver but were induced in the telencephalon. This study demonstrates that (1) tissues exhibit different gene responses to low EE2 exposure; (2) there are pronounced genomic effects in the liver and (3) multi-tissue gene profiling is needed to improve understanding of the effects of human pharmaceuticals on aquatic organisms.

Gonadal intersex in smallmouth bass Micropterus dolomieu from northern Indiana with correlations to molecular biomarkers and anthropogenic chemicals.

Science.gov (United States)

Abdel-Moneim, Ahmed; Deegan, Daragh; Gao, Jiejun; De Perre, Chloe; Doucette, Jarrod S; Jenkinson, Byron; Lee, Linda; Sepúlveda, Maria S

2017-11-01

Over the past decade, studies have shown that exposure to endocrine disrupting chemicals (EDCs) can cause gonadal intersex in fish. Smallmouth bass (Micropterus dolomieu) males appear to be highly susceptible to developing testicular oocytes (TO), the most prevalent form of gonadal intersex, as observed in various areas across the U.S. In this study, prevalence and severity of TO was quantified for smallmouth bass sampled from the St. Joseph River in northern Indiana, intersex biomarkers were developed, and association between TO prevalence and organic contaminants were explored. At some sites, TO prevalence reached maximum levels before decreasing significantly after the spawning season. We examined the relationship between TO presence and expression of gonadal and liver genes involved in sex differentiation and reproductive functions (esr1, esr2, foxl2, fshr, star, lhr and vtg). We found that vitellogenin (vtg) transcript levels were significantly higher in the liver of males with TO, but only when sampled during the spawning season. Further, we identified a positive correlation between plasma VTG levels and vtg transcript levels, suggesting its use as a non-destructive biomarker of TO in this species. Finally, we evaluated 43 contaminants in surface water at representative sites using passive sampling to look for contaminants with possible links to the observed TO prevalence. No quantifiable levels of estrogens or other commonly agreed upon EDCs such as the bisphenols were observed in our contaminant assessment; however, we did find high levels of herbicides as well as consistent quantifiable levels of PFOS, PFOA, and triclosan in the watershed where high TO prevalence was exhibited. Our findings suggest that the observed TO prevalence may be the result of exposures to mixtures of nonsteroidal EDCs. Copyright © 2017 Elsevier Ltd. All rights reserved.

Hepatocyte polyploidization and its association with pathophysiological processes.

Science.gov (United States)

Wang, Min-Jun; Chen, Fei; Lau, Joseph T Y; Hu, Yi-Ping

2017-05-18

A characteristic cellular feature of the mammalian liver is the progressive polyploidization of the hepatocytes, where individual cells acquire more than two sets of chromosomes. Polyploidization results from cytokinesis failure that takes place progressively during the course of postnatal development. The proportion of polyploidy also increases with the aging process or with cellular stress such as surgical resection, toxic stimulation, metabolic overload, or oxidative damage, to involve as much as 90% of the hepatocytes in mice and 40% in humans. Hepatocyte polyploidization is generally considered an indicator of terminal differentiation and cellular senescence, and related to the dysfunction of insulin and p53/p21 signaling pathways. Interestingly, the high prevalence of hepatocyte polyploidization in the aged mouse liver can be reversed when the senescent hepatocytes are serially transplanted into young mouse livers. Here we review the current knowledge on the mechanism of hepatocytes polyploidization during postnatal growth, aging, and liver diseases. The biologic significance of polyploidization in senescent reversal, within the context of new ways to think of liver aging and liver diseases is considered.

Integrin-linked kinase is involved in matrix-induced hepatocyte differentiation

International Nuclear Information System (INIS)

Gkretsi, Vasiliki; Bowen, William C.; Yang, Yu; Wu, Chuanyue; Michalopoulos, George K.

2007-01-01

Hepatocytes have restricted proliferative capacity in culture and when cultured without matrix, lose the hepatocyte-specific gene expression and characteristic cellular micro-architecture. Overlay of matrix-preparations on de-differentiated hepatocytes restores differentiation. Integrin-linked kinase (ILK) is a cell-matrix-adhesion protein crucial in fundamental processes such as differentiation and survival. In this study, we investigated the role of ILK, and its binding partners PINCH, α-parvin, and Mig-2 in matrix-induced hepatocyte differentiation. We report here that ILK is present in the liver and localizes at cell-matrix adhesions of cultured hepatocytes. We also show that ILK, PINCH, α-parvin, and Mig-2 expression level is dramatically reduced in the re-differentiated hepatocytes. Interestingly, hepatocytes lacking ILK undergo matrix-induced differentiation but their differentiation is incomplete, as judged by monitoring cell morphology and production of albumin. Our results show that ILK and cell-matrix adhesion proteins play an important role in the process of matrix-induced hepatocyte differentiation

Seasonal and size-related variation of subcellular biomarkers in quagga mussels (Dreissena bugensis) inhabiting sites affected by moderate contamination with complex mixtures of pollutants.

Science.gov (United States)

Ács, A; Vehovszky, Á; Győri, J; Farkas, A

2016-07-01

The size-related differences in subcellular biomarker responses were assessed in Dreissena bugensis mussels inhabiting harbours moderately affected by pollution with complex mixtures of heavy metals and polycyclic aromatic hydrocarbons (PAHs). Adult D. bugensis samples were collected from three harbours of Lake Balaton (Hungary) characterized by moderate shipping activity, and as reference site, from a highly protected remote area of the lake. Biomarkers of exposure (metallothioneins (MTs), ethoxyresorufin-o-deethylase (EROD)), oxidative stress (lipid peroxidation (LPO), DNA strand breaks (DNAsb)) and possible endocrine disruption (vitellogenin-like proteins (VTG)) were analysed in whole-tissue homogenates of differently sized groups of mussels in relation to environmental parameters and priority pollutants (heavy metals and polycyclic aromatic hydrocarbons). Integrated biomarker response (IBR) indices were calculated for biomarker responses gained through in situ measurements to signalize critical sites and to better distinguish natural tendencies from biological effects of contaminants. Biomarker responses showed close positive correlation in case of MT, EROD, LPO, and DNAsb and negative correlation with VTG levels with mussel shell length in autumn, when higher levels of biomarkers appeared, possibly due to natural lifecycle changes of animals.

Effects of increasing temperature due to aquatic climate change on the self-fertility and the sexual development of the hermaphrodite fish, Kryptolebias marmoratus.

Science.gov (United States)

Park, Chang-Beom; Kim, Young Jun; Soyano, Kiyoshi

2017-01-01

In order to assess the effects of increasing temperature on the reproductive performance of fish, different thermal conditions (i.e., 25.0, 26.5, 27.5, 28.5, 30.0 °C) were used in this study and the self-fertilizing hermaphrodite fish, Kryptolebias marmoratus, was exposed to these different thermal conditions. During an exposure period of 30 to 150 days, the gonadosomatic index (GSI), gonadal development, the levels of plasma 17β-estradial (E2) and testosterone (T), hepatic vitellogenin (VTG) mRNA abundance, and the number of self-fertilized eggs were analyzed. This study confirmed that a high water temperature above 27.5 °C led to the suppression of self-fertility of hermaphroditic fish from 30 days after exposure. The oocyte quality and maturation would be affected by the disruption of hepatic VTG synthesis at a high water temperature of 30 °C, which resulted in the reduced the self-fertility in K. marmoratus. Consequently, this study suggests that elevated water temperature due to aquatic climate change prior to sexual maturation and the onset of spawning can lead to the reproductive dysfunction of hermaphroditic K. marmoratus.

Differential expression of largemouth bass (Micropterus salmoides) estrogen receptor isotypes alpha, beta, and gamma by estradiol.

Science.gov (United States)

Sabo-Attwood, Tara; Kroll, Kevin J; Denslow, Nancy D

2004-04-15

The expression levels of three estrogen receptor (ER) isotypes alpha, beta, and gamma were quantified in female largemouth bass (Micropterus salmoides) (LMB) liver, ovary, brain, and pituitary tissues. ER alpha and beta expression predominated in the liver, while ERs beta and gamma predominated in the other tissues. Temporally in females, ER alpha was highly up-regulated, ER gamma was slightly up-regulated, and ER beta levels remained unchanged in the liver when plasma 17-beta estradiol (E2) and vitellogenin (Vtg) levels were elevated in the spring. In ovarian tissue from these same fish, all three ERs were maximally expressed in the fall, during early oocyte development and prior to peak plasma E2 levels. When males were injected with E2, ER alpha was highly inducible, ER gamma was moderately up-regulated, and ER beta levels were not affected. None of the ER isotypes were induced by E2 in gonadal tissues. These results combined suggest that the ERs themselves are not regulated in the same manner by E2, and furthermore, do not contribute equally to the transcriptional regulation of genes involved in fish reproduction such as Vtg.

Long-term culture and expansion of primary human hepatocytes

NARCIS (Netherlands)

Levy, G.; Bomze, D.; Heinz, S.; Ramachandran, S.D.; Noerenberg, A.; Cohen, M.; Shibolet, O.; Sklan, E.; Braspenning, J.C.; Nahmias, Y.

2015-01-01

Hepatocytes have a critical role in metabolism, but their study is limited by the inability to expand primary hepatocytes in vitro while maintaining proliferative capacity and metabolic function. Here we describe the oncostatin M (OSM)-dependent expansion of primary human hepatocytes by low

The potential of induced pluripotent stem cell derived hepatocytes.

Science.gov (United States)

Hannoun, Zara; Steichen, Clara; Dianat, Noushin; Weber, Anne; Dubart-Kupperschmitt, Anne

2016-07-01

Orthotopic liver transplantation remains the only curative treatment for liver disease. However, the number of patients who die while on the waiting list (15%) has increased in recent years as a result of severe organ shortages; furthermore the incidence of liver disease is increasing worldwide. Clinical trials involving hepatocyte transplantation have provided encouraging results. However, transplanted cell function appears to often decline after several months, necessitating liver transplantation. The precise aetiology of the loss of cell function is not clear, but poor engraftment and immune-mediated loss appear to be important factors. Also, primary human hepatocytes (PHH) are not readily available, de-differentiate, and die rapidly in culture. Hepatocytes are available from other sources, such as tumour-derived human hepatocyte cell lines and immortalised human hepatocyte cell lines or porcine hepatocytes. However, all these cells suffer from various limitations such as reduced or differences in functions or risk of zoonotic infections. Due to their significant potential, one possible inexhaustible source of hepatocytes is through the directed differentiation of human induced pluripotent stem cells (hiPSCs). This review will discuss the potential applications and existing limitations of hiPSC-derived hepatocytes in regenerative medicine, drug screening, in vitro disease modelling and bioartificial livers. Copyright © 2016 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Toxicogenomic responses in rainbow trout (Oncorhynchus mykiss) hepatocytes exposed to model chemicals and a synthetic mixture

International Nuclear Information System (INIS)

Finne, E.F.; Cooper, G.A.; Koop, B.F.; Hylland, K.; Tollefsen, K.E.

2007-01-01

As more salmon gene expression data has become available, the cDNA microarray platform has emerged as an appealing alternative in ecotoxicological screening of single chemicals and environmental samples relevant to the aquatic environment. This study was performed to validate biomarker gene responses of in vitro cultured rainbow trout (Oncorhynchus mykiss) hepatocytes exposed to model chemicals, and to investigate effects of mixture toxicity in a synthetic mixture. Chemicals used for 24 h single chemical- and mixture exposures were 10 nM 17α-ethinylestradiol (EE2), 0.75 nM 2,3,7,8-tetrachloro-di-benzodioxin (TCDD), 100 μM paraquat (PQ) and 0.75 μM 4-nitroquinoline-1-oxide (NQO). RNA was isolated from exposed cells, DNAse treated and quality controlled before cDNA synthesis, fluorescent labelling and hybridisation to a 16k salmonid microarray. The salmonid 16k cDNA array identified differential gene expression predictive of exposure, which could be verified by quantitative real time PCR. More precisely, the responses of biomarker genes such as cytochrome p4501A and UDP-glucuronosyl transferase to TCDD exposure, glutathione reductase and gammaglutamyl cysteine synthetase to paraquat exposure, as well as vitellogenin and vitelline envelope protein to EE2 exposure validated the use of microarray applied to RNA extracted from in vitro exposed hepatocytes. The mutagenic compound NQO did not result in any change in gene expression. Results from exposure to a synthetic mixture of the same four chemicals, using identical concentrations as for single chemical exposures, revealed combined effects that were not predicted by results for individual chemicals alone. In general, the response of exposure to this mixture led to an average loss of approximately 60% of the transcriptomic signature found for single chemical exposure. The present findings show that microarray analyses may contribute to our mechanistic understanding of single contaminant mode of action as well as

Toxicogenomic responses in rainbow trout (Oncorhynchus mykiss) hepatocytes exposed to model chemicals and a synthetic mixture

Energy Technology Data Exchange (ETDEWEB)

Finne, E.F. [Norwegian Institute for Water Research, Gaustadalleen 21, N-0349 Oslo (Norway) and University of Oslo, Department of Biology, P.O. Box 1066, Blindern, N-0316 Oslo (Norway)]. E-mail: eivind.finne@niva.no; Cooper, G.A. [Centre for Biomedical Research, University of Victoria, BC V8P5C2 (Canada); Koop, B.F. [Centre for Biomedical Research, University of Victoria, BC V8P5C2 (Canada); Hylland, K. [Norwegian Institute for Water Research, Gaustadalleen 21, N-0349 Oslo (Norway); University of Oslo, Department of Biology, P.O. Box 1066, Blindern, N-0316 Oslo (Norway); Tollefsen, K.E. [Norwegian Institute for Water Research, Gaustadalleen 21, N-0349 Oslo (Norway)

2007-03-10

As more salmon gene expression data has become available, the cDNA microarray platform has emerged as an appealing alternative in ecotoxicological screening of single chemicals and environmental samples relevant to the aquatic environment. This study was performed to validate biomarker gene responses of in vitro cultured rainbow trout (Oncorhynchus mykiss) hepatocytes exposed to model chemicals, and to investigate effects of mixture toxicity in a synthetic mixture. Chemicals used for 24 h single chemical- and mixture exposures were 10 nM 17{alpha}-ethinylestradiol (EE2), 0.75 nM 2,3,7,8-tetrachloro-di-benzodioxin (TCDD), 100 {mu}M paraquat (PQ) and 0.75 {mu}M 4-nitroquinoline-1-oxide (NQO). RNA was isolated from exposed cells, DNAse treated and quality controlled before cDNA synthesis, fluorescent labelling and hybridisation to a 16k salmonid microarray. The salmonid 16k cDNA array identified differential gene expression predictive of exposure, which could be verified by quantitative real time PCR. More precisely, the responses of biomarker genes such as cytochrome p4501A and UDP-glucuronosyl transferase to TCDD exposure, glutathione reductase and gammaglutamyl cysteine synthetase to paraquat exposure, as well as vitellogenin and vitelline envelope protein to EE2 exposure validated the use of microarray applied to RNA extracted from in vitro exposed hepatocytes. The mutagenic compound NQO did not result in any change in gene expression. Results from exposure to a synthetic mixture of the same four chemicals, using identical concentrations as for single chemical exposures, revealed combined effects that were not predicted by results for individual chemicals alone. In general, the response of exposure to this mixture led to an average loss of approximately 60% of the transcriptomic signature found for single chemical exposure. The present findings show that microarray analyses may contribute to our mechanistic understanding of single contaminant mode of action as

Metabolism of para-aminophenol by rat hepatocytes.

Science.gov (United States)

Yan, Z; Nikelly, J G; Killmer, L; Tarloff, J B

2000-08-01

Autoxidation of para-aminophenol (PAP) has been proposed to account for the selective nephrotoxicity of this compound. However, other studies suggest that hepatic metabolites of PAP rather than the parent compound may be responsible for renal damage. These studies were designed to investigate PAP metabolism in isolated hepatocytes. We synthesized several proposed metabolites for analysis by HPLC/mass spectrometry and compared those results with HPLC/mass spectrometric analyses of metabolites found after incubating hepatocytes with PAP. Hepatocytes prepared from male Sprague-Dawley rats were incubated in Krebs-Henseleit buffer at 37 degrees C for 5 h with 2.3 mM PAP under an atmosphere of 5% CO2/95% O2. Aliquots were withdrawn at 0.1 h of incubation and then hourly through 5 h of incubation. Reactions were terminated by the addition of acetonitrile. Hepatocyte viability was unaltered with PAP present in the incubation medium. We found that hepatocytes converted PAP to two major metabolites (PAP-GSH conjugates and PAP-N-acetylcysteine conjugates) and several minor metabolites [PAP-O-glucuronide, acetaminophen (APAP), APAP-O-glucuronide, APAP-GSH conjugates, and 4-hydroxyformanilide]. Preincubating hepatoyctes with 1-aminobenzotriazole, an inhibitor of cytochromes P450, did not alter the pattern of PAP metabolism. In conclusion, we found that PAP was metabolized in hepatocytes predominantly to PAP-GSH conjugates and PAP-N-acetylcysteine conjugates in sufficient quantities to account for the nephrotoxicity of PAP.

Short-term responses of selected endocrine parameters in juvenile rainbow trout (Oncorhynchus mykiss) exposed to 4-nonylphenol.

Science.gov (United States)

Naderi, Mohammad; Zargham, Davood; Asadi, Asad; Bashti, Tayebeh; Kamayi, Kianoosh

2015-12-01

The synthetic organic compound 4-nonylphenol (4-NP) has been shown to have a wide range of adverse effects on the endocrine system of various animals including fish. The present study evaluated the potential effects of 4-NP on vitellogenin (VTG) synthesis, steroid, and thyroid hormone concentrations in both juvenile male and female rainbow trout (Oncorhynchus mykiss). Fish were exposed by intraperitoneal injection to different doses of 4-NP (1, 10, 50, and 100 μg g(-1)) or vehicle (coconut oil) over a period of 14 days. Blood samples were collected 7 and 14 days after initiation of treatment. Plasma VTG levels in 4-NP-treated fish were detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis as a high molecular weight protein band of 180 KDa. In addition, plasma VTG concentrations were quantified indirectly using plasma alkali-labile phosphate (ALP) and plasma calcium. Both ALP and calcium levels in plasma showed similar and parallel increase patterns after exposure to 4-NP that were significantly higher compared with controls. The analysis of plasma sex steroid levels revealed a significant increase in 17β-estradiol and testosterone in plasma of juvenile males and females, respectively. Furthermore, a significant increase was observed in plasma cortisol levels. On the other hand, 4-NP decreased both plasma triiodothyronine and thyroxine after 7 and 14 days of treatment. These results suggest that 4-NP can affect different parts of the endocrine system, which may lead to serious impairments in physiological homeostasis of juvenile rainbow trout. © The Author(s) 2012.

Water and nonelectrolyte permeability of isolated rat hepatocytes

International Nuclear Information System (INIS)

Alpini, G.; Garrick, R.A.; Jones, M.J.; Nunes, R.; Tavoloni, N.

1986-01-01

We have measured the diffusive permeability coefficients of isolated rat hepatocytes to 3 H 2 O, [ 14 C]urea, [ 14 C]erythritol, [ 14 C]mannitol, [ 3 H]sucrose, and [ 3 H]inulin, employing a technique previously developed for erythrocytes (Redwood et al., J. Gen. Physiol 64:706-729, 1974). Diffusion coefficients for the tracer molecules were measured in packed hepatocytes, supernatant fluid, and intracellular medium (lysed hepatocytes) and were calculated assuming one-dimensional semi-infinite diffusion through a homogeneous medium. By applying the series-parallel pathway model, the following permeability coefficients (10(-5) cm/sec) for the hepatocyte plasma membrane were obtained. 3 H 2 O, 98.6 +/- 18.4; [ 14 C]urea, 18.2 +/- 5.3; [ 14 C]erythritol, 4.8 +/- 1.6; [ 14 C]mannitol, 3.1 +/- 1.4; [ 3 H]sucrose, 0; [ 3 H]inulin, 0. These results indicate that isolated rat hepatocytes are highly permeable to water and polar nonelectrolytes, when compared with other transporting epithelia. This relatively high cellular permeability is consistent with a model in which nonelectrolyte permeation is via an aqueous pathway of equivalent pore diameter of 8-12 A. The finding that [ 14 C]erythritol and [ 14 C]mannitol cross the hepatocyte plasma membrane indicates that these molecules enter the bile canaliculus through the transcellular route. Conversely, the failure of [ 3 H]sucrose and [ 3 H]inulin to permeate the hepatocyte in the isolated condition supports the concept that biliary entry of these large carbohydrates, at least that fraction which cannot be accounted for by a vesicular mechanism, must occur via the transjunctional shunt pathway

Transcriptome of extracellular vesicles released by hepatocytes.

Directory of Open Access Journals (Sweden)

Felix Royo

Full Text Available The discovery that the cells communicate through emission of vesicles has opened new opportunities for better understanding of physiological and pathological mechanisms. This discovery also provides a novel source for non-invasive disease biomarker research. Our group has previously reported that hepatocytes release extracellular vesicles with protein content reflecting the cell-type of origin. Here, we show that the extracellular vesicles released by hepatocytes also carry RNA. We report the messenger RNA composition of extracellular vesicles released in two non-tumoral hepatic models: primary culture of rat hepatocytes and a progenitor cell line obtained from a mouse foetal liver. We describe different subpopulations of extracellular vesicles with different densities and protein and RNA content. We also show that the RNA cargo of extracellular vesicles released by primary hepatocytes can be transferred to rat liver stellate-like cells and promote their activation. Finally, we provide in vitro and in vivo evidence that liver-damaging drugs galactosamine, acetaminophen, and diclofenac modify the RNA content of these vesicles. To summarize, we show that the extracellular vesicles secreted by hepatocytes contain various RNAs. These vesicles, likely to be involved in the activation of stellate cells, might become a new source for non-invasive identification of the liver toxicity markers.

Inhibition of hepatocyte gap junctional intercellular communication by tumor promoters

International Nuclear Information System (INIS)

Ruch, R.J.

1988-01-01

The mechanisms by which tumor promoters enhance neoplasia are poorly understood. One effect common to most tumor promoters is their ability to inhibit the cell-to-cell exchange of small molecules and ions through gap junctions, i.e., gap junctional intercellular communication (IC). IC maybe necessary for normal growth control and the loss of IC may predispose cells to enhanced growth. In the present studies, the effects of liver tumor promoters and other agents on IC between rodent hepatocytes in primary culture has been studied. IC was detected between hepatocytes: (1) autoradiographically following the passage and incorporation of [5- 3 H]uridine nucleotides from pre-labeled donor hepatocytes to non-labeled, adjacent recipient hepatocytes and (2) by fluorescence microscopy after microinjection of fluorescent Lucifer Yellow CH dye into hepatocytes and visualizing dye spread into adjacent hepatocytes

Whole-body γ-irradiation decelerates rat hepatocyte polyploidization.

Science.gov (United States)

Ikhtiar, Adnan M

2015-07-01

To characterize hepatocyte polyploidization induced by intermediate dose of γ-ray. Male Wistar strain rats were whole-body irradiated (WBI) with 2 Gy of γ-ray at the age of 1 month, and 5-6 rats were sacrificed monthly at 0-25 months after irradiation. The nuclear DNA content of individual hepatocytes was measured by flow cytometry, then hepatocytes were classified into various ploidy classes. Survival percentage, after exposure up to the end of the study, did not indicate any differences between the irradiated groups and controls. The degree of polyploidization in hepatocytes of irradiated rats, was significantly lower than that for the control after 1 month of exposure, and it continued to be lower after up to 8 months. Thereafter, the degree of polyploidization in the irradiated group slowly returned to the control level when the irradiated rats reached the age of 10 months. Intermediate dose of ionizing radiation, in contrast to high doses, decelerate hepatocyte polyploidization, which may coincides with the hypothesis of the beneficial effects of low doses of ionizing radiation.

Property of hepatitis B virus replication in Tupaia belangeri hepatocytes

International Nuclear Information System (INIS)

Sanada, Takahiro; Tsukiyama-Kohara, Kyoko; Yamamoto, Naoki; Ezzikouri, Sayeh; Benjelloun, Soumaya; Murakami, Shuko; Tanaka, Yasuhito; Tateno, Chise; Kohara, Michinori

2016-01-01

The northern treeshrew (Tupaia belangeri) has been reported to be an effective candidate for animal infection model with hepatitis B virus (HBV). The objective of our study was to analyze the growth characteristics of HBV in tupaia hepatocytes and the host response to HBV infection. We established primary tupaia hepatocytes (3–6-week old tupaia) and infected them with HBV genotypes A, B and C, and all the genotypes proliferated as well as those in human primary hepatocytes (>10"5 copies/ml in culture supernatant). We next generated a chimeric mouse with tupaia liver by transplantation of tupaia primary hepatocytes to urokinase-type plasminogen activator cDNA (cDNA-uPA)/severe combined immunodeficient (SCID) mice and the replacement ratio with tupaia hepatocytes was found to be more than 95%. Infection of chimeric mice with HBV (genotypes B, C, and D) resulted in HBV-DNA level of 10"4-10"6 copies/ml after 8 weeks of infection, which were almost similar to that in humanized chimeric mouse. In contrast, serum HBV level in adult tupaia (1-year-old tupaia) was quite low (<10"3 copies/ml). Understanding the differences in the response to HBV infection in primary tupaia hepatocytes, chimeric mouse, and adult tupaia will contribute to elucidating the mechanism of persistent HBV infection and viral eradication. Thus, T. belangeri was found to be efficient for studying the host response to HBV infection, thereby providing novel insight into the pathogenesis of HBV. - Highlights: • Primary hepatocytes were established from tupaia that is a novel HBV infection model. • Tupaia primary hepatocytes were susceptible for HBV infection. • The immunodeficient chimeric mice with tupaia hepatocytes were established. • The chimeric mice with tupaia hepatocytes were susceptible for HBV infection.

Property of hepatitis B virus replication in Tupaia belangeri hepatocytes

Energy Technology Data Exchange (ETDEWEB)

Sanada, Takahiro [Department of Microbiology and Cell Biology, Tokyo Metropolitan Institute of Medical Science, 2-1-6, Kamikitazawa, Setagaya-ku, Tokyo 156-8506 (Japan); Tsukiyama-Kohara, Kyoko, E-mail: kkohara@vet.kagoshima-u.ac.jp [Transboundary Animal Diseases Centre, Joint Faculty of Veterinary Medicine, Kagoshima University, 1-21-24, Korimoto, Kagoshima-city, Kagoshima 890-0065 (Japan); Laboratory of Animal Hygiene, Joint Faculty of Veterinary Medicine, Kagoshima University, 1-21-24, Korimoto, Kagoshima, Kagoshima 890-0065 (Japan); Yamamoto, Naoki [Department of Microbiology and Cell Biology, Tokyo Metropolitan Institute of Medical Science, 2-1-6, Kamikitazawa, Setagaya-ku, Tokyo 156-8506 (Japan); Ezzikouri, Sayeh; Benjelloun, Soumaya [Viral Hepatitis Laboratory, Virology Unit, Institut Pasteur du Maroc, 1, Louis Pasteur, Casablanca 20360 (Morocco); Murakami, Shuko; Tanaka, Yasuhito [Department of Virology and Liver Unit, Nagoya City University Graduate School of Medical Sciences, Kawasumi 1, Mizuho-ku, Nagoya, Aichi 467-8601 (Japan); Tateno, Chise [PhoenixBio Co. Ltd., 3-4-1, Kagamiyama, Higashi-Hiroshima, Hiroshima 739-0046 (Japan); Kohara, Michinori, E-mail: kohara-mc@igakuken.or.jp [Department of Microbiology and Cell Biology, Tokyo Metropolitan Institute of Medical Science, 2-1-6, Kamikitazawa, Setagaya-ku, Tokyo 156-8506 (Japan)

2016-01-08

The northern treeshrew (Tupaia belangeri) has been reported to be an effective candidate for animal infection model with hepatitis B virus (HBV). The objective of our study was to analyze the growth characteristics of HBV in tupaia hepatocytes and the host response to HBV infection. We established primary tupaia hepatocytes (3–6-week old tupaia) and infected them with HBV genotypes A, B and C, and all the genotypes proliferated as well as those in human primary hepatocytes (>10{sup 5} copies/ml in culture supernatant). We next generated a chimeric mouse with tupaia liver by transplantation of tupaia primary hepatocytes to urokinase-type plasminogen activator cDNA (cDNA-uPA)/severe combined immunodeficient (SCID) mice and the replacement ratio with tupaia hepatocytes was found to be more than 95%. Infection of chimeric mice with HBV (genotypes B, C, and D) resulted in HBV-DNA level of 10{sup 4}-10{sup 6} copies/ml after 8 weeks of infection, which were almost similar to that in humanized chimeric mouse. In contrast, serum HBV level in adult tupaia (1-year-old tupaia) was quite low (<10{sup 3} copies/ml). Understanding the differences in the response to HBV infection in primary tupaia hepatocytes, chimeric mouse, and adult tupaia will contribute to elucidating the mechanism of persistent HBV infection and viral eradication. Thus, T. belangeri was found to be efficient for studying the host response to HBV infection, thereby providing novel insight into the pathogenesis of HBV. - Highlights: • Primary hepatocytes were established from tupaia that is a novel HBV infection model. • Tupaia primary hepatocytes were susceptible for HBV infection. • The immunodeficient chimeric mice with tupaia hepatocytes were established. • The chimeric mice with tupaia hepatocytes were susceptible for HBV infection.

Functional assessment of hepatocytes after transplantation into rat spleen

International Nuclear Information System (INIS)

Woods, R.J.; Fuller, B.J.; Attenburrow, V.D.; Nutt, L.H.; Hobbs, K.E.

1982-01-01

The retention of structural integrity and metabolic function by isolated hepatocytes after ectopic transplantation has been investigated in autografted rats. Rats were partially hepatectomized and isolated hepatocytes prepared from the excised liver lobes were implanted into their spleens. Histological examination of the spleens 7 or more weeks after implantation revealed aggregates of hepatocytes in the red pulp. Two tests of biochemical function were applied to the hepatocytes after transplantation. In the first the hepatobiliary imaging agent technetium-99m N-[N'-(2, 6-dimethylphenyl)carbamoylmethyl]iminodiacetic acid (99mTc HIDA), which was shown to be avidly taken up by isolated hepatocytes in vitro, was infused into the tail veins of autograft and control rats. Radioactivity accumulating in the spleens of autografted rats was markedly greater than that in controls implanted with lethally damaged cells or in nontransplanted rats. In the second the presence of bilirubin metabolites was sought in autograft spleens after intravenous infusion of bilirubin. Both mono- and diglucuronides of bilirubin were recovered from the spleens of autograft rats but no conjugates were recovered from the spleens of unoperated controls. We conclude that after autotransplantation isolated hepatocytes retain their morphology and at least some of their functional activities

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Billion-scale production of hepatocyte-like cells from human induced pluripotent stem cells.

Science.gov (United States)

Yamashita, Tomoki; Takayama, Kazuo; Sakurai, Fuminori; Mizuguchi, Hiroyuki

2018-02-19

Human induced pluripotent stem (iPS) cell-derived hepatocyte-like cells are expected to be utilized in drug screening and regenerative medicine. However, hepatocyte-like cells have not been fully used in such applications because it is difficult to produce such cells on a large scale. In this study, we tried to establish a method to mass produce hepatocyte-like cells using a three-dimensional (3D) cell culture bioreactor called the Rotary Cell Culture System (RCCS). RCCS enabled us to obtain homogenous hepatocyte-like cells on a billion scale (>10 9  cells). The gene expression levels of some hepatocyte markers (alpha-1 antitrypsin, cytochrome (CYP) 1A2, CYP2D6, and hepatocyte nuclear factor 4alpha) were higher in 3D-cultured hepatocyte-like cells than in 2D-cultured hepatocyte-like cells. This result suggests that RCCS could provide more suitable conditions for hepatocyte maturation than the conventional 2D cell culture conditions. In addition, more than 90% of hepatocyte-like cells were positive for albumin and could uptake low-density lipoprotein in the culture medium. We succeeded in the large-scale production of homogenous and functional hepatocyte-like cells from human iPS cells. This technology will be useful in drug screening and regenerative medicine, which require enormous numbers of hepatocyte-like cells. Copyright © 2018 Elsevier Inc. All rights reserved.

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Role of macrophages in the immune response to hepatocytes

International Nuclear Information System (INIS)

Bumgardner, G.L.; Chen, S.; Almond, S.P.; Ascher, N.L.; Payne, W.D.; Matas, A.J.

1990-01-01

The purpose of this study was to determine the role of host macrophages in the development of allospecific cytolytic T cells (allo-CTLs) in response to purified allogeneic MHC Class I+, Class II- hepatocytes in vivo in hepatocyte sponge matrix allografts (HC-SMA). Depletion of antigen-presenting cells (APCs) from responder splenocytes in mixed lymphocyte hepatocyte culture (MLHC) inhibits the development of allo-CTLs in response to purified hepatocytes. First the ability of sponge macrophages to function as accessory cells in indirect presentation of hepatocyte Class I antigen was tested in MLHC. We found that addition of irradiated sponge cells (a source of sponge macrophages) restored the development of allo-CTLs in MLHC depleted of responder APCs. Therefore, radioresistant sponge macrophages can function as accessory cells in MLHC. We next employed silica as an immunotherapy targeted against host macrophages and assessed the effect on development of allo-CTLs in HC-SMA. We found that local (intrasponge) silica treatment completely inhibited the development of allo-CTLs in HC-SMA. Combined local and systemic silica treatment resulted in inhibition of allocytotoxicity comparable to local silica treatment alone in the doses tested. We conclude that host macrophages which infiltrate HC-SMA can function as accessory cells in vitro in MLHC and that both infiltrating host macrophages and lymphocytes participate in the development of an alloimmune response to purified hepatocytes in vivo. This interaction may involve indirect antigen presentation of hepatocyte Class I antigen by macrophages to host lymphocytes which accumulate in HC-SMA

Histological and immunohistochemical investigation on ovarian development and plasma estradiol levels in the swordfish (Xiphias gladius L.

Directory of Open Access Journals (Sweden)

A Corriero

2009-06-01

Full Text Available The paper reports a histological and immunohistochemical description of oocyte growth and ultrastructural aspects of zona radiata (ZR formation as well as the relationship between plasma estradiol-17b‚ (E2 levels and ovarian development in swordfish (Xiphias gladius L. from the Mediterranean Sea. Ovaries were inactive during March to mid April; maturation occurred during late April to June and spawning in June and July. Zona radiata formation starts, as Pas positive material, in oocytes at the lipid stage. In this stage a deposit of electrondense material between oolemma and follicular cells appears. In the cortical alveoli stage and through the early vitellogenic stage, the deposition of a moderately electrondense material occurred on the inner side of the ZR. Finally, in late vitellogenic oocytes a third layer, made of microfibrillar material, appeared. The immunohistochemical analyses revealed that the initial internalisation of hepatic zona radiata proteins (Zrp in the swordfish oocyte starts before the uptake of vitellogenin (Vtg and that it is associated with the low previtellogenic E2 plasma levels, while a significant E2 increase in plasma is associated with the beginning of Vtg uptake. This would appear to confirm the hypothesis that the differential and sequential induction of zonagenesis and vitellogenesis may reflect a general feature of teleost oogenesis.

Sperm quality biomarkers complement reproductive and endocrine parameters in investigating environmental contaminants in common carp (Cyprinus carpio) from the Lake Mead National Recreation Area

Science.gov (United States)

Jenkins, Jill A.; Rosen, Michael R.; Dale, Rassa O.; Echols, Kathy R.; Torres, Leticia; Wieser, Carla M.; Kersten, Constance A.; Goodbred, Steven L.

2018-01-01

Lake Mead National Recreational Area (LMNRA) serves as critical habitat for several federally listed species and supplies water for municipal, domestic, and agricultural use in the Southwestern U.S. Contaminant sources and concentrations vary among the sub-basins within LMNRA. To investigate whether exposure to environmental contaminants is associated with alterations in male common carp (Cyprinus carpio) gamete quality and endocrine- and reproductive parameters, data were collected among sub-basins over 7 years (1999–2006). Endpoints included sperm quality parameters of motility, viability, mitochondrial membrane potential, count, morphology, and DNA fragmentation; plasma components were vitellogenin (VTG), 17ß-estradiol, 11-keto-testosterone, triiodothyronine, and thyroxine. Fish condition factor, gonadosomatic index, and gonadal histology parameters were also measured. Diminished biomarker effects were noted in 2006, and sub-basin differences were indicated by the irregular occurrences of contaminants and by several associations between chemicals (e.g., polychlorinated biphenyls, hexachlorobenzene, galaxolide, and methyl triclosan) and biomarkers (e.g., plasma thyroxine, sperm motility and DNA fragmentation). By 2006, sex steroid hormone and VTG levels decreased with subsequent reduced endocrine disrupting effects. The sperm quality bioassays developed and applied with carp complemented endocrine and reproductive data, and can be adapted for use with other species.

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Sex-specific vitellogenin production in immature rainbow trout

Energy Technology Data Exchange (ETDEWEB)

Carlson, D.B.; Williams, D.E.

1999-10-01

Many xenobiotics interact with hormone systems of animals, potentially leading to a phenomenon commonly called endocrine disruption. Much attention has focused on steroid hormone systems and corresponding receptor proteins, particularly estrogens. Vitellogenin (Vg) was measured in sexually immature rainbow trout (Oncorhynchus mykiss) exposed to 17{beta}-estradiol (E{sub 2}) in the diet. Mixed-sex populations of trout aged 3, 6, 12, or 18 months were maintained separately and fed E{sub 2} at 0.05 or 2.5 mg/kg for 7d. Females fed E{sub 2} at 0.05 mg/kg consistently produced three- to fourfold greater amounts of Vg than similarly aged males. Age- and sex-matched fish fed E{sub 2} at 2.5 mg/kg produced equivalent amounts of Vg. Sex differences in Vg production were apparent only at a dose of E{sub 2} (0.05 mg/kg) that results in submaximal Vg induction. Their results document the importance of considering the sex of juvenile fish when using Vg production as a marker of xenoestrogen exposure.

High throughput micro-well generation of hepatocyte micro-aggregates for tissue engineering.

Directory of Open Access Journals (Sweden)

Elien Gevaert

Full Text Available The main challenge in hepatic tissue engineering is the fast dedifferentiation of primary hepatocytes in vitro. One successful approach to maintain hepatocyte phenotype on the longer term is the cultivation of cells as aggregates. This paper demonstrates the use of an agarose micro-well chip for the high throughput generation of hepatocyte aggregates, uniform in size. In our study we observed that aggregation of hepatocytes had a beneficial effect on the expression of certain hepatocyte specific markers. Moreover we observed that the beneficial effect was dependent on the aggregate dimensions, indicating that aggregate parameters should be carefully considered. In a second part of the study, the selected aggregates were immobilized by encapsulation in methacrylamide-modified gelatin. Phenotype evaluations revealed that a stable hepatocyte phenotype could be maintained during 21 days when encapsulated in the hydrogel. In conclusion we have demonstrated the beneficial use of micro-well chips for hepatocyte aggregation and the size-dependent effects on hepatocyte phenotype. We also pointed out that methacrylamide-modified gelatin is suitable for the encapsulation of these aggregates.

High throughput micro-well generation of hepatocyte micro-aggregates for tissue engineering.

Science.gov (United States)

Gevaert, Elien; Dollé, Laurent; Billiet, Thomas; Dubruel, Peter; van Grunsven, Leo; van Apeldoorn, Aart; Cornelissen, Ria

2014-01-01

The main challenge in hepatic tissue engineering is the fast dedifferentiation of primary hepatocytes in vitro. One successful approach to maintain hepatocyte phenotype on the longer term is the cultivation of cells as aggregates. This paper demonstrates the use of an agarose micro-well chip for the high throughput generation of hepatocyte aggregates, uniform in size. In our study we observed that aggregation of hepatocytes had a beneficial effect on the expression of certain hepatocyte specific markers. Moreover we observed that the beneficial effect was dependent on the aggregate dimensions, indicating that aggregate parameters should be carefully considered. In a second part of the study, the selected aggregates were immobilized by encapsulation in methacrylamide-modified gelatin. Phenotype evaluations revealed that a stable hepatocyte phenotype could be maintained during 21 days when encapsulated in the hydrogel. In conclusion we have demonstrated the beneficial use of micro-well chips for hepatocyte aggregation and the size-dependent effects on hepatocyte phenotype. We also pointed out that methacrylamide-modified gelatin is suitable for the encapsulation of these aggregates.

Vitellogenin from the silkworm, Bombyx mori: an effective anti-bacterial agent.

Science.gov (United States)

Singh, Nitin Kumar; Pakkianathan, Britto Cathrin; Kumar, Manish; Prasad, Tulika; Kannan, Mani; König, Simone; Krishnan, Muthukalingan

2013-01-01

Silkworm, Bombyx mori, vitellogenin (Vg) was isolated from perivisceral fat body of day 3 of pupa. Both Vg subunits were co-purified as verified by mass spectrometry and immunoblot. Purified Vg responded to specific tests for major posttranslational modifications on native gels indicating its nature as lipo-glyco-phosphoprotein. The Vg fraction had strong antibacterial activity against Gram negative bacterium Escherichia coli and Gram positive bacterium Bacillus subtilis. Microscopic images showed binding of Vg to bacterial cells and their destruction. When infected silkworm larvae were treated with purified Vg they survived the full life cycle in contrast to untreated animals. This result showed that Vg has the ability to inhibit the proliferation of bacteria in the silkworm fluid system without disturbing the regular metabolism of the host.

Hepatocyte and keratinocyte growth factors and their receptors in human lung emphysema

Directory of Open Access Journals (Sweden)

Marchal Joëlle

2005-10-01

Full Text Available Abstract Background Hepatocyte and keratinocyte growth factors are key growth factors in the process of alveolar repair. We hypothesized that excessive alveolar destruction observed in lung emphysema involves impaired expression of hepatocyte and keratinocyte growth factors or their respective receptors, c-met and keratinocyte growth factor receptor. The aim of our study was to compare the expression of hepatocyte and keratinocyte growth factors and their receptors in lung samples from 3 groups of patients: emphysema; smokers without emphysema and non-smokers without emphysema. Methods Hepatocyte and keratinocyte growth factor proteins were analysed by immunoassay and western blot; mRNA expression was measured by real time quantitative polymerase chain reaction. Results Hepatocyte and keratinocyte growth factors, c-met and keratinocyte growth factor receptor mRNA levels were similar in emphysema and non-emphysema patients. Hepatocyte growth factor mRNA correlated negatively with FEV1 and the FEV1/FVC ratio both in emphysema patients and in smokers with or without emphysema. Hepatocyte and keratinocyte growth factor protein concentrations were similar in all patients' groups. Conclusion The expression of hepatocyte and keratinocyte growth factors and their receptors is preserved in patients with lung emphysema as compared to patients without emphysema. Hepatocyte growth factor mRNA correlates with the severity of airflow obstruction in smokers.

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Lifescience Database Archive (English)

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File list: ALL.Liv.10.AllAg.Hepatocytes [Chip-atlas[Archive

Lifescience Database Archive (English)

Full Text Available ALL.Liv.10.AllAg.Hepatocytes hg19 All antigens Liver Hepatocytes SRX815538,SRX81553...RX1013893,SRX1013888,SRX1013889,SRX1013891 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/ALL.Liv.10.AllAg.Hepatocytes.bed ...

Vitellogenin, a Marker of Estrogen Mimicking Contaminants in Fishes: Characterization, Quantification and Interference by Anti-Estrogens

OpenAIRE

Palumbo, Amanda J.

2008-01-01

Vitellogenin (Vg), the estrogen inducible protein precursor to egg yolk, serves as an indicator of exposure to estrogen mimicking environmental contaminants. Vg was isolated by size exclusion and ion exchange chromatography from plasma of California halibut (Paralichthys californicus) treated with estrogen. MALDI TOF mass spectrometry (MS) analysis resulted in a molecular mass of 188 kDa. MS/MS de novo sequencing provided evidence that California halibut has more than one form of Vg. Similar ...

Gel entrapment culture of rat hepatocytes for investigation of tetracycline-induced toxicity

International Nuclear Information System (INIS)

Shen Chong; Meng Qin; Schmelzer, Eva; Bader, Augustinus

2009-01-01

This paper aimed to explore three-dimensionally cultured hepatocytes for testing drug-induced nonalcoholic steatohepatitis. Gel entrapped rat hepatocytes were applied for investigation of the tetracycline-induced steatohepatitis, while hepatocyte monolayer was set as a control. The toxic responses of hepatocytes were systematically evaluated by measuring cell viability, liver-specific function, lipid accumulation, oxidative stress, adenosine triphosphate content and mitochondrial membrane potential. The results suggested that gel entrapped hepatocytes showed cell death after 96 h of tetracycline treatment at 25 μM which is equivalent to toxic serum concentration in rats, while hepatocyte monolayer showed cell death at a high dose of 200 μM. The concentration-dependent accumulation of lipid as well as mitochondrial damage were regarded as two early events for tetracycline hepatotoxicity in gel entrapment culture due to their detectability ahead of subsequent increase of oxidative stress and a final cell death. Furthermore, the potent protection of fenofibrate and fructose-1,6-diphosphate were evidenced in only gel entrapment culture with higher expressions on the genes related to β-oxidation than hepatocyte monolayer, suggesting the mediation of lipid metabolism and mitochondrial damage in tetracycline toxicity. Overall, gel entrapped hepatocytes in three-dimension reflected more of the tetracycline toxicity in vivo than hepatocyte monolayer and thus was suggested as a more relevant system for evaluating steatogenic drugs.

TGFbeta Induces Binucleation/Polyploidization in Hepatocytes through a Src-Dependent Cytokinesis Failure.

Science.gov (United States)

De Santis Puzzonia, Marco; Cozzolino, Angela Maria; Grassi, Germana; Bisceglia, Francesca; Strippoli, Raffaele; Guarguaglini, Giulia; Citarella, Franca; Sacchetti, Benedetto; Tripodi, Marco; Marchetti, Alessandra; Amicone, Laura

2016-01-01

In all mammals, the adult liver shows binucleated as well as mononucleated polyploid hepatocytes. The hepatic polyploidization starts after birth with an extensive hepatocyte binucleation and generates hepatocytes of several ploidy classes. While the functional significance of hepatocyte polyploidy is becoming clearer, how it is triggered and maintained needs to be clarified. Aim of this study was to identify a major inducer of hepatocyte binucleation/polyploidization and the cellular and molecular mechanisms involved. We found that, among several cytokines analyzed, known to be involved in early liver development and/or mass control, TGFbeta1 was capable to induce, together with the expected morphological changes, binucleation in hepatocytes in culture. Most importantly, the pharmacological inhibition of TGFbeta signaling in healthy mice during weaning, when the physiological binucleation occurs, induced a significant decrease of hepatocyte binucleation rate, without affecting cell proliferation and hepatic index. The TGFbeta-induced hepatocyte binucleation resulted from a cytokinesis failure, as assessed by video microscopy, and is associated with a delocalization of the cytokinesis regulator RhoA-GTPase from the mid-body of dividing cells. The use of specific chemical inhibitors demonstrated that the observed events are Src-dependent. Finally, the restoration of a fully epithelial phenotype by TGFbeta withdrawal gave rise to a cell progeny capable to maintain the polyploid state. In conclusion, we identified TGFbeta as a major inducer of hepatocyte binucleation both in vitro and in vivo, thus ascribing a novel role to this pleiotropic cytokine. The production of binucleated/tetraploid hepatocytes is due to a cytokinesis failure controlled by the molecular axis TGFbeta/Src/RhoA.

Alterations of gene expression indicating effects on estrogen signaling and lipid homeostasis in seabream hepatocytes exposed to extracts of seawater sampled from a coastal area of the central Adriatic Sea (Italy).

Science.gov (United States)

Cocci, Paolo; Capriotti, Martina; Mosconi, Gilberto; Campanelli, Alessandra; Frapiccini, Emanuela; Marini, Mauro; Caprioli, Giovanni; Sagratini, Gianni; Aretusi, Graziano; Palermo, Francesco Alessandro

2017-02-01

Recent evidences suggest that the toxicological effects of endocrine disrupting chemicals (EDCs) involve multiple nuclear receptor-mediated pathways, including estrogen receptor (ER) and peroxisome proliferator-activated receptor (PPAR) signaling systems. Thus, our objective in this study was to detect the summated endocrine effects of EDCs with metabolic activity in coastal waters of the central Adriatic Sea by means of a toxicogenomic approach using seabream hepatocytes. Gene expression patterns were also correlated with seawater levels of polychlorinated biphenyls (PCBs) and polycyclic aromatic hydrocarbons (PAHs). We found that seawater extracts taken at certain areas induced gene expression profiles of ERα/vitellogenin, PPARα/Stearoyl-CoA desaturase 1A, cytochrome P4501A (CYP1A) and metallothionein. These increased levels of biomarkers responses correlated with spatial distribution of PAHs/PCBs concentrations observed by chemical analysis in the different study areas. Collectively, our data give a snapshot of the presence of complex EDC mixtures that are able to perturb metabolic signaling in coastal marine waters. Copyright © 2016 Elsevier Ltd. All rights reserved.

File list: Oth.Liv.50.AllAg.Hepatocytes [Chip-atlas[Archive

Lifescience Database Archive (English)

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File list: Oth.Liv.20.AllAg.Hepatocytes [Chip-atlas[Archive

Lifescience Database Archive (English)

Full Text Available Oth.Liv.20.AllAg.Hepatocytes mm9 TFs and others Liver Hepatocytes ERX204070,SRX0190...04059,SRX116909,ERX204065,ERX204063,SRX116906,SRX555532,SRX019006,ERX204058 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.Liv.20.AllAg.Hepatocytes.bed ...

File list: Oth.Liv.05.AllAg.Hepatocytes [Chip-atlas[Archive

Lifescience Database Archive (English)

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Effects of edaravone, a radical scavenger, on hepatocyte transplantation.

Science.gov (United States)

Hayashi, Chihiro; Ito, Masahiro; Ito, Ryoutaro; Murakumo, Akiko; Yamamoto, Naoki; Hiramatsu, Noriko; Fox, Ira J; Horiguchi, Akihiko

2014-12-01

Hepatocyte transplantation (HTx) has yielded significant improvements in liver function and survival in experimentally induced acute liver failure and liver-based metabolic disease. However, transplantation is inefficient, and it is thought that transplanted hepatocytes have a shortened lifespan because of inflammation involving excess nitric oxide (NO). The present study aimed to clarify whether edaravone, a free radical scavenger used to treat ischemic stroke, could reduce ischemic changes in hepatocyte-transplanted livers. Edaravone (3 mg/kg) was administered intravenously 24 h before HTx to Nagase analbuminemic rats (NARs). Hepatocytes were isolated, and 30 × 10(6) cells were injected in a 1.0-ml volume directly into the spleens of NARs. All experimental groups studied received FK506 to control rejection. Animals in Group A received medium-only; Group B received HTx only; and Group C received HTx and edaravone. Forty-eight hours after transplantation, the hepatocytes from animals were isolated and analyzed for staining with propidium iodide- and annexin-V using flow cytometry. Liver sections were also studied by immunostaining for albumin, and TUNEL. Peripheral blood serum albumin levels were measured on post-transplant days 0, 3, 5, 7, 10 and 14 using ELISA. The edaravone-treated animals demonstrated an increased number of engrafted donor hepatocytes in the liver. The edaravone-treated liver sections also contained fewer TUNEL-positive cells and animals that received edaravone had higher serum albumin levels post-transplantation. Hepatocytes were also found to have increased in numbers 2 weeks following treatment with edaravone. Edaravone administration during HTx can suppress apoptosis near the transplanted cells, increasing engraftment. These studies indicate its potential usefulness for future clinical application. © 2014 Japanese Society of Hepato-Biliary-Pancreatic Surgery.

alpha-Amanitin induced apoptosis in primary cultured dog hepatocytes.

Directory of Open Access Journals (Sweden)

Adam Szelag

2010-06-01

Full Text Available Amatoxin poisoning is caused by mushroom species belonging to the genera Amanita, Galerina and Lepiota with the majority of lethal mushroom exposures attributable to Amanita phalloides. High mortality rate in intoxications with these mushrooms is principally a result of the acute liver failure following significant hepatocyte damage due to hepatocellular uptake of amatoxins. A wide variety of amatoxins have been isolated; however, alpha-amanitin (alpha-AMA appears to be the primary toxin. Studies in vitro and in vivo suggest that alpha-AMA does not only cause hepatocyte necrosis, but also may lead to apoptotic cell death. The objective of this study was to evaluate the complex hepatocyte apoptosis in alpha-AMA cytotoxicity. All experiments were performed on primary cultured canine hepatocytes. The cells were incubated for 12 h with alpha-AMA at a final concentration of 1, 5, 10 and 20 microM. Viability test (MTT assay, apoptosis evaluation (TUNEL reaction, detection of DNA laddering and electron microscopy were performed at 6 and 12 h of exposure to alpha-AMA. There was a clear correlation between hepatocyte viability, concentration of alpha-AMA and time of exposure to this toxin. The decline in cultured dog hepatocyte viability during the exposure to alpha-AMA is most likely preceded by enhanced cellular apoptosis. Our results demonstrate that apoptosis might contribute to pathogenesis of the severe liver injury in the course of amanitin intoxication, particularly during the early phase of poisoning.

RNA synthesis in primary cultures of adult rat hepatocytes

International Nuclear Information System (INIS)

Fugassa, E.; Gallo, G.; Voci, A.; Cordone, A.

1983-01-01

The ability of hepatocyte monolayers to synthesize RNA was investigated by measuring [3H]orotic acid incorporation into RNA and the total nuclear RNA polymerase activity as a function of the time in culture. The results demonstrate that primary cultures of hepatocytes maintained in a chemically defined serum- and hormone-free medium are able to synthesize RNA actively. This ability increases within the first 2 d of culture, despite the concomitant decrease in [3H]orotic acid uptake, and decreases only after 3 d. Factors such as serum, insulin, and dexamethasone, known to improve maintenance of functional hepatocytes, markedly stimulate the uptake of labeled precursor without apparently affecting the rate of RNA synthesis by cultured cells. It is suggested that the culture of adult rat hepatocytes provides a useful experimental model for the studies of hormonal regulation of transcription in liver

TGFbeta Induces Binucleation/Polyploidization in Hepatocytes through a Src-Dependent Cytokinesis Failure.

Directory of Open Access Journals (Sweden)

Marco De Santis Puzzonia

Full Text Available In all mammals, the adult liver shows binucleated as well as mononucleated polyploid hepatocytes. The hepatic polyploidization starts after birth with an extensive hepatocyte binucleation and generates hepatocytes of several ploidy classes. While the functional significance of hepatocyte polyploidy is becoming clearer, how it is triggered and maintained needs to be clarified. Aim of this study was to identify a major inducer of hepatocyte binucleation/polyploidization and the cellular and molecular mechanisms involved. We found that, among several cytokines analyzed, known to be involved in early liver development and/or mass control, TGFbeta1 was capable to induce, together with the expected morphological changes, binucleation in hepatocytes in culture. Most importantly, the pharmacological inhibition of TGFbeta signaling in healthy mice during weaning, when the physiological binucleation occurs, induced a significant decrease of hepatocyte binucleation rate, without affecting cell proliferation and hepatic index. The TGFbeta-induced hepatocyte binucleation resulted from a cytokinesis failure, as assessed by video microscopy, and is associated with a delocalization of the cytokinesis regulator RhoA-GTPase from the mid-body of dividing cells. The use of specific chemical inhibitors demonstrated that the observed events are Src-dependent. Finally, the restoration of a fully epithelial phenotype by TGFbeta withdrawal gave rise to a cell progeny capable to maintain the polyploid state. In conclusion, we identified TGFbeta as a major inducer of hepatocyte binucleation both in vitro and in vivo, thus ascribing a novel role to this pleiotropic cytokine. The production of binucleated/tetraploid hepatocytes is due to a cytokinesis failure controlled by the molecular axis TGFbeta/Src/RhoA.

File list: InP.Liv.20.AllAg.Hepatocytes [Chip-atlas[Archive

Lifescience Database Archive (English)

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File list: InP.Liv.50.AllAg.Hepatocytes [Chip-atlas[Archive

Lifescience Database Archive (English)

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High efficient differentiation of functional hepatocytes from porcine induced pluripotent stem cells.

Directory of Open Access Journals (Sweden)

Ying Ao

Full Text Available Hepatocyte transplantation is considered to be a promising therapy for patients with liver diseases. Induced pluripotent stem cells (iPSCs provide an unlimited source for the generation of functional hepatocytes. In this study, we generated iPSCs from porcine ear fibroblasts (PEFs by overexpressing Sox2, Klf4, Oct4, and c-Myc (SKOM, and developed a novel strategy for the efficient differentiation of hepatocyte-like cells from porcine iPSCs by following the processes of early liver development. The differentiated cells displayed the phenotypes of hepatocytes, exhibited classic hepatocyte-associated bio-functions, such as LDL uptake, glycogen storage and urea secretion, as well as possessed the metabolic activities of cytochrome P-450 (CYP 3A and 2C. Furthermore, we compared the hepatocyte differentiation efficacy of our protocol with another published method, and the results demonstrated that our differentiation strategy could significantly improve the generation of morphological and functional hepatocyte-like cells from porcine iPSCs. In conclusion, this study establishes an efficient method for in vitro generation of functional hepatocytes from porcine iPSCs, which could represent a promising cell source for preclinical testing of cell-based therapeutics for liver failure and for pharmacological applications.

A tapeworm molecule manipulates vitellogenin expression in the beetle Tenebrio molitor

Science.gov (United States)

Warr, E.; Meredith, J. M.; Nimmo, D. D.; Basu, S.; Hurd, H.; Eggleston, P.

2006-01-01

Metacestodes of Hymenolepis diminuta secrete a molecule that decreases vitellogenin (Vg) synthesis in the beetle host, Tenebrio molitor. The 5608 bp T. molitor Vg cDNA represents a single-copy gene encoding a single open reading frame of 1821 amino acids with a predicted molecular mass of 206 kDa. Northern blot analysis revealed detectable levels of transcripts only in adult females. In vivo, Vg mRNA abundance was significantly higher in fat bodies from infected females compared with control females at all but the earliest time point. In vitro, Vg mRNA abundance was significantly increased in fat bodies incubated with live stage I–II parasites. The apparent conflict between increased Vg mRNA abundance and decreased Vg protein in fat bodies from infected females is discussed. PMID:16907836

Vitellogenin from the silkworm, Bombyx mori: an effective anti-bacterial agent.

Directory of Open Access Journals (Sweden)

Nitin Kumar Singh

Full Text Available Silkworm, Bombyx mori, vitellogenin (Vg was isolated from perivisceral fat body of day 3 of pupa. Both Vg subunits were co-purified as verified by mass spectrometry and immunoblot. Purified Vg responded to specific tests for major posttranslational modifications on native gels indicating its nature as lipo-glyco-phosphoprotein. The Vg fraction had strong antibacterial activity against Gram negative bacterium Escherichia coli and Gram positive bacterium Bacillus subtilis. Microscopic images showed binding of Vg to bacterial cells and their destruction. When infected silkworm larvae were treated with purified Vg they survived the full life cycle in contrast to untreated animals. This result showed that Vg has the ability to inhibit the proliferation of bacteria in the silkworm fluid system without disturbing the regular metabolism of the host.

File list: InP.Liv.05.AllAg.Hepatocytes [Chip-atlas[Archive

Lifescience Database Archive (English)

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File list: InP.Liv.50.AllAg.Hepatocytes [Chip-atlas[Archive

Lifescience Database Archive (English)

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File list: InP.Liv.20.AllAg.Hepatocytes [Chip-atlas[Archive

Lifescience Database Archive (English)

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File list: InP.Liv.10.AllAg.Hepatocytes [Chip-atlas[Archive

Lifescience Database Archive (English)

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Estrogenicity and intersex in juvenile rainbow trout (Oncorhynchus mykiss) exposed to Pine/Eucalyptus pulp and paper production effluent in Chile

Energy Technology Data Exchange (ETDEWEB)

Chiang, Gustavo, E-mail: gchiang@centromeri.cl [Canadian Rivers Institute and Department of Biology, University of New Brunswick, Saint John, NB E2L 4L5 (Canada); Barra, Ricardo [Aquatic Systems Research Unit, EULA–Chile Environmental Sciences Centre, University of Concepción, Concepcion (Chile); Díaz-Jaramillo, Mauricio [Laboratorio de Ecotoxicología y Contaminación Ambiental (ECoA), IIMyC-CONICET-UNMdP, Mar del Plata (Argentina); Rivas, Meyling [Department of Zoology, Faculty of Natural and Oceanographic Sciences, University of Concepcion, Concepcion (Chile); Bahamonde, Paulina; Munkittrick, Kelly R. [Canadian Rivers Institute and Department of Biology, University of New Brunswick, Saint John, NB E2L 4L5 (Canada)

2015-07-15

Highlights: • Juvenile rainbow were exposed to Pine and Eucalyptus PPME along with an in situ bioassay downstream of the combined discharge. • Fish exposed to PPME showed induced levels of plasma vitellogenin and female gonad maturation. • Male fish showed intersex characteristics in laboratory and in situ assays. • Tertiary treated PPME from Eucalyptus production have stronger estrogenic effects on juvenile fish. - Abstract: Pulp and paper mill effluents (PPMEs) have been shown to increase gonad size, cause early maturation, and disrupt hormone functions in native and non-native Chilean fish. In this study, we assessed reproductive (plasma vitellogenin; VTG, gonad development) and metabolic (ethoxyresorufin-O-deethylase activity; EROD) end points, relative liver size (LSI) and condition factor (K) of juvenile female and male rainbow trout exposed to effluents. Unlike previous studies, which have focus either on the specific effects of effluent on fish in laboratory exposures or biotic population statuses downstream of discharge sites, we simultaneously assessed the impacts of PPMES on trout using two approaches: (1) laboratory exposures of tertiary treated PPME produced from processing Eucalyptus globulus or Pinus radiata; and (2) in situ bioassay downstream of the combined discharge of the same pulp mill. Despite an increase in the average gonadosomatic index (GSI) in exposed fish, no statistical differences in gonad size between exposed and unexposed individuals was detected. However, both female and male fish exposed to effluents showed significantly higher concentrations of plasma VTG, so more in fish exposed to Eucalyptus-based effluent when compared to Pinus PPME. In addition, male fish showed intersex characteristics in all exposure assays (Eucaliptus and Pinus) and, despite the low concentration of effluent in the river (<1% [v/v]), similar responses were observed in the caged fish. Finally, EROD activity was induced in both in situ exposures and

Estrogenicity and intersex in juvenile rainbow trout (Oncorhynchus mykiss) exposed to Pine/Eucalyptus pulp and paper production effluent in Chile

International Nuclear Information System (INIS)

Chiang, Gustavo; Barra, Ricardo; Díaz-Jaramillo, Mauricio; Rivas, Meyling; Bahamonde, Paulina; Munkittrick, Kelly R.

2015-01-01

Highlights: • Juvenile rainbow were exposed to Pine and Eucalyptus PPME along with an in situ bioassay downstream of the combined discharge. • Fish exposed to PPME showed induced levels of plasma vitellogenin and female gonad maturation. • Male fish showed intersex characteristics in laboratory and in situ assays. • Tertiary treated PPME from Eucalyptus production have stronger estrogenic effects on juvenile fish. - Abstract: Pulp and paper mill effluents (PPMEs) have been shown to increase gonad size, cause early maturation, and disrupt hormone functions in native and non-native Chilean fish. In this study, we assessed reproductive (plasma vitellogenin; VTG, gonad development) and metabolic (ethoxyresorufin-O-deethylase activity; EROD) end points, relative liver size (LSI) and condition factor (K) of juvenile female and male rainbow trout exposed to effluents. Unlike previous studies, which have focus either on the specific effects of effluent on fish in laboratory exposures or biotic population statuses downstream of discharge sites, we simultaneously assessed the impacts of PPMES on trout using two approaches: (1) laboratory exposures of tertiary treated PPME produced from processing Eucalyptus globulus or Pinus radiata; and (2) in situ bioassay downstream of the combined discharge of the same pulp mill. Despite an increase in the average gonadosomatic index (GSI) in exposed fish, no statistical differences in gonad size between exposed and unexposed individuals was detected. However, both female and male fish exposed to effluents showed significantly higher concentrations of plasma VTG, so more in fish exposed to Eucalyptus-based effluent when compared to Pinus PPME. In addition, male fish showed intersex characteristics in all exposure assays (Eucaliptus and Pinus) and, despite the low concentration of effluent in the river (<1% [v/v]), similar responses were observed in the caged fish. Finally, EROD activity was induced in both in situ exposures and

Renal medullary AA amyloidosis, hepatocyte dissociation and multinucleated hepatocytes in a 14-year-old free-ranging lioness (Panthera leo

Directory of Open Access Journals (Sweden)

J.H. Williams

2005-06-01

Full Text Available A 14-year-old lioness, originating from Etosha in Namibia, and a member of a pride in Pilanesberg National Park since translocation in 1994, was euthanased due to fight-related vertebral fracture and spinal injury, incurred approximately 6-8 weeks previously. Blood specimens collected at the time of death showed mild anaemia and a leukogram reflecting stress and chronic infection. Necropsy conducted within 2 hours of death was on a dehydrated, emaciated animal with hindquarter wasting and chronic traumatic friction injuries from dragging her hindlegs. There was cellulitis in the region of bite-wounds adjacent to the thoraco-lumbar vertebral fracture, at which site there was spinal cord compression, and there was marked intestinal helminthiasis. The outer renal medullae appeared pale and waxy and the liver was macroscopically unremarkable. Histopathology and electron microscopy of the kidneys revealed multifocal to coalescing deposits of proximal medullary interstitial amyloid, which fluoresced strongly with thioflavine T, and was sensitive to potassium permanganate treatment prior to Congo Red staining, thus indicating inflammatory (AA origin. There was diffuse hepatocyte dissociation, as well as numerous binucleated and scattered multinucleated (up to 8 nuclei/cell hepatocytes, with swollen hepatocyte mitochondria, in liver examined light microscopically. Ultrastructurally, the mono-, bi- and multinucleated hepatocytes contained multifocal irregular membrane-bound accumulations of finely-granular, amorphous material both intra-cytoplasmically and intra-nuclearly, as well as evidence of irreversible mitochondrial injury. The incidence and relevance in cats and other species of amyloidosis, particularly with renal medullary distribution, as well as of hepatocyte dissociation and multinucleation, as reported in selected literature, is briefly overviewed and their occurrence in this lioness is discussed.

Cholesterol Enhances the Toxic Effect of Ethanol and Acetaldehyde in Primary Mouse Hepatocytes

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Anayelly López-Islas

2016-01-01

Full Text Available Obesity and alcohol consumption are risk factors for hepatic steatosis, and both commonly coexist. Our objective was to evaluate the effect of ethanol and acetaldehyde on primary hepatocytes obtained from mice fed for two days with a high cholesterol (HC diet. HC hepatocytes increased lipid and cholesterol content. HC diet sensitized hepatocytes to the toxic effect of ethanol and acetaldehyde. Cyp2E1 content increased with HC diet, as well as in those treated with ethanol or acetaldehyde, while the activity of this enzyme determined in microsomes increased in the HC and in all ethanol treated hepatocytes, HC and CW. Oxidized proteins were increased in the HC cultures treated or not with the toxins. Transmission electron microscopy showed endoplasmic reticulum (ER stress and megamitochondria in hepatocytes treated with ethanol as in HC and the ethanol HC treated hepatocytes. ER stress determined by PERK content was increased in ethanol treated hepatocytes from HC mice and CW. Nuclear translocation of ATF6 was observed in HC hepatocytes treated with ethanol, results that indicate that lipids overload and ethanol treatment favor ER stress. Oxidative stress, ER stress, and mitochondrial damage underlie potential mechanisms for increased damage in steatotic hepatocyte treated with ethanol.

Profiling of benzophenone derivatives using fish and human estrogen receptor-specific in vitro bioassays

International Nuclear Information System (INIS)

Molina-Molina, Jose-Manuel; Escande, Aurelie; Pillon, Arnaud; Gomez, Elena; Pakdel, Farzad; Cavailles, Vincent; Olea, Nicolas; Ait-Aissa, Selim; Balaguer, Patrick

2008-01-01

Benzophenone (BP) derivatives, BP1 (2,4-dihydroxybenzophenone), BP2 (2,2',4,4'-tetrahydroxybenzophenone), BP3 (2-hydroxy-4-methoxybenzophenone), and THB (2,4,4'-trihydroxybenzophenone) are UV-absorbing chemicals widely used in pharmaceutical, cosmetics, and industrial applications, such as topical sunscreens in lotions and hair sprays to protect skin and hair from UV irradiation. Studies on their endocrine disrupting properties have mostly focused on their interaction with human estrogen receptor alpha (hERα), and there has been no comprehensive analysis of their potency in a system allowing comparison between hERα and hERβ activities. The objective of this study was to provide a comprehensive ER activation profile of BP derivatives using ER from human and fish origin in a battery of in vitro tests, i.e., competitive binding, reporter gene based assays, vitellogenin (Vtg) induction in isolated rainbow trout hepatocytes, and proliferation based assays. The ability to induce human androgen receptor (hAR)-mediated reporter gene expression was also examined. All BP derivatives tested except BP3 were full hERα and hERβ agonists (BP2 > THB > BP1) and displayed a stronger activation of hERβ compared with hERα, the opposite effect to that of estradiol (E 2 ). Unlike E 2 , BPs were more active in rainbow trout ERα (rtERα) than in hERα assay. All four BP derivatives showed anti-androgenic activity (THB > BP2 > BP1 > BP3). Overall, the observed anti-androgenic potencies of BP derivatives, together with their proposed greater effect on ERβ versus ERα activation, support further investigation of their role as endocrine disrupters in humans and wildlife

File list: NoD.Liv.50.AllAg.Hepatocytes [Chip-atlas[Archive

Lifescience Database Archive (English)

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Lifescience Database Archive (English)

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File list: NoD.Liv.20.AllAg.Hepatocytes [Chip-atlas[Archive

Lifescience Database Archive (English)

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File list: NoD.Liv.05.AllAg.Hepatocytes [Chip-atlas[Archive

Lifescience Database Archive (English)

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Hepatocyte produced matrix metalloproteinases are regulated by CD147 in liver fibrogenesis.

Science.gov (United States)

Calabro, Sarah R; Maczurek, Annette E; Morgan, Alison J; Tu, Thomas; Wen, Victoria W; Yee, Christine; Mridha, Auvro; Lee, Maggie; d'Avigdor, William; Locarnini, Stephen A; McCaughan, Geoffrey W; Warner, Fiona J; McLennan, Susan V; Shackel, Nicholas A

2014-01-01

The classical paradigm of liver injury asserts that hepatic stellate cells (HSC) produce, remodel and turnover the abnormal extracellular matrix (ECM) of fibrosis via matrix metalloproteinases (MMPs). In extrahepatic tissues MMP production is regulated by a number of mechanisms including expression of the glycoprotein CD147. Previously, we have shown that CD147 is expressed on hepatocytes but not within the fibrotic septa in cirrhosis [1]. Therefore, we investigated if hepatocytes produce MMPs, regulated by CD147, which are capable of remodelling fibrotic ECM independent of the HSC. Non-diseased, fibrotic and cirrhotic livers were examined for MMP activity and markers of fibrosis in humans and mice. CD147 expression and MMP activity were co-localised by in-situ zymography. The role of CD147 was studied in-vitro with siRNA to CD147 in hepatocytes and in-vivo in mice with CCl4 induced liver injury using ãCD147 antibody intervention. In liver fibrosis in both human and mouse tissue MMP expression and activity (MMP-2, -9, -13 and -14) increased with progressive injury and localised to hepatocytes. Additionally, as expected, MMPs were abundantly expressed by activated HSC. Further, with progressive fibrosis there was expression of CD147, which localised to hepatocytes but not to HSC. Functionally significant in-vitro regulation of hepatocyte MMP production by CD147 was demonstrated using siRNA to CD147 that decreased hepatocyte MMP-2 and -9 expression/activity. Further, in-vivo α-CD147 antibody intervention decreased liver MMP-2, -9, -13, -14, TGF-β and α-SMA expression in CCl4 treated mice compared to controls. We have shown that hepatocytes produce active MMPs and that the glycoprotein CD147 regulates hepatocyte MMP expression. Targeting CD147 regulates hepatocyte MMP production both in-vitro and in-vivo, with the net result being reduced fibrotic matrix turnover in-vivo. Therefore, CD147 regulation of hepatocyte MMP is a novel pathway that could be targeted by

Hepatocyte produced matrix metalloproteinases are regulated by CD147 in liver fibrogenesis.

Directory of Open Access Journals (Sweden)

Sarah R Calabro

Full Text Available The classical paradigm of liver injury asserts that hepatic stellate cells (HSC produce, remodel and turnover the abnormal extracellular matrix (ECM of fibrosis via matrix metalloproteinases (MMPs. In extrahepatic tissues MMP production is regulated by a number of mechanisms including expression of the glycoprotein CD147. Previously, we have shown that CD147 is expressed on hepatocytes but not within the fibrotic septa in cirrhosis [1]. Therefore, we investigated if hepatocytes produce MMPs, regulated by CD147, which are capable of remodelling fibrotic ECM independent of the HSC.Non-diseased, fibrotic and cirrhotic livers were examined for MMP activity and markers of fibrosis in humans and mice. CD147 expression and MMP activity were co-localised by in-situ zymography. The role of CD147 was studied in-vitro with siRNA to CD147 in hepatocytes and in-vivo in mice with CCl4 induced liver injury using ãCD147 antibody intervention.In liver fibrosis in both human and mouse tissue MMP expression and activity (MMP-2, -9, -13 and -14 increased with progressive injury and localised to hepatocytes. Additionally, as expected, MMPs were abundantly expressed by activated HSC. Further, with progressive fibrosis there was expression of CD147, which localised to hepatocytes but not to HSC. Functionally significant in-vitro regulation of hepatocyte MMP production by CD147 was demonstrated using siRNA to CD147 that decreased hepatocyte MMP-2 and -9 expression/activity. Further, in-vivo α-CD147 antibody intervention decreased liver MMP-2, -9, -13, -14, TGF-β and α-SMA expression in CCl4 treated mice compared to controls.We have shown that hepatocytes produce active MMPs and that the glycoprotein CD147 regulates hepatocyte MMP expression. Targeting CD147 regulates hepatocyte MMP production both in-vitro and in-vivo, with the net result being reduced fibrotic matrix turnover in-vivo. Therefore, CD147 regulation of hepatocyte MMP is a novel pathway that could be

File list: NoD.Liv.50.AllAg.Hepatocytes [Chip-atlas[Archive

Lifescience Database Archive (English)

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File list: NoD.Liv.05.AllAg.Hepatocytes [Chip-atlas[Archive

Lifescience Database Archive (English)

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Lifescience Database Archive (English)

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Reversibility of endocrine disruption in zebrafish (Danio rerio) - comparison of different effect levels

DEFF Research Database (Denmark)

Baumann, Lisa; Holbech, Henrik; Schiller, V.S.

: the androgen trenbolone binds directly and very effectively to the androgen receptor. Ethinylestradiol, a synthetic derivative of estradiol, causes feminization in wildlife and humans. The fungicide prochloraz acts as an aromatase inhibitor by direct interference with the aromatization of androgens......Endocrine disrupting chemicals (EDCs) exert effects at very low concentrations and can cause serious problems for the hormonal balance of various organisms. Exposure of wildlife to EDCs is not necessarily continuous, but may often occur in pulses. Consequently for the evaluation of the long......-term effects on populations, it is essential to know whether such EDC-related effects are reversible. Three different substances selected for different modes of action were tested for their long-term impact on sex ratio, gonadal development, vitellogenin (VTG) induction and aromatase activity in zebrafish...

Endocrine-disruptor molecular responses, occurrence of intersex and gonado-histopathological changes in tilapia species from a tropical freshwater dam (Awba Dam) in Ibadan, Nigeria.

Science.gov (United States)

Adeogun, Aina O; Onibonoje, Kolawole; Ibor, Oju R; Omiwole, Roseline A; Chukwuka, Azubuike V; Ugwumba, Alex O; Ugwumba, Adiaha A A; Arukwe, Augustine

2016-05-01

In the present study, the occurrence of endocrine disruptive responses in Tilapia species from Awba Dam has been investigated, and compared to a reference site (Modete Dam). The Awba Dam is a recipient of effluents from University of Ibadan (Nigeria) and several other anthropogenic sources. A total of 132 Tilapia species (Sarotherodon malenotheron (n=57 and 32, males and females, respectively) and Tilapia guineensis (n=23 and 20, males and females, respectively)) were collected from June to September 2014. At the reference site, samples of adult male and female S. melanotheron (48 males and 47 females) and T. guineensis (84 males and 27 females) were collected. Gonads were morphologically and histologically examined and gonadosomatic index (GSI) was calculated. Hepatic mRNA transcriptions of vitellogenin (Vtg) and zona radiata protein (Zrp) genes were analyzed using validated RT-qPCR. Significant increase in Vtg and Zrp transcripts were observed in male tilapias from Awba Dam, compared to males from the reference site. In addition, male tilapias from Awba Dam produced significantly higher Vtg and Zrp mRNA, compared to females in June and July. However, at the natural peak spawning period in August and September, females produced, significantly higher Vtg and Zrp mRNA, compared to males. Fish gonads revealed varying incidence of intersex with a striking presence of two (2) pairs of testes and a pair of ovary in S. melanotheron from Awba Dam. The entire fish population examined at Awba Dam showed a high prevalence of intersex (34.8%), involving phenotypic males and females of both species. Analysis of sediment contaminant levels revealed that As, Cd, Pb, Hg and Ni (heavy metals), monobutyltin cation, 4-iso-nonyphenol and PCB congeners (138, 153 and 180) were significantly higher in Awba Dam, compared to the reference site. Principal component analysis (PCA) showed that fish variables were positively correlated with sediment contaminant burden at Awba Dam, indicating

Scintigraphic evidence of transplanted hepatocytes in spleen and liver

International Nuclear Information System (INIS)

Henne-Bruns, D.; Kremer, B.; Gramminger, K.; Broelsch, C.

1986-01-01

In rats suffering from hepatic enzymatic deficiency transplanted hepatocytes could be evidenced scintigraphically in liver, spleen and granulomas. In pigs, however, it is very difficult to demonstrate transplanted hepatocytes by scintiscanning because of the thickness of the tissues and the high background radiation in large animals

Transplant of Hepatocytes, Undifferentiated Mesenchymal Stem Cells, and In Vitro Hepatocyte-Differentiated Mesenchymal Stem Cells in a Chronic Liver Failure Experimental Model: A Comparative Study.

Science.gov (United States)

El Baz, Hanan; Demerdash, Zeinab; Kamel, Manal; Atta, Shimaa; Salah, Faten; Hassan, Salwa; Hammam, Olfat; Khalil, Heba; Meshaal, Safa; Raafat, Inas

2018-02-01

Liver transplant is the cornerstone line of treatment for chronic liver diseases; however, the long list of complications and obstacles stand against this operation. Searching for new modalities for treatment of chronic liver illness is a must. In the present research, we aimed to compare the effects of transplant of undifferentiated human mesenchymal stem cells, in vitro differentiated mesenchymal stem cells, and adult hepatocytes in an experimental model of chronic liver failure. Undifferentiated human cord blood mesenchymal stem cells were isolated, pro-pagated, and characterized by morphology, gene expression analysis, and flow cytometry of surface markers and in vitro differentiated into hepatocyte-like cells. Rat hepatocytes were isolated by double perfusion technique. An animal model of chronic liver failure was developed, and undifferentiated human cord blood mesenchymal stem cells, in vitro hepato-genically differentiated mesenchymal stem cells, or freshly isolated rat hepatocytes were transplanted into a CCL4 cirrhotic experimental model. Animals were killed 3 months after transplant, and liver functions and histopathology were assessed. Compared with the cirrhotic control group, the 3 cell-treated groups showed improved alanine aminotransferase, aspartate aminotransferase, albumin, and bilirubin levels, with best results shown in the hepatocyte-treated group. Histopathologic examination of the treated groups showed improved fibrosis, with best results obtained in the undifferentiated mesenchymal stem cell-treated group. Both adult hepatocytes and cord blood mesenchymal stem cells proved to be promising candidates for cell-based therapy in liver regeneration on an experimental level. Improved liver function was evident in the hepatocyte-treated group, and fibrosis control was more evident in the undifferentiated mesenchymal stem cell-treated group.

A refined characterisation of the NeoHepatocyte phenotype necessitates a reappraisal of the transdifferentiation hypothesis.

Science.gov (United States)

Riquelme, Paloma; Wundt, Judith; Hutchinson, James A; Brulport, Marc; Jun, Yu; Sotnikova, Anna; Girreser, Ulrich; Braun, Felix; Gövert, Felix; Soria, Bernat; Nüssler, Andreas; Clement, Bernd; Hengstler, Jan G; Fändrich, Fred

2009-03-01

Under certain culture conditions human peripheral blood monocytes may be induced to express phenotypic markers of non-haematopoietic lineages, including hepatocyte-defining traits. One such example, the NeoHepatocyte, was previously shown to express a broad panel of hepatocyte-like marker antigens and metabolic activities, both in vitro and following engraftment in the liver of immunodeficient mice. In this report, a refined description of NeoHepatocytes, with regard to their expression of xenobiotic-metabolising enzymes, morphology, hepatocyte marker expression and cell surface phenotype, is presented in comparison with human macrophages in defined states of activation. Contrary to prior assertions, it would seem more likely that NeoHepatocytes express particular hepatocyte-defining genes during a normal programme of macrophage differentiation rather than undergoing a process of transdifferentiation to become hepatocyte-like cells.

In vitro culture of functionally active buffalo hepatocytes isolated by using a simplified manual perfusion method.

Directory of Open Access Journals (Sweden)

Santanu Panda

Full Text Available In farm animals, there is no suitable cell line available to understand liver-specific functions. This has limited our understanding of liver function and metabolism in farm animals. Culturing and maintenance of functionally active hepatocytes is difficult, since they survive no more than few days. Establishing primary culture of hepatocytes can help in studying cellular metabolism, drug toxicity, hepatocyte specific gene function and regulation. Here we provide a simple in vitro method for isolation and short-term culture of functionally active buffalo hepatocytes.Buffalo hepatocytes were isolated from caudate lobes by using manual enzymatic perfusion and mechanical disruption of liver tissue. Hepatocyte yield was (5.3 ± 0.66×107 cells per gram of liver tissue with a viability of 82.3 ± 3.5%. Freshly isolated hepatocytes were spherical with well contrasted border. After 24 hours of seeding onto fibroblast feeder layer and different extracellular matrices like dry collagen, matrigel and sandwich collagen coated plates, hepatocytes formed confluent monolayer with frequent clusters. Cultured hepatocytes exhibited typical cuboidal and polygonal shape with restored cellular polarity. Cells expressed hepatocyte-specific marker genes or proteins like albumin, hepatocyte nuclear factor 4α, glucose-6-phosphatase, tyrosine aminotransferase, cytochromes, cytokeratin and α1-antitrypsin. Hepatocytes could be immunostained with anti-cytokeratins, anti-albumin and anti α1-antitrypsin antibodies. Abundant lipid droplets were detected in the cytosol of hepatocytes using oil red stain. In vitro cultured hepatocytes could be grown for five days and maintained for up to nine days on buffalo skin fibroblast feeder layer. Cultured hepatocytes were viable for functional studies.We developed a convenient and cost effective technique for hepatocytes isolation for short-term culture that exhibited morphological and functional characteristics of active hepatocytes

Lack of direct mitogenic activity of dichloroacetate and trichloroacetate in cultured rat hepatocytes

International Nuclear Information System (INIS)

Walgren, Jennie L.; Kurtz, David T.; McMillan, JoEllyn M.

2005-01-01

Dichloroacetate (DCA) and trichloroacetate (TCA) are hepatocarcinogenic metabolites of the common groundwater contaminant, 1,1,2-trichloroethylene. DCA and TCA have been shown to induce hepatocyte proliferation in vivo, but it is not known if this response is the result of direct mitogenic activity or whether cell replication occurs indirectly in response to tissue injury or inflammation. In this study we used primary cultures of rat hepatocytes, a species susceptible to DCA- but not TCA-induced hepatocarcinogenesis, to determine whether DCA and TCA are direct hepatocyte mitogens. Rat hepatocytes, cultured in growth factor-free medium, were treated with 0.01-1.0 mM DCA or TCA for 10-40 h; cell replication was then assessed by measuring incorporation of 3 H-thymidine into DNA and by cell counts. DCA or TCA treatment did not alter 3 H-thymidine incorporation in the cultured hepatocytes. Although an increase in cell number was not observed, DCA treatment significantly abrogated the normal background cell loss, suggesting an ability to inhibit apoptotic cell death in primary hepatocyte cultures. Furthermore, treatment with DCA synergistically enhanced the mitogenic response to epidermal growth factor. The data indicate that DCA and TCA are not direct mitogens in hepatocyte cultures, which is of interest in view of their ability to stimulate hepatocyte replication in vivo. Nevertheless, the synergistic enhancement of epidermal growth factor-induced hepatocyte replication by DCA is of particular interest and warrants further study

Immortalized human hepatocytes as a tool for the study of hepatocytic (de-)differentiation

NARCIS (Netherlands)

Schippers, IJ; Moshage, H; Roelofsen, H; Muller, M; Heymans, HSA; Ruiters, M; Kuipers, F

Primary human hepatocytes were immortalized by stable transfection with a recombinant plasmid containing the early region of simian virus (SV) 40. The cells were cultured in serum-free, hormonally defined medium during the immortalization procedure. Foci of dividing cells were seen after 3 months.

Hypoxia-inducible factor-dependent production of profibrotic mediators by hypoxic hepatocytes.

Science.gov (United States)

Copple, Bryan L; Bustamante, Juan J; Welch, Timothy P; Kim, Nam Deuk; Moon, Jeon-Ok

2009-08-01

During the development of liver fibrosis, mediators are produced that stimulate cells in the liver to differentiate into myofibroblasts and to produce collagen. Recent studies demonstrated that the transcription factor, hypoxia-inducible factor-1alpha (HIF-1alpha), is critical for upregulation of profibrotic mediators, such as platelet-derived growth factor-A (PDGF-A), PDGF-B and plasminogen activator inhibitor-1 (PAI-1) in the liver, during the development of fibrosis. What remains unknown is the cell type-specific regulation of these genes by HIF-1alpha in liver cell types. Accordingly, the hypothesis was tested that HIF-1alpha is activated in hypoxic hepatocytes and regulates the production of profibrotic mediators by these cells. In this study, hepatocytes were isolated from the livers of control and HIF-1alpha- or HIF-1beta-deficient mice and exposed to hypoxia. Exposure of primary mouse hepatocytes to 1% oxygen stimulated nuclear accumulation of HIF-1alpha and upregulated PAI-1, vascular endothelial cell growth factor and the vasoactive peptides adrenomedullin-1 (ADM-1) and ADM-2. In contrast, the levels of PDGF-A and PDGF-B mRNAs were unaffected in these cells by hypoxia. Exposure of HIF-1alpha-deficient hepatocytes to 1% oxygen only partially prevented upregulation of these genes, suggesting that other hypoxia-regulated transcription factors, such as HIF-2alpha, may also regulate these genes. In support of this, HIF-2alpha was activated in hypoxic hepatocytes, and exposure of HIF-1beta-deficient hepatocytes to 1% oxygen completely prevented upregulation of PAI-1, vascular endothelial cell growth factor and ADM-1, suggesting that HIF-2alpha may also contribute to upregulation of these genes in hypoxic hepatocytes. Collectively, our results suggest that HIFs may be important regulators of profibrotic and vasoactive mediators by hypoxic hepatocytes.

Vitellogenin knockdown strongly affects cotton boll weevil egg viability but not the number of eggs laid by females.

Science.gov (United States)

Coelho, Roberta R; de Souza Júnior, José Dijair Antonino; Firmino, Alexandre A P; de Macedo, Leonardo L P; Fonseca, Fernando C A; Terra, Walter R; Engler, Gilbert; de Almeida Engler, Janice; da Silva, Maria Cristina M; Grossi-de-Sa, Maria Fatima

2016-09-01

Vitellogenin (Vg), a yolk protein precursor, is the primary egg nutrient source involved in insect reproduction and embryo development. The Cotton Boll weevil (CBW) Anthonomus grandis Boheman, the most important cotton pest in Americas, accumulates large amounts of Vg during reproduction. However, the precise role of this protein during embryo development in this insect remains unknown. Herein, we investigated the effects of vitellogenin (AgraVg) knockdown on the egg-laying and egg viability in A. grandis females, and also characterized morphologically the unviable eggs. AgraVg transcripts were found during all developmental stages of A. grandis, with highest abundance in females. Silencing of AgraVg culminated in a significant reduction in transcript amount, around 90%. Despite this transcriptional reduction, egg-laying was not affected in dsRNA-treated females but almost 100% of the eggs lost their viability. Eggs from dsRNA-treated females showed aberrant embryos phenotype suggesting interference at different stages of embryonic development. Unlike for other insects, the AgraVg knockdown did not affect the egg-laying ability of A. grandis, but hampered A. grandis reproduction by perturbing embryo development. We concluded that the Vg protein is essential for A. grandis reproduction and a good candidate to bio-engineer the resistance against this devastating cotton pest.

Comparison of para-aminophenol cytotoxicity in rat renal epithelial cells and hepatocytes.

Science.gov (United States)

Li, Ying; Bentzley, Catherine M; Tarloff, Joan B

2005-04-01

Several chemicals, including para-aminophenol (PAP), produce kidney damage in the absence of hepatic damage. Selective nephrotoxicity may be related to the ability of the kidney to reabsorb filtered water, thereby raising the intraluminal concentration of toxicants and exposing tubular epithelial cells to higher concentrations than would be present in other tissues. The present experiments tested the hypothesis that hepatocytes and renal epithelial cells exposed to equivalent concentrations of PAP would be equally susceptible to toxicity. Hepatocytes and renal epithelial cells were prepared by collagenase digestion of tissues obtained from female Sprague-Dawley rats. Toxicity was monitored using trypan blue exclusion, oxygen consumption and ATP content. We measured the rate of PAP clearance and formation of PAP-glutathione conjugate by HPLC. We found that renal epithelial cells accumulated trypan blue and showed declines in oxygen consumption and ATP content at significantly lower concentrations of PAP and at earlier time points than hepatocytes. The half-life of PAP in hepatocyte incubations was significantly shorter (0.71+/-0.07 h) than in renal epithelial cell incubations (1.33+/-0.23 h), suggesting that renal epithelial cells were exposed to PAP for longer time periods than hepatocytes. Renal epithelial cells formed significantly less glutathione conjugates of PAP (PAP-SG) than did hepatocytes, consistent with less efficient detoxification of reactive PAP intermediates by renal epithelial cells. Finally, hepatocytes contained significant more reduced glutathione (NPSH) than did renal epithelial cells, possibly explaining the enhanced formation of PAP-SG by this cell population. In conclusion, our data indicates that renal epithelial cells are intrinsically more susceptible to PAP cytotoxicity than are hepatocytes. This enhanced cytotoxicity may be due to longer exposure to PAP and/or reduced detoxification of reactive intermediates due to lower concentrations

YAP Inhibition Restores Hepatocyte Differentiation in Advanced HCC, Leading to Tumor Regression

Directory of Open Access Journals (Sweden)

Julien Fitamant

2015-03-01

Full Text Available Defective Hippo/YAP signaling in the liver results in tissue overgrowth and development of hepatocellular carcinoma (HCC. Here, we uncover mechanisms of YAP-mediated hepatocyte reprogramming and HCC pathogenesis. YAP functions as a rheostat in maintaining metabolic specialization, differentiation, and quiescence within the hepatocyte compartment. Increased or decreased YAP activity reprograms subsets of hepatocytes to different fates associated with deregulation of the HNF4A, CTNNB1, and E2F transcriptional programs that control hepatocyte quiescence and differentiation. Importantly, treatment with small interfering RNA-lipid nanoparticles (siRNA-LNPs targeting YAP restores hepatocyte differentiation and causes pronounced tumor regression in a genetically engineered mouse HCC model. Furthermore, YAP targets are enriched in an aggressive human HCC subtype characterized by a proliferative signature and absence of CTNNB1 mutations. Thus, our work reveals Hippo signaling as a key regulator of the positional identity of hepatocytes, supports targeting of YAP using siRNA-LNPs as a paradigm of differentiation-based therapy, and identifies an HCC subtype that is potentially responsive to this approach.

Environmental pollution affects molecular and biochemical responses during gonadal maturation of Astyanax fasciatus (Teleostei: Characiformes: Characidae).

Science.gov (United States)

Tolussi, Carlos E; Gomes, Aline D Olio; Kumar, Anupama; Ribeiro, Cristiele S; Nostro, Fabiana L Lo; Bain, Peter A; de Souza, Gabriela B; Cuña, Rodrigo Da; Honji, Renato M; Moreira, Renata G

2018-01-01

Endocrine disrupting compounds (EDCs) have the potential to alter fish reproduction at various levels of organization. The aim of this study was to assess the impact of a natural environment with heavily anthropogenic influence on the physiological processes involved in reproduction in the freshwater fish lambari (Astyanax fasciatus) using different biomarkers. Adult males and females were collected in different seasons from two distinct sites in the same watershed: Ponte Nova Reservoir (PN) considered a pristine or small anthropogenic influence reference point; and Billings Reservoir (Bil), subjected to a large anthropogenic impact. Biological indices, such as hepatosomatic index and gonadosomatic index (GSI), gonadal histomorphology, fecundity, and biomarkers such as plasma levels of estradiol (E2) as well as hepatic gene expression of its alfa nuclear receptor (ERα), were analyzed. Hepatic vitellogenin (VTG) gene expression was evaluated in both sexes, as an indicator of xenoestrogen exposure. Females collected at PN presented a typical annual variation reflected in GSI, whereas for those sampled at Bil the index did not change through the seasons. The higher concentration of E2 in males collected at Bil during spring/2013, together with the detection of VTG gene expression, suggest the presence of EDCs in the water. These EDCs may have also influenced fecundity of females from Bil, which was higher during winter and spring/2013. Gene expression of ERα and ovarian morphology did not differ between fish from both sites. Water conditions from Bil reservoir impacted by anthropic activity clearly interfered mainly with biomarkers of biological effect such as plasma E2 levels and absolute and relative fecundity, but also altered biomarkers of exposure as VTG gene expression. These facts support the notion that waterborne EDCs are capable of causing estrogenic activity in A. fasciatus. Copyright © 2017 Elsevier Inc. All rights reserved.

Gender differences in methionine accumulation and metabolism in freshly isolated mouse hepatocytes: Potential roles in toxicity

International Nuclear Information System (INIS)

Dever, Joseph T.; Elfarra, Adnan A.

2009-01-01

L-Methionine (Met) is hepatotoxic at high concentrations. Because Met toxicity in freshly isolated mouse hepatocytes is gender-dependent, the goal of this study was to assess the roles of Met accumulation and metabolism in the increased sensitivity of male hepatocytes to Met toxicity compared with female hepatocytes. Male hepatocytes incubated with Met (30 mM) at 37 o C exhibited higher levels of intracellular Met at 0.5, 1.0, and 1.5 h, respectively, compared to female hepatocytes. Conversely, female hepatocytes had higher levels of S-adenosyl-L-methionine compared to male hepatocytes. Female hepatocytes also exhibited higher L-methionine-L-sulfoxide levels relative to control hepatocytes, whereas the increases in L-methionine-D-sulfoxide (Met-D-O) levels were similar in hepatocytes of both genders. Addition of aminooxyacetic acid (AOAA), an inhibitor of Met transamination, significantly increased Met levels at 1.5 h and increased Met-D-O levels at 1.0 and 1.5 h only in Met-exposed male hepatocytes. No gender differences in cytosolic Met transamination activity by glutamine transaminase K were detected. However, female mouse liver cytosol exhibited higher methionine-DL-sulfoxide (MetO) reductase activity than male mouse liver cytosol at low (0.25 and 0.5 mM) MetO concentrations. Collectively, these results suggest that increased cellular Met accumulation, decreased Met transmethylation, and increased Met and MetO transamination in male mouse hepatocytes may be contributing to the higher sensitivity of the male mouse hepatocytes to Met toxicity in comparison with female mouse hepatocytes.

Quantitative Adverse Outcome Pathways and Their ...

Science.gov (United States)

A quantitative adverse outcome pathway (qAOP) consists of one or more biologically based, computational models describing key event relationships linking a molecular initiating event (MIE) to an adverse outcome. A qAOP provides quantitative, dose–response, and time-course predictions that can support regulatory decision-making. Herein we describe several facets of qAOPs, including (a) motivation for development, (b) technical considerations, (c) evaluation of confidence, and (d) potential applications. The qAOP used as an illustrative example for these points describes the linkage between inhibition of cytochrome P450 19A aromatase (the MIE) and population-level decreases in the fathead minnow (FHM; Pimephales promelas). The qAOP consists of three linked computational models for the following: (a) the hypothalamic-pitutitary-gonadal axis in female FHMs, where aromatase inhibition decreases the conversion of testosterone to 17β-estradiol (E2), thereby reducing E2-dependent vitellogenin (VTG; egg yolk protein precursor) synthesis, (b) VTG-dependent egg development and spawning (fecundity), and (c) fecundity-dependent population trajectory. While development of the example qAOP was based on experiments with FHMs exposed to the aromatase inhibitor fadrozole, we also show how a toxic equivalence (TEQ) calculation allows use of the qAOP to predict effects of another, untested aromatase inhibitor, iprodione. While qAOP development can be resource-intensive, the quan

Pulp and paper mill effluent treatments have differential endocrine-disrupting effects on rainbow trout.

Science.gov (United States)

Orrego, Rodrigo; Guchardi, John; Hernandez, Victor; Krause, Rachelle; Roti, Lucia; Armour, Jeffrey; Ganeshakumar, Mathumai; Holdway, Douglas

2009-01-01

Endocrine disruption (ED) effects due to pulp and paper mill effluents extracts involving different industrial procedures and effluent treatments (nontreated, primary, and secondary treated) were evaluated using immature triploid rainbow trout in a pulse-exposure toxicity experiment. The protocol involved the use of intraperitoneal injection of mill extracts (solid-phase extraction [SPE]) corrected for individual fish weight and included several laboratory standards (steroidal hormones and phytosterols). Biological endpoints at two different levels of biological organization were analyzed (molecular and individual organism). Results indicated that nonsignificant changes were observed in the individual physiological indices represented by condition factor, liver somatic index, and gonad somatic index during the experiment. Significant induction of liver ethoxyresorufin-O-deethylase activity was observed between different effluent treatments and experimental controls. Significant endocrine-disrupting effects at the reproductive level were observed in all effluent treatments involving significant increments in plasma vitellogenin (VTG) levels. Fish exposed to untreated effluent extracts had significantly higher VTG levels compared to fish exposed to primary and secondary treatment effluent extracts, indicating a decrease of the estrogenic effect due to the effluent treatment. The present study has shown that for the Chilean pulp and paper mill SPE extracts evaluated, an endocrine disruption effect was induced in immature triploid rainbow, reaffirming the significant estrogenic effects demonstrated previously in laboratory and field experiments.

Distinctive metabolite profiles in in-migrating Sockeye salmon suggest sex-linked endocrine perturbation.

Science.gov (United States)

Benskin, Jonathan P; Ikonomou, Michael G; Liu, Jun; Veldhoen, Nik; Dubetz, Cory; Helbing, Caren C; Cosgrove, John R

2014-10-07

The health of Skeena River Sockeye salmon (Onchorhychus nerka) has been of increasing concern due to declining stock returns over the past decade. In the present work, in-migrating Sockeye from the 2008 run were evaluated using a mass spectrometry-based, targeted metabolomics platform. Our objectives were to (a) investigate natural changes in a subset of the hepatic metabolome arising from migration-associated changes in osmoregulation, locomotion, and gametogenesis, and (b) compare the resultant profiles with animals displaying altered hepatic vitellogenin A (vtg) expression at the spawning grounds, which was previously hypothesized as a marker of xenobiotic exposure. Of 203 metabolites monitored, 95 were consistently observed in Sockeye salmon livers and over half of these changed significantly during in-migration. Among the most dramatic changes in both sexes were a decrease in concentrations of taurine (a major organic osmolyte), carnitine (involved in fatty acid transport), and two major polyunsaturated fatty acids (eicosapentaenoic acid and docosahexaenoic acid). In females, an increase in amino acids was attributed to protein catabolism associated with vitellogenesis. Animals with atypical vtg mRNA expression demonstrated unusual hepatic amino acid, fatty acid, taurine, and carnitine profiles. The cause of these molecular perturbations remains unclear, but may include xenobiotic exposure, natural senescence, and/or interindividual variability. These data provide a benchmark for further investigation into the long-term health of migrating Skeena Sockeye.

Interspecies differences in metabolism of arsenic by cultured primary hepatocytes

International Nuclear Information System (INIS)

Drobna, Zuzana; Walton, Felecia S.; Harmon, Anne W.; Thomas, David J.; Styblo, Miroslav

2010-01-01

Biomethylation is the major pathway for the metabolism of inorganic arsenic (iAs) in many mammalian species, including the human. However, significant interspecies differences have been reported in the rate of in vivo metabolism of iAs and in yields of iAs metabolites found in urine. Liver is considered the primary site for the methylation of iAs and arsenic (+3 oxidation state) methyltransferase (As3mt) is the key enzyme in this pathway. Thus, the As3mt-catalyzed methylation of iAs in the liver determines in part the rate and the pattern of iAs metabolism in various species. We examined kinetics and concentration-response patterns for iAs methylation by cultured primary hepatocytes derived from human, rat, mice, dog, rabbit, and rhesus monkey. Hepatocytes were exposed to [ 73 As]arsenite (iAs III ; 0.3, 0.9, 3.0, 9.0 or 30 nmol As/mg protein) for 24 h and radiolabeled metabolites were analyzed in cells and culture media. Hepatocytes from all six species methylated iAs III to methylarsenic (MAs) and dimethylarsenic (DMAs). Notably, dog, rat and monkey hepatocytes were considerably more efficient methylators of iAs III than mouse, rabbit or human hepatocytes. The low efficiency of mouse, rabbit and human hepatocytes to methylate iAs III was associated with inhibition of DMAs production by moderate concentrations of iAs III and with retention of iAs and MAs in cells. No significant correlations were found between the rate of iAs methylation and the thioredoxin reductase activity or glutathione concentration, two factors that modulate the activity of recombinant As3mt. No associations between the rates of iAs methylation and As3mt protein structures were found for the six species examined. Immunoblot analyses indicate that the superior arsenic methylation capacities of dog, rat and monkey hepatocytes examined in this study may be associated with a higher As3mt expression. However, factors other than As3mt expression may also contribute to the interspecies differences

No evidence for protective erythropoietin alpha signalling in rat hepatocytes

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Frede Stilla

2009-04-01

Full Text Available Abstract Background Recombinant human erythropoietin alpha (rHu-EPO has been reported to protect the liver of rats and mice from ischemia-reperfusion injury. However, direct protective effects of rHu-EPO on hepatocytes and the responsible signalling pathways have not yet been described. The aim of the present work was to study the protective effect of rHu-EPO on warm hypoxia-reoxygenation and cold-induced injury to hepatocytes and the rHu-EPO-dependent signalling involved. Methods Loss of viability of isolated rat hepatocytes subjected to hypoxia/reoxygenation or incubated at 4°C followed by rewarming was determined from released lactate dehydrogenase activity in the absence and presence of rHu-EPO (0.2–100 U/ml. Apoptotic nuclear morphology was assessed by fluorescence microscopy using the nuclear fluorophores H33342 and propidium iodide. Erythropoietin receptor (EPOR, EPO and Bcl-2 mRNAs were quantified by real time PCR. Activation of JAK-2, STAT-3 and STAT-5 in hepatocytes and rat livers perfused in situ was assessed by Western blotting. Results In contrast to previous in vivo studies on ischemia-reperfusion injury to the liver, rHu-EPO was without any protective effect on hypoxic injury, hypoxia-reoxygenation injury and cold-induced apoptosis to isolated cultured rat hepatocytes. EPOR mRNA was identified in these cells but specific detection of the EPO receptor protein was not possible due to the lack of antibody specificity. Both, in the cultured rat hepatocytes (10 U/ml for 15 minutes and in the rat liver perfused in situ with rHu-EPO (8.9 U/ml for 15 minutes no evidence for EPO-dependent signalling was found as indicated by missing effects of rHu-EPO on phosphorylation of JAK-2, STAT-3 and STAT-5 and on the induction of Bcl-2 mRNA. Conclusion Together, these results indicate the absence of any protective EPO signalling in rat hepatocytes. This implies that the protection provided by rHu-EPO in vivo against ischemia-reperfusion and

Cell swelling and glycogen metabolism in hepatocytes from fasted rats

NARCIS (Netherlands)

Gustafson, L. A.; Jumelle-Laclau, M. N.; van Woerkom, G. M.; van Kuilenburg, A. B.; Meijer, A. J.

1997-01-01

Cell swelling is known to increase net glycogen production from glucose in hepatocytes from fasted rats by activating glycogen synthase. Since both active glycogen synthase and phosphorylase are present in hepatocytes, suppression of flux through phosphorylase may also contribute to the net increase

Sodium-independent, bicuculline-sensitive [3H]GABA binding to isolated rat hepatocytes

International Nuclear Information System (INIS)

Minuk, G.Y.; Bear, C.E.; Sarjeant, E.J.

1987-01-01

To determine whether hepatocytes possess specific receptor sites for gamma-aminobutyric acid (GABA), a potent amino acid neurotransmitter, [ 3 H]GABA, was added to sodium-free suspensions of Percoll-purified hepatocytes derived from collagenase-perfused rat livers under various experimental conditions and in the presence or absence of specific GABA receptor agonists (muscimol) and antagonists (bicuculline). The effects of GABA, muscimol, and bicuculline on hepatocyte resting membrane potentials were also determined. Specific binding of [ 3 H]GABA to hepatocytes was a consistent finding. GABA-hepatocyte interactions were reversible and temperature dependent. Muscimol and bicuculline inhibited binding in a dose-dependent manner (IC50, 30 nM and 50 microM, respectively), whereas strychnine (1.0-100 microM), a nonspecific central nervous system stimulant, had no appreciable effect. Both GABA and muscimol (100 microM) caused significant hyperpolarization of hepatocyte resting membrane potential (delta PD 5.4 +/- 3.1 and 22.2 +/- 16.2 mV, respectively, means +/- SD, P less than 0.0005). Bicuculline (100 microM) inhibited the effect of muscimol (P less than 0.05). The results of this study suggest that specific GABA receptor sites exist on the surface of isolated rat hepatocytes. The presence of such sites raises the possibility that, in addition to adrenergic and cholinergic innervation, hepatic function may be influenced by GABA-ergic neurotransmitter mechanisms

Controlled cell morphology and liver-specific function of engineered primary hepatocytes by fibroblast layer cell densities.

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Sakai, Yusuke; Koike, Makiko; Kawahara, Daisuke; Hasegawa, Hideko; Murai, Tomomi; Yamanouchi, Kosho; Soyama, Akihiko; Hidaka, Masaaki; Takatsuki, Mitsuhisa; Fujita, Fumihiko; Kuroki, Tamotsu; Eguchi, Susumu

2018-03-05

Engineered primary hepatocytes, including co-cultured hepatocyte sheets, are an attractive to basic scientific and clinical researchers because they maintain liver-specific functions, have reconstructed cell polarity, and have high transplantation efficiency. However, co-culture conditions regarding engineered primary hepatocytes were suboptimal in promoting these advantages. Here we report that the hepatocyte morphology and liver-specific function levels are controlled by the normal human diploid fibroblast (TIG-118 cell) layer cell density. Primary rat hepatocytes were plated onto TIG-118 cells, previously plated 3 days before at 1.04, 5.21, and 26.1×10 3  cells/cm 2 . Hepatocytes plated onto lower TIG-118 cell densities expanded better during the early culture period. The hepatocytes gathered as colonies and only exhibited small adhesion areas because of the pushing force from proliferating TIG-118 cells. The smaller areas of each hepatocyte result in the development of bile canaliculi. The highest density of TIG-118 cells downregulated albumin synthesis activity of hepatocytes. The hepatocytes may have undergone apoptosis associated with high TGF-β1 concentration and necrosis due to a lack of oxygen. These occurrences were supported by apoptotic chromatin condensation and high expression of both proteins HIF-1a and HIF-1b. Three types of engineered hepatocyte/fibroblast sheets comprising different TIG-118 cell densities were harvested after 4 days of hepatocyte culture and showed a complete cell sheet format without any holes. Hepatocyte morphology and liver-specific function levels are controlled by TIG-118 cell density, which helps to design better engineered hepatocytes for future applications such as in vitro cell-based assays and transplantable hepatocyte tissues. Copyright © 2018 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Property of hepatitis B virus replication in Tupaia belangeri hepatocytes.

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Sanada, Takahiro; Tsukiyama-Kohara, Kyoko; Yamamoto, Naoki; Ezzikouri, Sayeh; Benjelloun, Soumaya; Murakami, Shuko; Tanaka, Yasuhito; Tateno, Chise; Kohara, Michinori

2016-01-08

The northern treeshrew (Tupaia belangeri) has been reported to be an effective candidate for animal infection model with hepatitis B virus (HBV). The objective of our study was to analyze the growth characteristics of HBV in tupaia hepatocytes and the host response to HBV infection. We established primary tupaia hepatocytes (3-6-week old tupaia) and infected them with HBV genotypes A, B and C, and all the genotypes proliferated as well as those in human primary hepatocytes (>10(5) copies/ml in culture supernatant). We next generated a chimeric mouse with tupaia liver by transplantation of tupaia primary hepatocytes to urokinase-type plasminogen activator cDNA (cDNA-uPA)/severe combined immunodeficient (SCID) mice and the replacement ratio with tupaia hepatocytes was found to be more than 95%. Infection of chimeric mice with HBV (genotypes B, C, and D) resulted in HBV-DNA level of 10(4)-10(6) copies/ml after 8 weeks of infection, which were almost similar to that in humanized chimeric mouse. In contrast, serum HBV level in adult tupaia (1-year-old tupaia) was quite low (<10(3) copies/ml). Understanding the differences in the response to HBV infection in primary tupaia hepatocytes, chimeric mouse, and adult tupaia will contribute to elucidating the mechanism of persistent HBV infection and viral eradication. Thus, T. belangeri was found to be efficient for studying the host response to HBV infection, thereby providing novel insight into the pathogenesis of HBV. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Effect of hepatocyte growth factor on radiation response of HeLa, V79, CHO and primary cultured parenchymal hepatocyte in vitro

International Nuclear Information System (INIS)

Yamazaki, Hideya; Inoue, Takehiro; Nose, Takayuki; Murayama, Shigeyuki; Teshima, Teruki; Ozeki, Syuji; Koizumi, Masahiko; Inoue, Toshihiko.

1996-01-01

Hepatocyte growth factor (HGF) is a multipotent cytokine enhancing regeneration of injured organs as liver, kidney and lung after injury. HGF enhances proliferation of various type of cells, inhibits proliferation of carcinoma cells, enhances motility of epithelial cells. We examined three cell lines (CHO, HeLa, V79) and primary cultured normal rat parenchymal hepatocytes to determine the effect of HGF on radiation response. HGF diminished survival of CHO and V79 cells determined by colony formation assay, whereas no significant change of survival was found in HeLa cells. No synergistic changes of survival were found when these three cell lines were irradiated with the addition of HGF. Thus, HGF did not enhance the radiation effect. We also analyzed the impact of irradiation with HGF on primary cultured normal rat parenchymal hepatocytes. At first, the release of glutamic-oxaloacetic amino-transaminase (GOT) in the supernatant was estimated. Irradiation (40 Gy) with or without HGF did not change GOT release in acute phase by 4 days after irradiation compared with the unirradiated control. Second, the DNA synthesis of rat parenchymal hepatocytes was analyzed using radioactive iodine-labeled deoxyuridine incorporation. HGF counteracted the suppression of DNA synthesis induced by irradiation. Thus, HGF may act as a mitogen even for irradiation-damaged normal cells. (author)

Extracellular matrix components influence DNA synthesis of rat hepatocytes in primary culture

International Nuclear Information System (INIS)

Sawada, N.; Tomomura, A.; Sattler, C.A.; Sattler, G.L.; Kleinman, H.K.; Pitot, H.C.

1986-01-01

The effects of several extracellular matrix components (EMCs) - fibronectin (Fn), laminin (Ln), type I (C-I) and type IV (C-IV) collagen - on DNA synthesis in rat hepatocytes in primary culture were examined by both quantitative scintillation spectrometry and autoradiography of [ 3 H]thymidine incorporation. Hepatocytes cultured on Fn showed the most active DNA synthesis initiated by epidermal growth factor (EGF) with decreasing levels of [ 3 H]thymidine uptake exhibited in the cell cultured on C-IV, C-I, and Ln, respectively. The decreasing level of DNA synthesis in hepatocytes cultured on Fn, C-IV, C-I, and Ln respectively was not influenced by cell density. The number of EGF receptors of hepatocytes was also not influenced by EMCs. These data suggest that EMCs modify hepatocyte DNA synthesis by means of post-EGF-receptor mechanisms which are regulated by both growth factors and cell density

Differential Impacts of Soybean and Fish Oils on Hepatocyte Lipid Droplet Accumulation and Endoplasmic Reticulum Stress in Primary Rabbit Hepatocytes

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Xueping Zhu

2016-01-01

Full Text Available Parenteral nutrition-associated liver disease (PNALD is a severe ailment associated with long-term parenteral nutrition. Soybean oil-based lipid emulsions (SOLE are thought to promote PNALD development, whereas fish oil-based lipid emulsions (FOLE are thought to protect against PNALD. This study aimed to investigate the effects of SOLE and FOLE on primary rabbit hepatocytes. The results reveal that SOLE caused significant endoplasmic reticulum (ER and mitochondrial damage, ultimately resulting in lipid droplets accumulation and ER stress. While these deleterious events induce hepatocyte injury, FOLE at high doses cause only minor ER and mitochondrial damage, which has no effect on hepatic function. SOLE also significantly upregulated glucose-regulated protein 94 mRNA and protein expression. These data indicate that SOLE, but not FOLE, damage the ER and mitochondria, resulting in lipid droplets accumulation and ER stress and, finally, hepatocyte injury. This likely contributes to the differential impacts of SOLE and FOLE on PNALD development and progression.

Metabolism of lipoproteins by human fetal hepatocytes

International Nuclear Information System (INIS)

Carr, B.R.

1987-01-01

The rate of clearance of lipoproteins from plasma appears to play a role in the development of atherogenesis. The liver may account for as much as two thirds of the removal of low-density lipoprotein and one third of the clearance of high-density lipoprotein in certain animal species and humans, mainly by receptor-mediated pathways. The purpose of the present investigation was to determine if human fetal hepatocytes maintained in vitro take up and degrade lipoproteins. We first determined that the maximal binding capacity of iodine 125-iodo-LDL was approximately 300 ng of low-density lipoprotein protein/mg of membrane protein and an apparent dissociation constant of approximately 60 micrograms low-density lipoprotein protein/ml in membranes prepared from human fetal liver. We found that the maximal uptake of [ 125 I]iodo-LDL and [ 125 I]iodo-HDL by fetal hepatocytes occurred after 12 hours of incubation. Low-density lipoprotein uptake preceded the appearance of degradation products by 4 hours, and thereafter the degradation of low-density lipoprotein increased linearly for at least 24 hours. In contrast, high-density lipoprotein was not degraded to any extent by fetal hepatocytes. [ 125 I]Iodo-LDL uptake and degradation were inhibited more than 75% by preincubation with low-density lipoprotein but not significantly by high-density lipoprotein, whereas [ 125 I]iodo-HDL uptake was inhibited 70% by preincubation with high-density lipoprotein but not by low-density lipoprotein. In summary, human fetal hepatocytes take up and degrade low-density lipoprotein by a receptor-mediated process similar to that described for human extrahepatic tissues

Role of CYP2B in Phenobarbital-Induced Hepatocyte Proliferation in Mice.

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Li, Lei; Bao, Xiaochen; Zhang, Qing-Yu; Negishi, Masahiko; Ding, Xinxin

2017-08-01

Phenobarbital (PB) promotes liver tumorigenesis in rodents, in part through activation of the constitutive androstane receptor (CAR) and the consequent changes in hepatic gene expression and increases in hepatocyte proliferation. A typical effect of CAR activation by PB is a marked induction of Cyp2b10 expression in the liver; the latter has been suspected to be vital for PB-induced hepatocellular proliferation. This hypothesis was tested here by using a Cyp2a(4/5)bgs -null (null) mouse model in which all Cyp2b genes are deleted. Adult male and female wild-type (WT) and null mice were treated intraperitoneally with PB at 50 mg/kg once daily for 5 successive days and tested on day 6. The liver-to-body weight ratio, an indicator of liver hypertrophy, was increased by 47% in male WT mice, but by only 22% in male Cyp2a(4/5)bgs -null mice, by the PB treatment. The fractions of bromodeoxyuridine-positive hepatocyte nuclei, assessed as a measure of the rate of hepatocyte proliferation, were also significantly lower in PB-treated male null mice compared with PB-treated male WT mice. However, whereas few proliferating hepatocytes were detected in saline-treated mice, many proliferating hepatocytes were still detected in PB-treated male null mice. In contrast, female WT mice were much less sensitive than male WT mice to PB-induced hepatocyte proliferation, and PB-treated female WT and PB-treated female null mice did not show significant difference in rates of hepatocyte proliferation. These results indicate that CYP2B induction plays a significant, but partial, role in PB-induced hepatocyte proliferation in male mice. U.S. Government work not protected by U.S. copyright.

Lipopolysaccharide stimulates p62-dependent autophagy-like aggregate clearance in hepatocytes.

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Chen, Christine; Deng, Meihong; Sun, Qian; Loughran, Patricia; Billiar, Timothy R; Scott, Melanie J

2014-01-01

Impairment of autophagy has been associated with liver injury. TLR4-stimulation by LPS upregulates autophagy in hepatocytes, although the signaling pathways involved remain elusive. The objective of this study was to determine the signaling pathway leading to LPS-stimulated autophagy in hepatocytes. Cell lysates from livers of wild type (WT; C57BL/6) mice given LPS (5 mg/kg-IP) and hepatocytes from WT, TLR4ko, and MyD88ko mice treated with LPS (100 ng/mL) up to 24 h were collected. LC3II, p62/SQSTM1, Nrf2, and beclin1 levels were determined by immunoblot, immunofluorescence, and qPCR. Autophagy-like activation was measured by GFP-LC3-puncta formation and LC3II-expression. Beclin1, Nrf2, p62, MyD88, and TIRAP were knocked-down using siRNA. LC3II-expression increased in both liver and hepatocytes after LPS and was dependent on TLR4. Beclin1 expression did not increase after LPS in hepatocytes and beclin1-knockdown did not affect LC3II levels. In hepatocytes given LPS, expression of p62 increased and p62 colocalized with LC3. p62-knockdown prevented LC3II puncta formation. LPS-induced LC3II/p62-puncta also required MyD88/TIRAP signaling and localization of both Nrf2 and NF κ B transcription factors to the nucleus to upregulate p62-expression. Therefore, TLR4-activation by LPS in hepatocytes induces a p62-mediated, not beclin1-mediated, autophagy-like clearance pathway that is hepatoprotective by clearing aggregate-prone or misfolded proteins from the cytosol and preserving energy homeostasis under stress.

DNA synthesis in periportal and perivenous hepatocytes of intact and hepatectomized young mice.

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Fernández-Blanco, A; Inda, A M; Errecalde, A L

2015-01-01

DNA synthesis of hepatocytes in two areas of Intact and Hepatectomized young mice liver along a circadian period was studied. DNA synthesis was significantly different at all analyzed time points in Intact and Hepatectomized animals. Differences between periportal and perivenous hepatocytes were found in hepatectomized animals at 04/42 and 08/46 hr of day/hour post-hepatectomy. DNAs peak in periportal hepatocytes regenerating liver occurs 4 hr earlier than in perivenous hepatocytes, probably reflecting their shorter G1 phase. Besides, daily mean values of regenerating livers were higher than those observed in Intact animals, as a consequence of surgical removal.

Induction of hepatocyte polyploidization in rats of different age by ionizing radiation of different LET

International Nuclear Information System (INIS)

Gil'yano, N.Ya.; Malinovskij, O.V.; Khair, M.B.

1992-01-01

A decrease in the effectiveness of neutron-irradiation with respect to fusion of nonproliferating hepatocytes of animals with age was shown by the method of flow cytometry. There was an inverse relationship between the effectiveness of induction of non-proliferating hepatocytes fusion and neutron energy. The process of hepatocyte fusion induced by neutrons was inhibited by uranyl acetate. No age-dependent changes were noted in the induction of polyploidization of proliferating hepatocytes by sparsely ionizing radiation. A hypothesis is proposed concerning a membrane nature of the target responsible for hepatocyte polyploidization induced by densely ionizing radiation. (authors). 8 refs., 4 figs., 5 tabs

Induction of hepatocyte polyploidization in rats of different age by ionizing radiation of different LET

International Nuclear Information System (INIS)

Gil'yano, N.Ya.; Malinovskij, O.V.; Khair, M.B.

1990-01-01

A decrease in the effectiveness of neutron-irradiation with respect to fusion of nonproliferating hepatocytes of animals with age was shown by the method of flow cytometry. There was an inverse relationship between the effectiveness of induction of non-proliferating hepatocytes fusion and neutron energy. The process of hepatocyte fusion induced by neutrons was inhibited by uranyl acetate. No age-dependent changes were noted in the induction of polyploidization of proliferating hepatocytes by sparsely ionizing radiation. A hypothesis is proposed concerning a membrane nature of the target responsible for hepatocyte polyploidization induced by densely ionizing radiation

Effects of eutrophication on vitellogenin gene expression in male fathead minnows (Pimephales promelas) exposed to 17α-ethynylestradiol in field mesocosms

International Nuclear Information System (INIS)

Gordon, Denise A.; Toth, Gregory P.; Graham, David W.; Lazorchak, James M.; Reddy, Tirumuru V.; Knapp, Charles W.; Noyelles, Frank de; Campbell, Scott; Lattier, David L.

2006-01-01

This study evaluated the effect of aquatic secondary nutrient supply levels (nitrogen and phosphorus) on the subcellular response of adult male fathead minnows (Pimephales promelas) exposed to a single nominal concentration of 17α-ethynylestradiol (EE2), a potent synthetic estrogen, under quasi-natural field conditions. Outdoor mesocosms were maintained under low, medium, and high nutrient supply conditions as categorized by total phosphorus (TP) level (nominal 0.012, 0.025, and 0.045 mg TP/L, respectively), and treated with EE2 with and without a carrier solvent. Using reverse transcription-polymerase chain reaction methods, vitellogenin gene (Vg) expression was determined in the fish collected at 0 h, 8 h, 24 h, 4 d, 7 d, and 14 d post-exposure. Induction of Vg was detected as early as 8 h post-exposure, with and without the carrier solvent, and persisted through Day 14. Results showed Vg to be significantly greater at low nutrient levels (p < 0.05), suggesting that EE2 bioavailability to the fish was likely greater under less-turbid water conditions. - Nutrient condition in surface waters strongly influences vitellogenin gene expression in male fathead minnows

Hepatocyte transplantation and advancements in alternative cell sources for liver-based regenerative medicine.

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Lee, Charlotte A; Sinha, Siddharth; Fitzpatrick, Emer; Dhawan, Anil

2018-06-01

Human hepatocyte transplantation has been actively perused as an alternative to liver replacement for acute liver failure and liver-based metabolic defects. Current challenges in this field include a limited cell source, reduced cell viability following cryopreservation and poor engraftment of cells into the recipient liver with consequent limited life span. As a result, alternative stem cell sources such as pluripotent stem cells, fibroblasts, hepatic progenitor cells, amniotic epithelial cells and mesenchymal stem/stromal cells (MSCs) can be used to generate induced hepatocyte like cells (HLC) with each technique exhibiting advantages and disadvantages. HLCs may have comparable function to primary human hepatocytes and could offer patient-specific treatment. However, long-term functionality of transplanted HLCs and the potential oncogenic risks of using stem cells have yet to be established. The immunomodulatory effects of MSCs are promising, and multiple clinical trials are investigating their effect in cirrhosis and acute liver failure. Here, we review the current status of hepatocyte transplantation, alternative cell sources to primary human hepatocytes and their potential in liver regeneration. We also describe recent clinical trials using hepatocytes derived from stem cells and their role in improving the phenotype of several liver diseases.

Functional properties of hepatocytes in vitro are correlated with cell polarity maintenance.

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Zeigerer, Anja; Wuttke, Anne; Marsico, Giovanni; Seifert, Sarah; Kalaidzidis, Yannis; Zerial, Marino

2017-01-01

Exploring the cell biology of hepatocytes in vitro could be a powerful strategy to dissect the molecular mechanisms underlying the structure and function of the liver in vivo. However, this approach relies on appropriate in vitro cell culture systems that can recapitulate the cell biological and metabolic features of the hepatocytes in the liver whilst being accessible to experimental manipulations. Here, we adapted protocols for high-resolution fluorescence microscopy and quantitative image analysis to compare two primary hepatocyte culture systems, monolayer and collagen sandwich, with respect to the distribution of two distinct populations of early endosomes (APPL1 and EEA1-positive), endocytic capacity, metabolic and signaling activities. In addition to the re-acquisition of hepatocellular polarity, primary hepatocytes grown in collagen sandwich but not in monolayer culture recapitulated the apico-basal distribution of EEA1 endosomes observed in liver tissue. We found that such distribution correlated with the organization of the actin cytoskeleton in vitro and, surprisingly, was dependent on the nutritional state in vivo. Hepatocytes in collagen sandwich also exhibited faster kinetics of low-density lipoprotein (LDL) and epidermal growth factor (EGF) internalization, showed improved insulin sensitivity and preserved their ability for glucose production, compared to hepatocytes in monolayer cultures. Although no in vitro culture system can reproduce the exquisite structural features of liver tissue, our data nevertheless highlight the ability of the collagen sandwich system to recapitulate key structural and functional properties of the hepatocytes in the liver and, therefore, support the usage of this system to study aspects of hepatocellular biology in vitro. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

A fat option for the pig: Hepatocytic differentiated mesenchymal stem cells for translational research

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Brückner, Sandra, E-mail: sandra.brueckner@medizin.uni-leipzig.de [University Hospital Leipzig, Department of Visceral, Transplantation, Thoracic and Vascular Surgery, Liebigstraße 21, Leipzig D-04103 (Germany); Tautenhahn, Hans-Michael, E-mail: hans-michael.tautenhahn@medizin.uni-leipzig.de [University Hospital Leipzig, Department of Visceral, Transplantation, Thoracic and Vascular Surgery, Liebigstraße 21, Leipzig D-04103 (Germany); TRM, Translational Centre for Regenerative Medicine, Philipp-Rosenthal-Str. 55, Leipzig D-04103 (Germany); Winkler, Sandra, E-mail: sandra.pelz@medizin.uni-leipzig.de [University Hospital Leipzig, Department of Visceral, Transplantation, Thoracic and Vascular Surgery, Liebigstraße 21, Leipzig D-04103 (Germany); Stock, Peggy, E-mail: peggy.stock@medizin.uni-leipzig.de [University Hospital Leipzig, Department of Visceral, Transplantation, Thoracic and Vascular Surgery, Liebigstraße 21, Leipzig D-04103 (Germany); Dollinger, Matthias, E-mail: matthias.dollinger@uniklinik-ulm.de [University Hospital Ulm, First Department of Medicine, Albert-Einstein-Allee 23, Ulm D-89081 (Germany); Christ, Bruno, E-mail: bruno.christ@medizin.uni-leipzig.de [University Hospital Leipzig, Department of Visceral, Transplantation, Thoracic and Vascular Surgery, Liebigstraße 21, Leipzig D-04103 (Germany); TRM, Translational Centre for Regenerative Medicine, Philipp-Rosenthal-Str. 55, Leipzig D-04103 (Germany)

2014-02-15

Study background: Extended liver resection is the only curative treatment option of liver cancer. Yet, the residual liver may not accomplish the high metabolic and regenerative capacity needed, which frequently leads to acute liver failure. Because of their anti-inflammatory and -apoptotic as well as pro-proliferative features, mesenchymal stem cells differentiated into hepatocyte-like cells might provide functional and regenerative compensation. Clinical translation of basic research requires pre-clinical approval in large animals. Therefore, we characterized porcine mesenchymal stem cells (MSC) from adipose tissue and bone marrow and their hepatocyte differentiation potential for future assessment of functional liver support after surgical intervention in the pig model. Methods: Mesenchymal surface antigens and multi-lineage differentiation potential of porcine MSC isolated by collagenase digestion either from bone marrow or adipose tissue (subcutaneous/visceral) were assessed by flow cytometry. Morphology and functional properties (urea-, glycogen synthesis and cytochrome P450 activity) were determined during culture under differentiation conditions and compared with primary porcine hepatocytes. Results: MSC from porcine adipose tissue and from bone marrow express the typical mesenchymal markers CD44, CD29, CD90 and CD105 but not haematopoietic markers. MSC from both sources displayed differentiation into the osteogenic as well as adipogenic lineage. After hepatocyte differentiation, expression of CD105 decreased significantly and cells adopted the typical polygonal morphology of hepatocytes. Glycogen storage was comparable in adipose tissue- and bone marrow-derived cells. Urea synthesis was about 35% lower in visceral than in subcutaneous adipose tissue-derived MSC. Cytochrome P450 activity increased significantly during differentiation and was twice as high in hepatocyte-like cells generated from bone marrow as from adipose tissue. Conclusion: The hepatocyte

A fat option for the pig: Hepatocytic differentiated mesenchymal stem cells for translational research

International Nuclear Information System (INIS)

Brückner, Sandra; Tautenhahn, Hans-Michael; Winkler, Sandra; Stock, Peggy; Dollinger, Matthias; Christ, Bruno

2014-01-01

Study background: Extended liver resection is the only curative treatment option of liver cancer. Yet, the residual liver may not accomplish the high metabolic and regenerative capacity needed, which frequently leads to acute liver failure. Because of their anti-inflammatory and -apoptotic as well as pro-proliferative features, mesenchymal stem cells differentiated into hepatocyte-like cells might provide functional and regenerative compensation. Clinical translation of basic research requires pre-clinical approval in large animals. Therefore, we characterized porcine mesenchymal stem cells (MSC) from adipose tissue and bone marrow and their hepatocyte differentiation potential for future assessment of functional liver support after surgical intervention in the pig model. Methods: Mesenchymal surface antigens and multi-lineage differentiation potential of porcine MSC isolated by collagenase digestion either from bone marrow or adipose tissue (subcutaneous/visceral) were assessed by flow cytometry. Morphology and functional properties (urea-, glycogen synthesis and cytochrome P450 activity) were determined during culture under differentiation conditions and compared with primary porcine hepatocytes. Results: MSC from porcine adipose tissue and from bone marrow express the typical mesenchymal markers CD44, CD29, CD90 and CD105 but not haematopoietic markers. MSC from both sources displayed differentiation into the osteogenic as well as adipogenic lineage. After hepatocyte differentiation, expression of CD105 decreased significantly and cells adopted the typical polygonal morphology of hepatocytes. Glycogen storage was comparable in adipose tissue- and bone marrow-derived cells. Urea synthesis was about 35% lower in visceral than in subcutaneous adipose tissue-derived MSC. Cytochrome P450 activity increased significantly during differentiation and was twice as high in hepatocyte-like cells generated from bone marrow as from adipose tissue. Conclusion: The hepatocyte

Modulation of vitellogenin synthesis through estrogen receptor beta-1 in goldfish (Carassius auratus) juveniles exposed to 17-β estradiol and nonylphenol

International Nuclear Information System (INIS)

Soverchia, L.; Ruggeri, B.; Palermo, F.; Mosconi, G.; Cardinaletti, G.; Scortichini, G.; Gatti, G.; Polzonetti-Magni, A.M.

2005-01-01

Many synthetic chemicals, termed xenoestrogens, have been shown to interact as agonists with the estrogen receptor (ER) to elicit biological responses similar to those of natural hormones. To date, the regulation of vitellogenesis in oviparous vertebrates has been widely used for evaluation of estrogenic effects. Therefore, Carassius auratus juveniles were chosen as a fish model for studying the effects of estradiol-17β and different concentrations (10 -6 and 10 -7 M) of 4-nonylphenol (4-NP) on the expression of liver ERβ-1 subtype; plasma vitellogenin and sex steroids (androgens and estradiol-17β) were also evaluated together with the bioaccumulation process, through mass-spectrometry. C. auratus is a species widespread in the aquatic environment and, on the toxicological point of view, can be considered a good 'sentinel' species. Juveniles of goldfish were maintained in tanks with only tap water or water with different concentrations (10 -6 and 10 -7 M) of 4-nonylphenol (4-NP), or 10 -7 M of estradiol-17β. After 3 weeks of treatment, animals were anesthetized within 5 min after capture, and blood was immediately collected into heparinized syringes by cardiac puncture and stored at -70 deg. C; the gonads were fixed, then frozen and stored at -70 deg. C; the whole fish, liver, and muscle tissues were harvested and immediately stored at -70 deg. C for molecular biology experiments and bioaccumulation measurements. The estrogenic effects of 4-NP were evidenced by the presence of plasma vitellogenin in juveniles exposed both to estradiol-17β and the two doses of 4-NP; moreover, exposure to 4-NP also increased aromatization of androgens, as suggested by decreasing androgens and increasing estradiol-17β plasma levels. The changes of these parameters were in agreement with the increasing transcriptional rate of ERβ-1 mRNA in the liver, demonstrating that both estradiol-17β and 4-NP modulate the vitellogenin rate through interaction with the ERβ-1 subtype. The

Circadian rhythms of Per2::Luc in individual primary mouse hepatocytes and cultures.

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Casey J Guenthner

Full Text Available BACKGROUND: Hepatocytes, the parenchymal cells of the liver, express core clock genes, such as Period2 and Cryptochrome2, which are involved in the transcriptional/translational feedback loop of the circadian clock. Whether or not the liver is capable of sustaining rhythms independent of a central pacemaker is controversial. Whether and how circadian information may be shared among cells in the liver in order to sustain oscillations is currently unknown. RESULTS: In this study we isolated primary hepatocytes from transgenic Per2(Luc mice and used bioluminescence as a read-out of the state of the circadian clock. Hepatocytes cultured in a collagen gel sandwich configuration exhibited persistent circadian rhythms for several weeks. The amplitude of the rhythms damped, but medium changes consistently reset the phase and amplitude of the cultures. Cry2(-/- Per2(Luc cells oscillated robustly and expressed a longer period. Co-culturing with wildtype cells did not significantly shorten the period, indicating that coupling among hepatocytes is insufficient to synchronize cells with significantly differing periods. However, spatial patterns revealed by cellular imaging of wildtype cultures provided evidence of weak local coupling among the hepatocytes. CONCLUSIONS: Our results with primary hepatocyte cultures demonstrate that cultured hepatocytes are weakly coupled. While this coupling is not sufficient to sustain global synchrony, it does increase local synchrony, which may stabilize the circadian rhythms of peripheral oscillators, such as the liver, against noise in the entraining signals.

Vitellogenin concentrations in feral Danish brown trout have decreased: An effect of improved sewage treatment in rural areas?

DEFF Research Database (Denmark)

Morthorst, Jane Ebsen; Mathiesen, Karsten Korsholm; Holbech, Henrik

2018-01-01

biomarkers for estrogenic exposure, and feminized fish have been observed downstream WWTP outlets in many countries. Danish central WWTPs reduce effluent estrogenicity effectively by advanced sewage treatment, and feminizations have not been observed downstream central WWTP outlets. However, between 2000......Feminization of male and juvenile fish caused by exposure to estrogens or estrogenic chemicals in effluents from central wastewater treatment plants (WWTP) is a worldwide issue of concern. Intersex and induction of the female yolk protein, vitellogenin, in male and juvenile fish are robust...

Effects of 17α-ethynylestradiol on hormonal responses and xenobiotic biotransformation system of Atlantic salmon (Salmo salar)

International Nuclear Information System (INIS)

Mortensen, Anne S.; Arukwe, Augustine

2007-01-01

Pharmaceuticals are ubiquitous pollutants in the aquatic environment where their potential effects on non-target species like fish has only recently become subject of systematic investigations. In the present study, experiments were undertaken to examine the effects of a synthetic pharmaceutical endocrine disruptor, ethynylestradiol (EE2), given in water at 5 or 50 ng/L and sampled at days 0 (control), 3 and 7 after exposure, on hepatic phase I and II biotransformation and hormonal pathways of juvenile salmon using quantitative (real-time) polymerase chain reaction (qPCR), Vtg ELISA and 7-ethoxyresorufin O-deethylase (EROD) catalytic activity. Our data show that EE2 produced time- and concentration-specific modulation of estrogen receptor isoforms (ERα, ERβ) and androgen receptor-β (ARβ). EE2 produced a concentration-specific induction of vitellogenin (Vtg) and zona radiata protein (Zr-protein) at day 3 after exposure. At day 7, Vtg and Zr-protein mRNA (and plasma Vtg protein) expression were significantly decreased in the group given 5 ng EE2/L, compared to dimethyl sulfoxide (DMSO) control group. In the xenobiotic biotransformation pathway, EE2 produced a significant increase of aryl hydrocarbon receptor-α (AhRα) at day 3 in the group given 5 ng EE2/L and AhRβ was decreased at the same concentration at day 7. While CYP3A was not significantly affected by EE2 exposure, the CYP1A1, AhR nuclear translocator (Arnt) and AhR repressor (AhRR) mRNA showed an apparent EE2 concentration and time-dependent decrease. The expression of uridine diphosphoglucuronosyl transferase (UGT) and glutathione S-transferase class pi-like (GSTpi-like) mRNA were decreased after exposure to 50 ng EE2/L at both day 3 and 7 after exposure. The effect of EE2 on the CYP1A1 gene expressions paralleled effect on EROD and AhRR mRNA, suggesting a direct role of EE2 in controlling cellular detoxification machinery. Interestingly, the carrier vehicle, DMSO produced significant time

Endocrine-disruptor molecular responses, occurrence of intersex and gonado-histopathological changes in tilapia species from a tropical freshwater dam (Awba Dam) in Ibadan, Nigeria

International Nuclear Information System (INIS)

Adeogun, Aina O.; Onibonoje, Kolawole; Ibor, Oju R.; Omiwole, Roseline A.; Chukwuka, Azubuike V.; Ugwumba, Alex O.; Ugwumba, Adiaha A.A.; Arukwe, Augustine

2016-01-01

Highlights: • Occurrence and severity of intersex in Nigerian aquatic environment. • Estrogenic and reproductive developmental effects of effluents from a University community. • Biomarker of endocrine disruption in fish from a developing country. • Relationship between estrogenic responses and sediment contaminants burden in a dam used for University domestic water supply and for fisheries. • Possible health consequences of environmental contamination - Abstract: In the present study, the occurrence of endocrine disruptive responses in Tilapia species from Awba Dam has been investigated, and compared to a reference site (Modete Dam). The Awba Dam is a recipient of effluents from University of Ibadan (Nigeria) and several other anthropogenic sources. A total of 132 Tilapia species (Sarotherodon malenotheron (n = 57 and 32, males and females, respectively) and Tilapia guineensis (n = 23 and 20, males and females, respectively)) were collected from June to September 2014. At the reference site, samples of adult male and female S. melanotheron (48 males and 47 females) and T. guineensis (84 males and 27 females) were collected. Gonads were morphologically and histologically examined and gonadosomatic index (GSI) was calculated. Hepatic mRNA transcriptions of vitellogenin (Vtg) and zona radiata protein (Zrp) genes were analyzed using validated RT-qPCR. Significant increase in Vtg and Zrp transcripts were observed in male tilapias from Awba Dam, compared to males from the reference site. In addition, male tilapias from Awba Dam produced significantly higher Vtg and Zrp mRNA, compared to females in June and July. However, at the natural peak spawning period in August and September, females produced, significantly higher Vtg and Zrp mRNA, compared to males. Fish gonads revealed varying incidence of intersex with a striking presence of two (2) pairs of testes and a pair of ovary in S. melanotheron from Awba Dam. The entire fish population examined at Awba Dam

Endocrine-disruptor molecular responses, occurrence of intersex and gonado-histopathological changes in tilapia species from a tropical freshwater dam (Awba Dam) in Ibadan, Nigeria

Energy Technology Data Exchange (ETDEWEB)

Adeogun, Aina O.; Onibonoje, Kolawole; Ibor, Oju R.; Omiwole, Roseline A.; Chukwuka, Azubuike V.; Ugwumba, Alex O.; Ugwumba, Adiaha A.A. [Department of Zoology, University of Ibadan, Ibadan (Nigeria); Arukwe, Augustine, E-mail: arukwe@bio.ntnu.no [Department of Biology, Norwegian University of Science and Technology (NTNU), 7491 Trondheim (Norway)

2016-05-15

Highlights: • Occurrence and severity of intersex in Nigerian aquatic environment. • Estrogenic and reproductive developmental effects of effluents from a University community. • Biomarker of endocrine disruption in fish from a developing country. • Relationship between estrogenic responses and sediment contaminants burden in a dam used for University domestic water supply and for fisheries. • Possible health consequences of environmental contamination - Abstract: In the present study, the occurrence of endocrine disruptive responses in Tilapia species from Awba Dam has been investigated, and compared to a reference site (Modete Dam). The Awba Dam is a recipient of effluents from University of Ibadan (Nigeria) and several other anthropogenic sources. A total of 132 Tilapia species (Sarotherodon malenotheron (n = 57 and 32, males and females, respectively) and Tilapia guineensis (n = 23 and 20, males and females, respectively)) were collected from June to September 2014. At the reference site, samples of adult male and female S. melanotheron (48 males and 47 females) and T. guineensis (84 males and 27 females) were collected. Gonads were morphologically and histologically examined and gonadosomatic index (GSI) was calculated. Hepatic mRNA transcriptions of vitellogenin (Vtg) and zona radiata protein (Zrp) genes were analyzed using validated RT-qPCR. Significant increase in Vtg and Zrp transcripts were observed in male tilapias from Awba Dam, compared to males from the reference site. In addition, male tilapias from Awba Dam produced significantly higher Vtg and Zrp mRNA, compared to females in June and July. However, at the natural peak spawning period in August and September, females produced, significantly higher Vtg and Zrp mRNA, compared to males. Fish gonads revealed varying incidence of intersex with a striking presence of two (2) pairs of testes and a pair of ovary in S. melanotheron from Awba Dam. The entire fish population examined at Awba Dam

Angiotensin II protects primary rat hepatocytes against bile salt-induced apoptosis.

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Golnar Karimian

Full Text Available UNLABELLED: Angiotensin II (AT-II is a pro-fibrotic compound that acts via membrane-bound receptors (AT-1R/AT-2R and thereby activates hepatic stellate cells (HSCs. AT-II receptor blockers (ARBs are thus important candidates in the treatment of liver fibrosis. However, multiple case reports suggest that AT-1R blockers may induce hepatocyte injury. Therefore, we investigated the effect of AT-II and its receptor blockers on cytokine-, oxidative stress- and bile salt-induced cell death in hepatocytes. Primary rat hepatocytes were exposed to TNF-α/Actinomycin D, the ROS-generating agent menadione or the bile salts: glycochenodeoxycholic acid (GCDCA and tauro-lithocholic acid-3 sulfate (TLCS, to induce apoptosis. AT-II (100 nmol/L was added 10 minutes prior to the cell death-inducing agent. AT-1R antagonists (Sartans and the AT-2R antagonist PD123319 were used at 1 µmol/L. Apoptosis (caspase-3 activity, acridine orange staining and necrosis (Sytox green staining were quantified. Expression of CHOP (marker for ER stress and AT-II receptor mRNAs were quantified by Q-PCR. AT-II dose-dependently reduced GCDCA-induced apoptosis of hepatocytes (-50%, p<0.05 without inducing necrosis. In addition, AT-II reduced TLCS-induced apoptosis of hepatocytes (-50%, p<0.05. However, AT-II did not suppress TNF/Act-D and menadione-induced apoptosis. Only the AT-1R antagonists abolished the protective effect of AT-II against GCDCA-induced apoptosis. AT-II increased phosphorylation of ERK and a significant reversal of the protective effect of AT-II was observed when signaling kinases, including ERK, were inhibited. Moreover, AT-II prevented the GCDCA-induced expression of CHOP (the marker of the ER-mediated apoptosis. CONCLUSION: Angiotensin II protects hepatocytes from bile salt-induced apoptosis through a combined activation of PI3-kinase, MAPKs, PKC pathways and inhibition of bile salt-induced ER stress. Our results suggest a mechanism for the observed hepatocyte

Expression analysis of vitellogenins in the workers of the red imported fire ant (Solenopsis invicta

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Chloe Hawkings

2018-05-01

Full Text Available Vitellogenin has been proposed to regulate division of labor and social organization in social insects. The red imported fire ant (Solenopsis invicta harbors four distinct, adjacent vitellogenin genes (Vg1, Vg2, Vg3, and Vg4. Contrary to honey bees that have a single Vg ortholog as well as potentially fertile nurses, and to other ant species that lay trophic eggs, S. invicta workers completely lack ovaries or the ability to lay eggs. This provides a unique model to investigate whether Vg duplication in S. invicta was followed by subfunctionalization to acquire non-reproductive functions and whether Vg was co-opted to regulate behavior within the worker caste. To investigate these questions, we compared the expression patterns of S. invicta Vg genes among workers from different morphological subcastes or performing different tasks. RT-qPCRs revealed higher relative expression of Vg1 in major workers compared to both medium and minor workers, and of Vg2 in major workers when compared to minor workers. Relative expression of Vg1 was also higher in carbohydrate foragers when compared to nurses and protein foragers. By contrast, the level of expression of Vg2, Vg3, and Vg4 were not significantly different among the workers performing the specific tasks. Additionally, we analyzed the relationship between the expression of the Vg genes and S-hydroprene, a juvenile hormone analog. No changes in Vg expression were recorded in workers 12 h after application of the analog. Our results suggest that in S. invicta the Vg gene underwent subfunctionalization after duplication to new functions based on the expression bias observed in these data. This may suggest an alternative and still unknown function for Vg in the workers that needs to be investigated further.

Cell proliferation studies in rodent hepatocytes during 1,4-dichlorobenzene administration

International Nuclear Information System (INIS)

Eldridge, S.R.; Tilbury, L.F.; Randall, H.; Goldsworthy, T.L.; Butterworth, B.E.

1990-01-01

In the NTP bioassay, 1,4-dichlorobenzene (DCB) induced hepatocellular carcinomas in mice, but not in rats. Because DCB is not DNA reactive, a cell proliferation study under conditions of the bioassay was undertaken to determine whether increased cell proliferation might play a role in DCB-induced hepatocarcinogenicity. DCB was administered in corn oil by gavage at the highest bioassay dose to male B6C3F1 mice (600 mg/kg) and male F344 rats (300 mg/kg) for five consecutive days. Cell proliferation was detected by labeling hepatocytes with either 5-bromo-2'-deoxyuridine (BRDU) or 3 H-thymidine delivered during the entire treatment period by subcutaneously implanted osmotic pumps. An increase in liver weight as a percentage of body weight was observed in treated mice (6.7±0.6 vs. 5.9±0.2) and rats (4.7±0.1 vs. 4.0±0.2) compared to controls. No significant elevations in plasma enzymes were found in either treated species, indicating a lack of overt hepatotoxicity. Histopathological evaluation revealed no evidence of hepatotoxicity in either species. The percentage of hepatocytes in S-phase was increased approximately 10-fold in both treated mice and rats compared to the respective control animals. Mice exhibited a centrilobular pattern of labeled hepatocytes, whereas rat hepatocytes were labeled hepatocytes, whereas rat hepatocytes were labeled throughout the lobules. These data demonstrate the hepatic mitogenic activity of DCB in mice and rats. However, this response dose not correlate with DCB-induced hepatocarcinogenicity. Further studies are required to examine the extent, duration and nature of the proliferative response in order to understand the species-specific effects of DCB

Hepatocyte transplants improve liver function and encephalopathy in portacaval shunted rats.

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Fogel, Wieslawa Agnieszka; Stasiak, Anna; Maksymowicz, Michał; Kobos, Jozef; Unzeta, Mercedes; Mussur, Miroslaw

2014-07-01

Rats with portacaval shunt (PCS) are useful experimental models of human hepatic encephalopathy in chronic liver dysfunction. We have previously shown that PCS modifies amine neurotransmitter systems in the CNS and increases voluntary alcohol intake by rats. Hepatocyte transplantation, used in acute liver failure, has recently also been applied to chronic liver diseases, which prompted us to investigate whether the altered brain amine system and the drinking behavior in long-term shunted rats could be normalized by hepatocyte transplants. Hepatocytes, isolated from syngeneic donors by collagenase digestion, were injected (3 × 10(6) cells/rat) into the pancreatic tail region, 6 months after PCS. Hepatic function was evaluated by measuring urine urea and plasma L-histidine concentrations. A free choice test with two bottles (tap water and 10% ethyl alcohol) was performed for 3 days to assess the rats' preference for alcohol. The rats were euthanized 2 months posttransplantation. Brain histamine and 5-hydroxyindoleacetic acid (5-HIAA) levels were measured by radioenzymatic assay and by HPLC-EC, respectively, N-tele-methylhistamine by GC/MS while MAOA and MAOB activities by isotopic procedures. Portacaval shunt rats with hepatocyte transplants gave more urea than before transplantation, with lower plasma L-His levels and higher body weight versus the PCS counterparts. Also, those rats consumed less alcohol. The CNS amines and 5-HIAA concentrations, as well as MAO-B activity, being abnormally high in untreated PCS rats, significantly reduced after PCS hepatocyte treatment. The results support the therapeutic values of hepatocyte transplants in chronic liver diseases and the temporary character of PCS-exerted CNS dysfunctions. © 2014 John Wiley & Sons Ltd.

DISTINCT FUNCTIONS OF JNK AND C-JUN IN OXIDANT-INDUCED HEPATOCYTE DEATH

Science.gov (United States)

Amir, Muhammad; Liu, Kun; Zhao, Enpeng; Czaja, Mark J.

2013-01-01

Overactivation of c-Jun N-terminal kinase (JNK)/c-Jun signaling is a central mechanism of hepatocyte injury and death including that from oxidative stress. However, the functions of JNK and c-Jun are still unclear, and this pathway also inhibits hepatocyte death. Previous studies of menadione-induced oxidant stress demonstrated that toxicity resulted from sustained JNK/c-Jun activation as death was blocked by the c-Jun dominant negative TAM67. To further delineate the function of JNK/c-Jun signaling in hepatocyte injury from oxidant stress, the effects of direct JNK inhibition on menadione-induced death were examined. In contrast to the inhibitory effect of TAM67, pharmacological JNK inhibition by SP600125 sensitized the rat hepatocyte cell line RALA255-10G to death from menadione. SP600125 similarly sensitized mouse primary hepatocytes to menadione toxicity. Death from SP600125/menadione was c-Jun dependent as it was blocked by TAM67, but independent of c-Jun phosphorylation. Death occurred by apoptosis and necrosis and activation of the mitochondrial death pathway. Short hairpin RNA knockdowns of total JNK or JNK2 sensitized to death from menadione, whereas a jnk1 knockdown was protective. Jnk2 null mouse primary hepatocytes were also sensitized to menadione death. JNK inhibition magnified decreases in cellular ATP content and β-oxidation induced by menadione. This effect mediated cell death as chemical inhibition of β-oxidation also sensitized cells to death from menadione, and supplementation with the β-oxidation substrate oleate blocked death. Components of the JNK/c-Jun signaling pathway have opposing functions in hepatocyte oxidant stress with JNK2 mediating resistance to cell death and c-Jun promoting death. PMID:22644775

β-Adrenergic induction of lipolysis in hepatocytes is inhibited by ethanol exposure.

Science.gov (United States)

Schott, Micah B; Rasineni, Karuna; Weller, Shaun G; Schulze, Ryan J; Sletten, Arthur C; Casey, Carol A; McNiven, Mark A

2017-07-14

In liver steatosis ( i.e. fatty liver), hepatocytes accumulate many large neutral lipid storage organelles known as lipid droplets (LDs). LDs are important in the maintenance of energy homeostasis, but the signaling mechanisms that stimulate LD metabolism in hepatocytes are poorly defined. In adipocytes, catecholamines target the β-adrenergic (β-AR)/cAMP pathway to activate cytosolic lipases and induce their recruitment to the LD surface. Therefore, the goal of this study was to determine whether hepatocytes, like adipocytes, also undergo cAMP-mediated lipolysis in response to β-AR stimulation. Using primary rat hepatocytes and human hepatoma cells, we found that treatment with the β-AR agent isoproterenol caused substantial LD loss via activation of cytosolic lipases adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL). β-Adrenergic stimulation rapidly activated PKA, which led to the phosphorylation of ATGL and HSL and their recruitment to the LD surface. To test whether this β-AR-dependent lipolysis pathway was altered in a model of alcoholic fatty liver, primary hepatocytes from rats fed a 6-week EtOH-containing Lieber-DeCarli diet were treated with cAMP agonists. Compared with controls, EtOH-exposed hepatocytes showed a drastic inhibition in β-AR/cAMP-induced LD breakdown and the phosphorylation of PKA substrates, including HSL. This observation was supported in VA-13 cells, an EtOH-metabolizing human hepatoma cell line, which displayed marked defects in both PKA activation and isoproterenol-induced ATGL translocation to the LD periphery. In summary, these findings suggest that β-AR stimulation mobilizes cytosolic lipases for LD breakdown in hepatocytes, and perturbation of this pathway could be a major consequence of chronic EtOH insult leading to fatty liver.

Oxidative stress is involved in Dasatinib-induced apoptosis in rat primary hepatocytes

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Xue, Tao; Luo, Peihua; Zhu, Hong; Zhao, Yuqin [Institute of Pharmacology and Toxicology, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058 (China); Wu, Honghai; Gai, Renhua; Wu, Youping [Center for Drug Safety Evaluation and Research of Zhejiang University, Hangzhou 310058 (China); Yang, Bo [Institute of Pharmacology and Toxicology, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058 (China); Yang, Xiaochun, E-mail: yangxiaochun@zju.edu.cn [Institute of Pharmacology and Toxicology, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058 (China); Center for Drug Safety Evaluation and Research of Zhejiang University, Hangzhou 310058 (China); He, Qiaojun, E-mail: qiaojunhe@zju.edu.cn [Institute of Pharmacology and Toxicology, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058 (China); Center for Drug Safety Evaluation and Research of Zhejiang University, Hangzhou 310058 (China)

2012-06-15

Dasatinib, a multitargeted inhibitor of BCR–ABL and SRC kinases, exhibits antitumor activity and extends the survival of patients with chronic myeloid leukemia (CML) and Philadelphia chromosome-positive acute lymphoblastic leukemia (ALL). However, some patients suffer from hepatotoxicity, which occurs through an unknown mechanism. In the present study, we found that Dasatinib could induce hepatotoxicity both in vitro and in vivo. Dasatinib reduced the cell viability of rat primary hepatocytes, induced the release of alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) in vitro, and triggered the ballooning degeneration of hepatocytes in Sprague–Dawley rats in vivo. Apoptotic markers (chromatin condensation, cleaved caspase-3 and cleaved PARP) were detected to indicate that the injury induced by Dasatinib in hepatocytes in vitro was mediated by apoptosis. This result was further validated in vivo using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assays. Here we found that Dasatinib dramatically increased the level of reactive oxygen species (ROS) in hepatocytes, reduced the intracellular glutathione (GSH) content, attenuated the activity of superoxide dismutase (SOD), generated malondialdehyde (MDA), a product of lipid peroxidation, decreased the mitochondrial membrane potential, and activated nuclear factor erythroid 2-related factor 2 (Nrf2) and mitogen-activated protein kinases (MAPK) related to oxidative stress and survival. These results confirm that oxidative stress plays a pivotal role in Dasatinib-mediated hepatotoxicity. N-acetylcysteine (NAC), a typical antioxidant, can scavenge free radicals, attenuate oxidative stress, and protect hepatocytes against Dasatinib-induced injury. Thus, relieving oxidative stress is a viable strategy for reducing Dasatinib-induced hepatotoxicity. -- Highlights: ►Dasatinib shows potential hepatotoxicity both in vitro and in vivo. ►Apoptosis plays a vital role in Dasatinib

What is the main driver of ageing in long-lived winter honeybees: antioxidant enzymes, innate immunity, or vitellogenin?

Science.gov (United States)

Aurori, Cristian M; Buttstedt, Anja; Dezmirean, Daniel S; Mărghitaş, Liviu A; Moritz, Robin F A; Erler, Silvio

2014-06-01

To date five different theories compete in explaining the biological mechanisms of senescence or ageing in invertebrates. Physiological, genetical, and environmental mechanisms form the image of ageing in individuals and groups. Social insects, especially the honeybee Apis mellifera, present exceptional model systems to study developmentally related ageing. The extremely high phenotypic plasticity for life expectancy resulting from the female caste system provides a most useful system to study open questions with respect to ageing. Here, we used long-lived winter worker honeybees and measured transcriptional changes of 14 antioxidative enzyme, immunity, and ageing-related (insulin/insulin-like growth factor signaling pathway) genes at two time points during hibernation. Additionally, worker bees were challenged with a bacterial infection to test ageing- and infection-associated immunity changes. Gene expression levels for each group of target genes revealed that ageing had a much higher impact than the bacterial challenge, notably for immunity-related genes. Antimicrobial peptide and antioxidative enzyme genes were significantly upregulated in aged worker honeybees independent of bacterial infections. The known ageing markers vitellogenin and IlP-1 were opposed regulated with decreasing vitellogenin levels during ageing. The increased antioxidative enzyme and antimicrobial peptide gene expression may contribute to a retardation of senescence in long-lived hibernating worker honeybees. © The Author 2013. Published by Oxford University Press on behalf of The Gerontological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Determination of metabolic stability using cryopreserved hepatocytes from rainbow trout (Oncorhynchus mykiss)

Science.gov (United States)

Standard protocols for isolating, cryopreserving, and thawing rainbow trout hepatocytes are described, along with procedures for using fresh or cryopreserved hepatocytes to assess chemical metabolic stability in fish by means of a substrate depletion approach. Variations on thes...

Evaluation of perfluoroalkyl acid activity using primary mouse and human hepatocytes

International Nuclear Information System (INIS)

Rosen, Mitchell B.; Das, Kaberi P.; Wood, Carmen R.; Wolf, Cynthia J.; Abbott, Barbara D.; Lau, Christopher

2013-01-01

While perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS) have been studied at length, less is known about the biological activity of other perfluoroalkyl acids (PFAAs) detected in the environment. Using a transient transfection assay developed in COS-1 cells, our group has previously evaluated a variety of PFAAs for activity associated with activation of peroxisome proliferator-activated receptor alpha (PPARα). Here we use primary heptatocytes to further assess the biological activity of a similar group of PFAAs using custom designed Taqman Low Density Arrays. Primary mouse and human hepatoyctes were cultured for 48 h in the presence of varying concentrations of 12 different PFAAs or Wy14,643, a known activator of PPARα. Total RNA was collected and the expression of 48 mouse or human genes evaluated. Gene selection was based on either in-house liver microarray data (mouse) or published data using primary hepatocytes (human). Gene expression in primary mouse hepatocytes was more restricted than expected. Genes typically regulated in whole tissue by PPARα agonists were not altered in mouse cells including Acox1, Me1, Acaa1a, Hmgcs1, and Slc27a1. Cyp2b10, a gene regulated by the constitutive androstane receptor and a transcript normally up-regulated by in vivo exposure to PFAAs, was also unchanged in cultured mouse hepatocytes. Cyp4a14, Ehhadh, Pdk4, Cpt1b, and Fabp1 were regulated as expected in mouse cells. A larger group of genes were differentially expressed in human primary hepatocytes, however, little consistency was observed across compounds with respect to which genes produced a significant dose response making the determination of relative biological activity difficult. This likely reflects weaker activation of PPARα in human versus rodent cells as well as variation among individual cell donors. Unlike mouse cells, CYP2B6 was up-regulated in human hepatocytes by a number of PFAAs as was PPARδ. Rankings were conducted on the limited

Bidirectional transport of iminodiacetic organic anion analogues between plasma and hepatocyte

International Nuclear Information System (INIS)

Peters, A.M.; Myers, M.J.; Mohammadtaghi, S.; Mubashar, M.; Mathie, R.T.

1998-01-01

The kinetics of organic anions are well described and back-diffusion from hepatocyte to plasma is accepted. Although iminodiacetic (IDA) analogues, as organic anions, should also show bidirectional transport between hepatocyte and plasma, this has not been directly demonstrated heretofore. The aim of this study was to directly demonstrate back-diffusion and to quantify it in terms of its fractional rate constant. Kinetics of diethyl IDA were studied in three anaesthetised dogs in which femoral arterial and hepatic venous samples were obtained after injection of tracer into (a) a peripheral vein or (b) hepatic artery or portal vein. Arterial time-concentration curves were also compared between peripheral venous and either hepatic arterial or portal venous injections. Time-activity curves were recorded from regions of interest over the cardiac blood pool and peripheral hepatic parenchyma in 30 patients undergoing routine IDA hepatobiliary imaging with diethyl IDA or mebrofenin and fractional rate constants of clearance of IDA from the hepatocyte compared between compartmental and deconvolution analyses. After peripheral injection in dogs, there was an early arteriovenous concentration gradient across the liver indicating an hepatocyte extraction fraction in the three animals of 0.9, 0.8 and 0.6. The net extraction fraction decreased exponentially over 40 min. Time-concentration curves from hepatic vein and femoral artery were virtually superimposed following intrahepatic injections. Peripheral arterial curves, however, had different shapes according to whether injections were intrahepatic or peripheral, and were consistent with significant back-diffusion. In clinical studies, the blood disappearance curves were fitted as the sum of two exponentials and the liver curves as the difference of two exponentials (with rate constants denoted α 1 h and α 2 h ). Based on compartmental analysis of the blood curves, the sum of the fractional rate constants of tracer movement

Micropatterned co-culture of hepatocyte spheroids layered on non-parenchymal cells to understand heterotypic cellular interactions

International Nuclear Information System (INIS)

Otsuka, Hidenori; Sasaki, Kohei; Okimura, Saya; Nagamura, Masako; Nakasone, Yuichi

2013-01-01

Microfabrication and micropatterning techniques in tissue engineering offer great potential for creating and controlling cellular microenvironments including cell–matrix interactions, soluble stimuli and cell–cell interactions. Here, we present a novel approach to generate layered patterning of hepatocyte spheroids on micropatterned non-parenchymal feeder cells using microfabricated poly(ethylene glycol) (PEG) hydrogels. Micropatterned PEG-hydrogel-treated substrates with two-dimensional arrays of gelatin circular domains (ϕ = 100 μm) were prepared by photolithographic method. Only on the critical structure of PEG hydrogel with perfect protein rejection, hepatocytes were co-cultured with non-parenchymal cells to be led to enhanced hepatocyte functions. Then, we investigated the mechanism of the functional enhancement in co-culture with respect to the contributions of soluble factors and direct cell–cell interactions. In particular, to elucidate the influence of soluble factors on hepatocyte function, hepatocyte spheroids underlaid with fibroblasts (NIH/3T3 mouse fibroblasts) or endothelial cells (BAECs: bovine aortic endothelial cells) were compared with physically separated co-culture of hepatocyte monospheroids with NIH3T3 or BAEC using trans-well culture systems. Our results suggested that direct heterotypic cell-to-cell contact and soluble factors, both of these between hepatocytes and fibroblasts, significantly enhanced hepatocyte functions. In contrast, direct heterotypic cell-to-cell contact between hepatocytes and endothelial cells only contributed to enhance hepatocyte functions. This patterning technique can be a useful experimental tool for applications in basic science, drug screening and tissue engineering, as well as in the design of artificial liver devices. (paper)

Acidosis-induced downregulation of hepatocyte mitochondrial aquaporin-8 and ureagenesis from ammonia.

Science.gov (United States)

Molinas, Sara M; Soria, Leandro R; Marrone, Julieta; Danielli, Mauro; Trumper, Laura; Marinelli, Raúl A

2015-08-01

It has been proposed that, during metabolic acidosis, the liver downregulates mitochondrial ammonia detoxification via ureagenesis, a bicarbonate-consuming process. Since we previously demonstrated that hepatocyte mitochondrial aquaporin-8 channels (mtAQP8) facilitate the uptake of ammonia and its metabolism into urea, we studied whether mtAQP8 is involved in the liver adaptive response to acidosis. Primary cultured rat hepatocytes were adapted to acidosis by exposing them to culture medium at pH 7.0 for 40 h. Control cells were exposed to pH 7.4. Hepatocytes exposed to acid medium showed a decrease in mtAQP8 protein expression (-30%, p ammonia was assessed by incubating the cells with (15)N-labeled ammonia and measuring (15)N-labeled urea synthesis by nuclear magnetic resonance. Reduced ureagenesis was found in acidified hepatocytes (-31%, p ammonia in response to acidosis.

Hepatocyte heterogeneity in the metabolism of carbohydrates.

Science.gov (United States)

Jungermann, K; Thurman, R G

1992-01-01

Periportal and perivenous hepatocytes possess different amounts and activities of the rate-generating enzymes of carbohydrate and oxidative energy metabolism and thus different metabolic capacities. This is the basis of the model of metabolic zonation, according to which periportal cells catalyze predominantly the oxidative catabolism of fatty and amino acids as well as glucose release and glycogen formation via gluconeogenesis, and perivenous cells carry out preferentially glucose uptake for glycogen synthesis and glycolysis coupled to liponeogenesis. The input of humoral and nervous signals into the periportal and perivenous zones is different; gradients of oxygen, substrates and products, hormones and mediators and nerve densities exist which are important not only for the short-term regulation of carbohydrate metabolism but also for the long-term regulation of zonal gene expression. The specialization of periportal and perivenous hepatocytes in carbohydrate metabolism has been well characterized. In vivo evidence is provided by the complex metabolic situation termed the 'glucose paradox' and by zonal flux differences calculated on the basis of the distribution of enzymes and metabolites. In vitro evidence is given by the different flux rates determined with classical invasive techniques, e.g. in periportal-like and perivenous-like hepatocytes in cell culture, in periportal- and perivenous-enriched hepatocyte populations and in perfused livers during orthograde and retrograde flow, as well as with noninvasive techniques using miniature oxygen electrodes, e.g. in livers perfused in either direction. Differences of opinion in the interpretation of studies with invasive and noninvasive techniques by the authors are discussed. The declining gradient in oxygen concentrations, the decreasing glucagon/insulin ratio and the different innervation could be important factors in the zonal expression of the genes of carbohydrate-metabolizing enzymes. While it is clear that

Live cell imaging of cytosolic NADH/NAD+ ratio in hepatocytes and liver slices.

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Masia, Ricard; McCarty, William J; Lahmann, Carolina; Luther, Jay; Chung, Raymond T; Yarmush, Martin L; Yellen, Gary

2018-01-01

Fatty liver disease (FLD), the most common chronic liver disease in the United States, may be caused by alcohol or the metabolic syndrome. Alcohol is oxidized in the cytosol of hepatocytes by alcohol dehydrogenase (ADH), which generates NADH and increases cytosolic NADH/NAD + ratio. The increased ratio may be important for development of FLD, but our ability to examine this question is hindered by methodological limitations. To address this, we used the genetically encoded fluorescent sensor Peredox to obtain dynamic, real-time measurements of cytosolic NADH/NAD + ratio in living hepatocytes. Peredox was expressed in dissociated rat hepatocytes and HepG2 cells by transfection, and in mouse liver slices by tail-vein injection of adeno-associated virus (AAV)-encoded sensor. Under control conditions, hepatocytes and liver slices exhibit a relatively low (oxidized) cytosolic NADH/NAD + ratio as reported by Peredox. The ratio responds rapidly and reversibly to substrates of lactate dehydrogenase (LDH) and sorbitol dehydrogenase (SDH). Ethanol causes a robust dose-dependent increase in cytosolic NADH/NAD + ratio, and this increase is mitigated by the presence of NAD + -generating substrates of LDH or SDH. In contrast to hepatocytes and slices, HepG2 cells exhibit a relatively high (reduced) ratio and show minimal responses to substrates of ADH and SDH. In slices, we show that comparable results are obtained with epifluorescence imaging and two-photon fluorescence lifetime imaging (2p-FLIM). Live cell imaging with Peredox is a promising new approach to investigate cytosolic NADH/NAD + ratio in hepatocytes. Imaging in liver slices is particularly attractive because it allows preservation of liver microanatomy and metabolic zonation of hepatocytes. NEW & NOTEWORTHY We describe and validate a new approach for measuring free cytosolic NADH/NAD + ratio in hepatocytes and liver slices: live cell imaging with the fluorescent biosensor Peredox. This approach yields dynamic, real

IL-6 modulates hepatocyte proliferation via induction of HGF/p21cip1: Regulation by SOCS3

International Nuclear Information System (INIS)

Sun Rui; Jaruga, Barbara; Kulkarni, Shailin; Sun Haoyu; Gao Bin

2005-01-01

The precise role of IL-6 in liver regeneration and hepatocyte proliferation is controversial and the role of SOCS3 in liver regeneration remains unknown. Here we show that in vitro treatment with IL-6 inhibited primary mouse hepatocyte proliferation. IL-6 induced p21 cip1 protein expression in primary mouse hepatocytes. Disruption of the p21 cip1 gene abolished the inhibitory effect of IL-6 on cell proliferation. Co-culture with nonparenchymal liver cells diminished IL-6 inhibition of hepatocyte proliferation, which was likely due to IL-6 stimulation of nonparenchymal cells to produce HGF. Finally, IL-6 induced higher levels of p21 cip1 protein expression and a slightly stronger inhibition of cell proliferation in SOCS3 +/- mouse hepatocytes compared to wild-type hepatocytes, while liver regeneration was enhanced and prolonged in SOCS3 +/- mice. Our findings suggest that IL-6 directly inhibits hepatocyte proliferation via a p21 cip1 -dependent mechanism and indirectly enhances hepatocyte proliferation via stimulating nonparenchymal cells to produce HGF. SOCS3 negatively regulates liver regeneration

Evaluation of potential mechanisms of atrazine-induced reproductive impairment in fathead minnow (Pimephales promelas) and Japanese medaka (Oryzias latipes)

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Richter, Cathy; Papoulias, Diana M.; Whyte, Jeffrey J.; Tillitt, Donald E.

2016-01-01

Atrazine has been implicated in reproductive dysfunction of exposed organisms, and previous studies documented decreased egg production in Japanese medaka (Oryzias latipes) and fathead minnows (Pimephales promelas) during 30-d to 38-d exposures to 0.5 µg/L, 5 µg/L, and 50 µg/L atrazine. The authors evaluated possible mechanisms underlying the reduction in egg production. Gene expression in steroidogenesis pathways and the hypothalamus–pituitary–gonad axis of male and female fish was measured. Atrazine did not significantly induce gonad aromatase (cyp19a1a) expression. An atrazine-induced shift in the number of females in an active reproductive state was observed. Expression of the egg maturation genes vitellogenin 1 (vtg1) and zona pellucida glycoprotein 3.1 (zp3.1) in medaka females was correlated and had a bimodal distribution. In both species, females with low vtg1 or zp3.1 expression also had low expression of steroidogenesis genes in the gonad, estrogen receptor in the liver, and gonadotropins in the brain. In the medaka, the number of females per tank that had high expression of zp3.1 was significantly correlated with egg production per tank. The number of medaka females with low expression of zp3.1 increased significantly with atrazine exposure. Thus, the decline in egg production observed in response to atrazine exposure may be the result of a coordinated downregulation of genes required for reproduction in a subset of females.

Effects of atrazine on hepatic metabolism and endocrine homeostasis in rainbow trout (Oncorhynchus mykiss)

International Nuclear Information System (INIS)

Salaberria, Iurgi; Hansen, Bjorn Henrik; Asensio, Vega; Olsvik, Pal A.; Andersen, Rolf A.; Jenssen, Bjorn Munro

2009-01-01

The herbicide atrazine (ATZ) is one of the most widely used pesticides in the world and is now under scrutiny for its alleged capacity to disrupt the endocrine system. Exhibiting negligible interaction with the estrogen receptor (ER), ATZ's mode of action remains to be elucidated. ATZ may act as an inducer of the enzyme aromatase, which converts androgens to estrogens, although other mechanisms should also be taken into consideration such as impairment of hepatic metabolism. Therefore we administered juvenile rainbow trout (Oncorhynchus mykiss) a dose of either 2 or 200 μg ATZ/kg, or of carrier control phosphate buffered saline (PBS) and we measured plasma concentrations of testosterone (T), 17beta-estradiol (E2) and vitellogenin (Vtg) 6 days after exposure. Simultaneously we analyzed hepatic gene expression of cytochrome P450 (CYP) 1A and pi-class glutathione S-transferase (GST-P), and catalase (CAT) activity. Although sex steroid levels showed no significant alterations, we found a dose-dependent increase in Vtg and a concomitant decrease in CYP1A. There was no effect of ATZ on GST-P mRNA levels but GST-P was positively correlated with CYP1A. Also, CYP1A was negatively correlated with liver CAT and E2, and varied with T concentrations in a hormetic manner. The results showed that ATZ can alter hepatic metabolism, induce estrogenic effects and oxidative stress in vivo, and that these effects are linked

Application of endocrine disruptor screening program fish short-term reproduction assay: Reproduction and endocrine function in fathead minnow (Pimephales promelas) and killifish (Fundulus heteroclitus) exposed to Bermuda pond sediment.

Science.gov (United States)

Fort, Douglas J; Mathis, Michael; Fort, Chelsea E; Fort, Hayley M; Bacon, Jamie P

2015-06-01

A modified tier 1 Endocrine Disruptor Screening Program (EDSP) 21-d fish short-term reproduction assay (FSTRA) was used to evaluate the effects of sediment exposure from freshwater and brackish ponds in Bermuda on reproductive fecundity and endocrine function in fathead minnow (Pimephales promelas) and killifish (Fundulus heteroclitus). Reproductively active male and female fish were exposed to control sediment and sediment from 2 freshwater ponds (fathead minnow) and 2 marine ponds (killifish) contaminated with polyaromatic hydrocarbons and metals via flow-through exposure for 21 d. Reproductive fecundity was monitored daily. At termination, the status of the reproductive endocrine system was assessed by the gonadosomatic index, gonadal histology, plasma steroids (estrogen [E2], testosterone [T], and 11-ketotestosterone [11-KT]), steroidogenic enzymes (aromatase and combined 3β/17β -hydroxysteroid dehydrogenase [3β/17β-HSD]), and plasma vitellogenin (VTG). Decreased reproductive fecundity, lower male body weight, and altered endocrinological measures of reproductive status were observed in both species. Higher plasma T levels in female minnows and 11-KT levels in both male and female minnows and female killifish exposed to freshwater and brackish sediments, respectively. Decreased female E2 and VTG levels and gonadal cytochrome P19 (aromatase) activity were also found in sediment exposed females from both species. No effect on female 3β/17β-HSD activity was found in either species. The FSTRA provided a robust model capable of modification to evaluate reproductive effects of sediment exposure in fish. © 2015 SETAC.

Functional characterization of the vitellogenin promoter in the silkworm, Bombyx mori.

Science.gov (United States)

Xu, J; Wang, Y Q; Li, Z Q; Ling, L; Zeng, B S; You, L; Chen, Y Z; Aslam, A F M; Huang, Y P; Tan, A J

2014-10-01

Genetic transformation and genome editing technologies have been successfully established in the lepidopteran insect model, the domesticated silkworm, Bombyx mori, providing great potential for functional genomics and practical applications. However, the current lack of cis-regulatory elements in B. mori gene manipulation research limits further exploitation in functional gene analysis. In the present study, we characterized a B. mori endogenous promoter, Bmvgp, which is a 798-bp DNA sequence adjacent to the 5'-end of the vitellogenin gene (Bmvg). PiggyBac-based transgenic analysis shows that Bmvgp precisely directs expression of a reporter gene, enhanced green fluorescent protein (EGFP), in a sex-, tissue- and stage-specific manner. In transgenic animals, EGFP expression can be detected in the female fat body from larval-pupal ecdysis to the following pupal and adult stage. Furthermore, in vitro and in vivo experiments revealed that EGFP expression can be activated by 20-hydroxyecdysone, which is consistent with endogenous Bmvg expression. These data indicate that Bmvgp is an effective endogenous cis-regulatory element in B. mori. © 2014 The Royal Entomological Society.

Assessment of mitochondrial dysfunction-related, drug-induced hepatotoxicity in primary rat hepatocytes

International Nuclear Information System (INIS)

Liu, Cong; Sekine, Shuichi; Ito, Kousei

2016-01-01

Evidence that mitochondrial dysfunction plays a central role in drug-induced liver injury is rapidly accumulating. In contrast to physiological conditions, in which almost all adenosine triphosphate (ATP) in hepatocytes is generated in mitochondria via aerobic respiration, the high glucose content and limited oxygen supply of conventional culture systems force primary hepatocytes to generate most ATP via cytosolic glycolysis. Thus, such anaerobically poised cells are resistant to xenobiotics that impair mitochondrial function, and are not suitable to identify drugs with mitochondrial liabilities. In this study, primary rat hepatocytes were cultured in galactose-based medium, instead of the conventional glucose-based medium, and in hyperoxia to improve the reliance of energy generation on aerobic respiration. Activation of mitochondria was verified by diminished cellular lactate release and increased oxygen consumption. These conditions improved sensitivity to the mitochondrial complex I inhibitor rotenone. Since oxidative stress is also a general cause of mitochondrial impairment, cells were exposed to test compounds in the presence of transferrin to increase the generation of reactive oxygen species via increased uptake of iron. Finally, 14 compounds with reported mitochondrial liabilities were tested to validate this new drug-induced mitochondrial toxicity assay. Overall, the culture of primary rat hepatocytes in galactose, hyperoxia and transferrin is a useful model for the identification of mitochondrial dysfunction-related drug-induced hepatotoxicity. - Highlights: • Drug-induced mitochondrial toxicity was evaluated using primary rat hepatocytes. • Galactose and hyperoxia could activate OXPHOS in primary rat hepatocytes. • Cells with enhanced OXPHOS exhibit improved sensitivity to mitochondrial toxins. • Transferrin potentiate mitochondrial toxicity via increased ROS production.

Assessment of mitochondrial dysfunction-related, drug-induced hepatotoxicity in primary rat hepatocytes

Energy Technology Data Exchange (ETDEWEB)

Liu, Cong; Sekine, Shuichi, E-mail: ssekine@faculty.chiba-u.jp; Ito, Kousei

2016-07-01

Evidence that mitochondrial dysfunction plays a central role in drug-induced liver injury is rapidly accumulating. In contrast to physiological conditions, in which almost all adenosine triphosphate (ATP) in hepatocytes is generated in mitochondria via aerobic respiration, the high glucose content and limited oxygen supply of conventional culture systems force primary hepatocytes to generate most ATP via cytosolic glycolysis. Thus, such anaerobically poised cells are resistant to xenobiotics that impair mitochondrial function, and are not suitable to identify drugs with mitochondrial liabilities. In this study, primary rat hepatocytes were cultured in galactose-based medium, instead of the conventional glucose-based medium, and in hyperoxia to improve the reliance of energy generation on aerobic respiration. Activation of mitochondria was verified by diminished cellular lactate release and increased oxygen consumption. These conditions improved sensitivity to the mitochondrial complex I inhibitor rotenone. Since oxidative stress is also a general cause of mitochondrial impairment, cells were exposed to test compounds in the presence of transferrin to increase the generation of reactive oxygen species via increased uptake of iron. Finally, 14 compounds with reported mitochondrial liabilities were tested to validate this new drug-induced mitochondrial toxicity assay. Overall, the culture of primary rat hepatocytes in galactose, hyperoxia and transferrin is a useful model for the identification of mitochondrial dysfunction-related drug-induced hepatotoxicity. - Highlights: • Drug-induced mitochondrial toxicity was evaluated using primary rat hepatocytes. • Galactose and hyperoxia could activate OXPHOS in primary rat hepatocytes. • Cells with enhanced OXPHOS exhibit improved sensitivity to mitochondrial toxins. • Transferrin potentiate mitochondrial toxicity via increased ROS production.

Possibility of Undifferentiated Human Thigh Adipose Stem Cells Differentiating into Functional Hepatocytes

Directory of Open Access Journals (Sweden)

Jong Hoon Lee

2012-11-01

Full Text Available BackgroundThis study aimed to investigate the possibility of isolating mesenchymal stem cells (MSCs from human thigh adipose tissue and the ability of human thigh adipose stem cells (HTASCs to differentiate into hepatocytes.MethodsThe adipose-derived stem cells (ADSCs were isolated from thigh adipose tissue. Growth factors, cytokines, and hormones were added to the collagen coated dishes to induce the undifferentiated HTASCs to differentiate into hepatocyte-like cells. To confirm the experimental results, the expression of hepatocyte-specific markers on undifferentiated and differentiated HTASCs was analyzed using reverse transcription polymerase chain reaction and immunocytochemical staining. Differentiation efficiency was evaluated using functional tests such as periodic acid schiff (PAS staining and detection of the albumin secretion level using enzyme-linked immunosorbent assay (ELISA.ResultsThe majority of the undifferentiated HTASCs were changed into a more polygonal shape showing tight interactions between the cells. The differentiated HTASCs up-regulated mRNA of hepatocyte markers. Immunocytochemical analysis showed that they were intensely stained with anti-albumin antibody compared with undifferentiated HTASCs. PAS staining showed that HTASCs submitted to the hepatocyte differentiation protocol were able to more specifically store glycogen than undifferentiated HTASCs, displaying a purple color in the cytoplasm of the differentiated HTASCs. ELISA analyses showed that differentiated HTASCs could secrete albumin, which is one of the hepatocyte markers.ConclusionsMSCs were islolated from human thigh adipose tissue differentiate to heapatocytes. The source of ADSCs is not only abundant abdominal adipose tissue, but also thigh adipose tissue for cell therapy in liver regeneration and tissue regeneration.

3D hepatic cultures simultaneously maintain primary hepatocyte and liver sinusoidal endothelial cell phenotypes.

Directory of Open Access Journals (Sweden)

Yeonhee Kim

Full Text Available Developing in vitro engineered hepatic tissues that exhibit stable phenotype is a major challenge in the field of hepatic tissue engineering. However, the rapid dedifferentiation of hepatic parenchymal (hepatocytes and non-parenchymal (liver sinusoidal endothelial, LSEC cell types when removed from their natural environment in vivo remains a major obstacle. The primary goal of this study was to demonstrate that hepatic cells cultured in layered architectures could preserve or potentially enhance liver-specific behavior of both cell types. Primary rat hepatocytes and rat LSECs (rLSECs were cultured in a layered three-dimensional (3D configuration. The cell layers were separated by a chitosan-hyaluronic acid polyelectrolyte multilayer (PEM, which served to mimic the Space of Disse. Hepatocytes and rLSECs exhibited several key phenotypic characteristics over a twelve day culture period. Immunostaining for the sinusoidal endothelial 1 antibody (SE-1 demonstrated that rLSECs cultured in the 3D hepatic model maintained this unique feature over twelve days. In contrast, rLSECs cultured in monolayers lost their phenotype within three days. The unique stratified structure of the 3D culture resulted in enhanced heterotypic cell-cell interactions, which led to improvements in hepatocyte functions. Albumin production increased three to six fold in the rLSEC-PEM-Hepatocyte cultures. Only rLSEC-PEM-Hepatocyte cultures exhibited increasing CYP1A1/2 and CYP3A activity. Well-defined bile canaliculi were observed only in the rLSEC-PEM-Hepatocyte cultures. Together, these data suggest that rLSEC-PEM-Hepatocyte cultures are highly suitable models to monitor the transformation of toxins in the liver and their transport out of this organ. In summary, these results indicate that the layered rLSEC-PEM-hepatocyte model, which recapitulates key features of hepatic sinusoids, is a potentially powerful medium for obtaining comprehensive knowledge on liver metabolism

Pretreatment with mixed-function oxidase inducers increases the sensitivity of the hepatocyte/DNA repair assay

International Nuclear Information System (INIS)

Shaddock, J.G.; Heflich, R.H.; McMillan, D.C.; Hinson, J.A.; Casciano, D.A.

1989-01-01

A recent National Toxicology Program evaluation indicates that the rat hepatocyte/DNA repair assay has a high false-negative rate and that it is insensitive to some genotoxic hepatocarcinogens as well as other species and organ-specific carcinogens. In this study, the authors examined whether the sensitivity of the hepatocyte/DNA repair assay might be increased through animal pretreatment with various hepatic mixed-function oxidase inducers, i.e., Aroclor 1254, phenobarbital, and 3,3',4,4'-tetrachloroazobenzene (TCAB). The effects on unscheduled DNA synthesis (UDS), a measured of DNA damage and repair, were studied in cultures exposed to known and/or potential carcinogens that had been evaluated as negative or questionable or that produced conflicting results with hepatocytes isolated from uninduced animals. 4,4'-Oxydianiline, 1-nitropy-rene, and TCAB produced concentration-dependent increases in UDS in hepatocytes from rats pretreated with Aroclor 1254. 4,4'-Oxydianiline and TCAB also induced a dose-dependent increase in DNA repair in hepatocytes from rats pretreated with phenobarbital, whereas 1-nitropyrene was negative. These data indicate that the limited sensitivity to chemical carcinogens displayed by the hepatocyte/DNA repair assay may be increased by using hepatocytes isolated from animals exposed to hepatic mixed-function oxidase inducers

Pretreatment with mixed-function oxidase inducers increases the sensitivity of the hepatocyte/DNA repair assay

Energy Technology Data Exchange (ETDEWEB)

Shaddock, J.G.; Heflich, R.H.; McMillan, D.C.; Hinson, J.A.; Casciano, D.A. (National Center for Toxicological Research, Jefferson, AK (USA) Univ. of Arkansas for Medical Sciences, Little Rock (USA))

1989-01-01

A recent National Toxicology Program evaluation indicates that the rat hepatocyte/DNA repair assay has a high false-negative rate and that it is insensitive to some genotoxic hepatocarcinogens as well as other species and organ-specific carcinogens. In this study, the authors examined whether the sensitivity of the hepatocyte/DNA repair assay might be increased through animal pretreatment with various hepatic mixed-function oxidase inducers, i.e., Aroclor 1254, phenobarbital, and 3,3{prime},4,4{prime}-tetrachloroazobenzene (TCAB). The effects on unscheduled DNA synthesis (UDS), a measured of DNA damage and repair, were studied in cultures exposed to known and/or potential carcinogens that had been evaluated as negative or questionable or that produced conflicting results with hepatocytes isolated from uninduced animals. 4,4{prime}-Oxydianiline, 1-nitropy-rene, and TCAB produced concentration-dependent increases in UDS in hepatocytes from rats pretreated with Aroclor 1254. 4,4{prime}-Oxydianiline and TCAB also induced a dose-dependent increase in DNA repair in hepatocytes from rats pretreated with phenobarbital, whereas 1-nitropyrene was negative. These data indicate that the limited sensitivity to chemical carcinogens displayed by the hepatocyte/DNA repair assay may be increased by using hepatocytes isolated from animals exposed to hepatic mixed-function oxidase inducers.

Metabolism of methyl-branched iodo palmitic acids in cultured hepatocytes

International Nuclear Information System (INIS)

Thomas, G.; Pepin, D.; Loriette, C.; Chambaz, J.; Bereziat, G.; Vidal, M.; Apparu, M.; Coornaert, S.

1989-01-01

The metabolic fate of methyl-branched iodo fatty acids was studied in primary culture of rat hepatocytes. We compared 16-iodo-2-R,S-methyl palmitic acid (2-Me), which can be β oxidized, with 16-iodo-3-R,S-methyl palmitic acid (3-Me) which can be β oxidized only after an initial α oxydation and with 16-iodo-2,2-dimethyl palmitic acid (2,2-Me 2 ) and 16-iodo-3,3-dimethyl palmitic acid (3,3-Me 2 ) which cannot be β oxidized at all. The normal fate of natural fatty acids was given by comparative experiments with [1- 14 C] palmitic acid. Monomethyl-branched iodo fatty acids were taken up in the same range as palmitic acid but more than dimethyl-branched iodo fatty acids. After a 15-h incubation, acido-soluble products (ASP) accounted for 75% of the radioactivity taken up as 16-iodo-2-methyl palmitic acid, 50% as other methyl-branched iodo fatty acids and only 30% as palmitic acid. Cultured hepatocytes, labelled for 3 h with the various fatty acids and reincubated for 12 h without fatty acid, secreted large amounts of free dimethyl-branched iodo fatty acids as compared to the monomethyl ones and palmitic acid. Only hepatocytes prelabelled with 16-[ 125 I]iodo-2,2-dimethyl palmitic acid exhibited an appreciable secretion of labeled triglycerides, but at a lower rate than with [1- 14 C] palmitic acid. Conversely, the 16-iodo-monomethyl palmitic acids remained chiefly in hepatocyte triglycerides. Minute amounts of 16-iodo-methyl-branched palmitic acids were found in hepatocyte or secreted phospholipids as compared with palmitic acid. (orig.)

Insulin-induced CARM1 upregulation facilitates hepatocyte proliferation

Energy Technology Data Exchange (ETDEWEB)

Yeom, Chul-gon; Kim, Dong-il; Park, Min-jung; Choi, Joo-hee [College of Veterinary Medicine, Chonnam National University, Gwangju 500-757 (Korea, Republic of); Jeong, Jieun; Wi, Anjin; Park, Whoashig [Jeollanamdo Forest Resources Research Institute, Naju 520-833 (Korea, Republic of); Han, Ho-jae [College of Veterinary Medicine, Seoul National University, Seoul 151-741 (Korea, Republic of); Park, Soo-hyun, E-mail: parksh@chonnam.ac.kr [College of Veterinary Medicine, Chonnam National University, Gwangju 500-757 (Korea, Republic of)

2015-06-05

Previously, we reported that CARM1 undergoes ubiquitination-dependent degradation in renal podocytes. It was also reported that CARM1 is necessary for fasting-induced hepatic gluconeogenesis. Based on these reports, we hypothesized that treatment with insulin, a hormone typically present under the ‘fed’ condition, would inhibit gluconeogenesis via CARM1 degradation. HepG2 cells, AML-12 cells, and rat primary hepatocytes were treated with insulin to confirm CARM1 downregulation. Surprisingly, insulin treatment increased CARM1 expression in all cell types examined. Furthermore, treatment with insulin increased histone 3 methylation at arginine 17 and 26 in HepG2 cells. To elucidate the role of insulin-induced CARM1 upregulation, the HA-CARM1 plasmid was transfected into HepG2 cells. CARM1 overexpression did not increase the expression of lipogenic proteins generally increased by insulin signaling. Moreover, CARM1 knockdown did not influence insulin sensitivity. Insulin is known to facilitate hepatic proliferation. Like insulin, CARM1 overexpression increased CDK2 and CDK4 expression. In addition, CARM1 knockdown reduced the number of insulin-induced G2/M phase cells. Moreover, GFP-CARM1 overexpression increased the number of G2/M phase cells. Based on these results, we concluded that insulin-induced CARM1 upregulation facilitates hepatocyte proliferation. These observations indicate that CARM1 plays an important role in liver pathophysiology. - Highlights: • Insulin treatment increases CARM1 expression in hepatocytes. • CARM1 overexpression does not increase the expression of lipogenic proteins. • CARM1 knockdown does not influence insulin sensitivity. • Insulin-induced CARM1 upregulation facilitates hepatocyte proliferation.

Insulin-induced CARM1 upregulation facilitates hepatocyte proliferation

International Nuclear Information System (INIS)

Yeom, Chul-gon; Kim, Dong-il; Park, Min-jung; Choi, Joo-hee; Jeong, Jieun; Wi, Anjin; Park, Whoashig; Han, Ho-jae; Park, Soo-hyun

2015-01-01

Previously, we reported that CARM1 undergoes ubiquitination-dependent degradation in renal podocytes. It was also reported that CARM1 is necessary for fasting-induced hepatic gluconeogenesis. Based on these reports, we hypothesized that treatment with insulin, a hormone typically present under the ‘fed’ condition, would inhibit gluconeogenesis via CARM1 degradation. HepG2 cells, AML-12 cells, and rat primary hepatocytes were treated with insulin to confirm CARM1 downregulation. Surprisingly, insulin treatment increased CARM1 expression in all cell types examined. Furthermore, treatment with insulin increased histone 3 methylation at arginine 17 and 26 in HepG2 cells. To elucidate the role of insulin-induced CARM1 upregulation, the HA-CARM1 plasmid was transfected into HepG2 cells. CARM1 overexpression did not increase the expression of lipogenic proteins generally increased by insulin signaling. Moreover, CARM1 knockdown did not influence insulin sensitivity. Insulin is known to facilitate hepatic proliferation. Like insulin, CARM1 overexpression increased CDK2 and CDK4 expression. In addition, CARM1 knockdown reduced the number of insulin-induced G2/M phase cells. Moreover, GFP-CARM1 overexpression increased the number of G2/M phase cells. Based on these results, we concluded that insulin-induced CARM1 upregulation facilitates hepatocyte proliferation. These observations indicate that CARM1 plays an important role in liver pathophysiology. - Highlights: • Insulin treatment increases CARM1 expression in hepatocytes. • CARM1 overexpression does not increase the expression of lipogenic proteins. • CARM1 knockdown does not influence insulin sensitivity. • Insulin-induced CARM1 upregulation facilitates hepatocyte proliferation

Ammonia-induced energy disorders interfere with bilirubin metabolism in hepatocytes.

Science.gov (United States)

Wang, Qiongye; Wang, Yanfang; Yu, Zujiang; Li, Duolu; Jia, Bin; Li, Jingjing; Guan, Kelei; Zhou, Yubing; Chen, Yanling; Kan, Quancheng

2014-08-01

Hyperammonemia and jaundice are the most common clinical symptoms of hepatic failure. Decreasing the level of ammonia in the blood is often accompanied by a reduction in bilirubin in patients with hepatic failure. Previous studies have shown that hyperammonemia can cause bilirubin metabolism disorders, however it is unclear exactly how hyperammonemia interferes with bilirubin metabolism in hepatocytes. The purpose of the current study was to determine the mechanism or mechanisms by which hyperammonemia interferes with bilirubin metabolism in hepatocytes. Cell viability and apoptosis were analyzed in primary hepatocytes that had been exposed to ammonium chloride. Mitochondrial morphology and permeability were observed and analyzed, intermediates of the tricarboxylic acid (TCA) cycle were determined and changes in the expression of enzymes related to bilirubin metabolism were analyzed after ammonia exposure. Hyperammonemia inhibited cell growth, induced apoptosis, damaged the mitochondria and hindered the TCA cycle in hepatocytes. This led to a reduction in energy synthesis, eventually affecting the expression of enzymes related to bilirubin metabolism, which then caused further problems with bilirubin metabolism. These effects were significant, but could be reversed with the addition of adenosine triphosphate (ATP). This study demonstrates that ammonia can cause problems with bilirubin metabolism by interfering with energy synthesis. Copyright © 2014 Elsevier Inc. All rights reserved.

Optimization of the isolation and cultivation of Cyprinus carpio primary hepatocytes.

Science.gov (United States)

Yanhong, Fan; Chenghua, He; Guofang, Liu; Haibin, Zhang

2008-10-01

The aquatic environment is affected by numerous chemical contaminants. There is an increasing need to identify these chemicals and to evaluate their potential toxicity towards aquatic life. In this research we optimized techniques for primary cell culture of Cyprinus carpio hepatocytes as one adjunct model for ecotoxicological evaluation of the potential hazards of xenobiotics in the aquatic environment. In this study, Cyprinus carpio hepatocytes were isolated by mechanical separation, two-step collagenase perfusion, and pancreatin digestion. The hepatocytes or parenchymal cells could be separated from cell debris and from non-parenchymal cells by low-speed centrifugation (Percoll gradient centrifugation). The harvested hepatocytes were suspended in DMEM, M199 (cultured in 5% CO(2)), or L-15 (cultured without 5% CO(2)) medium then cultured at 17, 27, or 37 degrees C. Cell yield was counted by use of a hemocytometer, and the viability of the cells was assessed by use of the Trypan blue exclusion test. Results from these studies showed that the best method of isolation was pancreatin digestion (the cell yield was 2.7 x 10(8) per g (liver weight) and the viability was 98.4%) and the best medium was M199 (cultured in 5% CO(2)) or L-15 (cultured without 5% CO(2)). The optimum culture temperature was 27 degrees C. The primary hepatocytes culture of Cyprimus carpio grew well and satisfied requirements for most toxicological experiments in this condition.

Inducibility of carbamoylphosphate synthetase (ammonia) in cultures of embryonic hepatocytes: ontogenesis of the responsiveness to hormones

NARCIS (Netherlands)

Lamers, W. H.; Zonneveld, D.; Charles, R.

1984-01-01

Glucocorticosteroids and cyclic AMP induce carbamoylphosphate synthetase (ammonia) (CPS) in rat hepatocytes. Using an enzyme immunoassay applied to hepatocyte cultures fixed in situ, it has been demonstrated that the capacity of hepatocytes to synthesize CPS in the presence of both hormones is

Alternative Cell Sources to Adult Hepatocytes for Hepatic Cell Therapy.

Science.gov (United States)

Pareja, Eugenia; Gómez-Lechón, María José; Tolosa, Laia

2017-01-01

Adult hepatocyte transplantation is limited by scarce availability of suitable donor liver tissue for hepatocyte isolation. New cell-based therapies are being developed to supplement whole-organ liver transplantation, to reduce the waiting-list mortality rate, and to obtain more sustained and significant metabolic correction. Fetal livers and unsuitable neonatal livers for organ transplantation have been proposed as potential useful sources of hepatic cells for cell therapy. However, the major challenge is to use alternative cell sources for transplantation that can be derived from reproducible methods. Different types of stem cells with hepatic differentiation potential are eligible for generating large numbers of functional hepatocytes for liver cell therapy to treat degenerative disorders, inborn hepatic metabolic diseases, and organ failure. Clinical trials are designed to fully establish the safety profile of such therapies and to define target patient groups and standardized protocols.

High Throughput Micro-Well Generation of Hepatocyte Micro-Aggregates for Tissue Engineering

NARCIS (Netherlands)

Gevaert, Elien; Dollé, Laurent; Billiet, Thomas; Dubruel, Peter; van Grunsven, Leo; van Apeldoorn, Aart A.; Cornelissen, Ria

2014-01-01

The main challenge in hepatic tissue engineering is the fast dedifferentiation of primary hepatocytes in vitro. One successful approach to maintain hepatocyte phenotype on the longer term is the cultivation of cells as aggregates. This paper demonstrates the use of an agarose micro-well chip for the

Subtoxic Alterations in Hepatocyte-Derived Exosomes: An Early Step in Drug-Induced Liver Injury?

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Holman, Natalie S; Mosedale, Merrie; Wolf, Kristina K; LeCluyse, Edward L; Watkins, Paul B

2016-06-01

Drug-induced liver injury (DILI) is a significant clinical and economic problem in the United States, yet the mechanisms that underlie DILI remain poorly understood. Recent evidence suggests that signaling molecules released by stressed hepatocytes can trigger immune responses that may be common across DILI mechanisms. Extracellular vesicles released by hepatocytes, principally hepatocyte-derived exosomes (HDEs), may constitute one such signal. To examine HDE alterations as a function of drug-induced stress, this work utilized prototypical hepatotoxicant acetaminophen (APAP) in male Sprague-Dawley (SD) rats, SD rat hepatocytes, and primary human hepatocytes. HDE were isolated using ExoQuick precipitation reagent and analyzed by quantification of the liver-specific RNAs albumin and microRNA-122 (miR-122). In vivo, significant elevations in circulating exosomal albumin mRNA were observed at subtoxic APAP exposures. Significant increases in exosomal albumin mRNA were also observed in primary rat hepatocytes at subtoxic APAP concentrations. In primary human hepatocytes, APAP elicited increases in both exosomal albumin mRNA and exosomal miR-122 without overt cytotoxicity. However, the number of HDE produced in vitro in response to APAP did not increase with exosomal RNA quantity. We conclude that significant drug-induced alterations in the liver-specific RNA content of HDE occur at subtoxic APAP exposures in vivo and in vitro, and that these changes appear to reflect selective packaging rather than changes in exosome number. The current findings demonstrate that translationally relevant HDE alterations occur in the absence of overt hepatocellular toxicity, and support the hypothesis that HDE released by stressed hepatocytes may mediate early immune responses in DILI. © The Author 2016. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Hepatocyte polarization is essential for the productive entry of the hepatitis B virus.

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Schulze, Andreas; Mills, Kerry; Weiss, Thomas S; Urban, Stephan

2012-02-01

Human hepatitis B virus (HBV) is characterized by a high species specificity and a distinct liver tropism. Within the liver, HBV replication occurs in differentiated and polarized hepatocytes. Accordingly, the in vitro HBV infection of primary human hepatocytes (PHHs) and the human hepatoma cell line, HepaRG, is restricted to differentiated, hepatocyte-like cells. Though preparations of PHH contain up to 100% hepatic cells, cultures of differentiated HepaRG cells are a mixture of hepatocyte-like and biliary-like epithelial cells. We used PHH and HepaRG cells and compared the influence of virus inoculation dose, cell differentiation, and polarization on productive HBV infection. At multiplicities of genome equivalents (mge) >8,000, almost 100% of PHHs could be infected. In contrast, only a subset of HepaRG cells stained positive for HBcAg at comparable or even higher mge. Infection predominantly occurred at the edges of islands of hepatocyte-like HepaRG cells. This indicates a limited accessibility of the HBV receptor, possibly as a result of its polar sorting. Multidrug resistance protein 2 (MRP2), a marker selectively transported to the apical (i.e., canalicular) cell membrane, revealed two polarization phenotypes of HepaRG cells. HBV infection within the islands of hepatocyte-like HepaRG cells preferentially occurred in cells that resemble PHH, exhibiting canalicular structures. However, disruption of cell-cell junctions allowed the additional infection of cells that do not display a PHH-like polarization. HBV enters hepatocytes via the basolateral membrane. This model, at least partially, explains the difference of PHH and HepaRG cells in infection efficacy, provides insights into natural HBV infection, and establishes a basis for optimization of the HepaRG infection system. Copyright © 2011 American Association for the Study of Liver Diseases.

[The polyploidization characteristics of the hepatocytes of the mouse-like hamster Calomyscus mystax].

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Anatskaia, O V; Malikov, V G; Meĭer, M N; Kudriavtsev, B N

1995-01-01

A cytophotometric measurement of DNA content in hepatocytes of maturing mouse-like hamsters was made. Cells belonging to ordinary mammalian ploidy classes 2c, 2c x 2, 4c, and 4c x 2 made about 90% of the hepatocyte population. The share of binucleated cells wa high (about 80%), the majority of these cells being 2c X 2 hepatocytes. Binucleated cells with tetraploid and diploid nuclei occur in almost every animal. An average hepatocyte ploidy level in mouse-like hamster is 4.6c. The main peculiarity of parenchymal liver cell populations is that up 5% of hepatocytes contain 3--11 nuclei of different ploidy classes. Multinucleated cells increase in number from 1.5% to 4% within the period from one year (the age of maturation) to two years. Later on their percentage does not change. It is found that in binucleated and multinucleated hepatocytes DNA synthesis can proceed asynchronously. Asynchrony in DNA synthesis elevates as the number of nuclei increases. Among the 2c x 2 and 2c x 3 cells an uneven distribution of 3H-thymidine label can occur, respectively, in 5 and in 50% cases, whereas all the cells with more than 3 nuclei display an uneven an uneven 3H-thymidin label distribution. The formation of multinucleated cells is supposed to be associated with asynchrony in DNA-synthesis in binucleated cells and with the restitution of mitosis.

Dendritic cells and hepatocytes use distinct pathways to process protective antigen from plasmodium in vivo.

Directory of Open Access Journals (Sweden)

Ian A Cockburn

2011-03-01

Full Text Available Malaria-protective CD8+ T cells specific for the circumsporozoite (CS protein are primed by dendritic cells (DCs after sporozoite injection by infected mosquitoes. The primed cells then eliminate parasite liver stages after recognizing the CS epitopes presented by hepatocytes. To define the in vivo processing of CS by DCs and hepatocytes, we generated parasites carrying a mutant CS protein containing the H-2K(b epitope SIINFEKL, and evaluated the T cell response using transgenic and mutant mice. We determined that in both DCs and hepatocytes CS epitopes must reach the cytosol and use the TAP transporters to access the ER. Furthermore, we used endosomal mutant (3d and cytochrome c treated mice to address the role of cross-presentation in the priming and effector phases of the T cell response. We determined that in DCs, CS is cross-presented via endosomes while, conversely, in hepatocytes protein must be secreted directly into the cytosol. This suggests that the main targets of protective CD8+ T cells are parasite proteins exported to the hepatocyte cytosol. Surprisingly, however, secretion of the CS protein into hepatocytes was not dependent upon parasite-export (Pexel/VTS motifs in this protein. Together, these results indicate that the presentation of epitopes to CD8+ T cells follows distinct pathways in DCs when the immune response is induced and in hepatocytes during the effector phase.

Cell therapy from bench to bedside: Hepatocytes from fibroblasts - the truth and myth of transdifferentiation.

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Sanal, Madhusudana Girija

2015-06-07

Hepatocyte transplantation is an alternative to liver transplantation in certain disorders such as inherited liver diseases and liver failure. It is a relatively less complicated surgical procedure, and has the advantage that it can be repeated several times if unsuccessful. Another advantage is that hepatocytes can be isolated from partly damaged livers which are not suitable for liver transplantation. Despite these advantages hepatocyte transplantation is less popular. Important issues are poor engraftment of the transplanted cells and the scarcity of donor hepatocytes. Generation of "hepatocyte like cells"/iHeps from embryonic stem cells (ES) and induced pluripotent stem cells (iPSCs) by directed differentiation is an emerging solution to the latter issue. Direct conversation or trans-differentiation of fibroblasts to "hepatocyte like cells" is another way which is, being explored. However this method has several inherent and technical disadvantages compared to the directed differentiation from ES or iPSC. There are several methods claiming to be "highly efficient" for generating "highly functional" "hepatocyte like cells". Currently different groups are working independently and coming up with differentiation protocols and each group claiming an advantage for their protocol. Directed differentiation protocols need to be designed, compared, analyzed and tweaked systematically and logically than empirically. There is a need for a well-coordinated global initiative comparable to the Human Genome Project to achieve this goal in the near future.

Chronic alcohol feeding potentiates hormone-induced calcium signalling in hepatocytes.

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Bartlett, Paula J; Antony, Anil Noronha; Agarwal, Amit; Hilly, Mauricette; Prince, Victoria L; Combettes, Laurent; Hoek, Jan B; Gaspers, Lawrence D

2017-05-15

Chronic alcohol consumption causes a spectrum of liver diseases, but the pathogenic mechanisms driving the onset and progression of disease are not clearly defined. We show that chronic alcohol feeding sensitizes rat hepatocytes to Ca 2+ -mobilizing hormones resulting in a leftward shift in the concentration-response relationship and the transition from oscillatory to more sustained and prolonged Ca 2+ increases. Our data demonstrate that alcohol-dependent adaptation in the Ca 2+ signalling pathway occurs at the level of hormone-induced inositol 1,4,5 trisphosphate (IP 3 ) production and does not involve changes in the sensitivity of the IP 3 receptor or size of internal Ca 2+ stores. We suggest that prolonged and aberrant hormone-evoked Ca 2+ increases may stimulate the production of mitochondrial reactive oxygen species and contribute to alcohol-induced hepatocyte injury. ABSTRACT: 'Adaptive' responses of the liver to chronic alcohol consumption may underlie the development of cell and tissue injury. Alcohol administration can perturb multiple signalling pathways including phosphoinositide-dependent cytosolic calcium ([Ca 2+ ] i ) increases, which can adversely affect mitochondrial Ca 2+ levels, reactive oxygen species production and energy metabolism. Our data indicate that chronic alcohol feeding induces a leftward shift in the dose-response for Ca 2+ -mobilizing hormones resulting in more sustained and prolonged [Ca 2+ ] i increases in both cultured hepatocytes and hepatocytes within the intact perfused liver. Ca 2+ increases were initiated at lower hormone concentrations, and intercellular calcium wave propagation rates were faster in alcoholics compared to controls. Acute alcohol treatment (25 mm) completely inhibited hormone-induced calcium increases in control livers, but not after chronic alcohol-feeding, suggesting desensitization to the inhibitory actions of ethanol. Hormone-induced inositol 1,4,5 trisphosphate (IP 3 ) accumulation and phospholipase C

Comparison of trout hepatocytes and liver S9 fractions as in ...

Science.gov (United States)

Isolated hepatocytes and liver S9 fractions have been used to collect in vitro biotransformation data for fish as a means of improving modeled estimates of chemical bioaccumulation. To date, however, there have been few direct comparisons of these two methods. In the present study, cryopreserved trout hepatocytes were used to measure in vitro intrinsic clearance rates for 6 polycyclic aromatic hydrocarbons (PAHs). These rates were extrapolated to estimates of in vivo intrinsic clearance and used as inputs to a well-stirred liver model to predict hepatic clearance. Predicted rates of hepatic clearance were then evaluated by comparison to measured rates determined previously using isolated perfused livers. Hepatic clearance rates predicted using hepatocytes were in good agreement with measured values (fractions. For one compound (benzo[a]pyrene), the in vivo intrinsic clearance rate calculated using S9 data was 10-fold higher than that determined using hepatocytes, possibly due to a diffusion limitation on cellular uptake. Generally, however, there was good agreement between calculated in vivo intrinsic clearance rates obtained using either in vitro test system. These results suggest that both systems can be used to improve

Whole-body X-irradiation of mice accelerates polyploidization of hepatocytes

International Nuclear Information System (INIS)

Shima, A.; Egami, N.

1985-01-01

Male C57BL/6 mice were whole-body irradiated with 4.75 gy of X-rays at the age of 2 months and killed at 2, 6, 12 and 19 months after irradiation. The percentage survival began to decline earlier and faster in the irradiated group than the controls up to 19 months after exposure when the study was terminated. The nuclear DNA content of individual hepatocytes was measured by a Feulgen-DNA microfluorometric method, and hepatocytes were classified into various ploidy classes. In the irradiated mice, the degree of polyploidization was significantly higher than the controls by 2 months after exposure and steadily increased up to 6 months after exposure. Thereafter, however, a slow return to the control level was found up to 19 months after irradiation. These results appear to support a hypothesis that radiation accelerates the ageing process as judged from hepatocyte polyploidization. (author)

Augmenter of liver regeneration (ALR) protects human hepatocytes against apoptosis

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Ilowski, Maren [Liver Regeneration Group, Department of Surgery, Grosshadern Hospital, Ludwig Maximilians University, Munich (Germany); Kleespies, Axel [Department of Surgery, Grosshadern Hospital, Ludwig Maximilians University, Munich (Germany); Toni, Enrico N. de [Department of Medicine II, Grosshadern Hospital, Ludwig Maximilians University, Munich (Germany); Donabauer, Barbara [Liver Regeneration Group, Department of Surgery, Grosshadern Hospital, Ludwig Maximilians University, Munich (Germany); Jauch, Karl-Walter [Department of Surgery, Grosshadern Hospital, Ludwig Maximilians University, Munich (Germany); Hengstler, Jan G. [Leibniz Research Centre for Working Environment and Human Factors, Technical University, Dortmund (Germany); Thasler, Wolfgang E., E-mail: wolfgang.thasler@med.uni-muenchen.de [Liver Regeneration Group, Department of Surgery, Grosshadern Hospital, Ludwig Maximilians University, Munich (Germany); Department of Surgery, Grosshadern Hospital, Ludwig Maximilians University, Munich (Germany)

2011-01-07

Research highlights: {yields} ALR decreases cytochrome c release from mitochondria. {yields} ALR protects hepatocytes against apoptosis induction by ethanol, TRAIL, anti-Apo, TGF-{beta} and actinomycin D. {yields} ALR exerts a liver-specific anti-apoptotic effect. {yields} A possible medical usage of ALR regarding protection of liver cells during apoptosis inducing therapies. -- Abstract: Augmenter of liver regeneration (ALR) is known to support liver regeneration and to stimulate proliferation of hepatocytes. However, it is not known if ALR exerts anti-apoptotic effects in human hepatocytes and whether this protective effect is cell type specific. This is relevant, because compounds that protect the liver against apoptosis without undesired effects, such as protection of metastatic tumour cells, would be appreciated in several clinical settings. Primary human hepatocytes (phH) and organotypic cancer cell lines were exposed to different concentrations of apoptosis inducers (ethanol, TRAIL, anti-Apo, TGF-{beta}, actinomycin D) and cultured with or without recombinant human ALR (rhALR). Apoptosis was evaluated by the release of cytochrome c from mitochondria and by FACS with propidium iodide (PI) staining. ALR significantly decreased apoptosis induced by ethanol, TRAIL, anti-Apo, TGF-{beta} and actinomycin D. Further, the anti-apoptotic effect of ALR was observed in primary human hepatocytes and in HepG2 cells but not in bronchial (BC1), colonic (SW480), gastric (GC1) and pancreatic (L3.6PL) cell lines. Therefore, the hepatotrophic growth factor ALR acts in a liver specific manner with regards to both its mitogenic and its anti-apoptotic effect. Unlike the growth factors HGF and EGF, rhALR acts in a liver specific manner. Therefore, ALR is a promising candidate for further evaluation as a possible hepatoprotective factor in clinical settings.

Augmenter of liver regeneration (ALR) protects human hepatocytes against apoptosis

International Nuclear Information System (INIS)

Ilowski, Maren; Kleespies, Axel; Toni, Enrico N. de; Donabauer, Barbara; Jauch, Karl-Walter; Hengstler, Jan G.; Thasler, Wolfgang E.

2011-01-01

Research highlights: → ALR decreases cytochrome c release from mitochondria. → ALR protects hepatocytes against apoptosis induction by ethanol, TRAIL, anti-Apo, TGF-β and actinomycin D. → ALR exerts a liver-specific anti-apoptotic effect. → A possible medical usage of ALR regarding protection of liver cells during apoptosis inducing therapies. -- Abstract: Augmenter of liver regeneration (ALR) is known to support liver regeneration and to stimulate proliferation of hepatocytes. However, it is not known if ALR exerts anti-apoptotic effects in human hepatocytes and whether this protective effect is cell type specific. This is relevant, because compounds that protect the liver against apoptosis without undesired effects, such as protection of metastatic tumour cells, would be appreciated in several clinical settings. Primary human hepatocytes (phH) and organotypic cancer cell lines were exposed to different concentrations of apoptosis inducers (ethanol, TRAIL, anti-Apo, TGF-β, actinomycin D) and cultured with or without recombinant human ALR (rhALR). Apoptosis was evaluated by the release of cytochrome c from mitochondria and by FACS with propidium iodide (PI) staining. ALR significantly decreased apoptosis induced by ethanol, TRAIL, anti-Apo, TGF-β and actinomycin D. Further, the anti-apoptotic effect of ALR was observed in primary human hepatocytes and in HepG2 cells but not in bronchial (BC1), colonic (SW480), gastric (GC1) and pancreatic (L3.6PL) cell lines. Therefore, the hepatotrophic growth factor ALR acts in a liver specific manner with regards to both its mitogenic and its anti-apoptotic effect. Unlike the growth factors HGF and EGF, rhALR acts in a liver specific manner. Therefore, ALR is a promising candidate for further evaluation as a possible hepatoprotective factor in clinical settings.

Hydrodynamic Delivery of Cre Protein to Lineage-Mark or Time-Stamp Mouse Hepatocytes In situ

Science.gov (United States)

Sonsteng, Katherine M.; Prigge, Justin R.; Talago, Emily A.; June, Ronald K.; Schmidt, Edward E.

2014-01-01

Cre-responsive fluorescent marker alleles are powerful tools for cell lineage tracing in mice; however their utility is limited by regulation of Cre activity. When targeting hepatocytes, hydrodynamic delivery of a Cre-expression plasmid can convert Cre-responsive alleles without inducing the intracellular or systemic antiviral responses often associated with viral-derived Cre-expression vectors. In this method, rapid high-volume intravenous inoculation induces hepatocyte-targeted uptake of extracellular molecules. Here we tested whether hydrodynamic delivery of Cre protein or Cre fused to the HIV-TAT cell-penetrating peptide could convert Cre-responsive reporters in hepatocytes of mice. Hydrodynamic delivery of 2 nmol of either Cre or TAT-Cre protein converted the reporter allele in 5 to 20% of hepatocytes. Neither protein gave detectable Cre activity in endothelia, non-liver organs, or non-hepatocyte cells in liver. Using mice homozygous for a Cre-responsive marker that directs red- (Cre-naïve) or green- (Cre-converted) fluorescent proteins to the nucleus, we assessed sub-saturation Cre-activity. One month after hydrodynamic inoculation with Cre protein, 58% of hepatocyte nuclei that were green were also red, indicating that less than half of the hepatocytes that had obtained enough Cre to convert one marker allele to green were able to convert all alleles. For comparison, one month after hydrodynamic delivery of a Cre-expression plasmid with a weak promoter, only 26% of the green nuclei were also red. Our results show that hydrodynamic delivery of Cre protein allows rapid allelic conversion in hepatocytes, but Cre-activity is sub-saturating so many cells will not convert multiple Cre-responsive alleles. PMID:24626158

Intracellular pH regulation in hepatocytes isolated from three teleost species.

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Furimsky, M; Moon, T W; Perry, S F

1999-09-01

The mechanisms of intracellular pH (pH(i)) regulation were studied in hepatocytes isolated from three species of teleost: rainbow trout (Oncorhynchus mykiss), black bullhead (Ameiurus melas) and American eel (Anguilla rostrata). Intracellular pH was monitored over time using the pH-sensitive fluorescent dye BCECF in response to acid loading under control conditions and in different experimental media containing either low Na(+) or Cl(-) concentrations, the Na(+)-H(+) exchanger blocker amiloride or the blocker of the V-type H(+)-ATPase, bafilomycin A(1). In trout and bullhead hepatocytes, recovery to an intracellular acid load occurred principally by way of a Na(+)-dependent amiloride-sensitive Na(+)-H(+) exchanger. In eel hepatocytes, the Na(+)-H(+) exchanger did not contribute to recovery to an acid load though evidence suggests that it is present on the cell membrane and participates in the maintenance of steady-state pH(i). The V-type H(+)-ATPase did not participate in recovery to an acid load in any species. A Cl(-)-HCO(3)(-) exchanger may play a role in recovery to an acid load in eel hepatocytes by switching off and retaining base that would normally be tonically extruded. Thus, it is clear that hepatocytes isolated from the three species are capable of regulating pH(i), principally by way of a Na(+)-H(+) exchanger and a Cl(-)-HCO(3)(-) exchanger, but do not exploit identical mechanisms for pH(i) recovery. J. Exp. Zool. 284:361-367, 1999. Copyright 1999 Wiley-Liss, Inc.

Metabolism of cysteine by cyteinesulfinate-independent pathway(s) in rat hepatocytes

International Nuclear Information System (INIS)

Stipanuk, M.H.; De La Rosa, J.; Drake, M.R.

1986-01-01

The metabolism of cysteine (CYS) and that of cysteinesulfinate (CSA) were studied in freshly isolated hepatocytes from fed rats. In incubations of rat hepatocytes with either 1 or 25 mM CSA, over 90% of the 14 CO 2 formed from [1- 14 C]CSA could be accounted for by production of hypotaurine plus taurine. In similar incubations with 1 or 25 mM CYS, only 4% of 14 CO 2 evolution from [1- 14 C]CYS could be accounted for by production of hypotaurine plus taurine. Addition of unlabeled CSA inhibited recovery of label from [1- 14 C]CYS as 14 CO 2 by 33%. Metabolism of CYS and of CSA were affected differently by addition of α-ketoglutarate, a cosubstrate for transamination, or of propargylglycine, an inhibitor of cystathionase activity. These data suggest that a substantial proportion of CYS is catabolized by CSA-independent pathways in the rat hepatocyte. Although addition of α-ketoglutarate to incubations of hepatocytes with CSA resulted in a marked increase in CSA catabolism via the transamination pathway, addition of keto acids to incubation systems had little or no effect on production of any metabolite from CYS. Thus, CYS transamination does not appear to be a major pathway of CYS metabolism in the hepatocyte. Inhibition of cystathionase with propargylglycine reduced both 14 CO 2 production from [1- 14 C]CYS and ammonia plus urea nitrogen production from CYS by about 50%; CSA catabolism was not affected. Thus, cleavage of cyst(e)ine by cystathionase may be an important physiological pathway for CYS catabolism in the liver

Induced Mitogenic Activity in AML-12 Mouse Hepatocytes Exposed to Low-dose Ultra-Wideband Electromagnetic Radiation

Directory of Open Access Journals (Sweden)

P. B. Tchounwou

2005-04-01

Full Text Available Ultra–wideband (UWB technology has increased with the use of various civilian and military applications. In the present study, we hypothesized that low-dose UWB electromagnetic radiation (UWBR could elicit a mitogenic effect in AML-12 mouse hepatocytes, in vitro. To test this hypothesis, we exposed AML-12 mouse hepatocytes, to UWBR in a specially constructed gigahertz transverse electromagnetic mode (GTEM cell. Cells were exposed to UWBR for 2 h at a temperature of 23°C, a pulse width of 10 ns, a repetition rate of 1 kHz, and field strength of 5-20 kV/m. UWB pulses were triggered by an external pulse generator for UWBR exposure but were not triggered for the sham exposure. We performed an MTT Assay to assess cell viability for UWBR-treated and sham-exposed hepatocytes. Data from viability studies indicated a time-related increase in hepatocytes at time intervals from 8-24 h post exposure. UWBR exerted a statistically significant (p < 0.05 dose-dependent response in cell viability in both serum-treated and serum free medium (SFM -treated hepatocytes. Western blot analysis of hepatocyte lysates demonstrated that cyclin A protein was induced in hepatocytes, suggesting that increased MTT activity after UWBR exposure was due to cell proliferation. This study indicates that UWBR has a mitogenic effect on AML-12 mouse hepatocytes and implicates a possible role for UWBR in hepatocarcinoma.

Reproduction impairment and endocrine disruption in female zebrafish after long-term exposure to MC-LR: A life cycle assessment.

Science.gov (United States)

Hou, Jie; Li, Li; Wu, Ning; Su, Yujing; Lin, Wang; Li, Guangyu; Gu, Zemao

2016-01-01

Microcystin-LR (MC-LR) has been found to cause reproductive and developmental impairments as well as to disrupt sex hormone homeostasis of fish during acute and sub-chronic toxic experiments. However, fish in natural environments are continuously exposed to MC-LR throughout their entire life cycle as opposed to short-term exposure. Here, we tested the hypothesis that the mechanism by which MC-LR harms female fish reproduction and development within natural water bodies is through interference of the reproductive endocrine system. In the present study, zebrafish hatchlings (5 d post-fertilization) were exposed to 0, 0.3, 3 and 30 μg/L MC-LR for 90 d until reaching sexual maturity. Female zebrafish were selected, and the changes in growth and developmental indicators, ovarian ultrastructure as well as the levels of gonadal steroid hormones and vitellogenin (VTG) were examined along with the transcription of related genes in the hypothalamic-pituitary-gonadal-liver axis (HPGL-axis). The results showed for the first time, a life cycle exposure to MC-LR caused growth inhibition, decreased ovary weight and ovarian ultra-pathological lesions. Decreased ovarian testosterone levels indicated that MC-LR disrupted sex steroid hormone balance. Significantly up-regulated transcription of brain FSHβ and LHβ along with ovarian ERα, FSHR and LHR suggested positive feedback regulation in the HPGL-axis was induced as a compensatory mechanism for MC-LR damage. It was also noted that ovarian VTG content and hepatic ERα and VTG1 expression were all down-regulated, which might be responsible for reduced vitellus storage noted in our histological observations. Our findings indicate that a life cycle exposure to MC-LR impairs the development and reproduction of female zebrafish by disrupting the transcription of related HPGL-axis genes, suggesting that MC-LR has potential adverse effects on fish reproduction and thus population dynamics in MCs-contaminated aquatic environment

Effects of sewage effluent and ethynyl oestradiol upon molecular markers of oestrogenic exposure, maturation and reproductive success in the sand goby (Pomatoschistus minutus, Pallas)

International Nuclear Information System (INIS)

Robinson, Craig D.; Brown, Elaine; Craft, John A.; Davies, Ian M.; Moffat, Colin F.; Pirie, David; Robertson, Fiona; Stagg, Ronald M.; Struthers, Susan

2003-01-01

Male fish in several UK estuaries are known to be exposed to oestrogenic contamination, and whilst a limited number of studies have shown that exposure to oestrogens can reduce the reproductive success of fish, the impact of environmentally relevant exposures is less clear. The aim of this study was, therefore, to investigate the effects of exposure to environmentally realistic concentrations of a sewage effluent and the synthetic oestrogen 17α-ethynyl oestradiol (EE 2 ) upon the reproductive success of a marine fish. Sand goby (Pomatoschistus minutus) were exposed for 7 months to EE 2 or a sewage effluent containing known xeno-oestrogens (alkylphenol polyethoxylates) and bred using within treatment crosses. Nominal exposure concentrations were 6 ng l -1 EE 2 , 0.3 or 0.03% v/v sewage effluent. At the end of the breeding trials, expression of hepatic zona radiata protein (Zrp) and vitellogenin (Vtg) mRNA were determined using two recently developed cDNA probes. Exposure to 6 ng l -1 EE 2 induced Zrp and Vtg mRNA expression in male and female sand goby, impaired male maturation and reproductive behaviour, reduced female fecundity and reduced egg fertility. As a consequence, fertile egg production of the EE 2 -exposed population was reduced by 90%. Exposure to sewage effluent (0.3% v/v) increased adult mortality and female Zrp and Vtg mRNA expression, but did not induce male vitellogenesis. Exposure to EE 2 and 0.3% v/v sewage effluent impaired development of the male urogenital papilla. Fish exposed to 0.03% v/v sewage effluent produced more fertile eggs than those exposed to 0.3% effluent, or those receiving no effluent. It is concluded that male vitellogenesis in an oestrogenically exposed population may be accompanied by reduced reproductive success, but that it may not be indicative of altered reproductive output in a population exposed to an industrial sewage effluent

Lipopolysaccharide impairs hepatocyte ureagenesis from ammonia: involvement of mitochondrial aquaporin-8.

Science.gov (United States)

Soria, Leandro R; Marrone, Julieta; Molinas, Sara M; Lehmann, Guillermo L; Calamita, Giuseppe; Marinelli, Raúl A

2014-05-02

We recently reported that hepatocyte mitochondrial aquaporin-8 (mtAQP8) channels facilitate the uptake of ammonia and its metabolism into urea. Here we studied the effect of bacterial lipopolysaccharides (LPS) on ammonia-derived ureagenesis. In LPS-treated rats, hepatic mtAQP8 protein expression and diffusional ammonia permeability (measured utilizing ammonia analogues) of liver inner mitochondrial membranes were downregulated. NMR studies using 15N-labeled ammonia indicated that basal and glucagon-induced ureagenesis from ammonia were significantly reduced in hepatocytes from LPS-treated rats. Our data suggest that hepatocyte mtAQP8-mediated ammonia removal via ureagenesis is impaired by LPS, a mechanism potentially relevant to the molecular pathogenesis of defective hepatic ammonia detoxification in sepsis. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Regulation of lipid synthesis in hepatocytes from lean and obese Zucker rats

International Nuclear Information System (INIS)

Triscari, J.; Greenwood, M.R.; Sullivan, A.C.

1981-01-01

Fatty acid synthesis and CO 2 production were evaluated in hepatocytes from lean and obese Zucker rats in the presence of 3 H 2 O, and several carbon precursors. The incorporation of 3 H 2 O into fatty acids was greater in obese compared to lean rats in both the isolated hepatocyte and in vivo. The rates of incorporation of 3 H 2 O into fatty acids and cholesterol in hepatocytes of both lean and obese rats were linear for 2 hr, in the absence or presence of 16.7 mM glucose. Rates of fatty acid synthesis were higher in the presence of 16.7 mM glucose compared to the absence of glucose in both lean and obese while rates of cholesterol synthesis were similar. The incorporation of 3H2O into fatty acids, but not into cholesterol, was correlated with increasing glucose concentration and was 2 to three-fold higher in hepatocytes of obese compared to lean rats in the presence of several carbon precursors. Differences in CO 2 production between lean and obese rats suggested increased pentose phosphate shunt activity, decreased pyruvate dehydrogenase activity, and lower tricarboxylic acid cycle activity in obese rats. Fatty acid synthesis and CO 2 production from 3 H 2 O and [U- 14 C]glucose in hepatocytes of lean and obese rats was similarly elevated by insulin and depressed by glucagon at several concentrations, suggesting that hepatocytes of obese animals respond to these hormones. These data indicate that rates of hepatic fatty acid synthesis although higher in obese rats respond to modulation in a fashion which is similar to the response in lean rats. The present studies suggest that the oxidation of several carbon precursors in the tricarboxylic acid cycle is diminished in obese compared to lean rats, but pentose phosphate shunt activity is greater in the obese Zucker rats

Bile acid-induced necrosis in primary human hepatocytes and in patients with obstructive cholestasis

Energy Technology Data Exchange (ETDEWEB)

Woolbright, Benjamin L.; Dorko, Kenneth [Department of Pharmacology, Toxicology & Therapeutics, University of Kansas Medical Center, Kansas City, KS (United States); Antoine, Daniel J.; Clarke, Joanna I. [MRC Centre for Drug Safety Science, Department of Molecular and Clinical Pharmacology, Institute of Translational Medicine, University of Liverpool, Liverpool (United Kingdom); Gholami, Parviz [Department of Internal Medicine, University of Kansas Medical Center, Kansas City, KS (United States); Li, Feng [Department of Pharmacology, Toxicology & Therapeutics, University of Kansas Medical Center, Kansas City, KS (United States); Kumer, Sean C.; Schmitt, Timothy M.; Forster, Jameson [Department of Surgery, University of Kansas Medical Center, Kansas City, KS (United States); Fan, Fang [Department of Pathology, University of Kansas Medical Center, Kansas City, KS (United States); Jenkins, Rosalind E.; Park, B. Kevin [MRC Centre for Drug Safety Science, Department of Molecular and Clinical Pharmacology, Institute of Translational Medicine, University of Liverpool, Liverpool (United Kingdom); Hagenbuch, Bruno [Department of Pharmacology, Toxicology & Therapeutics, University of Kansas Medical Center, Kansas City, KS (United States); Olyaee, Mojtaba [Department of Internal Medicine, University of Kansas Medical Center, Kansas City, KS (United States); Jaeschke, Hartmut, E-mail: hjaeschke@kumc.edu [Department of Pharmacology, Toxicology & Therapeutics, University of Kansas Medical Center, Kansas City, KS (United States)

2015-03-15

Accumulation of bile acids is a major mediator of cholestatic liver injury. Recent studies indicate bile acid composition between humans and rodents is dramatically different, as humans have a higher percent of glycine conjugated bile acids and increased chenodeoxycholate content, which increases the hydrophobicity index of bile acids. This increase may lead to direct toxicity that kills hepatocytes, and promotes inflammation. To address this issue, this study assessed how pathophysiological concentrations of bile acids measured in cholestatic patients affected primary human hepatocytes. Individual bile acid levels were determined in serum and bile by UPLC/QTOFMS in patients with extrahepatic cholestasis with, or without, concurrent increases in serum transaminases. Bile acid levels increased in serum of patients with liver injury, while biliary levels decreased, implicating infarction of the biliary tracts. To assess bile acid-induced toxicity in man, primary human hepatocytes were treated with relevant concentrations, derived from patient data, of the model bile acid glycochenodeoxycholic acid (GCDC). Treatment with GCDC resulted in necrosis with no increase in apoptotic parameters. This was recapitulated by treatment with biliary bile acid concentrations, but not serum concentrations. Marked elevations in serum full-length cytokeratin-18, high mobility group box 1 protein (HMGB1), and acetylated HMGB1 confirmed inflammatory necrosis in injured patients; only modest elevations in caspase-cleaved cytokeratin-18 were observed. These data suggest human hepatocytes are more resistant to human-relevant bile acids than rodent hepatocytes, and die through necrosis when exposed to bile acids. These mechanisms of cholestasis in humans are fundamentally different to mechanisms observed in rodent models. - Highlights: • Cholestatic liver injury is due to cytoplasmic bile acid accumulation in hepatocytes. • Primary human hepatocytes are resistant to BA-induced injury

The role of hepatocyte nuclear factor 4 alpha in development and progression of liver diseases

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YANG Jinlian

2016-02-01

Full Text Available Hepatocyte nuclear factor 4 alpha (HNF4α, a member of the nuclear receptor superfamily, has a high expression level in mature hepatocytes. HNF4α can regulate hepatocyte-specific gene expression at a transcriptional level, promote hepatocyte development and differentiation, participate in establishment and maintenance of hepatocyte polarity, and enhance the synthetic, metabolic, and detoxifying functions of the liver. Through inhibiting the activation of hepatic stellate cells, reversing epithelial-mesenchymal transition, and inhibiting the proliferation, invasion, and metastasis of hepatoma cells, HNF4α may be involved in the development and progression of various liver diseases including liver fibrosis, liver cirrhosis, and hepatocellular carcinoma. This paper elaborates on the biological functions of HNF4α, and summarizes and analyzes the research advances in the mechanisms of action of HNF4α in the pathological process of liver diseases, in order to provide references for further investigation of the potential targeted therapies for liver diseases.

Glutamic Acid as Enhancer of Protein Synthesis Kinetics in Hepatocytes from Old Rats.

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Brodsky, V Y; Malchenko, L A; Butorina, N N; Lazarev Konchenko, D S; Zvezdina, N D; Dubovaya, T K

2017-08-01

Dense cultures of hepatocytes from old rats (~2 years old, body weight 530-610 g) are different from similar cultures of hepatocytes from young rats by the low amplitude of protein synthesis rhythm. Addition of glutamic acid (0.2, 0.4, or 0.6 mg/ml) into the culture medium with hepatocytes of old rats resulted in increase in the oscillation amplitudes of the protein synthesis rhythm to the level of young rats. A similar action of glutamic acid on the protein synthesis kinetics was observed in vivo after feeding old rats with glutamic acid. Inhibition of metabotropic receptors of glutamic acid with α-methyl-4-carboxyphenylglycine (0.01 mg/ml) abolished the effect of glutamic acid. The amplitude of oscillation of the protein synthesis rhythm in a cell population characterizes synchronization of individual oscillations caused by direct cell-cell communications. Hence, glutamic acid, acting as a receptor-dependent transmitter, enhanced direct cell-cell communications of hepatocytes that were decreased with aging. As differentiated from other known membrane signaling factors (gangliosides, norepinephrine, serotonin, dopamine), glutamic acid can penetrate into the brain and thus influence the communications and protein synthesis kinetics that are disturbed with aging not only in hepatocytes, but also in neurons.

Vitellogenin in the honey bee brain: Atypical localization of a reproductive protein that promotes longevity.

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Münch, Daniel; Ihle, Kate E; Salmela, Heli; Amdam, Gro V

2015-11-01

In comparative gerontology, highly social insects such as honey bees (Apis mellifera) receive much attention due to very different and flexible aging patterns among closely related siblings. While experimental strategies that manipulate socio-environmental factors suggest a causative link between aging and social signals and behaviors, the molecular underpinnings of this linkage are less well understood. Here we study the atypical localization of the egg-yolk protein vitellogenin (Vg) in the brain of the honey bee. Vg is known to influence honey bee social regulation and aging rate. Our findings suggest that Vg immunoreactivity in the brain is specifically localized within the class of non-neuronal glial cells. We discuss how these results can help explain the socially-dependent aging rate of honey bees. Copyright © 2015 Elsevier Inc. All rights reserved.

Tissue distribution and functional analysis of vitellogenin-6 of Toxocara canis.

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Zhu, Hong-Hong; Ma, Guang-Xu; Luo, Yong-Fang; Luo, Yong-Li; Yin, Sha-Sha; Xiong, Yi; Zhou, Rong-Qiong

2017-06-01

Toxocara canis is an common intestinal nematode of canids and the principal causative agent of human toxocariasis. Vitellogenin (Vg), a source of amino acids and lipids in the eggs, are considered to play an important role in embryo development of a wide range of organisms. In the present study, the transcriptional levels of Tc-vit-6 gene in male and female adult T. canis were determined by quantitative real-time PCR, which indicated high transcription of Tc-vit-6 in the intestine, reproductive tract and body wall of male and female adult T. canis. The fragment of Tc-vit-6 encoding a vWD domain, was cloned and expressed to produce a rabbit anti-TcvWD polyclonal antibody. Tissue distribution of TcVg6 was detected by immunohistochemical assays, which showed predominant distribution of TcVg6 in the tissues of intestine, as well as reproductive tract (including some of the germ cells) and musculature of male and female adult worms. Collectively, these results indicated multiple biological roles of TcVg6 apart from that in the reproduction of T. canis. Copyright © 2017 Elsevier Inc. All rights reserved.

Optimization of the isolation and cultivation of Cyprinus carpio primary hepatocytes

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Yanhong, Fan; Chenghua, He; Guofang, Liu

2008-01-01

The aquatic environment is affected by numerous chemical contaminants. There is an increasing need to identify these chemicals and to evaluate their potential toxicity towards aquatic life. In this research we optimized techniques for primary cell culture of Cyprinus carpio hepatocytes as one adjunct model for ecotoxicological evaluation of the potential hazards of xenobiotics in the aquatic environment. In this study, Cyprinus carpio hepatocytes were isolated by mechanical separation, two-step collagenase perfusion, and pancreatin digestion. The hepatocytes or parenchymal cells could be separated from cell debris and from non-parenchymal cells by low-speed centrifugation (Percoll gradient centrifugation). The harvested hepatocytes were suspended in DMEM, M199 (cultured in 5% CO2), or L-15 (cultured without 5% CO2) medium then cultured at 17, 27, or 37 °C. Cell yield was counted by use of a hemocytometer, and the viability of the cells was assessed by use of the Trypan blue exclusion test. Results from these studies showed that the best method of isolation was pancreatin digestion (the cell yield was 2.7 × 108 per g (liver weight) and the viability was 98.4%) and the best medium was M199 (cultured in 5% CO2) or L-15 (cultured without 5% CO2). The optimum culture temperature was 27 °C. The primary hepatocytes culture of Cyprimus carpio grew well and satisfied requirements for most toxicological experiments in this condition. PMID:19002769

Independent, parallel pathways to CXCL10 induction in HCV-infected hepatocytes.

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Brownell, Jessica; Wagoner, Jessica; Lovelace, Erica S; Thirstrup, Derek; Mohar, Isaac; Smith, Wesley; Giugliano, Silvia; Li, Kui; Crispe, I Nicholas; Rosen, Hugo R; Polyak, Stephen J

2013-10-01

The pro-inflammatory chemokine CXCL10 is induced by HCV infection in vitro and in vivo, and is associated with outcome of IFN (interferon)-based therapy. We studied how hepatocyte sensing of early HCV infection via TLR3 (Toll-like receptor 3) and RIG-I (retinoic acid inducible gene I) led to expression of CXCL10. CXCL10, type I IFN, and type III IFN mRNAs and proteins were measured in PHH (primary human hepatocytes) and hepatocyte lines harboring functional or non-functional TLR3 and RIG-I pathways following HCV infection or exposure to receptor-specific stimuli. HuH7 human hepatoma cells expressing both TLR3 and RIG-I produced maximal CXCL10 during early HCV infection. Neutralization of type I and type III IFNs had no impact on virus-induced CXCL10 expression in TLR3+/RIG-I+ HuH7 cells, but reduced CXCL10 expression in PHH. PHH cultures were positive for monocyte, macrophage, and dendritic cell mRNAs. Immunodepletion of non-parenchymal cells (NPCs) eliminated marker expression in PHH cultures, which then showed no IFN requirement for CXCL10 induction during HCV infection. Immunofluorescence studies also revealed a positive correlation between intracellular HCV Core and CXCL10 protein expression (r(2) = 0.88, p ≤ 0.001). While CXCL10 induction in hepatocytes during the initial phase of HCV infection is independent of hepatocyte-derived type I and type III IFNs, NPC-derived IFNs contribute to CXCL10 induction during HCV infection in PHH cultures. Copyright © 2013 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Impaired mitochondrial metabolism and protein synthesis in streptozotocin diabetic rat hepatocytes

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Memon, R.A.; Bessman, S.P.; Mohan, C.

1990-01-01

Isolated hepatocytes prepared from control, streptozotocin diabetic rats were incubated at 30 degrees C in Krebs-Henseleit bicarbonate buffer, pH 7.4, containing 0.5 mM concentration of each of the 20 natural amino acids. Effect of insulin on the oxidation of 2,3- 14 C and 1,4- 14 C succinate (suc) carbons and their incorporation into hepatocyte protein, lipid and various metabolic intermediates was studied. Mitochondrial oxidation of suc carbons and their incorporation into protein and lipid was significantly lower in diabetic and insulin treated diabetic rats. Diabetic rats failed to exhibit any significant insulin effect on the oxidation of either 2,3 or 1,4- 14 C suc carbons. Amphibolic channeling of 2,3- 14 C suc carbons into amino acids was significantly reduced in hepatocytes of diabetic rats, however, more of these carbons were diverted into the gluconeogenesis pathway. Diabetes caused a far greater decrease in the oxidation of 2,3- 14 C suc carbons as compared to 1,4- 14 C suc. Based on an earlier report that insulin stimulates only the intramitochondrial Krebs cycle reactions, the authors conclude that the diminished level of anabolic activities in the diabetic rat hepatocytes is due to the subsequent reduction in amphibolic channeling of metabolic intermediates

Requirements of glycerol and fatty acid for triglyceride synthesis and ketogenesis by hepatocytes from normal and triiodothyronine-treated rats

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Olubadewo, J.O.; Heimberg, M.

1985-01-01

Hepatocytes from T3-treated rats synthesized less triglyceride and more ketone bodies from [1- 14 C]oleate at all concentrations from 0-2 mM, than did hepatocytes from euthyroid animals; addition of 1.0 mM glycerol increased triglyceride synthesis and reduced ketogenesis in hepatocytes from T3-treated rats to the rates observed in euthyroid hepatocytes in the absence of added glycerol. Glycerol did not alter triglyceride synthesis, but reduced ketogenesis genesis by euthyroid hepatocytes. It is probable from these and other data that, in the hyperthyroid rat, glycero-3-P, and not fatty acid, is rate limiting for synthesis of triglyceride, and, secondarily for reducing rates of ketogenesis in the hepatocyte

Three cDNAs encoding vitellogenin homologs from Antarctic copepod, Tigriopus kingsejongensis: Cloning and transcriptional analysis in different maturation stages, temperatures, and putative reproductive hormones.

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Lee, Soo Rin; Lee, Ji-Hyun; Kim, Ah Ran; Kim, Sanghee; Park, Hyun; Baek, Hea Ja; Kim, Hyun-Woo

2016-02-01

Three full-length cDNAs encoding lipoprotein homologs were identified in Tigriopus kingsejongensis, a newly identified copepod from Antarctica. Structural and transcriptional analyses revealed homology with two vitellogenin-like proteins, Tik-Vg1 and Tik-Vg2, which were 1855 and 1795 amino acids in length, respectively, along with a third protein, Tik-MEP, which produced a 1517-residue protein with similarity to a melanin engaging protein (MEP) in insects Phylogenetic analysis showed that Vgs in Maxillopods including two Tik-Vgs belong to the arthropod vitellogenin-like clade, which includes clottable proteins (CPs) in decapod crustaceans and vitellogenins in insects. Tik-MEP clustered together with insect MEPs, which appear to have evolved before the apoB-like and arthropod Vg-like clades. Interestingly, no genes orthologous to those found in the apoB clade were identified in Maxillopoda, suggesting that functions of large lipid transfer proteins (LLTPs) in reproduction and lipid metabolism may be different from those in insect and decapod crustaceans. As suggested by phylogenetic analyses, the two Tik-Vgs belonging to the arthropod Vg-like clade appear to play major roles in oocyte maturation, while Vgs belonging to the apoB clade function primarily in the reproduction of decapod crustaceans. Transcriptional analysis of Tik-Vg expression revealed a 24-fold increase in mature and ovigerous females compared with immature female, whereas expression of Tik-MEP remained low through all reproductive stages. Acute temperature changes did not affect the transcription of Tik-Vg genes, whereas Tik-MEP appeared to be affected by temperature change. Among the three hormones thought to be involved in molting and reproduction in arthropods, only farnesoic acid (FA) induced transcription of the two Tik-Vg genes. Regardless of developmental stage and hormone treatment, Tik-Vg1 and Tik-Vg2 exhibited a strong positive correlation in expression, suggesting that expression of these

Preventing hepatocyte oxidative stress cytotoxicity with Mangifera indica L. extract (Vimang).

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Remirez, Diadelis; Tafazoli, Shahrzad; Delgado, Rene; Harandi, Asghar A; O'Brien, Peter J

2005-01-01

Vimang is an aqueous extract of Mangifera indica used in Cuba to improve the quality of life in patients suffering from inflammatory diseases. In the present study we evaluated the effects of Vimang at preventing reactive oxygen species (ROS) formation and lipid peroxidation in intact isolated rat hepatocytes. Vimang at 20, 50 and 100 microg/ml inhibited hepatocyte ROS formation induced by glucose-glucose oxidase. Hepatocyte cytotoxicity and lipid peroxidation induced by cumene hydroperoxide was also inhibited by Vimang in a dose and time dependent manner at the same concentration. Vimang also inhibited superoxide radical formation by xanthine oxidase and hypoxanthine. The superoxide radical scavenging and antioxidant activity of the Vimang extract was likely related to its gallates, catechins and mangiferin content. To our knowledge, this is the first report of cytoprotective antioxidant effects of Vimang in cellular oxidative stress models.

U.V.-enhanced reactivation of u.v.-irradiated herpes virus by primary cultures of rat hepatocytes

International Nuclear Information System (INIS)

Zurlo, J.; Yager, J.D.

1984-01-01

Carcinogen treatment of cultured mammalian cells prior to infection with u.v.-irradiated virus results in enhanced virus survival and mutagenesis suggesting the induction of SOS-type processes. In this paper, we report the development of a primary rat hepatocyte culture system to investigate cellular responses to DNA damage which may be relevant to hepatocarcinogenesis in vivo. We have obtained data demonstrating that enhanced reactivation of u.v.-irradiated Herpes simplex virus type 1 (HSV-1) occurs in hepatocytes irradiated with u.v. Cultured hepatocytes were pretreated with u.v. at the time of enhanced DNA synthesis. These treatments caused an inhibition followed by a recovery of DNA synthesis. At various times after pretreatment, the hepatocytes were infected with control or u.v.-irradiated HSV-1 at low multiplicity, and virus survival was measured by direct plaque assay. U.v.-irradiated HSV-1 exhibited the expected two-component survival curve in control or u.v. pretreated hepatocytes. The magnitude of enhanced reactivation of HSV-1 was dependent on the u.v. dose to the hepatocytes, the time of infection following u.v. pretreatment, and the level of DNA synthesis at the time of pretreatment. These results suggest that u.v. treatment of rat hepatocytes causes the induction of SOS-type functions that may have a role in the initiation of hepatocarcinogenesis

Vitellogenin-RNAi and ovariectomy each increase lifespan, increase protein storage, and decrease feeding, but are not additive in grasshoppers.

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Tetlak, Alicia G; Burnett, Jacob B; Hahn, Daniel A; Hatle, John D

2015-12-01

Reduced reproduction has been shown to increase lifespan in many animals, yet the mechanisms behind this trade-off are unclear. We addressed this question by combining two distinct, direct means of life-extension via reduced reproduction, to test whether they were additive. In the lubber grasshopper, Romalea microptera, ovariectomized (OVX) individuals had a ~20% increase in lifespan and a doubling of storage relative to controls (Sham operated). Similarly, young female grasshoppers treated with RNAi against vitellogenin (the precursor to egg yolk protein) had increased fat body mass and halted ovarian growth. In this study, we compared VgRNAi to two control groups that do not reduce reproduction, namely buffer injection (Buffer) and injection with RNAi against a hexameric storage protein (Hex90RNAi). Each injection treatment was tested with and without ovariectomy. Hence, we tested feeding, storage, and lifespans in six groups: OVX and Buffer, OVX and Hex90RNAi, OVX and VgRNAi, Sham and Buffer, Sham and Hex90RNAi, and Sham and VgRNAi. Ovariectomized grasshoppers and VgRNAi grasshoppers each had similar reductions in feeding (~40%), increases in protein storage in the hemolymph (150-300%), and extensions in lifespan (13-21%). Ovariectomized grasshoppers had higher vitellogenin protein levels than did VgRNAi grasshoppers. Last but not least, when ovariectomy and VgRNAi were applied together, there was no greater effect on feeding, protein storage, or longevity. Hence, feeding regulation, and protein storage in insects, may be conserved components of life-extension via reduced reproduction.

Human hepatocyte depletion in the presence of HIV-1 infection in dual reconstituted humanized mice

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Wang, Weimin; Cheng, Yan; Makarov, Edward; Ganesan, Murali; Gebhart, Catherine L.; Gorantla, Santhi; Osna, Natalia

2018-01-01

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) infection impairs liver function, and liver diseases have become a leading cause of morbidity in infected patients. The immunopathology of liver damage caused by HIV-1 remains unclear. We used chimeric mice dually reconstituted with a human immune system and hepatocytes to address the relevance of the model to pathobiology questions related to human hepatocyte survival in the presence of systemic infection. TK-NOG males were transplanted with mismatched human hematopoietic stem/progenitor cells and hepatocytes, human albumin concentration and the presence of human immune cells in blood were monitored for hepatocytes and immune reconstitution, and mice were infected with HIV-1. HIV-1-infected animals showed a decline in human albumin concentration with a significant reduction in percentage of human hepatocytes compared to uninfected mice. The decrease in human albumin levels correlated with a decline in CD4+ cells in the liver and with an increase in HIV-1 viral load. HIV-1 infection elicited proinflammatory response in the immunological milieu of the liver in HIV-infected mice compared to uninfected animals, as determined by upregulation of IL23, CXCL10 and multiple toll-like receptor expression. The inflammatory reaction associated with HIV-1 infection in vivo could contribute to the depletion and dysfunction of hepatocytes. The dual reconstituted TK-NOG mouse model is a feasible platform to investigate hepatocyte-related HIV-1 immunopathogenesis. This article has an associated First Person interview with the first author of the paper. PMID:29361613

Human hepatocyte depletion in the presence of HIV-1 infection in dual reconstituted humanized mice

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Raghubendra Singh Dagur

2018-02-01

Full Text Available Human immunodeficiency virus type 1 (HIV-1 infection impairs liver function, and liver diseases have become a leading cause of morbidity in infected patients. The immunopathology of liver damage caused by HIV-1 remains unclear. We used chimeric mice dually reconstituted with a human immune system and hepatocytes to address the relevance of the model to pathobiology questions related to human hepatocyte survival in the presence of systemic infection. TK-NOG males were transplanted with mismatched human hematopoietic stem/progenitor cells and hepatocytes, human albumin concentration and the presence of human immune cells in blood were monitored for hepatocytes and immune reconstitution, and mice were infected with HIV-1. HIV-1-infected animals showed a decline in human albumin concentration with a significant reduction in percentage of human hepatocytes compared to uninfected mice. The decrease in human albumin levels correlated with a decline in CD4+ cells in the liver and with an increase in HIV-1 viral load. HIV-1 infection elicited proinflammatory response in the immunological milieu of the liver in HIV-infected mice compared to uninfected animals, as determined by upregulation of IL23, CXCL10 and multiple toll-like receptor expression. The inflammatory reaction associated with HIV-1 infection in vivo could contribute to the depletion and dysfunction of hepatocytes. The dual reconstituted TK-NOG mouse model is a feasible platform to investigate hepatocyte-related HIV-1 immunopathogenesis. This article has an associated First Person interview with the first author of the paper.

[Current status and future perspectives of hepatocyte transplantation].

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Pareja, Eugenia; Cortés, Miriam; Gómez-Lechón, M José; Maupoey, Javier; San Juan, Fernando; López, Rafael; Mir, Jose

2014-02-01

The imbalance between the number of potential beneficiaries and available organs, originates the search for new therapeutic alternatives, such as Hepatocyte transplantation (HT).Even though this is a treatment option for these patients, the lack of unanimity of criteria regarding indications and technique, different cryopreservation protocols, as well as the different methodology to assess the response to this therapy, highlights the need of a Consensus Conference to standardize criteria and consider future strategies to improve the technique and optimize the results.Our aim is to review and update the current state of hepatocyte transplantation, emphasizing the future research attempting to solve the problems and improve the results of this treatment. Copyright © 2013 AEC. Published by Elsevier Espana. All rights reserved.

Effects of Cytochrome P 450 Inhibitors on Itraconazole and Fluconazole Induced Cytotoxicity in Hepatocytes

International Nuclear Information System (INIS)

Somchit, N.; Ngee, C.S.; Yaakob, A.; Ahmad, Z.; Zakaria, Z.A.

2009-01-01

Itraconazole and fluconazole have been reported to induce hepatotoxicity in patients. The present study was designed to investigate the role of cytochrome P450 inhibitors, SKF 525A, and curcumin pretreatment on the cytotoxicity of antifungal drugs fluconazole and itraconazole. For 3 consecutive days, female rats were administered daily SKF 525A or curcumin (5 and 25?mg/kg). Control rats received an equivalent amount of dosed vehicle. The animals were anaesthetised 24 hours after receiving the last dose for liver perfusion. Hepatocytes were then exposed to various concentrations of antifungal drugs. In vitro incubation of hepatocytes with itraconazole revealed significantly lower viability when compared to fluconazole as assessed by lactate dehydrogenase, aspartate aminotransferase and alanine aminotransferase activities. The cytotoxicity of itraconazole was enhanced when incubated with hepatocytes pretreated with SKF 525A. SKF 525A had no effects on the cytotoxicity of fluconazole. Curcumin failed to either increase or decrease the cytotoxicity of both antifungal drugs. ATP levels also showed significant decrease in both itraconazole and fluconazole incubated hepatocytes. However, SKF 525A pretreated hepatocytes had significantly lower ATP levels after itraconazole incubations. Collectively, these results confirm the involvement of cytochrome P450 in the cytoprotection in itraconazole induced hepatocyte toxicity. Differences of the effects of SKF 525A on the cytotoxicity induced by itraconazole and fluconazole may be due to the differences on the metabolism of each antifungal drug in vivo.

Effects of clofibric acid on mRNA expression profiles in primary cultures of rat, mouse and human hepatocytes.

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Richert, Lysiane; Lamboley, Christelle; Viollon-Abadie, Catherine; Grass, Peter; Hartmann, Nicole; Laurent, Stephane; Heyd, Bruno; Mantion, Georges; Chibout, Salah-Dine; Staedtler, Frank

2003-09-01

The mRNA expression profile in control and clofibric acid (CLO)-treated mouse, rat, and human hepatocytes was analyzed using species-specific oligonucleotide DNA microarrays (Affymetrix). A statistical empirical Bayes procedure was applied in order to select the significantly differentially expressed genes. Treatment with the peroxisome proliferator CLO induced up-regulation of genes involved in peroxisome proliferation and in cell proliferation as well as down-regulation of genes involved in apoptosis in hepatocytes of rodent but not of human origin. CLO treatment induced up-regulation of microsomal cytochrome P450 4a genes in rodent hepatocytes and in two of six human hepatocyte cultures. In addition, genes encoding phenobarbital-inducible cytochrome P450s were also up-regulated by CLO in rodent and human hepatocyte cultures. Up-regulation of phenobarbital-inducible UDP-glucuronosyl-transferase genes by CLO was observed in both rat and human but not in mouse hepatocytes. CLO treatment induced up-regulation of L-fatty acid binding protein (L-FABP) gene in hepatocytes of both rodent and human origin. However, while genes of the cytosolic, microsomal, and mitochondrial pathways involved in fatty acid transport and metabolism were up-regulated by CLO in both rodent and human hepatocyte cultures, genes of the peroxisomal pathway of lipid metabolism were up-regulated in rodents only. An up-regulation of hepatocyte nuclear factor 1alpha (HNF1alpha) by CLO was observed only in human hepatocyte cultures, suggesting that this trans-activating factor may play a key role in the regulation of fatty acid metabolism in human liver as well as in the nonresponsiveness of human liver to CLO-induced regulation of cell proliferation and apoptosis.

Effects of clofibric acid on mRNA expression profiles in primary cultures of rat, mouse and human hepatocytes

International Nuclear Information System (INIS)

Richert, Lysiane; Lamboley, Christelle; Viollon-Abadie, Catherine; Grass, Peter; Hartmann, Nicole; Laurent, Stephane; Heyd, Bruno; Mantion, Georges; Chibout, Salah-Dine; Staedtler, Frank

2003-01-01

The mRNA expression profile in control and clofibric acid (CLO)-treated mouse, rat, and human hepatocytes was analyzed using species-specific oligonucleotide DNA microarrays (Affymetrix). A statistical empirical Bayes procedure was applied in order to select the significantly differentially expressed genes. Treatment with the peroxisome proliferator CLO induced up-regulation of genes involved in peroxisome proliferation and in cell proliferation as well as down-regulation of genes involved in apoptosis in hepatocytes of rodent but not of human origin. CLO treatment induced up-regulation of microsomal cytochrome P450 4a genes in rodent hepatocytes and in two of six human hepatocyte cultures. In addition, genes encoding phenobarbital-inducible cytochrome P450s were also up-regulated by CLO in rodent and human hepatocyte cultures. Up-regulation of phenobarbital-inducible UDP-glucuronosyl-transferase genes by CLO was observed in both rat and human but not in mouse hepatocytes. CLO treatment induced up-regulation of L-fatty acid binding protein (L-FABP) gene in hepatocytes of both rodent and human origin. However, while genes of the cytosolic, microsomal, and mitochondrial pathways involved in fatty acid transport and metabolism were up-regulated by CLO in both rodent and human hepatocyte cultures, genes of the peroxisomal pathway of lipid metabolism were up-regulated in rodents only. An up-regulation of hepatocyte nuclear factor 1α (HNF1α) by CLO was observed only in human hepatocyte cultures, suggesting that this trans-activating factor may play a key role in the regulation of fatty acid metabolism in human liver as well as in the nonresponsiveness of human liver to CLO-induced regulation of cell proliferation and apoptosis

Aniline Induces Oxidative Stress and Apoptosis of Primary Cultured Hepatocytes

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Yue Wang

2016-11-01

Full Text Available The toxicity and carcinogenicity of aniline in humans and animals have been well documented. However, the molecular mechanism involved in aniline-induced liver toxicity and carcinogenesis remains unclear. In our research, primary cultured hepatocytes were exposed to aniline (0, 1.25, 2.50, 5.0 and 10.0 μg/mL for 24 h in the presence or absence of N-acetyl-l-cysteine (NAC. Levels of reactive oxygen species (ROS, malondialdehyde (MDA, and glutathione (GSH, activities of superoxide dismutase (SOD and catalase (CAT, mitochondrial membrane potential, DNA damage, cell viability, and apoptosis were detected. Levels of ROS and MDA were significantly increased and levels of GSH and CAT, activity of SOD, and mitochondrial membrane potential in hepatocytes were significantly decreased by aniline compared with the negative control group. The tail moment and DNA content of the tail in exposed groups were significantly higher than those in the negative control group. Cell viability was reduced and apoptotic death was induced by aniline in a concentration-dependent manner. The phenomena of ROS generation, oxidative damage, loss of mitochondrial membrane potential, DNA damage and apoptosis could be prevented if ROS inhibitor NAC was added. ROS generation is involved in the loss of mitochondrial membrane potential and DNA injury, which may play a role in aniline-induced apoptosis in hepatocytes. Our study provides insight into the mechanism of aniline-induced toxicity and apoptosis of hepatocytes.

EXPERIMENTAL SUBSTANTIATION OF PERMEABILIZED HEPATOCYTES MODEL FOR INVESTIGATION OF MITOCHONDRIA IN SITU RESPIRATION.

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Merlavsky, V M; Manko, B O; Ikkert, O V; Manko, V V

2015-01-01

To verify experimentally the model of permeabilized hepatocytes, the degree of cell permeability was assessed using trypan blue and polarographycally determined cell respiration rate upon succinate (0.35 mM) and a-ketoglutarate (1 mM) oxidation. Oxidative phosphorylation was stimulated by ADP (750 μM). Hepatocyte permeabilization depends on digitonin concentraion in medium and on the number of cells in suspension. Thus, the permeabilization of 0.9-1.7 million cells/ml was completed by 25 μg/ml of digitonin, permeabilization of 2.0-3.0 million cells/ml--by 50 μg/ml of digitonin and permeabilization of 4.0-5.6 million cells/ml--by 100 μg/ml. Thus, the higher is the suspension density, the higher digitonin concentration is required. Treatment of hepatocytes with digitonin resulted in a decrease of endogenous respiration rate to a minimum upon 20-22 μg of digitonin per 1 million cells. Supplementation of permeabilized hepatocytes with α-ketoglutarate maintained stable respiration rate, on the level higher than endogenous respiration at the corresponding digitonin concentration, unlike the intact cells. Respiration rate of permeabilized hepatocytes at the simultaneous addition of α-ketoglutarate and ADP increased to the level of intact cell respiration, irrespective of digitonin concentration. Addition of solely succinate and especially succinate plus ADP markedly intensified the respiration of permeabilized hepatocytes to the level higher than that of intact cells. The dependence of succinate-stimulated respiration on digitonin concentration reached maximum at 20-22 αg of digitonin per 1 million cells. Optimal ratio of digitonin amount and the cell number in suspension is expected to be different in various tissues.

Non-viral ex vivo hepatic gene transfer by in situ lipofection of liver and intraperitoneal transplantation of hepatocytes.

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Rangarajan, P N; Vatsala, P G; Ashok, M S; Srinivas, V K; Habibullah, C M; Padmanaban, G

1997-04-29

Perfusion of liver with plasmid DNA-lipofectin complexes via the portal vein results in efficient accumulation of the vector in hepatocytes. Such hepatocytes, when administered intraperitoneally into a hepatectomized rat, repopulate the liver and express the transgene efficiently. This procedure obviates the need for large-scale hepatocyte culture for ex vivo gene transfer. Further, intraperitoneal transplantation is a simple and cost-effective strategy of introducing genetically modified hepatocytes into liver. Thus, in situ lipofection of liver and intraperitoneal transfer of hepatocytes can be developed into a novel method of non-viral ex vivo gene transfer technique that has applications in the treatment of metabolic disorders of liver and hepatic gene therapy.

Microencapsulation of Hepatocytes and Mesenchymal Stem Cells for Therapeutic Applications.

Science.gov (United States)

Meier, Raphael P H; Montanari, Elisa; Morel, Philippe; Pimenta, Joël; Schuurman, Henk-Jan; Wandrey, Christine; Gerber-Lemaire, Sandrine; Mahou, Redouan; Bühler, Leo H

2017-01-01

Encapsulated hepatocyte transplantation and encapsulated mesenchymal stem cell transplantation are newly developed potential treatments for acute and chronic liver diseases, respectively. Cells are microencapsulated in biocompatible semipermeable alginate-based hydrogels. Microspheres protect cells against antibodies and immune cells, while allowing nutrients, small/medium size proteins and drugs to diffuse inside and outside the polymer matrix. Microencapsulated cells are assessed in vitro and designed for experimental transplantation and for future clinical applications.Here, we describe the protocol for microencapsulation of hepatocytes and mesenchymal stem cells within hybrid poly(ethylene glycol)-alginate hydrogels.

Hepatocyte transplantation improves early survival after partial hepatic resection and irradiation

International Nuclear Information System (INIS)

Guha, C.; Sharma, A.; Alfieri, A.; Guha, U.; Sokhi, R.; Gagandeep, S.; Gupta, S.; Vikram, B.; RoyChowdhury, J.

1997-01-01

Purpose: Radiation therapy (RT) is limited in its role as an adjuvant therapy of intrahepatic malignancies because of lower tolerance of human liver to irradiation (TD (5(5)) -TD (50(5)) ∼ 30-40 Gy). Although, surgical resection of primary or metastatic hepatic tumors has been shown to prolong survival, it is often limited by the presence of residual disease. RT could potentially improve survival of patients with positive surgical margins. However, radiation damage to the liver may be enhanced by hepatocellular proliferation induced by partial hepatic (PH) resection. We hypothesize that hepatocyte transplantation would be able to provide metabolic support and modulate the development of radiation-induced liver disease post-resection. The present study was designed to test the potential of hepatocyte transplantation in modifying the outcome of hepatocellular damage induced by PH and RT. Methods: Adult male Fischer 344 rats (Charles River) received hepatic irradiation of 50 Gy in a single fraction, after surgical exposure and shielding of the stomach and intestine, using a 320 MGC Philips orthovoltage unit. Immediately following irradiation, a two-third partial hepatectomy was performed. Four days post-radiation, the treatment group was injected with 5 x 10 6 syngeneic hepatocytes into the splenic pulp after a left subcostal incision, which allows homogeneous liver engraftment of the transplanted hepatocytes. Hematoxylin and eosin stains of liver biopsies, performed at various time points (3 days, 1, 2, 3 weeks or, anytime when animals died) were used for histologic evaluation. Time-adjusted survival was calculated from the date of irradiation by the product-limit Kaplan-Meier method, adjusting the denominator at every time point for the number of rats at risk. Results: Eight weeks after RT, 30% (n = 11) of the control animals (PH + 50 Gy) were alive compared to 100% (n = 9) of the transplant recipients (p <0.05). The median survival of the control group was 15

Homocysteine inhibits hepatocyte proliferation via endoplasmic reticulum stress.

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Xue Yu

Full Text Available Homocysteine is an independent risk factor for coronary, cerebral, and peripheral vascular diseases. Recent studies have shown that levels of homocysteine are elevated in patients with impaired hepatic function, but the precise role of homocysteine in the development of hepatic dysfunction is unclear. In this study, we examined the effect of homocysteine on hepatocyte proliferation in vitro. Our results demonstrated that homocysteine inhibited hepatocyte proliferation by up-regulating protein levels of p53 as well as mRNA and protein levels of p21(Cip1 in primary cultured hepatocytes. Homocysteine induced cell growth arrest in p53-positive hepatocarcinoma cell line HepG2, but not in p53-null hepatocarcinoma cell line Hep3B. A p53 inhibitor pifithrin-α inhibited the expression of p21(Cip1 and attenuated homocysteine-induced cell growth arrest. Homocysteine induced TRB3 expression via endoplasmic reticulum stress pathway, resulting in Akt dephosphorylation. Knock-down of endogenous TRB3 significantly suppressed the inhibitory effect of homocysteine on cell proliferation and the phosphorylation of Akt. LiCl reversed homocysteine-mediated cell growth arrest by inhibiting TRB3-mediated Akt dephosphorylation. These results demonstrate that both TRB3 and p21(Cip1 are critical molecules in the homocysteine signaling cascade and provide a mechanistic explanation for impairment of liver regeneration in hyperhomocysteinemia.

Evaluation and optimization of hepatocyte culture media factors by design of experiments (DoE) methodology.

Science.gov (United States)

Dong, Jia; Mandenius, Carl-Fredrik; Lübberstedt, Marc; Urbaniak, Thomas; Nüssler, Andreas K N; Knobeloch, Daniel; Gerlach, Jörg C; Zeilinger, Katrin

2008-07-01

Optimization of cell culture media based on statistical experimental design methodology is a widely used approach for improving cultivation conditions. We applied this methodology to refine the composition of an established culture medium for growth of a human hepatoma cell line, C3A. A selection of growth factors and nutrient supplements were systematically screened according to standard design of experiments (DoE) procedures. The results of the screening indicated that the medium additives hepatocyte growth factor, oncostatin M, and fibroblast growth factor 4 significantly influenced the metabolic activities of the C3A cell line. Surface response methodology revealed that the optimum levels for these factors were 30 ng/ml for hepatocyte growth factor and 35 ng/ml for oncostatin M. Additional experiments on primary human hepatocyte cultures showed high variance in metabolic activities between cells from different individuals, making determination of optimal levels of factors more difficult. Still, it was possible to conclude that hepatocyte growth factor, epidermal growth factor, and oncostatin M had decisive effects on the metabolic functions of primary human hepatocytes.

Hepatitis E Virus Induces Hepatocyte Apoptosis via Mitochondrial Pathway in Mongolian Gerbils

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Yifei Yang

2018-03-01

Full Text Available Previous studies demonstrated that Mongolian gerbils can be infected by hepatitis E virus (HEV, which induces the hepatic injury. Here, the mitochondria in hepatocytes from HEV-infected gerbils were considerably swollen, thin cristae. After HEV infection, the activity of superoxide dismutase significantly decreased (p < 0.01, while malondialdehyde concentrations significantly increased, compared with those in the control group (p < 0.01. Adenosine triphosphatase levels decreased significantly in the hepatocyte of the inoculated groups, compared with those in control group (p < 0.05 at days 21, 28, 42 post-inoculation (dpi as well. Furthermore, the levels of ATP synthetase ATP5A1 significantly decreased during HEV infection, compared with those in the control group (p < 0.05. According to the TdT mediated dUTP nick end labeling (TUNEL detection, TUNEL positive hepatocytes increased in the inoculated group, compared with that in the control group (p < 0.05. Up-regulation of the mitochondrion-mediated apoptosis regulating proteins, Bax and Bcl-2, in the HEV-infected gerbils (p < 0.05 was observed. However, cytochrome c levels in mitochondria decreased, while this molecule was detected in the cytoplasm of the infected animals, in contrast to that in the control group. Apaf-1, and active caspase-9 and -3 levels were shown to be significantly higher in the inoculated group compared with those in the control group (p < 0.05. Taken together, our results demonstrated that HEV infection induces hepatocyte injuries and activity of the mitochondrial apoptotic pathway, which trigger the hepatocyte apoptosis in Mongolian gerbils.

Glucose production and storage in hepatocytes isolated from normal versus diabetic rats

International Nuclear Information System (INIS)

Olivieri, M.C.; Dragland-Meserve, C.J.; Parker Botelho, L.H.

1987-01-01

The rates of glucose production and storage were compared in hepatocytes isolated from normal versus insulin-resistant diabetic rats. A single low-dose (40 mg/kg) IV injection of streptozotocin to 250 g rats resulted in a Type II diabetic animal model which was hyperglycemic with normal insulin levels. Addition of 8 mM 14 C-lactate and 2 mM pyruvate to hepatocytes resulted in a linear increase in total glucose production ( 14 C-glucose and unlabeled glucose) and incorporation into glycogen measured over 120 min. The rate of gluconeogenesis was estimated from the production of 14 C-glucose and the rate of glycogenolysis was estimated from the production of unlabeled glucose in cells incubated in the presence or absence of 14 C-labelled substrate. There was not significant difference in total glucose production in hepatocytes isolated from normal versus diabetic rats, however, the contribution from gluconeogenesis versus glycogenolysis was significantly different. Following a 1 h incubation of cells from normal rats, 42% of the total glucose production was due to gluconeogenesis and 58% was due to glycogenolysis. In cells from diabetic rats, 83% of total glucose production was from gluconeogenesis and 17% from glycogenolysis. Also, incubation with 14 C-lactate/pyruvate resulted in a 3.3-fold increase in 14 C-glucose incorporation into glycogen in hepatocytes isolated from normal rats compared to diabetic rats. These data suggest that alterations occur in the rate-limiting enzymes responsible for glucose production and storage in hepatocytes isolated from a rat model of insulin-resistant Type II diabetes

Endothelial cell-derived matrix promotes the metabolic functional maturation of hepatocyte via integrin-Src signalling.

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Guo, Xinyue; Li, Weihong; Ma, Minghui; Lu, Xin; Zhang, Haiyan

2017-11-01

The extracellular matrix (ECM) microenvironment is involved in the regulation of hepatocyte phenotype and function. Recently, the cell-derived extracellular matrix has been proposed to represent the bioactive and biocompatible materials of the native ECM. Here, we show that the endothelial cell-derived matrix (EC matrix) promotes the metabolic maturation of human adipose stem cell-derived hepatocyte-like cells (hASC-HLCs) through the activation of the transcription factor forkhead box protein A2 (FOXA2) and the nuclear receptors hepatocyte nuclear factor 4 alpha (HNF4α) and pregnane X receptor (PXR). Reducing the fibronectin content in the EC matrix or silencing the expression of α5 integrin in the hASC-HLCs inhibited the effect of the EC matrix on Src phosphorylation and hepatocyte maturation. The inhibition of Src phosphorylation using the inhibitor PP2 or silencing the expression of Src in hASC-HLCs also attenuated the up-regulation of the metabolic function of hASC-HLCs in a nuclear receptor-dependent manner. These data elucidate integrin-Src signalling linking the extrinsic EC matrix signals and metabolic functional maturation of hepatocyte. This study provides a model for studying the interaction between hepatocytes and non-parenchymal cell-derived matrix. © 2017 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

Protective effects of melittin on transforming growth factor-β1 injury to hepatocytes via anti-apoptotic mechanism

International Nuclear Information System (INIS)

Lee, Woo-Ram; Park, Ji-Hyun; Kim, Kyung-Hyun; Park, Yoon-Yub; Han, Sang-Mi; Park, Kwan-kyu

2011-01-01

Melittin is a cationic, hemolytic peptide that is the main toxic component in the venom of the honey bee (Apis mellifera). Melittin has multiple effects, including anti-bacterial, anti-viral and anti-inflammatory, in various cell types. However, the anti-apoptotic mechanisms of melittin have not been fully elucidated in hepatocytes. Apoptosis contributes to liver inflammation and fibrosis. Knowledge of the apoptotic mechanisms is important to develop new and effective therapies for treatment of cirrhosis, portal hypertension, liver cancer, and other liver diseases. In the present study, we investigated the anti-apoptotic effect of melittin on transforming growth factor (TGF)-β1-induced apoptosis in hepatocytes. TGF-β1-treated hepatocytes were exposed to low doses (0.5 and 1 μg/mL) and high dose (2 μg/mL) of melittin. The low doses significantly protected these cells from DNA damage in TGF-β1-induced apoptosis compared to the high dose. Also, melittin suppressed TGF-β1-induced apoptotic activation of the Bcl-2 family and caspase family of proteins, which resulted in the inhibition of poly-ADP-ribose polymerase (PARP) cleavage. These results demonstrate that TGF-β1 induces hepatocyte apoptosis and that an optimal dose of melittin exerts anti-apoptotic effects against TGF-β1-induced injury to hepatocytes via the mitochondrial pathway. These results suggest that an optimal dose of melittin can serve to protect cells against TGF-β1-mediated injury. - Highlights: → We investigated the anti-apoptotic effect of melittin on TGF-β1-induced hepatocyte. → TGF-β1 induces hepatocyte apoptosis. → TGF-β1-treated hepatocytes were exposed to low doses and high dose of melittin. → Optimal dose of melittin exerts anti-apoptotic effects to hepatocytes.

AR42J-B-13 cell: An expandable progenitor to generate an unlimited supply of functional hepatocytes

International Nuclear Information System (INIS)

Wallace, Karen; Fairhall, Emma A.; Charlton, Keith A.; Wright, Matthew C.

2010-01-01

Hepatocytes are the preparation of choice for Toxicological research in vitro. However, despite the fact that hepatocytes proliferate in vivo during liver regeneration, they are resistant to proliferation in vitro, do not tolerate sub-culture and tend to enter a de-differentiation program that results in a loss of hepatic function. These limitations have resulted in the search for expandable rodent and human cells capable of being directed to differentiate into functional hepatocytes. Research with stem cells suggests that it may be possible to provide the research community with hepatocytes in vitro although to date, significant challenges remain, notably generating a sufficiently pure population of hepatocytes with a quantitative functionality comparable with hepatocytes. This paper reviews work with the AR42J-B-13 (B-13) cell line. The B-13 cell was cloned from the rodent AR42J pancreatic cell line, express genes associated with pancreatic acinar cells and readily proliferates in simple culture media. When exposed to glucocorticoid, 75-85% of the cells trans-differentiate into hepatocyte-like (B-13/H) cells functioning at a level quantitatively similar to freshly isolated rat hepatocytes (with the remaining cells retaining the B-13 phenotype). Trans-differentiation of pancreatic acinar cells also appears to occur in vivo in rats treated with glucocorticoid; in mice with elevated circulating glucocorticoid and in humans treated for long periods with glucocorticoid. The B-13 response to glucocorticoid therefore appears to be related to a real pathophysiological response of a pancreatic cell to glucocorticoid. An understanding of how this process occurs and if it can be generated or engineered in human cells would result in a cell line with the ability to generate an unlimited supply of functional human hepatocytes in a cost effective manner.

Estrogen therapy offsets thermal impairment of vitellogenesis, but not zonagenesis, in maiden spawning female Atlantic salmon (Salmo salar

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Kelli Anderson

2017-11-01

Full Text Available In female Atlantic salmon (Salmo salar, exposure to warm summer temperatures causes a reduction in plasma 17β-estradiol (E2, which impairs downstream vitellogenesis and zonagenesis, and reduces egg fertility and embryo survival. The aim of the present study was to determine whether E2-treatment could offset thermal impairment of endocrine function and maintain egg quality in maiden (first-time-spawning S. salar reared at 22 °C. Treatment with E2 at 22 °C stimulated vitellogenin (vtg gene expression and subsequent protein synthesis which promoted oocyte growth and increased egg size relative to untreated fish at 14 and 22 °C. However, E2-treatment at 22 °C was not associated with an increase in egg fertility and embryo survival relative to untreated fish at 22 °C, despite the positive effects of E2-treatment on vitellogenesis and oocyte growth. As there was no evidence to suggest that the estrogen receptor alpha expression was suppressed by high temperature, this could be due to the lack of stimulation on zonagenesis by E2-treatment observed at high temperature during oocyte development. Our results demonstrate that treatment with E2 is not able to maintain zonagenesis or egg quality in maiden S. salar at high temperature, even when vtg gene expression, protein synthesis and subsequent oocyte growth is promoted. This implies that the mechanisms regulating zonagenesis, but not vitellogenesis are impaired at elevated temperature in female S. salar broodstock, and highlights the remarkable complexity of thermally induced endocrine disruption in fish.

An endocrine disrupting chemical changes courtship and parental care in the sand goby

International Nuclear Information System (INIS)

Saaristo, Minna; Craft, John A.; Lehtonen, Kari K.; Lindstroem, Kai

2010-01-01

Endocrine disrupting chemicals (EDCs) are a diverse group of compounds that can mimic, block or modulate the synthesis of natural hormones. They are known to cause impairment of reproduction of aquatic organisms at very low concentrations. The aim of this study was to examine how exposure from 10 to 31 days to 17α-ethinyl estradiol (EE2, 41 ng L -1 ) affects the courtship and parental care behaviour of male sand gobies (Pomatoschistus minutus). The sand goby exhibits a polygynous mating system, where males compete for females and provide paternal care. First, male courtship performance towards a stimulus female was recorded with video camera. Secondly, after the male had received eggs his parental care behaviour was video recorded. In addition to behavioural endpoints, we measured the expression of hepatic vitellogenin (Vtg) and zona radiata protein (Zrp) mRNA, as well as common somatic indices. Our study shows that exposure to EE2 affected male fanning behaviour during both courtship and parental care. Interestingly, small exposed males increased their courtship fanning to similar levels as larger control males. However, during parental care egg fanning was not related to male size, and all exposed males fanned more than control males. The EE2-exposure induced Vtg and Zrp mRNA expression in males and decreased hepatosomatic index (HSI), and increased gonadosomatic index (GSI). Females prefer males that fan more, which will favour the small EDC exposed males. This may lead to mating that favours males that are not strong enough to tend the eggs until they hatch, thus decreasing the reproductive success of individuals.

Evidence of estrogenic endocrine disruption in smallmouth and largemouth bass inhabiting Northeast U.S. National Wildlife Refuge waters: A reconnaissance study

Science.gov (United States)

Iwanowicz, Luke R.; Blazer, Vicki S.; Pinkney, A.E.; Guy, C.P.; Major, A.M.; Munney, K.; Mierzykowski, S.; Lingenfelser, S.; Secord, A.; Patnode, K.; Kubiak, T.J.; Stern, C.; Hahn, Cassidy M.; Iwanowicz, Deborah; Walsh, Heather L.; Sperry, Adam J.

2016-01-01

Intersex as the manifestation of testicular oocytes (TO) in male gonochoristic fishes has been used as an indicator of estrogenic exposure. Here we evaluated largemouth bass (Micropterus salmoides) or smallmouth bass (Micropterus dolomieu) form 19 National Wildlife Refuges (NWRs) in the Northeast U.S. inhabiting waters on or near NWR lands for evidence of estrogenic endocrine disruption. Waterbodies sampled included rivers, lakes, impoundments, ponds, and reservoirs. Here we focus on evidence of endocrine disruption in male bass evidenced by gonad histopathology including intersex or abnormal plasma vitellogenin (Vtg) concentrations. During the fall seasons of 2008–2010, we collected male smallmouth bass (n=118) from 12 sites and largemouth bass (n=173) from 27 sites. Intersex in male smallmouth bass was observed at all sites and ranged from 60% to 100%; in male largemouth bass the range was 0–100%. Estrogenicity, as measured using a bioluminescent yeast reporter, was detected above the probable no effects concentration (0.73 ng/L) in ambient water samples from 79% of the NWR sites. Additionally, the presence of androgen receptor and glucocorticoid receptor ligands were noted as measured via novel nuclear receptor translocation assays. Mean plasma Vtg was elevated (>0.2 mg/ml) in male smallmouth bass at four sites and in male largemouth bass at one site. This is the first reconnaissance survey of this scope conducted on US National Wildlife Refuges. The baseline data collected here provide a necessary benchmark for future monitoring and justify more comprehensive NWR-specific studies.

Assessment of reproductive effects in largemouth bass (Micropterus salmoides) exposed to bleached/unbleached kraft mill effluents.

Science.gov (United States)

Sepúlveda, M S; Ruessler, D S; Denslow, N D; Holm, S E; Schoeb, T R; Gross, T S

2001-11-01

This study evaluated the potential effects of different concentrations of bleached/unbleached kraft mill effluent (B/UKME) on several reproductive endpoints in adult largemouth bass (Micropterus salmoides). The kraft mill studied produces a 50/50 mix of bleached/unbleached market pulp with an estimated release of 36 million gal of effluent/day. Bleaching sequences were C90d10EopHDp and CEHD for softwood (pines) and hardwoods (mainly tupelo, gums, magnolia, and water oaks), respectively. Bass were exposed to different effluent concentrations (0 [controls, exposed to well water], 10, 20, 40, or 80%) for either 28 or 56 days. At the end of each exposure period, fish were euthanized, gonads collected for histological evaluation and determination of gonadosomatic index (GSI), and plasma was analyzed for 17beta-estradiol, 11-ketotestosterone, and vitellogenin (VTG). Largemouth bass exposed to B/UKME responded with changes at the biochemical level (decline in sex steroids in both sexes and VTG in females) that were usually translated into tissue/organ-level responses (declines in GSI in both sexes and in ovarian development in females). Although most of these responses occurred after exposing fish to 40% B/UKME concentrations or greater, some were observed after exposures to 20% B/UKME. These threshold concentrations fall within the 60% average yearly concentration of effluent that exists in the stream near the point of discharge (Rice Creek), but are above the <10% effluent concentration present in the St. Johns River. The chemical(s) responsible for such changes as well as their mode(s) of action remain unknown at this time.

Evidence of estrogenic endocrine disruption in smallmouth and largemouth bass inhabiting Northeast U.S. national wildlife refuge waters: A reconnaissance study.

Science.gov (United States)

Iwanowicz, L R; Blazer, V S; Pinkney, A E; Guy, C P; Major, A M; Munney, K; Mierzykowski, S; Lingenfelser, S; Secord, A; Patnode, K; Kubiak, T J; Stern, C; Hahn, C M; Iwanowicz, D D; Walsh, H L; Sperry, A

2016-02-01

Intersex as the manifestation of testicular oocytes (TO) in male gonochoristic fishes has been used as an indicator of estrogenic exposure. Here we evaluated largemouth bass (Micropterus salmoides) or smallmouth bass (Micropterus dolomieu) form 19 National Wildlife Refuges (NWRs) in the Northeast U.S. inhabiting waters on or near NWR lands for evidence of estrogenic endocrine disruption. Waterbodies sampled included rivers, lakes, impoundments, ponds, and reservoirs. Here we focus on evidence of endocrine disruption in male bass evidenced by gonad histopathology including intersex or abnormal plasma vitellogenin (Vtg) concentrations. During the fall seasons of 2008-2010, we collected male smallmouth bass (n=118) from 12 sites and largemouth bass (n=173) from 27 sites. Intersex in male smallmouth bass was observed at all sites and ranged from 60% to 100%; in male largemouth bass the range was 0-100%. Estrogenicity, as measured using a bioluminescent yeast reporter, was detected above the probable no effects concentration (0.73ng/L) in ambient water samples from 79% of the NWR sites. Additionally, the presence of androgen receptor and glucocorticoid receptor ligands were noted as measured via novel nuclear receptor translocation assays. Mean plasma Vtg was elevated (>0.2mg/ml) in male smallmouth bass at four sites and in male largemouth bass at one site. This is the first reconnaissance survey of this scope conducted on US National Wildlife Refuges. The baseline data collected here provide a necessary benchmark for future monitoring and justify more comprehensive NWR-specific studies. Published by Elsevier Inc.

Isolation of primary human hepatocytes from normal and diseased liver tissue: a one hundred liver experience.

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Ricky H Bhogal

2011-03-01

Full Text Available Successful and consistent isolation of primary human hepatocytes remains a challenge for both cell-based therapeutics/transplantation and laboratory research. Several centres around the world have extensive experience in the isolation of human hepatocytes from non-diseased livers obtained from donor liver surplus to surgical requirement or at hepatic resection for tumours. These livers are an important but limited source of cells for therapy or research. The capacity to isolate cells from diseased liver tissue removed at transplantation would substantially increase availability of cells for research. However no studies comparing the outcome of human hepatocytes isolation from diseased and non-diseased livers presently exist. Here we report our experience isolating human hepatocytes from organ donors, non-diseased resected liver and cirrhotic tissue. We report the cell yields and functional qualities of cells isolated from the different types of liver and demonstrate that a single rigorous protocol allows the routine harvest of good quality primary hepatocytes from the most commonly accessible human liver tissue samples.

Cytotoxic mechanisms of hydrosulfide anion and cyanide anion in primary rat hepatocyte cultures

International Nuclear Information System (INIS)

Thompson, Rodney W.; Valentine, Holly L.; Valentine, William M.

2003-01-01

Hydrogen sulfide and hydrogen cyanide are known to compromise mitochondrial respiration through inhibition of cytochrome c oxidase and this is generally considered to be their primary mechanism of toxicity. Experimental studies and the efficiency of current treatment protocols suggest that H 2 S may exert adverse physiological effects through additional mechanisms. To evaluate the role of alternative mechanisms in H 2 S toxicity, the relative contributions of electron transport inhibition, uncoupling of mitochondrial respiration, and opening of the mitochondrial permeability transition pore (MPTP) to hydrosulfide and cyanide anion cytotoxicity in primary hepatocyte cultures were examined. Supplementation of hepatocytes with the glycolytic substrate, fructose, rescued hepatocytes from cyanide anion induced toxicity, whereas fructose supplementation increased hydrosulfide anion toxicity suggesting that hydrosulfide anion may compromise glycolysis in hepatocytes. Although inhibitors of the MPTP opening were protective for hydrosulfide anion, they had no effect on cyanide anion toxicity, consistent with an involvement of the permeability transition pore in hydrosulfide anion toxicity but not cyanide anion toxicity. Exposure of isolated rat liver mitochondria to hydrosulfide did not result in large amplitude swelling suggesting that if H 2 S induces the permeability transition it does so indirectly through a mechanism requiring other cellular components. Hydrosulfide anion did not appear to be an uncoupler of mitochondrial respiration in hepatocytes based upon the inability of oligomycin and fructose to protect hepatocytes from hydrosulfide anion toxicity. These findings support mechanisms additional to inhibition of cytochrome c oxidase in hydrogen sulfide toxicity. Further investigations are required to assess the role of the permeability transition in H 2 S toxicity, determine whether similar affects occur in other cell types or in vivo and evaluate whether this may

[Changes in the chromatin structure of hepatocyte nuclei of rats trained to hypoxia].

Science.gov (United States)

Domkina, L K; Bresler, V M; Simanovskiĭ, L N

1976-03-01

Structure of chromatin in the nuclei of the isolated surviving hepatocytes and in the isolated nuclei of hepatocytes were studied by fluorochroming with acridine orange and by microfluorimetry of fluorescenc connected with the stain chromatin at 530 and 590 nm in intact rats and in the animals trained to hypoxia in a pressure chamber for 60 days. The nuclei of hepatocytes of intact rats were distributed by fluorescence at 530 nm into three classes with the intensity ratio of 1:2:4; as to the nuclei of hepatocytes of the rats trained to hypoxia - they formed a single class corresponding to the second class of control. In intact rats the ratio of the fluorescence intensity at 590 nm to such at 530 nm (alpha coefficient) formed normal distribution; in trained rats - a bimodal distribution with a shift of the maximum in the direction of reduction and increase of alpha in comparison with control. It is supposed that in hypoxia there is a repression of one and depression of other genes in the chromatine of the nuclei of the liver.

Deletion of hepatocyte nuclear factor-1-beta in an infant with prune belly syndrome.

Science.gov (United States)

Haeri, Sina; Devers, Patricia L; Kaiser-Rogers, Kathleen A; Moylan, Vincent J; Torchia, Beth S; Horton, Amanda L; Wolfe, Honor M; Aylsworth, Arthur S

2010-08-01

Prune belly syndrome is a rare congenital disorder characterized by deficiency of abdominal wall muscles, cryptorchidism, and urinary tract anomalies. We have had the opportunity to study a baby with prune belly syndrome associated with an apparently de novo 1.3-megabase interstitial 17q12 microdeletion that includes the hepatocyte nuclear factor-1-beta gene at 17q12. One previous patient, an adult, has been reported with prune belly syndrome and a hepatocyte nuclear factor-1-beta microdeletion. Hepatocyte nuclear factor-1-beta is a widely expressed transcription factor that regulates tissue-specific gene expression and is expressed in numerous tissues including mesonephric duct derivatives, the renal tubule of the metanephros, and the developing prostate of the mouse. Mutations in hepatocyte nuclear factor-1-beta cause the "renal cysts and diabetes syndrome," isolated renal cystic dysplasia, and a variety of other malformations. Based on its expression pattern and the observation of two affected cases, we propose that haploinsufficiency of hepatocyte nuclear factor-1-beta may be causally related to the production of the prune belly syndrome phenotype through a mechanism of prostatic and ureteral hypoplasia that results in severe obstructive uropathy with urinary tract and abdominal distension. Copyright Thieme Medical Publishers.

Swelling of rat hepatocytes stimulates glycogen synthesis

NARCIS (Netherlands)

Baquet, A.; Hue, L.; Meijer, A. J.; van Woerkom, G. M.; Plomp, P. J.

1990-01-01

In hepatocytes from fasted rats, several amino acids are known to stimulate glycogen synthesis via activation of glycogen synthase. The hypothesis that an increase in cell volume resulting from amino acid uptake may be involved in the stimulation of glycogen synthesis is supported by the following

The cytoskeleton of digitonin-treated rat hepatocytes.

Science.gov (United States)

Fiskum, G; Craig, S W; Decker, G L; Lehninger, A L

1980-06-01

Treatment of isolated rat hepatocptes with low concentrations of digitonin increases the permeability of the plsma membrane to cytosolic proteins without causing release of organelles such as mitochondria into the surrounding medium. Electron microscopy showed that treatment of the cells with increasing concentations of digitonin results in a progressive loss in the continuity of the plasma membrane, while most other aspects of cellular morphology remain normal. Depletion of background staining material from the cytosol by digitonin treatment of the cells greatly enhances the visualization of the cytoskeleton. The use of this technique, together with immunofluorescent light microscopy, has verified the presence of an actin-containing filamentous network at the hepatocyte cortex as well as intermediate filaments distributed throughout the cell. Digitonin is thus useful both for selectively permeabilizing the plasma membrane and for intensifying the appearance of intracellular structures such as microfilaments that are normally difficult to observe in cells such as hepatocytes.

Retroviral-mediated gene transfer and expression of human phenylalanine hydroxylase in primary mouse hepatocytes

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Peng, H.; Armentano, D.; Mackenzie-Graham, L.; Shen, R.F.; Darlington, G.; Ledley, F.D.; Woo, S.L.C. (Baylor College of Medicine, Houston, TX (USA))

1988-11-01

Genetic therapy for phenylketonuria (severe phenylalanine hydroxylase deficiency) may require introduction of a normal phenylalanine hydroxylase gene into hepatic cells of patients. The authors report development of a recombinant retrovirus based on the N2 vector for gene transfer and expression of human phenylalanine hydroxylase cDNA in primary mouse hepatocytes. This construct contains an internal promoter of the human {alpha}{sub 1}-antitrypsin gene driving transcription of the phenylalanine hydroxylase cDNA. Primary mouse hepatocytes were isolated from newborn mice, infected with the recombinant virus, and selected for expression of the neomycin-resistance gene. Hepatocytes transformed with the recombinant virus contained high levels of human phenylalanine hydroxylase mRNA transcripts originating from the retroviral and internal promoters. These results demonstrate that the transcriptional regulatory elements of the {alpha}{sub 1} antitrypsin gene retain their tissue-specific function in the recombinant provirus and establish a method for efficient transfer and high-level expression of human phenylalanine hydroxylase in primary hepatocytes.

Retroviral-mediated gene transfer and expression of human phenylalanine hydroxylase in primary mouse hepatocytes

International Nuclear Information System (INIS)

Peng, H.; Armentano, D.; Mackenzie-Graham, L.; Shen, R.F.; Darlington, G.; Ledley, F.D.; Woo, S.L.C.

1988-01-01

Genetic therapy for phenylketonuria (severe phenylalanine hydroxylase deficiency) may require introduction of a normal phenylalanine hydroxylase gene into hepatic cells of patients. The authors report development of a recombinant retrovirus based on the N2 vector for gene transfer and expression of human phenylalanine hydroxylase cDNA in primary mouse hepatocytes. This construct contains an internal promoter of the human α 1 -antitrypsin gene driving transcription of the phenylalanine hydroxylase cDNA. Primary mouse hepatocytes were isolated from newborn mice, infected with the recombinant virus, and selected for expression of the neomycin-resistance gene. Hepatocytes transformed with the recombinant virus contained high levels of human phenylalanine hydroxylase mRNA transcripts originating from the retroviral and internal promoters. These results demonstrate that the transcriptional regulatory elements of the α 1 antitrypsin gene retain their tissue-specific function in the recombinant provirus and establish a method for efficient transfer and high-level expression of human phenylalanine hydroxylase in primary hepatocytes

Ferritin expression in rat hepatocytes and Kupffer cells after lead nitrate treatment.

Science.gov (United States)

Fan, Yang; Yamada, Toshiyuki; Shimizu, Takeshi; Nanashima, Naoki; Akita, Miki; Suto, Kohji; Tsuchida, Shigeki

2009-02-01

Lead nitrate induces hepatocyte proliferation and subsequent apoptosis in rat livers. Iron is a constituent of heme and is also required for cell proliferation. In this study, the expression of ferritin light-chain (FTL), the major iron storage protein, was investigated in rat livers after a single intravenous injection of lead nitrate. Western blotting and immunohistochemistry revealed that FTL was increased in hepatocytes around the central veins and strongly expressed in nonparenchymal cells. Some FTL-positive nonparenchymal cells were identified as Kupffer cells that were positive for CD68. FTL-positive Kupffer cells occupied about 60% of CD68-positive cells in the periportal and perivenous areas. The relationships between FTL expression and apoptosis induction or the engulfment of apoptotic cells were examined. TUNEL-positive cells were increased in the treatment group, and enhanced expression of milk fat globule EGF-like 8 was demonstrated in some Kupffer cells and hepatocytes, indicating enhanced apoptosis induction and phagocytosis of apoptotic cells. FTL-positive Kupffer cells were not detected without lead nitrate treatment or in rat livers treated with clofibrate, which induces hepatocyte proliferation but not apoptosis. These results suggest that FTL expression in Kupffer cells after lead treatment is dependent on phagocytosis of apoptotic cells.

Resveratrol Differentially Regulates NAMPT and SIRT1 in Hepatocarcinoma Cells and Primary Human Hepatocytes

Science.gov (United States)

Schuster, Susanne; Penke, Melanie; Gorski, Theresa; Petzold-Quinque, Stefanie; Damm, Georg; Gebhardt, Rolf; Kiess, Wieland; Garten, Antje

2014-01-01

Resveratrol is reported to possess chemotherapeutic properties in several cancers. In this study, we wanted to investigate the molecular mechanisms of resveratrol-induced cell cycle arrest and apoptosis as well as the impact of resveratrol on NAMPT and SIRT1 protein function and asked whether there are differences in hepatocarcinoma cells (HepG2, Hep3B cells) and non-cancerous primary human hepatocytes. We found a lower basal NAMPT mRNA and protein expression in hepatocarcinoma cells compared to primary hepatocytes. In contrast, SIRT1 was significantly higher expressed in hepatocarcinoma cells than in primary hepatocytes. Resveratrol induced cell cycle arrest in the S- and G2/M- phase and apoptosis was mediated by activation of p53 and caspase-3 in HepG2 cells. In contrast to primary hepatocytes, resveratrol treated HepG2 cells showed a reduction of NAMPT enzymatic activity and increased p53 acetylation (K382). Resveratrol induced NAMPT release from HepG2 cells which was associated with increased NAMPT mRNA expression. This effect was absent in primary hepatocytes where resveratrol was shown to function as NAMPT and SIRT1 activator. SIRT1 inhibition by EX527 resembled resveratrol effects on HepG2 cells. Furthermore, a SIRT1 overexpression significantly decreased both p53 hyperacetylation and resveratrol-induced NAMPT release as well as S-phase arrest in HepG2 cells. We could show that NAMPT and SIRT1 are differentially regulated by resveratrol in hepatocarcinoma cells and primary hepatocytes and that resveratrol did not act as a SIRT1 activator in hepatocarcinoma cells. PMID:24603648

Resveratrol differentially regulates NAMPT and SIRT1 in Hepatocarcinoma cells and primary human hepatocytes.

Directory of Open Access Journals (Sweden)

Susanne Schuster

Full Text Available Resveratrol is reported to possess chemotherapeutic properties in several cancers. In this study, we wanted to investigate the molecular mechanisms of resveratrol-induced cell cycle arrest and apoptosis as well as the impact of resveratrol on NAMPT and SIRT1 protein function and asked whether there are differences in hepatocarcinoma cells (HepG2, Hep3B cells and non-cancerous primary human hepatocytes. We found a lower basal NAMPT mRNA and protein expression in hepatocarcinoma cells compared to primary hepatocytes. In contrast, SIRT1 was significantly higher expressed in hepatocarcinoma cells than in primary hepatocytes. Resveratrol induced cell cycle arrest in the S- and G2/M- phase and apoptosis was mediated by activation of p53 and caspase-3 in HepG2 cells. In contrast to primary hepatocytes, resveratrol treated HepG2 cells showed a reduction of NAMPT enzymatic activity and increased p53 acetylation (K382. Resveratrol induced NAMPT release from HepG2 cells which was associated with increased NAMPT mRNA expression. This effect was absent in primary hepatocytes where resveratrol was shown to function as NAMPT and SIRT1 activator. SIRT1 inhibition by EX527 resembled resveratrol effects on HepG2 cells. Furthermore, a SIRT1 overexpression significantly decreased both p53 hyperacetylation and resveratrol-induced NAMPT release as well as S-phase arrest in HepG2 cells. We could show that NAMPT and SIRT1 are differentially regulated by resveratrol in hepatocarcinoma cells and primary hepatocytes and that resveratrol did not act as a SIRT1 activator in hepatocarcinoma cells.

Optimization of the isolation and cultivation of Cyprinus carpio primary hepatocytes

OpenAIRE

Yanhong, Fan; Chenghua, He; Guofang, Liu; Haibin, Zhang

2008-01-01

The aquatic environment is affected by numerous chemical contaminants. There is an increasing need to identify these chemicals and to evaluate their potential toxicity towards aquatic life. In this research we optimized techniques for primary cell culture of Cyprinus carpio hepatocytes as one adjunct model for ecotoxicological evaluation of the potential hazards of xenobiotics in the aquatic environment. In this study, Cyprinus carpio hepatocytes were isolated by mechanical separation, two-st...

Magentic Cell labeling of primary and stem cell-derived pig hepatocytes for MRI-based cell tracking of heptocytes transplantation

Science.gov (United States)

Pig hepatocytes are an important investigational tool for optimizing hepatocyte transplantation schemes in both allogeneic and xenogeneic transplant scenarios. MRI can be used to serially monitor the transplanted cells, but only if the hepatocytes can be labeled with a magnetic particle. In this wo...

Prolongation of liver-specific function for primary hepatocytes maintenance in 3D printed architectures.

Science.gov (United States)

Kim, Yohan; Kang, Kyojin; Yoon, Sangtae; Kim, Ji Sook; Park, Su A; Kim, Wan Doo; Lee, Seung Bum; Ryu, Ki-Young; Jeong, Jaemin; Choi, Dongho

2018-01-23

Isolated primary hepatocytes from the liver are very similar to in vivo native liver hepatocytes, but they have the disadvantage of a limited lifespan in 2D culture. Although a sandwich culture and 3D organoids with mesenchymal stem cells (MSCs) as an attractive assistant cell source to extend lifespan can be used, it cannot fully reproduce the in vivo architecture. Moreover, long-term 3D culture leads to cell death because of hypoxic stress. Therefore, to overcome the drawback of 2D and 3D organoids, we try to use a 3D printing technique using alginate hydrogels with primary hepatocytes and MSCs. The viability of isolated hepatocytes was more than 90%, and the cells remained alive for 7 days without morphological changes in the 3D hepatic architecture with MSCs. Compared to a 2D system, the expression level of functional hepatic genes and proteins was higher for up to 7 days in the 3D hepatic architecture. These results suggest that both the 3D bio-printing technique and paracrine molecules secreted by MSCs supported long-term culture of hepatocytes without morphological changes. Thus, this technique allows for widespread expansion of cells while forming multicellular aggregates, may be applied to drug screening and could be an efficient method for developing an artificial liver.

Hanging Drop, A Best Three-Dimensional (3D) Culture Method for Primary Buffalo and Sheep Hepatocytes.

Science.gov (United States)

Shri, Meena; Agrawal, Himanshu; Rani, Payal; Singh, Dheer; Onteru, Suneel Kumar

2017-04-26

Livestock, having close resemblance to humans, could be a better source of primary hepatocytes than rodents. Herein, we successfully developed three-dimensional (3D) culturing system for primary sheep and buffalo hepatocytes. The 3D-structures of sheep hepatocytes were formed on the fifth-day and maintained until the tenth-day on polyHEMA-coated plates and in hanging drops with William's E media (HDW). Between the cultured and fresh cells, we observed a similar expression of GAPDH, HNF4α, ALB, CYP1A1, CK8 and CK18. Interestingly, a statistically significant increase was noted in the TAT, CPS, AFP, AAT, GSP and PCNA expression. In buffalo hepatocytes culture, 3D-like structures were formed on the third-day and maintained until the sixth-day on polyHEMA and HDW. The expression of HNF4α, GSP, CPS, AFP, AAT, PCNA and CK18 was similar between cultured and fresh cells. Further, a statistically significant increase in the TAT and CK8 expression, and a decrease in the GAPDH, CYP1A1 and ALB expression were noted. Among the culture systems, HDW maintained the liver transcript markers more or less similar to the fresh hepatocytes of the sheep and buffalo for ten and six days, respectively. Taken together, hanging drop is an efficient method for 3D culturing of primary sheep and buffalo hepatocytes.

Activation-dependent mitochondrial translocation of Foxp3 in human hepatocytes

International Nuclear Information System (INIS)

Rojas, Joselyn; Teran-Angel, Guillermo; Barbosa, Luisa; Peterson, Darrell L.; Berrueta, Lisbeth; Salmen, Siham

2016-01-01

Foxp3 is considered to be the master regulator for the development and function of regulatory T cells (Treg). Recently Foxp3, has been detected in extra lymphoid tissue, and in hepatocytes and has been associated with hepatocellular carcinoma (HCC), although its role has not been defined. Since it is expected that there is a relationship between protein localization, activity and cellular function, the aim of this study was to explore the subcellular localization of Foxp3 in resting and stimulated human hepatocytes. Foxp3 expression was measured by flow cytometry, subcellular fractioning, and immunofluorescence, and this data was used to track the shuttling of Foxp3 in different subcellular compartments in hepatocytes (HepG2 cell line), stimulated by using the PKC activators (PMA), core and preS1/2 antigen from hepatitis B virus (HBV). Our data shows that besides the nuclear location, mitochondrial translocation was detected after stimulation with PMA and at to a lesser extent, with preS1/2. In addition, Foxp3 is localizes at outer mitochondrial membrane. These results suggest a non-canonical role of Foxp3 in the mitochondrial compartment in human hepatocytes, and opens a new field about their role in liver damages during HBV infection. - Highlights: • The expression and subcellular distribution of Foxp3, is modulated by PMA and preS1/2. • PMA and preS1/2 increase Foxp3 expression on HepG2. • PMA and preS1/2 induce foxp3 enrichment at mitochondrial, microsomal and nuclear compartments. • Results suggest a non-canonical function of Foxp3 or a mitochondrial transcriptional activity.

Activation-dependent mitochondrial translocation of Foxp3 in human hepatocytes

Energy Technology Data Exchange (ETDEWEB)

Rojas, Joselyn; Teran-Angel, Guillermo; Barbosa, Luisa [Instituto de Inmunología Clínica, Facultad de Medicina, Universidad de Los Andes, Merida (Venezuela, Bolivarian Republic of); Peterson, Darrell L. [Department of Biochemistry and Molecular Biology, Virginia Commonwealth University, Richmond, VA (United States); Berrueta, Lisbeth, E-mail: lberruet@ula.ve [Instituto de Inmunología Clínica, Facultad de Medicina, Universidad de Los Andes, Merida (Venezuela, Bolivarian Republic of); Division of Preventive Medicine, Brigham and Women' s Hospital, Harvard Medical School, Boston, MA (United States); Salmen, Siham, E-mail: sihamsa@ula.ve [Instituto de Inmunología Clínica, Facultad de Medicina, Universidad de Los Andes, Merida (Venezuela, Bolivarian Republic of)

2016-05-01

Foxp3 is considered to be the master regulator for the development and function of regulatory T cells (Treg). Recently Foxp3, has been detected in extra lymphoid tissue, and in hepatocytes and has been associated with hepatocellular carcinoma (HCC), although its role has not been defined. Since it is expected that there is a relationship between protein localization, activity and cellular function, the aim of this study was to explore the subcellular localization of Foxp3 in resting and stimulated human hepatocytes. Foxp3 expression was measured by flow cytometry, subcellular fractioning, and immunofluorescence, and this data was used to track the shuttling of Foxp3 in different subcellular compartments in hepatocytes (HepG2 cell line), stimulated by using the PKC activators (PMA), core and preS1/2 antigen from hepatitis B virus (HBV). Our data shows that besides the nuclear location, mitochondrial translocation was detected after stimulation with PMA and at to a lesser extent, with preS1/2. In addition, Foxp3 is localizes at outer mitochondrial membrane. These results suggest a non-canonical role of Foxp3 in the mitochondrial compartment in human hepatocytes, and opens a new field about their role in liver damages during HBV infection. - Highlights: • The expression and subcellular distribution of Foxp3, is modulated by PMA and preS1/2. • PMA and preS1/2 increase Foxp3 expression on HepG2. • PMA and preS1/2 induce foxp3 enrichment at mitochondrial, microsomal and nuclear compartments. • Results suggest a non-canonical function of Foxp3 or a mitochondrial transcriptional activity.

A fast and robust hepatocyte quantification algorithm including vein processing

Directory of Open Access Journals (Sweden)

Homeyer André

2010-03-01

Full Text Available Abstract Background Quantification of different types of cells is often needed for analysis of histological images. In our project, we compute the relative number of proliferating hepatocytes for the evaluation of the regeneration process after partial hepatectomy in normal rat livers. Results Our presented automatic approach for hepatocyte (HC quantification is suitable for the analysis of an entire digitized histological section given in form of a series of images. It is the main part of an automatic hepatocyte quantification tool that allows for the computation of the ratio between the number of proliferating HC-nuclei and the total number of all HC-nuclei for a series of images in one processing run. The processing pipeline allows us to obtain desired and valuable results for a wide range of images with different properties without additional parameter adjustment. Comparing the obtained segmentation results with a manually retrieved segmentation mask which is considered to be the ground truth, we achieve results with sensitivity above 90% and false positive fraction below 15%. Conclusions The proposed automatic procedure gives results with high sensitivity and low false positive fraction and can be applied to process entire stained sections.

Stathmin Mediates Hepatocyte Resistance to Death from Oxidative Stress by down Regulating JNK

Science.gov (United States)

Zhao, Enpeng; Amir, Muhammad; Lin, Yu; Czaja, Mark J.

2014-01-01

Stathmin 1 performs a critical function in cell proliferation by regulating microtubule polymerization. This proliferative function is thought to explain the frequent overexpression of stathmin in human cancer and its correlation with a bad prognosis. Whether stathmin also functions in cell death pathways is unclear. Stathmin regulates microtubules in part by binding free tubulin, a process inhibited by stathmin phosphorylation from kinases including c-Jun N-terminal kinase (JNK). The involvement of JNK activation both in stathmin phosphorylation, and in hepatocellular resistance to oxidative stress, led to an examination of the role of stathmin/JNK crosstalk in oxidant-induced hepatocyte death. Oxidative stress from menadione-generated superoxide induced JNK-dependent stathmin phosphorylation at Ser-16, Ser-25 and Ser-38 in hepatocytes. A stathmin knockdown sensitized hepatocytes to both apoptotic and necrotic cell death from menadione without altering levels of oxidant generation. The absence of stathmin during oxidative stress led to JNK overactivation that was the mechanism of cell death as a concomitant knockdown of JNK1 or JNK2 blocked death. Hepatocyte death from JNK overactivation was mediated by the effects of JNK on mitochondria. Mitochondrial outer membrane permeabilization occurred in stathmin knockdown cells at low concentrations of menadione that triggered apoptosis, whereas mitochondrial β-oxidation and ATP homeostasis were compromised at higher, necrotic menadione concentrations. Stathmin therefore mediates hepatocyte resistance to death from oxidative stress by down regulating JNK and maintaining mitochondrial integrity. These findings demonstrate a new mechanism by which stathmin promotes cell survival and potentially tumor growth. PMID:25285524

Stathmin mediates hepatocyte resistance to death from oxidative stress by down regulating JNK.

Directory of Open Access Journals (Sweden)

Enpeng Zhao

Full Text Available Stathmin 1 performs a critical function in cell proliferation by regulating microtubule polymerization. This proliferative function is thought to explain the frequent overexpression of stathmin in human cancer and its correlation with a bad prognosis. Whether stathmin also functions in cell death pathways is unclear. Stathmin regulates microtubules in part by binding free tubulin, a process inhibited by stathmin phosphorylation from kinases including c-Jun N-terminal kinase (JNK. The involvement of JNK activation both in stathmin phosphorylation, and in hepatocellular resistance to oxidative stress, led to an examination of the role of stathmin/JNK crosstalk in oxidant-induced hepatocyte death. Oxidative stress from menadione-generated superoxide induced JNK-dependent stathmin phosphorylation at Ser-16, Ser-25 and Ser-38 in hepatocytes. A stathmin knockdown sensitized hepatocytes to both apoptotic and necrotic cell death from menadione without altering levels of oxidant generation. The absence of stathmin during oxidative stress led to JNK overactivation that was the mechanism of cell death as a concomitant knockdown of JNK1 or JNK2 blocked death. Hepatocyte death from JNK overactivation was mediated by the effects of JNK on mitochondria. Mitochondrial outer membrane permeabilization occurred in stathmin knockdown cells at low concentrations of menadione that triggered apoptosis, whereas mitochondrial β-oxidation and ATP homeostasis were compromised at higher, necrotic menadione concentrations. Stathmin therefore mediates hepatocyte resistance to death from oxidative stress by down regulating JNK and maintaining mitochondrial integrity. These findings demonstrate a new mechanism by which stathmin promotes cell survival and potentially tumor growth.

Hepatocyte membrane injury and bleb formation following low dose comfrey toxicity in rats.

Science.gov (United States)

Yeong, M L; Wakefield, S J; Ford, H C

1993-04-01

Comfrey, a popular herbal remedy, contains hepatotoxic pyrrolizidine alkaloids and has been implicated in recent human toxicity. Although alkaloids from other plant sources have been extensively researched, studies on the hepatotoxic effects of comfrey alkaloids are scant. The effects of high dose comfrey toxicity have been studied and the present investigation was undertaken to identify changes associated with relatively low dose toxicity. Eight young adult rats were dosed weekly for six weeks with 50 mg/kg of comfrey derived alkaloids. The animals were dissected one week after the last dose and the livers examined by light and electron microscopy. Changes at the light microscopic level showed vascular congestion, mild zone 3 necrosis and loss of definition of hepatocyte cellular membranes. Extensive ultrastructural abnormalities were identified in the form of endothelial sloughing and the loss of hepatocyte microvilli. A striking finding was florid bleb formation on the sinusoidal borders of hepatocytes. Many blebs were shed into the space of Disse and extruded to fill, and sometimes occlude, sinusoidal lumina. Platelets were frequently found in areas of bleb formation. There was evidence of late damage in collagenization of Disse's space. Hepatocyte bleb formation is known to occur under a variety of pathological conditions but there is little to no information in the literature on the effects, if any, of bleb formation on fibrogenesis and the microcirculation and its role in the pathogenesis of liver disease. The pyrrolizidine alkaloids of comfrey may serve as an experimental tool to study the process of bleb formation and the intimate relationship between hepatocyte and sinusoidal injury in the liver.

Hepatocyte-based in vitro model for assessment of drug-induced cholestasis

Energy Technology Data Exchange (ETDEWEB)

Chatterjee, Sagnik, E-mail: Sagnik.Chatterjee@pharm.kuleuven.be [Drug Delivery and Disposition, KU Leuven Department of Pharmaceutical and Pharmacological Sciences, O and N2, Herestraat 49 — bus 921, 3000 Leuven (Belgium); Richert, Lysiane, E-mail: l.richert@kaly-cell.com [KaLy-Cell, 20A rue du Général Leclerc, 67115 Plobsheim (France); Augustijns, Patrick, E-mail: Patrick.Augustijns@pharm.kuleuven.be [Drug Delivery and Disposition, KU Leuven Department of Pharmaceutical and Pharmacological Sciences, O and N2, Herestraat 49 — bus 921, 3000 Leuven (Belgium); Annaert, Pieter, E-mail: Pieter.Annaert@pharm.kuleuven.be [Drug Delivery and Disposition, KU Leuven Department of Pharmaceutical and Pharmacological Sciences, O and N2, Herestraat 49 — bus 921, 3000 Leuven (Belgium)

2014-01-01

Early detection of drug-induced cholestasis remains a challenge during drug development. We have developed and validated a biorelevant sandwich-cultured hepatocytes- (SCH) based model that can identify compounds causing cholestasis by altering bile acid disposition. Human and rat SCH were exposed (24–48 h) to known cholestatic and/or hepatotoxic compounds, in the presence or in the absence of a concentrated mixture of bile acids (BAs). Urea assay was used to assess (compromised) hepatocyte functionality at the end of the incubations. The cholestatic potential of the compounds was expressed by calculating a drug-induced cholestasis index (DICI), reflecting the relative residual urea formation by hepatocytes co-incubated with BAs and test compound as compared to hepatocytes treated with test compound alone. Compounds with clinical reports of cholestasis, including cyclosporin A, troglitazone, chlorpromazine, bosentan, ticlopidine, ritonavir, and midecamycin showed enhanced toxicity in the presence of BAs (DICI ≤ 0.8) for at least one of the tested concentrations. In contrast, the in vitro toxicity of compounds causing hepatotoxicity by other mechanisms (including diclofenac, valproic acid, amiodarone and acetaminophen), remained unchanged in the presence of BAs. A safety margin (SM) for drug-induced cholestasis was calculated as the ratio of lowest in vitro concentration for which was DICI ≤ 0.8, to the reported mean peak therapeutic plasma concentration. SM values obtained in human SCH correlated well with reported % incidence of clinical drug-induced cholestasis, while no correlation was observed in rat SCH. This in vitro model enables early identification of drug candidates causing cholestasis by disturbed BA handling. - Highlights: • Novel in vitro assay to detect drug-induced cholestasis • Rat and human sandwich-cultured hepatocytes (SCH) as in vitro models • Cholestatic compounds sensitize SCH to toxic effects of accumulating bile acids • Drug

Hepatocyte-based in vitro model for assessment of drug-induced cholestasis

International Nuclear Information System (INIS)

Chatterjee, Sagnik; Richert, Lysiane; Augustijns, Patrick; Annaert, Pieter

2014-01-01

Early detection of drug-induced cholestasis remains a challenge during drug development. We have developed and validated a biorelevant sandwich-cultured hepatocytes- (SCH) based model that can identify compounds causing cholestasis by altering bile acid disposition. Human and rat SCH were exposed (24–48 h) to known cholestatic and/or hepatotoxic compounds, in the presence or in the absence of a concentrated mixture of bile acids (BAs). Urea assay was used to assess (compromised) hepatocyte functionality at the end of the incubations. The cholestatic potential of the compounds was expressed by calculating a drug-induced cholestasis index (DICI), reflecting the relative residual urea formation by hepatocytes co-incubated with BAs and test compound as compared to hepatocytes treated with test compound alone. Compounds with clinical reports of cholestasis, including cyclosporin A, troglitazone, chlorpromazine, bosentan, ticlopidine, ritonavir, and midecamycin showed enhanced toxicity in the presence of BAs (DICI ≤ 0.8) for at least one of the tested concentrations. In contrast, the in vitro toxicity of compounds causing hepatotoxicity by other mechanisms (including diclofenac, valproic acid, amiodarone and acetaminophen), remained unchanged in the presence of BAs. A safety margin (SM) for drug-induced cholestasis was calculated as the ratio of lowest in vitro concentration for which was DICI ≤ 0.8, to the reported mean peak therapeutic plasma concentration. SM values obtained in human SCH correlated well with reported % incidence of clinical drug-induced cholestasis, while no correlation was observed in rat SCH. This in vitro model enables early identification of drug candidates causing cholestasis by disturbed BA handling. - Highlights: • Novel in vitro assay to detect drug-induced cholestasis • Rat and human sandwich-cultured hepatocytes (SCH) as in vitro models • Cholestatic compounds sensitize SCH to toxic effects of accumulating bile acids • Drug

Hepatocyte Aggregate Formation on Chitin-Based Anisotropic Microstructures of Butterfly Wings

Directory of Open Access Journals (Sweden)

Abdelrahman Elbaz

2018-01-01

Full Text Available Scaffold nanotopography plays the most significant role in the mimicry of the in vivo microenvironment of the hepatocytes. Several attempts have been made to develop methods and substrates suited to growing hepatocytes into aggregates. Functional biomaterials, particularly biodegradable polymers, have been used in several studies aimed to develop improved scaffolds with ordered geometry and nanofibrous architecture for tissue engineering. However, there are still some limitation in their fabrication: it is not cost-efficient, is time-consuming, and exhibits some technological complications. The synthetic scaffolds are usually non-biodegradable and can be non-biocompatible compared to the naturally derived biomaterials. Here, we utilized a simple, cost-effective, and green method with two-step chemical treatment to get more selected hydrophilic butterfly wings from Morpho menelaus, Papilio ulysses telegonus, and Ornithoptera croesus lydius as a chitin-based natural scaffolds to growing hepatocyte aggregates. We established a three-dimensional (3D in vitro model for culture of HepG2 cells and aggregate formation that maintained the hepatocytes function on these natural anisotropic microstructures. Cells cultured on these substrates show higher viability than those cultured on a two-dimensional (2D culture plate. Methylthiazolyldiphenyl-tetrazolium bromide (MTT assay results revealed excellent viability of HepG2 cells on P. u. telegonus wings (fibrous area. The results also demonstrated appropriate cell activity, cell retention, and stable and functional expression in terms of albumin secretion and urea synthesis activity compared to the 2D monolayer culture of hepatocytes on the culture dish surface. With a slightly different degree, the other substrates also shown similar results. We anticipate that these natural anisotropic, biodegradable, and biocompatible substrates can maintain long-term hepatic culture as an in vitro 3D model for potential

Oncostatin M induces upregulation of claudin-2 in rodent hepatocytes coinciding with changes in morphology and function of tight junctions

International Nuclear Information System (INIS)

Imamura, Masafumi; Kojima, Takashi; Lan, Mengdong; Son, Seiichi; Murata, Masaki; Osanai, Makoto; Chiba, Hideki; Hirata, Koichi; Sawada, Norimasa

2007-01-01

In rodent livers, integral tight junction (TJ) proteins claudin-1, -2, -3, -5 and -14 are detected and play crucial roles in the barrier to keep bile in bile canaculi away from the blood circulation. Claudin-2 shows a lobular gradient increasing from periportal to pericentral hepatocytes, whereas claudin-1 and -3 are expressed in the whole liver lobule. Although claudin-2 expression induces cation-selective channels in tight junctions of epithelial cells, the physiological functions and regulation of claudin-2 in hepatocytes remain unclear. Oncostatin M (OSM) is a multifunctional cytokine implicated in the differentiation of hepatocytes that induces formation of E-cadherin-based adherens junctions in fetal hepatocytes. In this study, we examined whether OSM could induce expression and function of claudin-2 in rodent hepatocytes, immortalized mouse and primary cultured proliferative rat hepatocytes. In the immortalized mouse and primary cultured proliferative rat hepatocytes, treatment with OSM markedly increased mRNA and protein of claudin-2 together with formation of developed networks of TJ strands. The increase of claudin-2 enhanced the paracellular barrier function which depended on molecular size. The increase of claudin-2 expression induced by OSM in rodent hepatocytes was regulated through distinct signaling pathways including PKC. These results suggest that expression of claudin-2 in rodent hepatocytes may play a specific role as controlling the size of paracellular permeability in the barrier to keep bile in bile canaculi

Acrolein cytotoxicity in hepatocytes involves endoplasmic reticulum stress, mitochondrial dysfunction and oxidative stress

International Nuclear Information System (INIS)

Mohammad, Mohammad K.; Avila, Diana; Zhang, Jingwen; Barve, Shirish; Arteel, Gavin; McClain, Craig; Joshi-Barve, Swati

2012-01-01

Acrolein is a common environmental, food and water pollutant and a major component of cigarette smoke. Also, it is produced endogenously via lipid peroxidation and cellular metabolism of certain amino acids and drugs. Acrolein is cytotoxic to many cell types including hepatocytes; however the mechanisms are not fully understood. We examined the molecular mechanisms underlying acrolein hepatotoxicity in primary human hepatocytes and hepatoma cells. Acrolein, at pathophysiological concentrations, caused a dose-dependent loss of viability of hepatocytes. The death was apoptotic at moderate and necrotic at high concentrations of acrolein. Acrolein exposure rapidly and dramatically decreased intracellular glutathione and overall antioxidant capacity, and activated the stress-signaling MAP-kinases JNK, p42/44 and p38. Our data demonstrate for the first time in human hepatocytes, that acrolein triggered endoplasmic reticulum (ER) stress and activated eIF2α, ATF-3 and -4, and Gadd153/CHOP, resulting in cell death. Notably, the protective/adaptive component of ER stress was not activated, and acrolein failed to up-regulate the protective ER-chaperones, GRP78 and GRP94. Additionally, exposure to acrolein disrupted mitochondrial integrity/function, and led to the release of pro-apoptotic proteins and ATP depletion. Acrolein-induced cell death was attenuated by N-acetyl cysteine, phenyl-butyric acid, and caspase and JNK inhibitors. Our data demonstrate that exposure to acrolein induces a variety of stress responses in hepatocytes, including GSH depletion, oxidative stress, mitochondrial dysfunction and ER stress (without ER-protective responses) which together contribute to acrolein toxicity. Our study defines basic mechanisms underlying liver injury caused by reactive aldehyde pollutants such as acrolein. -- Highlights: ► Human primary hepatocytes and cultured cell lines are used. ► Multiple cell death signaling pathways are activated by acrolein. ► Novel finding of

Acrolein cytotoxicity in hepatocytes involves endoplasmic reticulum stress, mitochondrial dysfunction and oxidative stress

Energy Technology Data Exchange (ETDEWEB)

Mohammad, Mohammad K. [Department of Medicine, University of Louisville (United States); Alcohol Research Center, University of Louisville (United States); Avila, Diana [Department of Medicine, University of Louisville (United States); Department of Pharmacology and Toxicology, University of Louisville (United States); Alcohol Research Center, University of Louisville (United States); Zhang, Jingwen [Department of Medicine, University of Louisville (United States); Alcohol Research Center, University of Louisville (United States); Barve, Shirish [Department of Medicine, University of Louisville (United States); Department of Pharmacology and Toxicology, University of Louisville (United States); Alcohol Research Center, University of Louisville (United States); Arteel, Gavin [Department of Pharmacology and Toxicology, University of Louisville (United States); Alcohol Research Center, University of Louisville (United States); McClain, Craig [Department of Medicine, University of Louisville (United States); Department of Pharmacology and Toxicology, University of Louisville (United States); Alcohol Research Center, University of Louisville (United States); Robley Rex VAMC, Louisville, KY (United States); Joshi-Barve, Swati, E-mail: s0josh01@louisville.edu [Department of Medicine, University of Louisville (United States); Department of Pharmacology and Toxicology, University of Louisville (United States); Alcohol Research Center, University of Louisville (United States)

2012-11-15

Acrolein is a common environmental, food and water pollutant and a major component of cigarette smoke. Also, it is produced endogenously via lipid peroxidation and cellular metabolism of certain amino acids and drugs. Acrolein is cytotoxic to many cell types including hepatocytes; however the mechanisms are not fully understood. We examined the molecular mechanisms underlying acrolein hepatotoxicity in primary human hepatocytes and hepatoma cells. Acrolein, at pathophysiological concentrations, caused a dose-dependent loss of viability of hepatocytes. The death was apoptotic at moderate and necrotic at high concentrations of acrolein. Acrolein exposure rapidly and dramatically decreased intracellular glutathione and overall antioxidant capacity, and activated the stress-signaling MAP-kinases JNK, p42/44 and p38. Our data demonstrate for the first time in human hepatocytes, that acrolein triggered endoplasmic reticulum (ER) stress and activated eIF2α, ATF-3 and -4, and Gadd153/CHOP, resulting in cell death. Notably, the protective/adaptive component of ER stress was not activated, and acrolein failed to up-regulate the protective ER-chaperones, GRP78 and GRP94. Additionally, exposure to acrolein disrupted mitochondrial integrity/function, and led to the release of pro-apoptotic proteins and ATP depletion. Acrolein-induced cell death was attenuated by N-acetyl cysteine, phenyl-butyric acid, and caspase and JNK inhibitors. Our data demonstrate that exposure to acrolein induces a variety of stress responses in hepatocytes, including GSH depletion, oxidative stress, mitochondrial dysfunction and ER stress (without ER-protective responses) which together contribute to acrolein toxicity. Our study defines basic mechanisms underlying liver injury caused by reactive aldehyde pollutants such as acrolein. -- Highlights: ► Human primary hepatocytes and cultured cell lines are used. ► Multiple cell death signaling pathways are activated by acrolein. ► Novel finding of

Serum Hepatocyte Growth Factor as A Non-Invasive Marker For Evaluation of Hepatocellular Carcinoma

International Nuclear Information System (INIS)

Abdelgawad, M.R.; Wahba, M.A.

2012-01-01

The change and the prognostic value of serum hepatocyte growth factor and AFP level in patients with cirrhosis and/or primary liver cancer (HCC) were investigated. The level of serum hepatocyte growth factor was determined by using enzyme-linked immunosorbent assay, and AFP was determined by using radioimmunoassay in 29 patients with cirrhosis. Twenty five patients with primary liver cancer (13 patients without nodular cirrhosis and 12 patients with nodular cirrhosis) were categorized according to tumour size (≤ or >5 cm) and the level of AFP (≤ or > 200 ng/dl). The correlation between serum AFP and hepatocyte growth factor were significantly increased (P 0.05). Serum AFP can significantly discriminate between all studied groups (P 0.001) except for the comparison between control and cirrhosis (P>0.05), and also between HCC and HCC without nodular cirrhosis and HCC with cirrhosis (P>0.05). Serum HGF and AFP levels were positively affected by tumour size and nodular cirrhosis (P<0.001). Also, serum HGF level was highly affected by the levels of serum AFP in HCC patients. Non-significant correlation was observed between serum hepatocyte growth factor and AFP in control, cirrhosis, cirrhosis and HCC patients with AFP ? 200 ng/dl. It could be concluded that the over expressions of the hepatocyte growth factor and AFP may indicate an adverse prognosis for patients with cirrhosis and/or liver cancer. The sustained high level of serum hepatocyte growth factor in cirrhosis and/or HCC could be considered a factor related to early tumour diagnosis, so, serum HGF level may be used as a non-invasive marker in diagnosis and prognosis of liver malignancy. However, further studies are highly recommended to evaluate the role of HGF or its constituents in diagnosis and/or therapy in the future in a larger cohort of patients with different stages of liver malignancy

Computational Topology Based Quantification Of Hepatocytes Nuclei In Lipopolysaccharide-Induced Liver Injury In Mice

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Rodrigo Rojas Moraleda

2016-06-01

The computational topology approach proposed successfully detected hepatocyte cells under several natural variations. We evaluated on a per-pixel basis how the segmentation performs on: i all nuclei in the images, ii big round nuclei considered belonging to hepatocytes cells (accuracy 87.2%, recall 80.3%, and iii nuclei regarded to non-parenchymal cells.  

In vitro differentiation and maturation of mouse embryonic stem cells into hepatocytes

International Nuclear Information System (INIS)

Ishii, Takamichi; Yasuchika, Kentaro; Fujii, Hideaki; Hoppo, Toshitaka; Baba, Shinji; Naito, Masato; Machimoto, Takafumi; Kamo, Naoko; Suemori, Hirofumi; Nakatsuji, Norio; Ikai, Iwao

2005-01-01

It is difficult to induce the maturation of embryonic stem (ES) cells into hepatocytes in vitro. We previously reported that Thy1-positive mesenchymal cells derived from the mouse fetal liver promote the maturation of hepatic progenitor cells. Here, we isolated alpha-fetoprotein (AFP)-producing cells from mouse ES cells for subsequent differentiation into hepatocytes in vitro by coculture with Thy1-positive cells. ES cells expressing green fluorescent protein (GFP) under the control of an AFP promoter were cultured under serum- and feeder layer-free culture conditions. The proportion of GFP-positive cells plateaued at 41.6 ± 12.2% (means ± SD) by day 7. GFP-positive cells, isolated by flow cytometry, were cultured in the presence or absence of Thy1-positive cells as a feeder layer. Isolated GFP-positive cells were stained for AFP, Foxa2, and albumin. The expression of mRNAs encoding tyrosine amino transferase, tryptophan 2,3-dioxygenase, and glucose-6-phosphatase were only detected following coculture with Thy1-positive cells. Following coculture with Thy1-positive cells, the isolated cells produced and stored glycogen. Ammonia clearance activity was also enhanced following coculture. Electron microscopic analysis indicated that the cocultured cells exhibited the morphologic features of mature hepatocytes. In conclusion, coculture with Thy1-positive cells in vitro induced the maturation of AFP-producing cells isolated from ES cell cultures into hepatocytes

Purification, characterisation and protective effects of polysaccharides from alfalfa on hepatocytes.

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Wang, Shaopu; Dong, Xiaofang; Ma, Hao; Cui, Yaoming; Tong, Jianming

2014-11-04

The objective of this study was to determine the preliminary characteristics and protective effects of alfalfa polysaccharides (APS) on hepatocytes in vitro. The crude APS was purified by DEAE-cellulose and Sephadex G-100 chromatography, resulting in the four purified fractions: APS-1, APS-2, APS-3 and APS-4. The results indicated that APS-3 had higher carbohydrate and uronic acid contents and that APS-4 had a more complicated monosaccharide composition compared to the other purified fractions. The average molecular weights of APS-1, APS-2, APS-3 and APS-4 were 48,536, 6,221, 66,559 and 13,076 Da, respectively. Furthermore, APS (crude and its purified fractions) restored the activities of antioxidant enzymes and increased the total antioxidant capacity of hepatocytes subjected to H2O2-induced oxidative stress. Furthermore, APS treatment counteracted the increases in lactic dehydrogenase and malonaldehyde in the culture supernatant. These results clearly demonstrate that APS possesses a protective effect against oxidative injury in hepatocytes. Copyright © 2014 Elsevier Ltd. All rights reserved.

The regulation of cytoskeletal and liver-specific gene expression during liver regeneration and primary hepatocyte culture

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Robinson, G.S.

1989-01-01

The focus of this dissertation is to determine what role(s) the extracellular matrix and expression of certain cytoskeletal genes play in the regulation of hepatocyte growth and the maintenance of a differential state. The expression of several cytoskeletal and liver-specific genes was examined during liver regeneration and in hepatocyte cultures maintained in a hormonally-defined, serum-free medium and plated on two different matrices: rat tail collagen and the EHS matrix. During liver regeneration and in hepatocytes cultured on rat tail collagen, there was a dramatic increase in tubulin mRNA levels coincident with but not linked to DNA synthesis. The message levels for other cytoskeletal genes similarly increased, while a decrease was observed in the mRNA levels of the liver-specific genes, serum albumin and alpha 1 inhibitor III. Hepatocytes cultured on the EHS matrix resulted in the maintenance of low levels of cytoskeletal gene expression and high levels of liver-specific gene expression, similar to that observed in the normal liver. Results from subcellar fractionation and two-dimensional gel electrophoresis of 35 S-labelled proteins paralleled the results seen at the mRNA level. Preliminary work suggests that microtubule organization may play a role in the expression of the liver-specific genes which encode secreted proteins. These studies, which compare hepatocytes cultured on collagen or the EHS matrix gel, reveal that both cell-cell and cell-matrix interactions play a major role in the maintenance of the differential phenotype in hepatocytes

Hepatic Stellate Cell-Derived Microvesicles Prevent Hepatocytes from Injury Induced by APAP/H2O2

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Renwei Huang

2016-01-01

Full Text Available Hepatic stellate cells (HSCs, previously described for liver-specific mesenchymal stem cells (MSCs, appear to contribute to liver regeneration. Microvesicles (MVs are nanoscale membrane fragments, which can regulate target cell function by transferring contents from their parent cells. The aim of this study was to investigate the effect of HSC-derived MVs on xenobiotic-induced liver injury. Rat and human hepatocytes, BRL-3A and HL-7702, were used to build hepatocytes injury models by n-acetyl-p-aminophenol n-(APAP or H2O2 treatment. MVs were prepared from human and rat HSCs, LX-2, and HST-T6 and, respectively, added to injured BRL-3A and HL-7702 hepatocytes. MTT assay was utilized to determine cell proliferation. Cell apoptosis was analyzed by flow cytometry and hoechst33258 staining. Western blot was used for analyzing the expression of activated caspase-3. Liver injury indicators, alanine aminotransferase (ALT, aspartate aminotransferase (AST, and lactate dehydrogenase (LDH in culture medium were also assessed. Results showed that (1 HSC-MVs derived from LX-2 and HST-T6 were positive to CD90 and annexin V surface markers; (2 HSC-MVs dose-dependently improved the viability of hepatocytes in both injury models; (3 HSC-MVs dose-dependently inhibited the APAP/H2O2 induced hepatocytes apoptosis and activated caspase-3 expression and leakage of LDH, ALT, and AST. Our results demonstrate that HSC-derived MVs protect hepatocytes from toxicant-induced injury.

Nor-ursodeoxycholic acid reverses hepatocyte-specific nemo-dependent steatohepatitis.

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Beraza, Naiara; Ofner-Ziegenfuss, Lisa; Ehedego, Haksier; Boekschoten, Mark; Bischoff, Stephan C; Mueller, Michael; Trauner, Michael; Trautwein, Christian

2011-03-01

Hepatocyte-specific NEMO/NF-κB deleted mice (NEMO(Δhepa)) develop spontaneous non-alcoholic steatohepatitis (NASH). Free fatty acids and bile acids promote DR5 expression. TRAIL/NK cell-mediated activation of TRAIL-R2/DR5 plays an important role during acute injury in NEMO(Δhepa) mice. To inhibit the progression of NASH in the absence of hepatocyte-NEMO/NF-kB signaling. NEMOf/f and NEMO(Δhepa) mice were fed with a low-fat diet, and with two anticholestatic diets; UDCA and NorUDCA. The impact of these treatments on the progression of NASH was evaluated. We show that high expression of DR5 in livers from NEMO(Δhepa) mice is accompanied by an abundant presence of bile acids (BAs), misregulation of BA transporters and significant alteration of lipid metabolism-related genes. Additionally, mice lacking NEMO in hepatocytes spontaneously showed ductular response at young age. Unexpectedly, feeding of NEMO(Δhepa) mice with low-fat diet failed to improve chronic liver injury. Conversely, anti-cholestatic treatment with nor-ursodeoxycholic acid (NorUDCA), but not with ursodeoxycholic acid (UDCA), led to a significant attenuation of liver damage in NEMO(Δhepa) mice. The strong therapeutic effect of NorUDCA relied on a significant downregulation of LXR-dependent lipogenesis and the normalisation of BA metabolism through mechanisms involving cross-talk between Cyp7a1 and SHP. This was associated with the significant improvement of liver histology, NEMO(Δhepa)/NorUDCA-treated mice showed lower apoptosis and reduced CyclinD1 expression, indicating attenuation of the compensatory proliferative response to hepatocellular damage. Finally, fibrosis and ductular reaction markers were significantly reduced in NorUDCA-treated NEMO(Δhepa) mice. Overall, our work demonstrates the contribution of bile acids metabolism to the progression of NASH in the absence of hepatocyte-NF-kB through mechanisms involving DR5-apoptosis, inflammation and fibrosis. Our work suggests a potential

Acrolein cytotoxicity in hepatocytes involves endoplasmic reticulum stress, mitochondrial dysfunction and oxidative stress

OpenAIRE

Mohammad, Mohammad K; Avila, Diana; Zhang, Jingwen; Barve, Shirish; Arteel, Gavin; McClain, Craig; Joshi-Barve, Swati

2012-01-01

Acrolein is a common environmental, food and water pollutant and a major component of cigarette smoke. Also, it is produced endogenously via lipid peroxidation and cellular metabolism of certain amino acids and drugs. Acrolein is cytotoxic to many cell types including hepatocytes; however the mechanisms are not fully understood. We examined the molecular mechanisms underlying acrolein hepatotoxicity in primary human hepatocytes and hepatoma cells. Acrolein, at pathophysiological concentration...

Generation of human pluripotent stem cell-derived hepatocyte-like cells for drug toxicity screening.

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Takayama, Kazuo; Mizuguchi, Hiroyuki

2017-02-01

Because drug-induced liver injury is one of the main reasons for drug development failures, it is important to perform drug toxicity screening in the early phase of pharmaceutical development. Currently, primary human hepatocytes are most widely used for the prediction of drug-induced liver injury. However, the sources of primary human hepatocytes are limited, making it difficult to supply the abundant quantities required for large-scale drug toxicity screening. Therefore, there is an urgent need for a novel unlimited, efficient, inexpensive, and predictive model which can be applied for large-scale drug toxicity screening. Human embryonic stem (ES) cells and induced pluripotent stem (iPS) cells are able to replicate indefinitely and differentiate into most of the body's cell types, including hepatocytes. It is expected that hepatocyte-like cells generated from human ES/iPS cells (human ES/iPS-HLCs) will be a useful tool for drug toxicity screening. To apply human ES/iPS-HLCs to various applications including drug toxicity screening, homogenous and functional HLCs must be differentiated from human ES/iPS cells. In this review, we will introduce the current status of hepatocyte differentiation technology from human ES/iPS cells and a novel method to predict drug-induced liver injury using human ES/iPS-HLCs. Copyright © 2016 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.

Acquisition of lipid metabolic capability in hepatocyte-like cells directly induced from mouse fibroblasts

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Shizuka eMiura

2014-08-01

Full Text Available Recently, the numbers of patients with non-alcoholic fatty liver disease (NAFLD and non-alcoholic steatohepatitis (NASH have increased worldwide. NAFLD and NASH are known as risk factors for liver cirrhosis and hepatocellular carcinoma. Because many factors can promote the progression of NAFLD and NASH, the treatment of these patients involves various strategies. Thus, it is desired that drugs for patients with NAFLD and NASH should be developed more easily and rapidly using cultures of primary hepatocytes. However, it is difficult to use hepatocytes as a tool for drug screening, because these cells cannot be functionally maintained in culture. Thus, in this study, we sought to examine whether induced hepatocyte-like (iHep cells, which were directly induced from mouse dermal fibroblasts by infection with a retrovirus expressing Hnf4α and Foxa3, possess the potential for lipid metabolism, similar to hepatocytes. Our data showed that iHep cells were capable of synthesizing lipids from a cis-unsaturated fatty acid, a trans-unsaturated fatty acid, and a saturated fatty acid, accumulating the synthesized lipids in cellular vesicles, and secreting the lipids into the culture medium. Moreover, the lipid synthesis in iHep cells was significantly inhibited in cultures with lipid metabolism improvers. These results demonstrate that iHep cells could be useful not only for screening of drugs for patients with NAFLD and NASH, but also for elucidation of the mechanisms underlying hereditary lipid metabolism disorders, as an alternative to hepatocytes.

Generation, characterization and potential therapeutic applications of mature and functional hepatocytes from stem cells.

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Zhang, Zhenzhen; Liu, Jianfang; Liu, Yang; Li, Zheng; Gao, Wei-Qiang; He, Zuping

2013-02-01

Liver cancer is the sixth most common tumor in the world and the majority of patients with this disease usually die within 1 year. The effective treatment for end-stage liver disease (also known as liver failure), including liver cancer or cirrhosis, is liver transplantation. However, there is a severe shortage of liver donors worldwide, which is the major handicap for the treatment of patients with liver failure. Scarcity of liver donors underscores the urgent need of using stem cell therapy to the end-stage liver disease. Notably, hepatocytes have recently been generated from hepatic and extra-hepatic stem cells. We have obtained mature and functional hepatocytes from rat hepatic stem cells. Here, we review the advancements on hepatic differentiation from various stem cells, including hepatic stem cells, embryonic stem cells, the induced pluripotent stem cells, hematopoietic stem cells, mesenchymal stem cells, and probably spermatogonial stem cells. The advantages, disadvantages, and concerns on differentiation of these stem cells into hepatic cells are highlighted. We further address the methodologies, phenotypes, and functional characterization on the differentiation of numerous stem cells into hepatic cells. Differentiation of stem cells into mature and functional hepatocytes, especially from an extra-hepatic stem cell source, would circumvent the scarcity of liver donors and human hepatocytes, and most importantly it would offer an ideal and promising source of hepatocytes for cell therapy and tissue engineering in treating liver disease. Copyright © 2012 Wiley Periodicals, Inc.

Hepatitis B virus evasion from cGAS sensing in human hepatocytes.

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Verrier, Eloi R; Yim, Seung-Ae; Heydmann, Laura; El Saghire, Houssein; Bach, Charlotte; Turon-Lagot, Vincent; Mailly, Laurent; Durand, Sarah C; Lucifora, Julie; Durantel, David; Pessaux, Patrick; Manel, Nicolas; Hirsch, Ivan; Zeisel, Mirjam B; Pochet, Nathalie; Schuster, Catherine; Baumert, Thomas F

2018-04-20

Chronic hepatitis B virus (HBV) infection is a major cause of chronic liver disease and cancer worldwide. The mechanisms of viral genome sensing and the evasion of innate immune responses by HBV infection are still poorly understood. Recently, the cyclic GMP-AMP synthase (cGAS) was identified as a DNA sensor. In this study, we aimed to investigate the functional role of cGAS in sensing of HBV infection and elucidate the mechanisms of viral evasion. We performed functional studies including loss- and gain-of-function experiments combined with cGAS effector gene expression profiling in an infectious cell culture model, primary human hepatocytes and HBV-infected human liver chimeric mice. Here we show that cGAS is expressed in the human liver, primary human hepatocytes and human liver chimeric mice. While naked relaxed-circular HBV DNA is sensed in a cGAS-dependent manner in hepatoma cell lines and primary human hepatocytes, host cell recognition of viral nucleic acids is abolished during HBV infection, suggesting escape from sensing, likely during packaging of the genome into the viral capsid. While the hepatocyte cGAS pathway is functionally active, as shown by reduction of viral cccDNA levels in gain-of-function studies, HBV infection suppressed cGAS expression and function in cell culture models and humanized mice. HBV exploits multiple strategies to evade sensing and antiviral activity of cGAS and its effector pathways. This article is protected by copyright. All rights reserved. © 2018 by the American Association for the Study of Liver Diseases.

Hyperinsulinemia is associated with increased soluble insulin receptors release from hepatocytes

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Marcia eHiriart

2014-06-01

Full Text Available It has been generally assumed that insulin circulates freely in blood. However it can also interact with plasma proteins. Insulin receptors are located in the membrane of target cells and consist of an alpha and beta subunits with a tyrosine kinase cytoplasmic domain. The ectodomain, called soluble insulin receptor (SIR has been found elevated in patients with diabetes mellitus. We explored if insulin binds to SIRs in circulation under physiological conditions and hypothesize that this SIR may be released by hepatocytes in response to high insulin concentrations. The presence of SIR in rat and human plasmas and the culture medium of hepatocytes was explored using Western blot analysis. A purification protocol was performed to isolated SIR using affinity, gel filtration and ion exchange chromatographies. A modified reverse hemolytic plaque assay was used to measure SIR release from cultured hepatocytes. Incubation with 1 nmol l-1 insulin induces the release of the insulin receptor ectodomains from normal rat hepatocytes. This effect can be partially prevented by blocking protease activity. Furthermore, plasma levels of SIR were higher in a model of metabolic syndrome, where rats are hyperinsulinemic. We also found increased SIR levels in hyperinsulinemic humans. SIR may be an important regulator of the amount of free insulin in circulation. In hyperinsulinemia the amount of this soluble receptor increases, this could lead to higher amounts of insulin bound to this receptor, rather than free insulin, which is the biologically active form of the hormone. This observation could enlighten the mechanisms of insulin resistance.

Identification and transcriptional modulation of the largemouth bass, Micropterus salmoides, vitellogenin receptor during oocyte development by insulin and sex steroids.

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Dominguez, Gustavo A; Quattro, Joseph M; Denslow, Nancy D; Kroll, Kevin J; Prucha, Melinda S; Porak, Wesley F; Grier, Harry J; Sabo-Attwood, Tara L

2012-09-01

Fish vitellogenin synthesized and released from the liver of oviparous animals is taken up into oocytes by the vitellogenin receptor. This is an essential process in providing nutrient yolk to developing embryos to ensure successful reproduction. Here we disclose the full length vtgr cDNA sequence for largemouth bass (LMB) that reveals greater than 90% sequence homology with other fish vtgr sequences. We classify LMB Vtgr as a member of the low density lipoprotein receptor superfamily based on conserved domains and categorize as the short variant that is devoid of the O-glycan segment. Phylogenetic analysis places LMB Vtgr sequence into a well-supported monophyletic group of fish Vtgr. Real-time PCR showed that the greatest levels of LMB vtgr mRNA expression occurred in previtellogenic ovarian tissues. In addition, we reveal the effects of insulin, 17beta-estradiol (E(2)), and 11-ketotestosterone (11-KT) in modulation of vtgr, esr, and ar mRNAs in previtellogenic oocytes. Insulin increased vtgr expression levels in follicles ex vivo while exposure to E(2) or 11-KT did not result in modulation of expression. However, both steroids were able to repress insulin-induced vtgr transcript levels. Coexposure with insulin and E(2) or of insulin and 11-KT increased ovarian esr2b and ar mRNA levels, respectively, which suggest a role for these nuclear receptors in insulin-mediated signaling pathways. These data provide the first evidence for the ordered stage-specific expression of LMB vtgr during the normal reproductive process and the hormonal influence of insulin and sex steroids on controlling vtgr transcript levels in ovarian tissues.

Hepatocytes in the development of liver support systems

NARCIS (Netherlands)

I.H.M. Borel Rinkes (Inne)

1993-01-01

textabstractThis thesis focuses on the development of alternative strategies in the treatment of patients with acute fulminant hepatic failure and inborn errors of metabolism, using hepatocytes as the basis of liver support. When compared with transplantation of the liver as an organ, the

Efficient derivation of functional hepatocytes from mouse induced pluripotent stem cells by a combination of cytokines and sodium butyrate

Institute of Scientific and Technical Information of China (English)

ZHANG Qi; YANG Yang; ZHANG Jian; WANG Guo-ying; LIU Wei; QIU Dong-bo; HEI Zi-qing; YING Qi-long; CHEN Gui-hua

2011-01-01

Background Hepatocyte transplantation has been proposed as an alternative to whole-organ transplantation to support many forms of hepatic insufficiency.Unfortunately,the lack of donor livers makes it difficult to obtain enough viable human hepatocytes for hepatocyte-based therapies.Therefore,it is urgent to find new ways to provide ample hepatocytes.Induced pluripotent stem (iPS) cells,a breakthrough in stem cell research,may terminate these hinders for cell transplantation.For the promise of iPS cells to be realized in liver diseases,it is necessary to determine if and how efficient they can be differentiated into functional hepatocytes.Methods In this study,we directly compared the hepatic-differentiation capacity of mouse iPS cells and embryonic stem (ES) cells with three different induction approaches:conditions via embryonic body (EB) formation plus cytokines,conditions by combination of dimethyl sulfoxide and sodium butyrate and chemically defined,serum free monolayer conditions.Among these three induction conditions,more homogenous populations can be promoted under chemically defined,serum free conditions.The cells generated under these conditions exhibited hepatic functions in vitro,including glycogen storage,indocynine green (ICG) uptake and release as well as urea secretion.Although efficient hepatocytes differentiation from mouse iPS cells were observed,mouse iPS cells showed relatively lower hepatic induction efficiency compared with mouse ES cells.Results Mouse iPS cells would be efficiently differentiated into functional hepatocytes in vitro,which may be helpful in facilitating the development of hepatocytes for transplantation and for research on drug discovery.Conclusion We demonstrate that mouse iPS cells retain full potential for fetal liver development and describe procedures that facilitates the efficient generation of highly differentiated human hepatocyte-like cells from iPS cells in vitro.

Palm kernel cake extract exerts hepatoprotective activity in heat-induced oxidative stress in chicken hepatocytes.

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Oskoueian, Ehsan; Abdullah, Norhani; Idrus, Zulkifli; Ebrahimi, Mahdi; Goh, Yong Meng; Shakeri, Majid; Oskoueian, Armin

2014-10-02

Palm kernel cake (PKC), the most abundant by-product of oil palm industry is believed to contain bioactive compounds with hepatoprotective potential. These compounds may serve as hepatoprotective agents which could help the poultry industry to alleviate adverse effects of heat stress on liver function in chickens. This study was performed to evaluate the hepatoprotective potential of PKC extract in heat-induced oxidative stress in chicken hepatocytes. The nature of the active metabolites and elucidation of the possible mechanism involved were also investigated. The PKC extract possessed free radical scavenging activity with values significantly (p < 0.05) lower than silymarin as the reference antioxidant. Heat-induced oxidative stress in chicken hepatocyte impaired the total protein, lipid peroxidation and antioxidant enzymes activity significantly (p < 0.05). Treatment of heat-induced hepatocytes with PKC extract (125 μg/ml) and silymarin as positive control increased these values significantly (p < 0.05). The real time PCR and western blot analyses revealed the significant (p < 0.05) up-regulation of oxidative stress biomarkers including TNF-like, IFN-γ and IL-1β genes; NF-κB, COX-2, iNOS and Hsp70 proteins expression upon heat stress in chicken hepatocytes. The PKC extract and silymarin were able to alleviate the expression of all of these biomarkers in heat-induced chicken hepatocytes. The gas chromatography-mass spectrometry analysis of PKC extract showed the presence of fatty acids, phenolic compounds, sugar derivatives and other organic compounds such as furfural which could be responsible for the observed hepatoprotective activity. Palm kernel cake extract could be a potential agent to protect hepatocytes function under heat induced oxidative stress.

Adenoviral-mediated correction of methylmalonyl-CoA mutase deficiency in murine fibroblasts and human hepatocytes

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Korson Mark

2007-04-01

Full Text Available Abstract Background Methylmalonic acidemia (MMA, a common organic aciduria, is caused by deficiency of the mitochondrial localized, 5'deoxyadenosylcobalamin dependent enzyme, methylmalonyl-CoA mutase (MUT. Liver transplantation in the absence of gross hepatic dysfunction provides supportive therapy and metabolic stability in severely affected patients, which invites the concept of using cell and gene delivery as future treatments for this condition. Methods To assess the effectiveness of gene delivery to restore the defective metabolism in this disorder, adenoviral correction experiments were performed using murine Mut embryonic fibroblasts and primary human methylmalonyl-CoA mutase deficient hepatocytes derived from a patient who harbored two early truncating mutations, E224X and R228X, in the MUT gene. Enzymatic and expression studies were used to assess the extent of functional correction. Results Primary hepatocytes, isolated from the native liver after removal subsequent to a combined liver-kidney transplantation procedure, or Mut murine fibroblasts were infected with a second generation recombinant adenoviral vector that expressed the murine methylmalonyl-CoA mutase as well as eGFP from distinct promoters. After transduction, [1-14C] propionate macromolecular incorporation studies and Western analysis demonstrated complete correction of the enzymatic defect in both cell types. Viral reconstitution of enzymatic expression in the human methylmalonyl-CoA mutase deficient hepatocytes exceeded that seen in fibroblasts or control hepatocytes. Conclusion These experiments provide proof of principle for viral correction in methylmalonic acidemia and suggest that hepatocyte-directed gene delivery will be an effective therapeutic treatment strategy in both murine models and in human patients. Primary hepatocytes from a liver that was unsuitable for transplantation provided an important resource for these studies.

Effect of UV-B (302 nm) irradiation on isolated rat hepatocytes

International Nuclear Information System (INIS)

Lahiri, S.; Habibullah, C.M.; Ayesha, Q.; Khan, A.A.; Srinivas, V.K.; Naithani, R.

1995-01-01

The present study aimed to evaluate the effect of UV-B irradiation on the functional integrity, and the metabolic and detoxifying capacity of isolated rat hepatocytes. Isolated rat hepatocytes were irradiated in various doses (400 Jm -2 , 600 Jm -2 , 800 Jm -2 and 1000 Jm -2 ). The cells were assayed for total lactate dehydrogenase, Na + -K + -ATPase, ATPase, ornithine carbamyltransferase activity (OCT) and urea production capacity. Lactate dehydrogenase and Na + -K + -ATPase activity were significantly decreased in all four irradiated groups (P<0.001), whereas viability, OCT and urea production capacity showed no alterations. (au) 22 refs

Acrolein cytotoxicity in hepatocytes involves endoplasmic reticulum stress, mitochondrial dysfunction and oxidative stress

Science.gov (United States)

Mohammad, Mohammad K; Avila, Diana; Zhang, Jingwen; Barve, Shirish; Arteel, Gavin; McClain, Craig; Joshi-Barve, Swati

2012-01-01

Acrolein is a common environmental, food and water pollutant and a major component of cigarette smoke. Also, it is produced endogenously via lipid peroxidation and cellular metabolism of certain amino acids and drugs. Acrolein is cytotoxic to many cell types including hepatocytes; however the mechanisms are not fully understood. We examined the molecular mechanisms underlying acrolein hepatotoxicity in primary human hepatocytes and hepatoma cells. Acrolein, at pathophysiological concentrations, caused a dose-dependent loss of viability of hepatocytes. The death was apoptotic at moderate and necrotic at high concentrations of acrolein. Acrolein exposure rapidly and dramatically decreased intracellular glutathione and overall antioxidant capacity, and activated the stress-signaling MAP-kinases JNK, p42/44 and p38. Our data demonstrate for the first time in human hepatocytes, that acrolein triggered endoplasmic reticulum (ER) stress and activated eIF2α, ATF-3 and -4, and Gadd153/CHOP, resulting in cell death. Notably, the protective/adaptive component of ER stress was not activated, and acrolein failed to up-regulate the protective ER-chaperones, GRP78 and GRP94. Additionally, exposure to acrolein disrupted mitochondrial integrity/function, and led to the release of pro-apoptotic proteins and ATP depletion. Acrolein-induced cell death was attenuated by N-acetyl cysteine, phenyl-butyric acid, and caspase and JNK inhibitors. Our data demonstrate that exposure to acrolein induces a variety of stress responses in hepatocytes, including GSH depletion, oxidative stress, mitochondrial dysfunction and ER stress (without ER-protective responses) which together contribute to acrolein toxicity. Our study defines basic mechanisms underlying liver injury caused by reactive aldehyde pollutants such as acrolein. PMID:23026831

Assessment of 17α-ethinylestradiol effects and underlying mechanisms in a continuous, multigeneration exposure of the Chinese rare minnow (Gobiocypris rarus)

International Nuclear Information System (INIS)

Zha Jinmiao; Sun Liwei; Zhou Yiqi; Spear, Philip A.; Ma, Mei; Wang Zijian

2008-01-01

17α-Ethinylestradiol (EE 2 ) is a synthetic estrogen used primarily in birth control pills and in hormone replacement therapy. Owing to its occurrence in surface waters at concentrations frequently greater than 1 ng/l and its projected future use, EE 2 is expected to pose a significant risk to aquatic organisms. This study was conducted to obtain long-term exposure data necessary for the establishment of water quality criteria and to investigate mechanisms associated with toxic effects. In a multigeneration experiment, Chinese rare minnows (Gobiocypris rarus) were constantly exposed to environmentally relevant concentrations of the synthetic estrogen EE 2 . Mortality, deformities, reproductive parameters, plasma vitellogenin and histopathology were assessed. The results showed that, in the F 0 generation, all endpoints were significantly affected at concentrations higher than 0.2 ng/l EE 2 . No F 1 phenotypic males developed to maturity at 0.2 ng/l and, when adult females of this exposure group were crossed with unexposed males, no F 2 fertile eggs were produced. Kidney histopathology and ultrastructure suggest anomalies possibly associated with increased vitellogenin accumulation. We concluded that the reproduction of the F 1 minnows was completely inhibited at the lowest concentration tested, 0.2 ng/l EE 2 , a concentration frequently detected in surface waters. Growth effects may be related to increased energy requirements including the energy used in VTG synthesis. Reproductive effects are presumably associated with male feminization and the occurrence of testis-ova in males; however, ovarian degeneration observed in females may also have contributed to reproductive failure

Clinical value of hemoglobin and its association with hepatocyte steatosis in chronic hepatitis B patients

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WANG Peng

2017-07-01

Full Text Available :ObjectiveTo investigate the clinical value of hemoglobin and its association with hepatocyte steatosis in chronic hepatitis B (CHB patients. MethodsA retrospective analysis was performed for the clinical and pathological data of 1580 CHB patients who were hospitalized in The First People′s Hospital of Shunde from January 2006 to December 2014 and underwent liver biopsy, among whom 216 (13.67% had hepatocyte steatosis (hepatocyte steatosis group and 1364 had no hepatocyte steatosis (non-hepatocyte steatosis group. The patients were divided into groups 1, 2, and 3 according to hemoglobin level, and the clinical and pathological features were analyzed and compared between the three groups. The t-test was used for comparison of continuous data between group; a one-way analysis of variance was used for comparision between multiple groups. The Mann-Whitney U test was used for ranked data between groups. The Kruskal-wallis H test was used for ranked data between multiple groups; the chi-square test was used for comparison of categorical data between groups. Spearman correlation analysis was also performed to determine the correlation between two variables. Univariate logistic regression analysis and multivariate stepwise regression analysis were used to identify the influencing factors for hepatocyte steatosis. ResultsBody mass index (BMI, systolic pressure, diastolic pressure, uric acid, total cholesterol, low-density lipoprotein, and HBV DNA load increased with the increase in hemoglobin level (F=12718,3024,4026,4624,38276,28108,7358, all P＜0.05. The incidence rates of hepatocyte steatosis in groups 1, 2, and 3 were 7.59%, 1176%,and 21.67%, respectively (χ2=44.23, P＜0.05. Hemoglobin was positively correlated with hepatic steatosis (rs=0.211, P＜0001. The multivariate logistic regression analysis showed that hemoglobin (odds ratio ［OR］=1.066, P＜0.05, BMI (OR=1576, P＜005, age (OR=1.041, P＜0.05, sex

Glycogen content in hepatocytes is related with their size in normal rat liver but not in cirrhotic one.

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Bezborodkina, Natalia N; Chestnova, Anna Yu; Vorobev, Mikhail L; Kudryavtsev, Boris N

2016-04-01

Hepatocytes differ from one another by the degree of the ploidy, size, position in the liver lobule, and level of the DNA-synthetic processes. It is believed, that the cell size exerts substantial influence on the metabolism of the hepatocytes and the glycogen content in them. The aim of the present study was to test this hypothesis. Dry weight of hepatocytes, their ploidy and glycogen content were determined in the normal and the cirrhotic rat liver. Liver cirrhosis in rats was produced by chronic inhalation of CCl4 vapours in the course of 6 months. A combined cytophotometric method was used. Dry weight of the cell, its glycogen and DNA content were successively measured on a mapped preparation. Hepatocytes of each ploidy class in the normal and the cirrhotic rat liver accumulated glycogen at the same rate. In the normal liver, there was a distinct correlation between the size of hepatocytes and glycogen content in them. This correlation was observed in each ploidy class, and was especially pronounced in the class of mononucleate tetraploid hepatocytes. In the cirrhotic liver, there was no correlation between the size of the cells and their glycogen content. The impairment of liver lobular structure probably explains the observed lack of correlation between hepatocyte size and their glycogen content in the cirrhotic liver. © 2016 International Society for Advancement of Cytometry. © 2016 International Society for Advancement of Cytometry.

Interleukin 6 regulates metallothionein gene expression and zinc metabolism in hepatocyte monolayer cultures

International Nuclear Information System (INIS)

Schroeder, J.J.; Cousins, R.J.

1990-01-01

Attention has focused on the cytokine interleukin 6 (IL-6) as a major mediator of acute-phase protein synthesis in hepatocytes in response to infection and tissue injury. The authors have evaluated the effects of IL-6 and IL-1α as well as extracellular zinc and glucocorticoid hormone on metal-lothionein gene expression and cellular zinc accumulation in rat hepatocyte monolayer cultures. Further, they have evaluated the teleological basis for cytokine mediation by examining cyto-protection from CCl 4 -induced damage. Incubation of hepatocytes with IL-6 led to concentration-dependent and time-dependent increases in metallothionein-1 and -2 mRNA and metallothionein protein. The level of each was increased within 3 hr after the addition of IL-6 at 10 ng/ml. Maximal increases the metallothionein mRNA and metallothionein protein were achieved after 12 hr and 36 hr, respectively. Concomitant with the up-regulation of metallothionein gene expression, IL-6 also increased cellular zinc. Responses to IL-6 required the synthetic glucocorticoid hormone dexamethasone and were optimized by increased extracellular zinc. Thus, IL-6 is a major cytokine mediator of metallothionein gene expression and zinc metabolism in hepatocytes and provides cytoprotection from CCl 4 -induced hepatotoxicity via a mode consistent with dependence upon increased cellular metallothionein synthesis and zinc accumulation

Insulin internalization in isolated rat hepatocytes

International Nuclear Information System (INIS)

Galan, J.; Trankina, M.; Noel, R.; Ward, W.

1990-01-01

This project was designed to determine whether neomycin, an aminoglycoside antibiotic, has a significant effect upon the pathways of ligand endocytosis in isolated rat hepatocytes. The pathways studied include receptor-mediated endocytosis and fluid-phase endocytosis. Neomycin causes a dose-dependent acceleration of 125 I-insulin internalization. Since fluid-phase endocytosis can also be a significant factor in 125 I-insulin internalization, lucifer yellow (LY), a marker for fluid-phase endocytosis, was incorporated into an assay similar to the 125 I-insulin internalization procedure. In the presence of 5 mM neomycin, a significant increase in LY uptake was evident at 0.2 and 0.4 mg/ml of LY. At 0.8 mg/ml, a decrease in LY uptake was observed. The increased rate of 125 I-insulin internalization in the presence of neomycin was intriguing. Since one action of neomycin is to inhibit phosphoinositidase C, it suggests that the phosphotidylinositol cycle may be involved in ligand internalization by hepatocytes. At low insulin concentrations, receptor-mediated uptake predominates. Fluid-phase uptake can become an important uptake route as insulin concentrations are increased. Since neomycin stimulates fluid-phase endocytosis, it must also be taken into account when measuring ligand internalization

A nuclear factor I-like activity and a liver-specific repressor govern estrogen-regulated in vitro transcription from the Xenopus laevis vitellogenin B1 promoter.

OpenAIRE

Corthésy, B; Cardinaux, J R; Claret, F X; Wahli, W

1989-01-01

A hormone-controlled in vitro transcription system derived from Xenopus liver nuclear extracts was exploited to identify novel cis-acting elements within the vitellogenin gene B1 promoter region. In addition to the already well-documented estrogen-responsive element (ERE), two elements were found within the 140 base pairs upstream of the transcription initiation site. One of them, a negative regulatory element, is responsible for the lack of promoter activity in the absence of the hormone and...

Proteomic Characterization of Primary Mouse Hepatocytes in Collagen Monolayer and Sandwich Culture.

Science.gov (United States)

Orsini, Malina; Sperber, Saskia; Noor, Fozia; Hoffmann, Esther; Weber, Susanne N; Hall, Rabea A; Lammert, Frank; Heinzle, Elmar

2018-01-01

Dedifferentiation of primary hepatocytes in vitro makes their application in long-term studies difficult. Embedding hepatocytes in a sandwich of extracellular matrix is reported to delay the dedifferentiation process to some extent. In this study, we compared the intracellular proteome of primary mouse hepatocytes (PMH) in conventional monolayer cultures (ML) to collagen sandwich culture (SW) after 1 day and 5 days of cultivation. Quantitative proteome analysis of PMH showed no differences between collagen SW and ML cultures after 1 day. Glycolysis and gluconeogenesis were strongly affected by long-term cultivation in both ML and SW cultures. Interestingly, culture conditions had no effect on cellular lipid metabolism. After 5 days, PMH in collagen SW and ML cultures exhibit characteristic indications of oxidative stress. However, in the SW culture the defense system against oxidative stress is significantly up-regulated to deal with this, whereas in the ML culture a down-regulation of these important enzymes takes place. Regarding the multiple effects of ROS and oxidative stress in cells, we conclude that the down-regulation of these enzymes seem to play a role in the loss of hepatic function observed in the ML cultivation. In addition, enzymes of the urea cycle were clearly down-regulated in ML culture. Proteomics confirms lack in oxidative stress defense mechanisms as the major characteristic of hepatocytes in monolayer cultures compared to sandwich cultures. J. Cell. Biochem. 119: 447-454, 2018. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Changes in Expression of Connexin 32, Bile Canaliculus-Like Structures, and Localization of Alkaline Phosphatase in Primary Cultures of Fetal Rat Hepatocytes

International Nuclear Information System (INIS)

Fukazawa, Shoko; Chida, Kohsuke; Taguchi, Meiko; Takeuchi, Akihiro; Ikeda, Noriaki

2013-01-01

We devised an experimental design in primary cultures of fetal rat hepatocytes for studying hepatocyte differentiation over a short period. In the present study, hepatocytes were first cultured for 3 days in dexamethasone-supplemented medium and then for an additional 3 days in dexamethasone- or epidermal growth factor-supplemented medium. In hepatocytes cultured continuously in dexamethasone-supplemented medium, the expression of connexin 32 increased and bile canaliculus-like structures and localization of alkaline phosphatase in the plasma membrane around bile canaliculus-like structures were maintained. Few cells incorporated bromodeoxyuridine. On the other hand, in most of the hepatocytes cultured in epidermal growth factor-supplemented medium, the expression of connexin 32 was minimally recognized, bile canaliculus-like structures were shortened or eliminated, and alkaline phosphatase was localized as numerous fine spots throughout the cytoplasm. More than 20% of all hepatocytes incorporated bromodeoxyuridine. The present study suggests that in hepatocytes, there is a close relationship among connexin 32 expression, the maintenance of bile canaliculus-like structures, and the localization of alkaline phosphatase to the plasma membrane around the bile canaliculus-like structures, and this indicates that the present experimental model is useful for studying hepatocyte differentiation over a short period

Adropin induction of lipoprotein lipase expression in tilapia hepatocytes.

Science.gov (United States)

Lian, Anji; Wu, Keqiang; Liu, Tianqiang; Jiang, Nan; Jiang, Quan

2016-01-01

The peptide hormone adropin plays a role in energy homeostasis. However, biological actions of adropin in non-mammalian species are still lacking. Using tilapia as a model, we examined the role of adropin in lipoprotein lipase (LPL) regulation in hepatocytes. To this end, the structural identity of tilapia adropin was established by 5'/3'-rapid amplification of cDNA ends (RACE). The transcripts of tilapia adropin were ubiquitously expressed in various tissues with the highest levels in the liver and hypothalamus. The prolonged fasting could elevate tilapia hepatic adropin gene expression, whereas no effect of fasting was observed on hypothalamic adropin gene levels. In primary cultures of tilapia hepatocytes, synthetic adropin was effective in stimulating LPL release, cellular LPL content, and total LPL production. The increase in LPL production also occurred with parallel rises in LPL gene levels. In parallel experiments, adropin could elevate cAMP production and up-regulate protein kinase A (PKA) and PKC activities. Using a pharmacological approach, cAMP/PKA and PLC/inositol trisphosphate (IP3)/PKC cascades were shown to be involved in adropin-stimulated LPL gene expression. Parallel inhibition of p38MAPK and Erk1/2, however, were not effective in these regards. Our findings provide, for the first time, evidence that adropin could stimulate LPL gene expression via direct actions in tilapia hepatocytes through the activation of multiple signaling mechanisms. © 2016 Society for Endocrinology.

Diclofenac inhibits tumor necrosis factor-a-induced nuclear factor-kB activation causing synergic hepatocyte apoptosis

NARCIS (Netherlands)

Frederiksson, L; Herpers, B; Benedetti, G; Matadin, Q; Puigvert, J.C.; de Bont, H; Dragovic, S.; Vermeulen, N.P.E.; Commandeur, J.N.M.; Danen, E; de Graauw, M; van de Water, B.

2011-01-01

Drug-induced liver injury (DILI) is an important clinical problem. It involves crosstalk between drug toxicity and the immune system, but the exact mechanism at the cellular hepatocyte level is not well understood. Here we studied the mechanism of crosstalk in hepatocyte apoptosis caused by

Differentiation of human umbilical cord mesenchymal stromal cells into low immunogenic hepatocyte-like cells.

Science.gov (United States)

Zhao, Qinjun; Ren, Hongying; Li, Xiyuan; Chen, Zhong; Zhang, Xiangyu; Gong, Wei; Liu, Yongjun; Pang, Tianxiang; Han, Zhong Chao

2009-01-01

Mesenchymal stromal cells (MSC) isolated from several human tissues have been known to differentiate into the hepatic lineage in vitro, but the immunogenicity of the differentiated hepatocyte-like cells (DHC) has not been reported. Umbilical cord (UC) MSC are thought to be an attractive cell source for cell therapy because of their young age and low infection rate compared with adult tissue MSC. Hepatic differentiation of UC-MSC was induced with a 2-step protocol. The expressions of hepatic markers were detected by RT-PCR and immunofluorescence staining. Albumin production and urea secretion were measured by ELISA and colorimetric assay respectively. The immunosuppressive properties of DHC was detected by mixed lymphocyte culture. After incubation with specific growth factors, including hepatocyte growth factor (HGF) and basic fibroblast growth factor (bFGF), UC MSC exhibited a high hepatic differentiation ability in an adherent culture condition. The differentiated UC MSC showed hepatocyte-like morphology and expressed several liver-specific markers at gene and protein levels. Furthermore, the DHC exhibited hepatocyte-specific functions, including albumin secretion, low-density lipoprotein uptake and urea production. More importantly, DHC did not express major histocompatibility complex (MHC) II antigen and were not able to induce lymphocyte proliferation in mixed lymphocyte culture, as undifferentiated UC MSC did. Our results indicate that UC MSC are able to differentiate into functional hepatocyte-like cells that still retain their low immunogenicity in vitro. More importantly, DHC incorporated into the parenchyma of liver when transplanted into mice with CCl(4)-induced liver injury. Therefore, DHC may be an ideal source for cell therapy of liver diseases.

Impaired mitochondrial functions contribute to 3-bromopyruvate toxicity in primary rat and mouse hepatocytes.

Science.gov (United States)

Sobotka, Ondřej; Endlicher, René; Drahota, Zdeněk; Kučera, Otto; Rychtrmoc, David; Raad, Marjan; Hakeem, Khurum; Červinková, Zuzana

2016-08-01

A compound with promising anticancer properties, 3-bromopyruvate (3-BP) is a synthetic derivative of a pyruvate molecule; however, its toxicity in non-malignant cells has not yet been fully elucidated. Therefore, we elected to study the effects of 3-BP on primary hepatocytes in monolayer cultures, permeabilized hepatocytes and isolated mitochondria. After a 1-h treatment with 100 μM 3-BP cell viability of rat hepatocytes was decreased by 30 % as measured by the WST-1 test (p < 0.001); after 3-h exposure to ≥200 μM 3-BP lactate dehydrogenase leakage was increased (p < 0.001). Reactive oxygen species production was increased in the cell cultures after a 1-h treatment at concentrations ≥100 μmol/l (p < 0.01), and caspase 3 activity was increased after a 20-h incubation with 150 μM and 200 μM 3-BP (p < 0.001). This toxic effect of 3-BP was also proved using primary mouse hepatocytes. In isolated mitochondria, 3-BP induced a dose- and time-dependent decrease of mitochondrial membrane potential during a 10-min incubation both with Complex I substrates glutamate + malate or Complex II substrate succinate, although this decrease was more pronounced with the latter. We also measured the effect of 3-BP on respiration of isolated mitochondria. ADP-activated respiration was inhibited by 20 μM 3-BP within 10 min. Similar effects were also found in permeabilized hepatocytes of both species.

Insulin-like growth factor-II receptors in cultured rat hepatocytes: regulation by cell density

International Nuclear Information System (INIS)

Scott, C.D.; Baxter, R.C.

1987-01-01

Insulin-like growth factor-II (IGF-II) receptors in primary cultures of adult rat hepatocytes were characterized and their regulation by cell density examined. In hepatocytes cultured at 5 X 10(5) cells per 3.8 cm2 plate [ 125 I]IGF-II bound to specific, high affinity receptors (Ka = 4.4 +/- 0.5 X 10(9) l/mol). Less than 1% cross-reactivity by IGF-I and no cross-reactivity by insulin were observed. IGF-II binding increased when cells were permeabilized with 0.01% digitonin, suggesting the presence of an intracellular receptor pool. Determined by Scatchard analysis and by polyacrylamide gel electrophoresis after affinity labeling, the higher binding was due solely to an increase in binding sites present on 220 kDa type II IGF receptors. In hepatocytes cultured at low densities, the number of cell surface receptors increased markedly, from 10-20,000 receptors per cell at a culture density of 6 X 10(5) cells/well to 70-80,000 receptors per cell at 0.38 X 10(5) cells/well. The increase was not due simply to the exposure of receptors from the intracellular pool, as a density-related increase in receptors was also seen in cells permeabilized with digitonin. There was no evidence that IGF binding proteins, either secreted by hepatocytes or present in fetal calf serum, had any effect on the measurement of receptor concentration or affinity. We conclude that rat hepatocytes in primary culture contain specific IGF-II receptors and that both cell surface and intracellular receptors are regulated by cell density

Differentiation of hepatocytes from induced pluripotent stem cells derived from human hair follicle mesenchymal stem cells.

Science.gov (United States)

Shi, Xu; Lv, Shuang; He, Xia; Liu, Xiaomei; Sun, Meiyu; Li, Meiying; Chi, Guangfan; Li, Yulin

2016-10-01

Due to the limitations of organ donors and immune rejection in severe liver diseases, stem cell-based therapy presents a promising application for tissue repair and regeneration. As a novel cell source, mesenchymal stem cells separated from human hair follicles (HF-MSCs) are convenient to obtain and have no age limit. To date, the differentiation of HF-MSCs into hepatocytes has not been reported. In this study, we explored whether HF-MSCs and HF-MSC-derived-induced pluripotent stem cells (HF-iPS) could differentiate into hepatocytes in vitro. Flow cytometry, Oil Red O stain and Alizarin Red stain were used to identify the characteristics of HF-MSCs. The expression of liver-specific gene was detected by immunofluorescence and Quantitative Polymerase Chain Reaction. Periodic Acid-Schiff stain, Indocyanine Green stain and Low-Density Lipoprotein stain were performed to evaluate the functions of induced hepatocyte-like cells (HLCs). HF-MSCs were unable to differentiate into HLCs using previously reported procedures for MSCs from other tissues. However, HF-iPS efficiently induced the generation of HLCs that expressed hepatocyte markers and drug metabolism-related genes. HF-iPS can be used as novel and alternative cellular tools for inducing hepatocytes in vitro, simultaneously benefiting from utilizing HF-MSCs as a noninvasive and convenient cell source for reprogramming.

Titanium Dioxide Nanoparticles Trigger Loss of Function and Perturbation of Mitochondrial Dynamics in Primary Hepatocytes.

Directory of Open Access Journals (Sweden)

Vaishaali Natarajan

Full Text Available Titanium dioxide (TiO2 nanoparticles are one of the most highly manufactured and employed nanomaterials in the world with applications in copious industrial and consumer products. The liver is a major accumulation site for many nanoparticles, including TiO2, directly through intentional exposure or indirectly through unintentional ingestion via water, food or animals and increased environmental contamination. Growing concerns over the current usage of TiO2 coupled with the lack of mechanistic understanding of its potential health risk is the motivation for this study. Here we determined the toxic effect of three different TiO2 nanoparticles (commercially available rutile, anatase and P25 on primary rat hepatocytes. Specifically, we evaluated events related to hepatocyte functions and mitochondrial dynamics: (1 urea and albumin synthesis using colorimetric and ELISA assays, respectively; (2 redox signaling mechanisms by measuring reactive oxygen species (ROS production, manganese superoxide dismutase (MnSOD activity and mitochondrial membrane potential (MMP; (3 OPA1 and Mfn-1 expression that mediates the mitochondrial dynamics by PCR; and (4 mitochondrial morphology by MitoTracker Green FM staining. All three TiO2 nanoparticles induced a significant loss (p < 0.05 in hepatocyte functions even at concentrations as low as 50 ppm with commercially used P25 causing maximum damage. TiO2 nanoparticles induced a strong oxidative stress in primary hepatocytes. TiO2 nanoparticles exposure also resulted in morphological changes in mitochondria and substantial loss in the fusion process, thus impairing the mitochondrial dynamics. Although this study demonstrated that TiO2 nanoparticles exposure resulted in substantial damage to primary hepatocytes, more in vitro and in vivo studies are required to determine the complete toxicological mechanism in primary hepatocytes and subsequently liver function.

Metabolism of six CYP probe substrates in fetal hepatocytes

Directory of Open Access Journals (Sweden)

Abdul Naveed Shaik

2016-06-01

Full Text Available Cytochrome P-450 (CYP are the most common drug metabolizing enzymes and are abundantly expressed in liver apart from kidney, lungs, intestine, brain etc. Their expression levels change with physiological conditions and disease states. The expression of these CYPs is less in human foetus and neonates compared to adults, which results in lower clearance of xenobiotics in infants and neonates compared to adults. Hepatocytes are the cells which are largely used to study these CYPs. We have isolated hepatocytes from aborted foetus to study the metabolism of six probe substrates: phenacetin, diclofenac, S-mephenytoin, dextromethorphan, nifedipine and testosterone. The results obtained show the expression of various CYPs (CYP1A2, CYP2C19, CYP2C9, CYP2D6, and CYP3A4 in human foetus and their involvement in metabolism of CYP probe substrates.

Intracellular cholesterol-binding proteins enhance HDL-mediated cholesterol uptake in cultured primary mouse hepatocytes.

Science.gov (United States)

Storey, Stephen M; McIntosh, Avery L; Huang, Huan; Landrock, Kerstin K; Martin, Gregory G; Landrock, Danilo; Payne, H Ross; Atshaves, Barbara P; Kier, Ann B; Schroeder, Friedhelm

2012-04-15

A major gap in our knowledge of rapid hepatic HDL cholesterol clearance is the role of key intracellular factors that influence this process. Although the reverse cholesterol transport pathway targets HDL to the liver for net elimination of free cholesterol from the body, molecular details governing cholesterol uptake into hepatocytes are not completely understood. Therefore, the effects of sterol carrier protein (SCP)-2 and liver fatty acid-binding protein (L-FABP), high-affinity cholesterol-binding proteins present in hepatocyte cytosol, on HDL-mediated free cholesterol uptake were examined using gene-targeted mouse models, cultured primary hepatocytes, and 22-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-amino]-23,24-bisnor-5-cholen-3β-ol (NBD-cholesterol). While SCP-2 overexpression enhanced NBD-cholesterol uptake, counterintuitively, SCP-2/SCP-x gene ablation also 1) enhanced the rapid molecular phase of free sterol uptake detectable in rate and maximal uptake of HDL free cholesterol and 2) differentially enhanced free cholesterol uptake mediated by the HDL3, rather than the HDL2, subfraction. The increased HDL free cholesterol uptake was not due to increased expression or distribution of the HDL receptor [scavenger receptor B1 (SRB1)], proteins regulating SRB1 [postsynaptic density protein (PSD-95)/Drosophila disk large tumor suppressor (dlg)/tight junction protein (ZO1) and 17-kDa membrane-associated protein], or other intracellular cholesterol trafficking proteins (steroidogenic acute response protein D, Niemann Pick C, and oxysterol-binding protein-related proteins). However, expression of L-FABP, the single most prevalent hepatic cytosolic protein that binds cholesterol, was upregulated twofold in SCP-2/SCP-x null hepatocytes. Double-immunogold electron microscopy detected L-FABP sufficiently close to SRB1 for direct interaction, similar to SCP-2. These data suggest a role for L-FABP in HDL cholesterol uptake, a finding confirmed with SCP-2/SCP-x/L-FABP null

[Study on the interface of human hepatocyte/micropore polypropylene ultrafiltration membrane].

Science.gov (United States)

Peng, Cheng-Hong; Han, Bao-San; Gao, Chang-You; Ma, Zu-Wei; Zhao, Zhi-Ming; Wang, Yong; Liu, Hong; Zhang, Gui-di; Yang, Mei-Juan

2004-09-02

To found a new interface of human hepatocyte/micropore polypropylene ultrafiltration membrane (MPP) with good cytocompatibility so as to construct bioartificial bioreactor with polypropylene hollow fibers in future. MPP ultrafiltration membrane underwent chemical grafting modification through ultraviolet irradiation and Fe(2+) reduction. The contact angles of MPP and the modified MPP membranes were measured. Human hepatic cells L-02 were cultured. MPP and modified MPP membranes were spread on the wells of culture plate and human hepatic cells and cytodex 3 were inoculated on them. Different kinds of microscopy were used to observe the morphology of these cells. The water contact angle of MPP and the modified MPP membranes decreased from 78 degrees +/- 5 degrees to 27 degrees +/- 4 degrees (P < 0.05), which indicated that the hydrophilicity of the membrane was improved obviously after the grafting modification. Human hepatocyte L-02 did not adhere to and spread on the modified MPP membrane surface, and only grew on the microcarrier cytodex 3 with higher density and higher proliferation ratio measured by MTT. Grafting modification of acrylamide on MPP membrane is a good method to improve the human hepatocyte cytocompatibility with MPP ultrafiltration membrane.

Enhancement of the predicted drug hepatotoxicity in gel entrapped hepatocytes within polysulfone-g-poly (ethylene glycol) modified hollow fiber

International Nuclear Information System (INIS)

Shen Chong; Zhang Guoliang; Meng Qin

2010-01-01

Collagen gel-based 3D cultures of hepatocytes have been proposed for evaluation of drug hepatotoxicity because of their more reliability than traditional monolayer culture. The collagen gel entrapment of hepatocytes in hollow fibers has been proven to well reflect the drug hepatotoxicity in vivo but was limited by adsorption of hydrophobic drugs onto hollow fibers. This study aimed to investigate the impact of hollow fibers on hepatocyte performance and drug hepatotoxicity. Polysulfone-g-poly (ethylene glycol) (PSf-g-PEG) hollow fiber was fabricated and applied for the first time to suppress the drug adsorption. Then, the impact of hollow fibers was evaluated by detecting the hepatotoxicity of eight selected drugs to gel entrapped hepatocytes within PSf and PSf-g-PEG hollow fibers, or without hollow fibers. The hepatocytes in PSf-g-PEG hollow fiber showed the highest sensitivity to drug hepatotoxicity, while those in PSf hollow fiber and cylindrical gel without hollow fiber underestimated the hepatotoxicity due to either drug adsorption or low hepatic functions. Therefore, the 3D culture of gel entrapped hepatocytes within PSf-g-PEG hollow fiber would be a promising tool for investigation of drug hepatotoxicity in vitro.

Early warning signs of endocrine disruption in adult fish from the ingestion of polyethylene with and without sorbed chemical pollutants from the marine environment

Energy Technology Data Exchange (ETDEWEB)

Rochman, Chelsea M., E-mail: cmrochman@ucdavis.edu; Kurobe, Tomofumi; Flores, Ida; Teh, Swee J.

2014-09-15

Plastic debris is associated with several chemical pollutants known to disrupt the functioning of the endocrine system. To determine if the exposure to plastic debris and associated chemicals promotes endocrine-disrupting effects in fish, we conducted a chronic two-month dietary exposure using Japanese medaka (Oryzias latipes) and environmentally relevant concentrations of microplastic (< 1 mm) and associated chemicals. We exposed fish to three treatments: a no-plastic (i.e. negative control), virgin-plastic (i.e. virgin polyethylene pre-production pellets) and marine-plastic treatment (i.e. polyethylene pellets deployed in San Diego Bay, CA for 3 months). Altered gene expression was observed in male fish exposed to the marine-plastic treatment, whereas altered gene expression was observed in female fish exposed to both the marine- and virgin-plastic treatment. Significant down-regulation of choriogenin (Chg H) gene expression was observed in males and significant down-regulation of vitellogenin (Vtg I), Chg H and the estrogen receptor (ERα) gene expression was observed in females. In addition, histological observation revealed abnormal proliferation of germ cells in one male fish from the marine-plastic treatment. Overall, our study suggests that the ingestion of plastic debris at environmentally relevant concentrations may alter endocrine system function in adult fish and warrants further research. - Highlights: • We saw down-regulation of Chg H in males exposed to marine plastic. • We saw down-regulation of Vtg I, Chg H and ERα in females exposed to plastic. • We saw abnormal proliferation of germ cells in a male exposed to marine plastic. • Our results suggest that the ingestion of plastic may alter endocrine system function.

Alteration in molecular markers of oocyte development and intersex condition in mullets impacted by wastewater treatment plant effluents.

Science.gov (United States)

Valencia, Ainara; Rojo-Bartolomé, Iratxe; Bizarro, Cristina; Cancio, Ibon; Ortiz-Zarragoitia, Maren

2017-05-01

Wastewater Treatment Plant (WWTP) discharges are an important source of endocrine disrupting chemicals (EDCs) into the aquatic environment. Fish populations inhabiting downstream of WWTP effluents show alterations in gonad and gamete development such as intersex condition, together with xenoestrogenic effects such as vitellogenin up-regulation. However, the molecular mechanisms participating in the development of intersex condition in fish are not elucidated. The aim of this study was to assess the impact of two WWTPs effluents (Gernika and Bilbao-Galindo situated in the South East Bay of Biscay) with different contaminant loads, in thicklip grey mullet (Chelon labrosus) populations inhabiting downstream, examining the presence and severity of intersex condition, during two seasons. Molecular markers of xenoestrogenicity and oocyte differentiation and development (vtgAa, cyp19a1a, cyp19a1b, cyp11b, foxl2, dmrt1 and gtf3a) were also studied. Intersex mullets were identified downstream of both WWTPs and vtgAa was upregulated in intersex and non intersex males. Sex dependent differential transcription levels of target genes were detected in mullets from Galindo. However, no such pattern was observed in mullets from Gernika, suggesting an attenuating effect over studied genes caused by a higher presence of EDCs in this site, as indicated by the elevated prevalence of intersex mullets in this population. In conclusion, no direct association between xenoestrogenic responses and intersex condition was established. Mullets from Gernika showed signs of severe EDC exposure compared to those from Galindo, as demonstrated by the higher prevalence of intersex males and the reduction in transcription profile differences between sexes of gametogenic gene markers. Copyright © 2016 Elsevier Inc. All rights reserved.

Early warning signs of endocrine disruption in adult fish from the ingestion of polyethylene with and without sorbed chemical pollutants from the marine environment

International Nuclear Information System (INIS)

Rochman, Chelsea M.; Kurobe, Tomofumi; Flores, Ida; Teh, Swee J.

2014-01-01

Plastic debris is associated with several chemical pollutants known to disrupt the functioning of the endocrine system. To determine if the exposure to plastic debris and associated chemicals promotes endocrine-disrupting effects in fish, we conducted a chronic two-month dietary exposure using Japanese medaka (Oryzias latipes) and environmentally relevant concentrations of microplastic (< 1 mm) and associated chemicals. We exposed fish to three treatments: a no-plastic (i.e. negative control), virgin-plastic (i.e. virgin polyethylene pre-production pellets) and marine-plastic treatment (i.e. polyethylene pellets deployed in San Diego Bay, CA for 3 months). Altered gene expression was observed in male fish exposed to the marine-plastic treatment, whereas altered gene expression was observed in female fish exposed to both the marine- and virgin-plastic treatment. Significant down-regulation of choriogenin (Chg H) gene expression was observed in males and significant down-regulation of vitellogenin (Vtg I), Chg H and the estrogen receptor (ERα) gene expression was observed in females. In addition, histological observation revealed abnormal proliferation of germ cells in one male fish from the marine-plastic treatment. Overall, our study suggests that the ingestion of plastic debris at environmentally relevant concentrations may alter endocrine system function in adult fish and warrants further research. - Highlights: • We saw down-regulation of Chg H in males exposed to marine plastic. • We saw down-regulation of Vtg I, Chg H and ERα in females exposed to plastic. • We saw abnormal proliferation of germ cells in a male exposed to marine plastic. • Our results suggest that the ingestion of plastic may alter endocrine system function

An endocrine disrupting chemical changes courtship and parental care in the sand goby

Energy Technology Data Exchange (ETDEWEB)

Saaristo, Minna, E-mail: Minna.Saaristo@helsinki.fi [Department of Bio- and Environmental Sciences, University of Helsinki, P.O. Box 65, FI-00014 Helsinki (Finland); Craft, John A. [Biological and Biomedical Sciences, Glasgow Caledonian University, Cowcaddens Road, Glasgow G4 0BA, Scotland (United Kingdom); Lehtonen, Kari K. [Finnish Environment Institute, Marine Research Centre, P.O. Box 140, FI-00251 Helsinki (Finland); Lindstroem, Kai [Environmental and Marine Biology, Abo Akademi University, FI-20520 Turku (Finland)

2010-05-10

Endocrine disrupting chemicals (EDCs) are a diverse group of compounds that can mimic, block or modulate the synthesis of natural hormones. They are known to cause impairment of reproduction of aquatic organisms at very low concentrations. The aim of this study was to examine how exposure from 10 to 31 days to 17{alpha}-ethinyl estradiol (EE2, 41 ng L{sup -1}) affects the courtship and parental care behaviour of male sand gobies (Pomatoschistus minutus). The sand goby exhibits a polygynous mating system, where males compete for females and provide paternal care. First, male courtship performance towards a stimulus female was recorded with video camera. Secondly, after the male had received eggs his parental care behaviour was video recorded. In addition to behavioural endpoints, we measured the expression of hepatic vitellogenin (Vtg) and zona radiata protein (Zrp) mRNA, as well as common somatic indices. Our study shows that exposure to EE2 affected male fanning behaviour during both courtship and parental care. Interestingly, small exposed males increased their courtship fanning to similar levels as larger control males. However, during parental care egg fanning was not related to male size, and all exposed males fanned more than control males. The EE2-exposure induced Vtg and Zrp mRNA expression in males and decreased hepatosomatic index (HSI), and increased gonadosomatic index (GSI). Females prefer males that fan more, which will favour the small EDC exposed males. This may lead to mating that favours males that are not strong enough to tend the eggs until they hatch, thus decreasing the reproductive success of individuals.

Responses of biomarkers of a standardized (Cyprinus carpio) and a native (Pimelodella laticeps) fish species after in situ exposure in a periurban zone of Luján river (Argentina).

Science.gov (United States)

Scarcia, Paola; Calamante, Gabriela; de la Torre, Fernando

2014-05-01

The Luján River basin, which is located in the northwest area of the province of Buenos Aires, Argentina, receives different anthropogenic inputs before reaching the Río de la Plata estuary. The aim of this study was to assess the adverse impact of the river in the middle part of the basin. To this end, an in situ cage assay was conducted in two sites of the river (S1 and S2) near Luján city, and the responses of hepatic biomarkers of both a standardized (Cyprinus carpio) and a native (Pimelodella laticeps) species were evaluated. The biomarkers studied were the condition factor and liver somatic indices (LSI), the enzymatic activities of catalase (CAT), superoxide dismutase (SOD), and glutathione-S-transferase (GST), lipid peroxidation levels (thiobarbituric acid reactive substances, TBARS) and the induction of hepatic cytochrome P450 1A (CYP1A) and vitellogenin (Vtg) proteins. After 14 days, LSI and GST activity increased, and TBARS levels decreased in both species exposed at S1 and S2. In addition, exposure at both sites promoted an increase in SOD activity and CYP1A induction in C. carpio, while Vtg expression was observed only at S1. A shorter exposure period (7 days) caused an initial response only at S2 mediated only by CAT in P. laticeps. Finally, our results demonstrate that a 14-day period of in situ exposure in Luján River could lead to antioxidant and biotransformation processes in C. carpio and to phase II biotransformation responses in P. laticeps. Copyright © 2012 Wiley Periodicals, Inc.

Isolation of Kupffer Cells and Hepatocytes from a Single Mouse Liver

DEFF Research Database (Denmark)

Aparicio-Vergara, Marcela; Tencerova, Michaela; Morgantini, Cecilia

2017-01-01

Liver perfusion is a common technique used to isolate parenchymal and non-parenchymal liver cells for in vitro experiments. This method allows hepatic cells to be separated based on their size and weight, by centrifugation using a density gradient. To date, other methods allow the isolation of only...... one viable hepatic cellular fraction from a single mouse; either parenchymal (hepatocytes) or non-parenchymal cells (i.e., Kupffer cells or hepatic stellate cells). Here, we describe a method to isolate both hepatocytes and Kupffer cells from a single mouse liver, thereby providing the unique...... advantage of studying different liver cell types that have been isolated from the same organism....

Metabolomics for informing adverse outcome pathways: Androgen receptor activation and the pharmaceutical spironolactone

International Nuclear Information System (INIS)

Davis, J.M.; Ekman, D.R.; Skelton, D.M.; LaLone, C.A.; Ankley, G.T.; Cavallin, J.E.; Villeneuve, D.L.; Collette, T.W.

2017-01-01

Highlights: • Metabolomics identified potential key events in an androgen receptor activation AOP. • Metabolomics indicate spironolactone may elicit effects via multiple nuclear receptors. • Spironolactone exposure may elicit interactive effects in multi-stressor environments. - Abstract: One objective in developing adverse outcome pathways (AOPs) is to connect biological changes that are relevant to risk assessors (i.e., fecundity) to molecular and cellular-level alterations that might be detectable at earlier stages of a chemical exposure. Here, we examined biochemical responses of fathead minnows (Pimephales promelas) to inform an AOP relevant to spironolactone’s activation of the androgen receptor, as well as explore other biological impacts possibly unrelated to this receptor. Liquid chromatography with high resolution mass spectrometry (LC–MS) was used to measure changes in endogenous polar metabolites in livers of male and female fish that were exposed to five water concentrations of spironolactone (0, 0.05, 0.5, 5, or 50 μg L"−"1) for 21 days. Metabolite profiles were affected at the two highest concentrations (5 and 50 μg L"−"1), but not in the lower-level exposures, which agreed with earlier reported results of reduced female fecundity and plasma vitellogenin (VTG) levels. We then applied partial least squares regression to assess whether metabolite alterations covaried with changes in fecundity, VTG gene expression and protein concentrations, and plasma 17β-estradiol and testosterone concentrations. Metabolite profiles significantly covaried with all measured endpoints in females, but only with plasma testosterone in males. Fecundity reductions occurred in parallel with changes in metabolites important in osmoregulation (e.g., betaine), membrane transport (e.g., L-carnitine), and biosynthesis of carnitine (e.g., methionine) and VTG (e.g., glutamate). Based on a network analysis program (i.e., mummichog), spironolactone also affected

Spatial variations in biomarkers of Mytilus edulis mussels at four polluted regions spanning the Northern Hemisphere

International Nuclear Information System (INIS)

Gagne, F.; Burgeot, T.; Hellou, J.; St-Jean, S.; Farcy, E.; Blaise, C.

2008-01-01

Economic and social developments have taken place at the expense of the health of the environment, both locally and on a global scale. In an attempt to better understand the large-scale effects of pollution and other stressors like climate change on the health status of Mytilus edulis, mussels were collected during the first two weeks of June 2005 at three sites (one pristine and two affected by pollution) located in each of the regions of the Canadian West Coast, the St. Lawrence estuary, the Atlantic East Coast and the northwestern coast of France, covering a total distance of some 11 000 km. The mussels were analyzed for morphologic integrity (condition factor), gametogenic activity (gonado-somatic and gonad maturation index, vitellogenin(Vtg)-like proteins), energy status (temperature-dependent mitochondrial electron transport activity and gonad lipid stores), defense mechanisms (glutathione S-transferase, metallothioneins, cytochrome P4503A activity and xanthine oxidoreductase-XOR), and tissue damage (lipid peroxidation-LPO and DNA strand breaks). The results showed that data from the reference sites in each region were usually not normally distributed, with discriminant factors reaching the number of regions (i.e. four), except for the biomarkers gonadal lipids, XOR and LPO in digestive gland. The integrated responses of the biomarkers revealed that biomarkers of stress were significantly more pronounced in mussels from the Seine estuary, suggesting that the impacts of pollution are more generalized in this area. Mussels from the Seine estuary and the Atlantic East Coast (Halifax Harbor) responded more strongly for Vtg-like proteins, but was not related to gonad maturation and gonado-somatic indexes, suggesting the presence of environmental estrogens. Moreover, these mussels displayed reduced DNA repair activity and increased LPO. Factorial analyses revealed that energy status, cytochrome P4503A activity and Vtg-like proteins were the most important

Does anti-androgen, flutamide cancel out the in vivo effects of the androgen, dihydrotestosterone on sexual development in juvenile Murray rainbowfish (Melanotaenia fluviatilis)?

Science.gov (United States)

Bhatia, Harpreet; Kumar, Anupama

2016-01-01

The aim of the present study was to investigate if the effects of the androgen, dihydrotestosterone (DHT) on the sexual development in juvenile Murray rainbowfish (Melanotaenia fluviatilis) are canceled out by the anti-androgen, flutamide. Fish (60 days post hatch) were exposed to 250ng/L of DHT, 25μg/L of flutamide (Flu-low), 250μg/L of flutamide (Flu-high), DHT+Flu low and DHT+Flu high. After 35 days of exposure, lengths and weights of the fish were measured and the condition factor (CF) calculated; vitellogenin (VTG) concentrations were measured in tail tissue; sex steroid hormones (17β-estradiol [E2] and 11-keto testosterone [11-KT]) were measured in the head tissue and abdominal regions were used in histological investigation of the gonads. Treatment with DHT reduced the body-length of both male and female fish, an effect which was canceled out by low and high concentrations of flutamide. However, flutamide (low or high) could not nullify the DHT-induced reduction in the CF in either sex. The E2 levels were reduced only in female fish after exposure to DHT but returned to normal after treatment with Flu-high. DHT increased the levels of 11-KT and decreased the E2/11-KT ratio in both sexes. Flu-high, but not Flu-low, could nullify these effects. Both DHT and flutamide (low or high) induced VTG production and this effect persisted when both chemicals were co-administered. Treatment with DHT did not affect gonadal cell development in the testes. However, the female fish treated with DHT contained ovaries in early-vitellogenic stage in comparison to the pre-vitellogenic ovaries in control fish. Co-treatment with flutamide (low or high) resulted in oocyte atresia. The results from the present study suggest that treatment with Flu-high could cancel out DHT-induced effects only on the hormonal profile and body-length in both male and female fish. Juvenile fish co-treated with DHT and flutamide (low or high) had high VTG levels and low CF. In addition, the ovaries

Intersex and alterations in reproductive development of a cichlid, Tilapia guineensis, from a municipal domestic water supply lake (Eleyele) in Southwestern Nigeria

International Nuclear Information System (INIS)

Adeogun, Aina O.; Ibor, Oju R.; Adeduntan, Sherifat D.; Arukwe, Augustine

2016-01-01

The objectives of this study were to develop and validate biomarker techniques for aquatic environmental monitoring of endocrine disrupting chemicals (EDCs) in Nigeria aquatic ecosystems, using the Eleyele Lake, which is a major source of domestic water supply to Ibadan and its surrounding towns, as a model aquatic environment and Tilapia guineensis, as a model organism. A total of 55 male and 28 female fish were used for this study. No significant difference in condition factor was observed between the sexes. Evaluation of gross gonadal morphology of the sampled fish showed 33% intersex prevalence in the sampled population, of which respective 71 and 29% were males and females, with visible testis and ovary developing alongside phenotypic females and males. Plasma concentrations of luteinizing hormone (LH), follicle-stimulating hormone (FSH), 11-ketotestosterone (11-KT) and 17β-estradiol (E2) were performed, showing that male fish had significantly higher plasma LH and E2 concentrations, compared to females. Vitellogenin (Vtg) and zona radiata proteins (Zrp) mRNA levels were significantly higher in males, compared to female fish. Contaminant analysis revealed that PCB 81, 123, 138 and 196 were the only PCB congeners detected in sediment and fish muscle (PCB153 in sediment), while dieldrin was the only organochlorine compound (OC) detected in Eleyele sediment. These responses were used in a multivariate analysis, showing that two principal components were extracted and accounted for 74% of total variation in the dataset. The principal component analysis (PCA) showed that male fish variables were positively correlated with PCB congeners 18 and 123, while female fish showed positive correlations with congener 81, 138, 189, 196, indicating sex-specific pattern of association between PCBs concentrations and biomarker expression. In addition, strong positive correlation between male fish and LH, E2, FSH and Vtg was observed, while female fish positively correlated with

Metabolomics for informing adverse outcome pathways: Androgen receptor activation and the pharmaceutical spironolactone

Energy Technology Data Exchange (ETDEWEB)

Davis, J.M., E-mail: davis.john@epa.gov [U.S. EPA, National Exposure Research Laboratory, 960 College Station Rd., Athens, GA 30605 (United States); Ekman, D.R., E-mail: ekman.drew@epa.gov [U.S. EPA, National Exposure Research Laboratory, 960 College Station Rd., Athens, GA 30605 (United States); Skelton, D.M. [U.S. EPA, National Exposure Research Laboratory, 960 College Station Rd., Athens, GA 30605 (United States); LaLone, C.A.; Ankley, G.T.; Cavallin, J.E.; Villeneuve, D.L. [U.S. EPA, National Health and Environmental Effects Research Laboratory, 6201 Congdon Blvd., Duluth, MN 55804 (United States); Collette, T.W. [U.S. EPA, National Exposure Research Laboratory, 960 College Station Rd., Athens, GA 30605 (United States)

2017-03-15

Highlights: • Metabolomics identified potential key events in an androgen receptor activation AOP. • Metabolomics indicate spironolactone may elicit effects via multiple nuclear receptors. • Spironolactone exposure may elicit interactive effects in multi-stressor environments. - Abstract: One objective in developing adverse outcome pathways (AOPs) is to connect biological changes that are relevant to risk assessors (i.e., fecundity) to molecular and cellular-level alterations that might be detectable at earlier stages of a chemical exposure. Here, we examined biochemical responses of fathead minnows (Pimephales promelas) to inform an AOP relevant to spironolactone’s activation of the androgen receptor, as well as explore other biological impacts possibly unrelated to this receptor. Liquid chromatography with high resolution mass spectrometry (LC–MS) was used to measure changes in endogenous polar metabolites in livers of male and female fish that were exposed to five water concentrations of spironolactone (0, 0.05, 0.5, 5, or 50 μg L{sup −1}) for 21 days. Metabolite profiles were affected at the two highest concentrations (5 and 50 μg L{sup −1}), but not in the lower-level exposures, which agreed with earlier reported results of reduced female fecundity and plasma vitellogenin (VTG) levels. We then applied partial least squares regression to assess whether metabolite alterations covaried with changes in fecundity, VTG gene expression and protein concentrations, and plasma 17β-estradiol and testosterone concentrations. Metabolite profiles significantly covaried with all measured endpoints in females, but only with plasma testosterone in males. Fecundity reductions occurred in parallel with changes in metabolites important in osmoregulation (e.g., betaine), membrane transport (e.g., L-carnitine), and biosynthesis of carnitine (e.g., methionine) and VTG (e.g., glutamate). Based on a network analysis program (i.e., mummichog), spironolactone also

Intersex and alterations in reproductive development of a cichlid, Tilapia guineensis, from a municipal domestic water supply lake (Eleyele) in Southwestern Nigeria

Energy Technology Data Exchange (ETDEWEB)

Adeogun, Aina O.; Ibor, Oju R.; Adeduntan, Sherifat D. [Department of Zoology, University of Ibadan, Ibadan (Nigeria); Arukwe, Augustine, E-mail: arukwe@bio.ntnu.no [Department of Biology, Norwegian University of Science and Technology (NTNU), Høgskoleringen 5, N-7491 Trondheim (Norway)

2016-01-15

The objectives of this study were to develop and validate biomarker techniques for aquatic environmental monitoring of endocrine disrupting chemicals (EDCs) in Nigeria aquatic ecosystems, using the Eleyele Lake, which is a major source of domestic water supply to Ibadan and its surrounding towns, as a model aquatic environment and Tilapia guineensis, as a model organism. A total of 55 male and 28 female fish were used for this study. No significant difference in condition factor was observed between the sexes. Evaluation of gross gonadal morphology of the sampled fish showed 33% intersex prevalence in the sampled population, of which respective 71 and 29% were males and females, with visible testis and ovary developing alongside phenotypic females and males. Plasma concentrations of luteinizing hormone (LH), follicle-stimulating hormone (FSH), 11-ketotestosterone (11-KT) and 17β-estradiol (E2) were performed, showing that male fish had significantly higher plasma LH and E2 concentrations, compared to females. Vitellogenin (Vtg) and zona radiata proteins (Zrp) mRNA levels were significantly higher in males, compared to female fish. Contaminant analysis revealed that PCB 81, 123, 138 and 196 were the only PCB congeners detected in sediment and fish muscle (PCB153 in sediment), while dieldrin was the only organochlorine compound (OC) detected in Eleyele sediment. These responses were used in a multivariate analysis, showing that two principal components were extracted and accounted for 74% of total variation in the dataset. The principal component analysis (PCA) showed that male fish variables were positively correlated with PCB congeners 18 and 123, while female fish showed positive correlations with congener 81, 138, 189, 196, indicating sex-specific pattern of association between PCBs concentrations and biomarker expression. In addition, strong positive correlation between male fish and LH, E2, FSH and Vtg was observed, while female fish positively correlated with

Liver system. V. Activation-extinction line of cyclic hepatocyte activities.

Science.gov (United States)

Dioguardi, N; Brambilla, F; Dell'Oca, M; Arosio, E; Parmeggiani, L

1991-01-01

The excitation-extintion line of hepatocytes from an inert state towards the stabilization of a given activity is described. Within the cell, the switching on of any given activity is a competitive process among different activities. The process is driven by the influence field created in the environment of the Rappaport acinus by sinusoidal blood which changes its characteristics during its passage from the portal zone to the central vein. Every step of the excitation-extintion pathway follows the so-called law of autoisodiasostasis (AIS), i.e. it is characterized by an oscillatory motion between restoring (homopoiesis or HP) and working (homeorhesis or HR) states. Since the cyclical bistable equilibrium of AIS characterizes all conditions of hepatocyte activities, the AIS cycle can be defined a limit cycle.

Hepatocytes Determine the Hypoxic Microenvironment and Radiosensitivity of Colorectal Cancer Cells Through Production of Nitric Oxide That Targets Mitochondrial Respiration

International Nuclear Information System (INIS)

Jiang, Heng; Verovski, Valeri N.; Leonard, Wim; Law, Ka Lun; Vermeersch, Marieke; Storme, Guy; Van den Berge, Dirk; Gevaert, Thierry; Sermeus, Alexandra; De Ridder, Mark

2013-01-01

Purpose: To determine whether host hepatocytes may reverse hypoxic radioresistance through nitric oxide (NO)-induced oxygen sparing, in a model relevant to colorectal cancer (CRC) liver metastases. Methods and Materials: Hepatocytes and a panel of CRC cells were incubated in a tissue-mimetic coculture system with diffusion-limited oxygenation, and oxygen levels were monitored by an oxygen-sensing fluorescence probe. To activate endogenous NO production, cocultures were exposed to a cytokine mixture, and the expression of inducible nitric oxide synthase was analyzed by reverse transcription–polymerase chain reaction, Western blotting, and NO/nitrite production. The mitochondrial targets of NO were examined by enzymatic activity. To assess hypoxic radioresponse, cocultures were irradiated and reseeded for colonies. Results: Resting hepatocytes consumed 10-40 times more oxygen than mouse CT26 and human DLD-1, HT29, HCT116, and SW480 CRC cells, and thus seemed to be the major effectors of hypoxic conditioning. As a result, hepatocytes caused uniform radioprotection of tumor cells at a 1:1 ratio. Conversely, NO-producing hepatocytes radiosensitized all CRC cell lines more than 1.5-fold, similar to the effect of selective mitochondrial inhibitors. The radiosensitizing effect was associated with a respiratory self-arrest of hepatocytes at the level of aconitase and complex II, which resulted in profound reoxygenation of tumor cells through oxygen sparing. Nitric oxide–producing hepatocytes were at least 10 times more active than NO-producing macrophages to reverse hypoxia-induced radioresistance. Conclusions: Hepatocytes were the major determinants of the hypoxic microenvironment and radioresponse of CRC cells in our model of metabolic hypoxia. We provide evidence that reoxygenation and radiosensitization of hypoxic CRC cells can be achieved through oxygen sparing induced by endogenous NO production in host hepatocytes

Hepatocytes Determine the Hypoxic Microenvironment and Radiosensitivity of Colorectal Cancer Cells Through Production of Nitric Oxide That Targets Mitochondrial Respiration

Energy Technology Data Exchange (ETDEWEB)

Jiang, Heng; Verovski, Valeri N.; Leonard, Wim; Law, Ka Lun; Vermeersch, Marieke; Storme, Guy; Van den Berge, Dirk; Gevaert, Thierry; Sermeus, Alexandra [Department of Radiotherapy, Universitair Ziekenhuis Brussel, Vrije Universiteit Brussel, Brussels (Belgium); De Ridder, Mark, E-mail: mark.deridder@uzbrussel.be [Department of Radiotherapy, Universitair Ziekenhuis Brussel, Vrije Universiteit Brussel, Brussels (Belgium)

2013-03-01

Purpose: To determine whether host hepatocytes may reverse hypoxic radioresistance through nitric oxide (NO)-induced oxygen sparing, in a model relevant to colorectal cancer (CRC) liver metastases. Methods and Materials: Hepatocytes and a panel of CRC cells were incubated in a tissue-mimetic coculture system with diffusion-limited oxygenation, and oxygen levels were monitored by an oxygen-sensing fluorescence probe. To activate endogenous NO production, cocultures were exposed to a cytokine mixture, and the expression of inducible nitric oxide synthase was analyzed by reverse transcription–polymerase chain reaction, Western blotting, and NO/nitrite production. The mitochondrial targets of NO were examined by enzymatic activity. To assess hypoxic radioresponse, cocultures were irradiated and reseeded for colonies. Results: Resting hepatocytes consumed 10-40 times more oxygen than mouse CT26 and human DLD-1, HT29, HCT116, and SW480 CRC cells, and thus seemed to be the major effectors of hypoxic conditioning. As a result, hepatocytes caused uniform radioprotection of tumor cells at a 1:1 ratio. Conversely, NO-producing hepatocytes radiosensitized all CRC cell lines more than 1.5-fold, similar to the effect of selective mitochondrial inhibitors. The radiosensitizing effect was associated with a respiratory self-arrest of hepatocytes at the level of aconitase and complex II, which resulted in profound reoxygenation of tumor cells through oxygen sparing. Nitric oxide–producing hepatocytes were at least 10 times more active than NO-producing macrophages to reverse hypoxia-induced radioresistance. Conclusions: Hepatocytes were the major determinants of the hypoxic microenvironment and radioresponse of CRC cells in our model of metabolic hypoxia. We provide evidence that reoxygenation and radiosensitization of hypoxic CRC cells can be achieved through oxygen sparing induced by endogenous NO production in host hepatocytes.

Induction of highly functional hepatocytes from human umbilical cord mesenchymal stem cells by HNF4α transduction.

Directory of Open Access Journals (Sweden)

Hualian Hang

Full Text Available To investigate the differentiation potential of human umbilical mesenchymal stem cells (HuMSCs and the key factors that facilitate hepatic differentiation.HuMSCs were induced to become hepatocyte-like cells according to a previously published protocol. The differentiation status of the hepatocyte-like cells was examined by observing the morphological changes under an inverted microscope and by immunofluorescence analysis. Hepatocyte nuclear factor 4 alpha (HNF4α overexpression was achieved by plasmid transfection of the hepatocyte-like cells. The expression of proteins and genes of interest was then examined by Western blotting and reverse transcription-polymerase chain reaction (RT-PCR or real-time RT-PCR methods.Our results demonstrated that HuMSCs can easily be induced into hepatocyte-like cells using a published differentiation protocol. The overexpression of HNF4α in the induced HuMSCs significantly enhanced the expression levels of hepatic-specific proteins and genes. HNF4α overexpression may be associated with liver-enriched transcription factor networks and the Wnt/β-Catenin pathway.The overexpression of HNF4α improves the hepatic differentiation of HuMSCs and is a simple way to improve cellular sources for clinical applications.

Insulin infusion reduces hepatocyte growth factor in lean humans

DEFF Research Database (Denmark)

de Courten, Barbora; de Courten, Maximilian; Dougherty, Sonia

2013-01-01

OBJECTIVE: Plasma Hepatocyte Growth Factor (HGF) is significantly elevated in obesity and may contribute to vascular disease, metabolic syndrome or cancer in obese individuals. The current studies were done to determine if hyperinsulinemia increases plasma HGF. MATERIALS/METHODS: Twenty-two parti...

Effects of dehydroepiandrosterone (DHEA) on glucose metabolism in isolated hepatocytes from Zucker rats

International Nuclear Information System (INIS)

Finan, A.; Cleary, M.P.

1986-01-01

DHEA has been shown to competitively inhibit the pentose phosphate shunt (PPS) enzyme glucose-6-phosphate dehydrogenase (G6PD) when added in vitro to supernatants or homogenates prepared from mammalian tissues. However, no consistent effect on G6PD activity has been determined in tissue removed from DHEA-treated rats. To explore the effects of DHEA on PPS, glucose utilization was measured in hepatocytes from lean and obese male Zucker rats (8 wks of age) following 1 wk of DHEA treatment (0.6% in diet). Incubation of isolated hepatocytes from treated lean Zucker rats with either [1- 14 C] glucose or [6- 14 C] glucose resulted in significant decreases in CO 2 production and total glucose utilization. DHEA-lean rats also had lowered fat pad weights. In obese rats, there was no effect of 1 wk of treatment on either glucose metabolism or fat pad weight. The calculated percent contribution of the PPS to glucose metabolism in hepatocytes was not changed for either DHEA-lean or obese rats when compared to control rats. In conclusion, 1 wk of DHEA treatment lowered overall glucose metabolism in hepatocytes of lean Zucker rats, but did not selectively affect the PPS. The lack of an effect of short-term treatment in obese rats may be due to differences in their metabolism or storage/release of DHEA in tissues in comparison to lean rats

Metabolism of the synthetic cannabinoid 5F-PY-PICA by human and rat hepatocytes and identification of biliary analytical targets by directional efflux in sandwich-cultured rat hepatocytes using UHPLC-HR-MS/MS

DEFF Research Database (Denmark)

Mardal, Marie; Annaert, Pieter; Noble, Carolina

2018-01-01

Analytical strategies for detecting drugs in biological samples rely on information on metabolism and elimination. 5F-PY-PICA belongs to the group of synthetic cannabinoids that are known to undergo excretion into the bile. The aims of this study were the in vitro identification of metabolites of 5......F-PY-PICA and to determine which analytical targets are excreted into the bile and urine. Metabolites identified after incubation of 5F-PY-PICA with pooled human liver microsomes (pHLM), pooled human hepatocytes (pHH), or suspended and sandwich-cultured rat hepatocytes (SCRH). Rat hepatocytes were......-PY-PICA, M4, and M22 are proposed as analytical targets for bile analysis in forensic screening protocols, whereas M6 should be one of the main urinary targets for 5F-PY-PICA analysis....

Identification and Transcriptional Modulation of the Largemouth Bass, Micropterus salmoides, Vitellogenin Receptor During Oocyte Development by Insulin and Sex Steroids1

Science.gov (United States)

Dominguez, Gustavo A.; Quattro, Joseph M.; Denslow, Nancy D.; Kroll, Kevin J.; Prucha, Melinda S.; Porak, Wesley F.; Grier, Harry J.; Sabo-Attwood, Tara L.

2012-01-01

ABSTRACT Fish vitellogenin synthesized and released from the liver of oviparous animals is taken up into oocytes by the vitellogenin receptor. This is an essential process in providing nutrient yolk to developing embryos to ensure successful reproduction. Here we disclose the full length vtgr cDNA sequence for largemouth bass (LMB) that reveals greater than 90% sequence homology with other fish vtgr sequences. We classify LMB Vtgr as a member of the low density lipoprotein receptor superfamily based on conserved domains and categorize as the short variant that is devoid of the O-glycan segment. Phylogenetic analysis places LMB Vtgr sequence into a well-supported monophyletic group of fish Vtgr. Real-time PCR showed that the greatest levels of LMB vtgr mRNA expression occurred in previtellogenic ovarian tissues. In addition, we reveal the effects of insulin, 17beta-estradiol (E2), and 11-ketotestosterone (11-KT) in modulation of vtgr, esr, and ar mRNAs in previtellogenic oocytes. Insulin increased vtgr expression levels in follicles ex vivo while exposure to E2 or 11-KT did not result in modulation of expression. However, both steroids were able to repress insulin-induced vtgr transcript levels. Coexposure with insulin and E2 or of insulin and 11-KT increased ovarian esr2b and ar mRNA levels, respectively, which suggest a role for these nuclear receptors in insulin-mediated signaling pathways. These data provide the first evidence for the ordered stage-specific expression of LMB vtgr during the normal reproductive process and the hormonal influence of insulin and sex steroids on controlling vtgr transcript levels in ovarian tissues. PMID:22786822

Implications of the simultaneous occurrence of glycolysis and gluconeogenesis in hepatocytes from normal and hyperthyroid rats

International Nuclear Information System (INIS)

Phillips, J.W.; Berry, M.N.

2001-01-01

The mammalian liver has the capability for both glycolysis and gluconeogenesis. In the fasting state, metabolites such as lactate and glycerol, generated in the peripheral tissues, are taken up by the liver and converted to glucose. However, hepatocytes from fasted animals are also capable of substantial rates of glycolysis. It is generally assumed that glycolysis and gluconeogenesis do not occur simultaneously in the same cell, but rather the metabolic conditions that facilitate flux through one pathway impair flow in the opposite direction. The actual direction of flow at any given moment is thought to be determined by regulatory mechanisms that control flux through the enzymatic steps specific to glycolysis and gluconeogenesis. The rates of glycolysis from [6- 3 H]glucose and gluconeogenesis from [U- 14 C]glycerol were determined in isolated hepatocytes from fasted normal and hyperthyroid rats. We observed that glycolysis from glucose and glucose synthesis from glycerol occurred simultaneously at substantial rates in hepatocytes from normal rats and that gluconeogenesis, but not glycolysis, was increased twofold in hepatocytes from thyroid treated rats. In the hyperthyroid state, the rate of glycolysis from glucose was approximately equal to the rate of glucose formation from glycerol. Hence, metabolism and ATP turnover were stimulated without substantially altering steady-state concentrations of glucose. The concomitant operation of hepatic glycolysis and gluconeogenesis may be a mechanism that accounts in part for the calorigenic effect of thyroid hormone. Since hepatocyles are generally impermeable to phosphorylated metabolites, our observations suggest that glycolysis, and phosphorylation of glycerol take place in the same cells, and that the occurrence of simultaneous glycolysis and gluconeogenesis is an indication of channelling within the hepatocyte cytoplasm of individual hepatocytes

Connexin 32 and connexin 43 are involved in lineage restriction of hepatic progenitor cells to hepatocytes

Directory of Open Access Journals (Sweden)

Haiyun Pei

2017-11-01

Full Text Available Abstract Background Bi-potential hepatic progenitor cells can give rise to both hepatocytes and cholangiocytes, which is the last phase and critical juncture in terms of sequentially hepatic lineage restriction from any kind of stem cells. If their differentiation can be controlled, it might access to functional hepatocytes to develop pharmaceutical and biotechnology industries as well as cell therapies for end-stage liver diseases. Methods In this study, we investigated the influence of Cx32 and Cx43 on hepatocyte differentiation of WB-F344 cells by in vitro gain and loss of function analyses. An inhibitor of Cx32 was also used to make further clarification. To reveal p38 MAPK pathway is closely related to Cxs, rats with 70% partial hepatectomy were injected intraperitoneally with a p38 inhibitor, SB203580. Besides, the effects of p38 MAPK pathway on differentiation of hepatoblasts isolated from fetal rat livers were evaluated by addition of SB203580 in culture medium. Results In vitro gain and loss of function analyses showed overexpression of Connexin 32 and knockdown of Connexin 43 promoted hepatocytes differentiation from hepatic progenitor cells. In addition, in vitro and ex vivo research revealed inhibition of p38 mitogen-activated protein kinase pathway can improve hepatocytes differentiation correlating with upregulation of Connexin 32 expression and downregulation of Connexin 43 expression. Conclusions Here we demonstrate that Connexins play crucial roles in facilitating differentiation of hepatic progenitors. Our work further implicates that regulators of Connexins and their related pathways might provide new insights to improve lineage restriction of stem cells to mature hepatocytes.

Xenobiotic-Metabolizing Enzyme and Transporter Gene Expression in Primary Cultures of Human Hepatocytes Modulated by Toxcast Chemicals

Science.gov (United States)

Primary human hepatocyte cultures are useful in vitro model systems of human liver because when cultured under appropriate conditions the hepatocytes retain liver-like functionality such as metabolism, transport, and cell signaling. This model system was used to characterize the ...

CXC chemokines function as a rheostat for hepatocyte proliferation and liver regeneration.

Directory of Open Access Journals (Sweden)

Gregory C Wilson

Full Text Available Our previous in vitro studies have demonstrated dose-dependent effects of CXCR2 ligands on hepatocyte cell death and proliferation. In the current study, we sought to determine if CXCR2 ligand concentration is responsible for the divergent effects of these mediators on liver regeneration after ischemia/reperfusion injury and partial hepatectomy.Murine models of partial ischemia/reperfusion injury and hepatectomy were used to study the effect of CXCR2 ligands on liver regeneration.We found that hepatic expression of the CXCR2 ligands, macrophage inflammatory protein-2 (MIP-2 and keratinocyte-derived chemokine (KC, was significantly increased after both I/R injury and partial hepatectomy. However, expression of these ligands after I/R injury was 30-100-fold greater than after hepatectomy. Interestingly, the same pattern of expression was found in ischemic versus non-ischemic liver lobes following I/R injury with expression significantly greater in the ischemic liver lobes. In both systems, lower ligand expression was associated with increased hepatocyte proliferation and liver regeneration in a CXCR2-dependent fashion. To confirm that these effects were related to ligand concentration, we administered exogenous MIP-2 and KC to mice undergoing partial hepatectomy. Mice received a "high" dose that replicated serum levels found after I/R injury and a "low" dose that was similar to that found after hepatectomy. Mice receiving the "high" dose had reduced levels of hepatocyte proliferation and regeneration whereas the "low" dose promoted hepatocyte proliferation and regeneration.Together, these data demonstrate that concentrations of CXC chemokines regulate the hepatic proliferative response and subsequent liver regeneration.

Tributyltin induces apoptotic signaling in hepatocytes through pathways involving the endoplasmic reticulum and mitochondria

International Nuclear Information System (INIS)

Grondin, Melanie; Marion, Michel; Denizeau, Francine; Averill-Bates, Diana A.

2007-01-01

Tri-n-butyltin is a widespread environmental toxicant, which accumulates in the liver. This study investigates whether tri-n-butyltin induces pro-apoptotic signaling in rat liver hepatocytes through pathways involving the endoplasmic reticulum and mitochondria. Tri-n-butyltin activated the endoplasmic reticulum pathway of apoptosis, which was demonstrated by the activation of the protease calpain, its translocation to the plasma membrane, followed by cleavage of the calpain substrates, cytoskeletal protein vinculin, and caspase-12. Caspase-12 is localized to the cytoplasmic side of the endoplasmic reticulum and is involved in apoptosis mediated by the endoplasmic reticulum. Tri-n-butyltin also caused translocation of the pro-apoptotic proteins Bax and Bad from the cytosol to mitochondria, as well as changes in mitochondrial membrane permeability, events which can activate the mitochondrial death pathway. Tri-n-butyltin induced downstream apoptotic events in rat hepatocytes at the nuclear level, detected by chromatin condensation and by confocal microscopy using acridine orange. We investigated whether the tri-n-butyltin-induced pro-apoptotic events in hepatocytes could be linked to perturbation of intracellular calcium homeostasis, using confocal microscopy. Tri-n-butyltin caused changes in intracellular calcium distribution, which were similar to those induced by thapsigargin. Calcium was released from a subcellular compartment, which is likely to be the endoplasmic reticulum, into the cytosol. Cytosolic acidification, which is known to trigger apoptosis, also occurred and involved the Cl - /HCO 3 - exchanger. Pro-apoptotic events in hepatocytes were inhibited by the calcium chelator, Bapta-AM, and by a calpain inhibitor, which suggests that changes in intracellular calcium homeostasis are involved in tri-n-butyltin-induced apoptotic signaling in rat hepatocytes

Activated Hepatic Stellate Cells Induce Tumor Progression of Neoplastic Hepatocytes in a TGF-β Dependent Fashion

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MIKULA, M.; PROELL, V.; FISCHER, A.N.M.; MIKULITS, W.

2010-01-01

The development of hepatocellular carcinomas from malignant hepatocytes is frequently associated with intra- and peritumoral accumulation of connective tissue arising from activated hepatic stellate cells. For both tumorigenesis and hepatic fibrogenesis, transforming growth factor (TGF)-β signaling executes key roles and therefore is considered as a hallmark of these pathological events. By employing cellular transplantation we show that the interaction of neoplastic MIM-R hepatocytes with the tumor microenvironment, containing either activated hepatic stellate cells (M1-4HSCs) or myofibroblasts derived thereof (M-HTs), induces progression in malignancy. Cotransplantation of MIM-R hepatocytes with M-HTs yielded strongest MIM-R generated tumor formation accompanied by nuclear localization of Smad2/3 as well as of β-catenin. Genetic interference with TGF-β signaling by gain of antagonistic Smad7 in MIM-R hepatocytes diminished epithelial dedifferentiation and tumor progression upon interaction with M1-4HSCs or M-HTs. Further analysis showed that tumors harboring disrupted Smad signaling are devoid of nuclear β-catenin accumulation, indicating a crosstalk between TGF-β and β-catenin signaling. Together, these data demonstrate that activated HSCs and myofibroblasts directly govern hepatocarcinogenesis in a TGF-β dependent fashion by inducing autocrine TGF-β signaling and nuclear β-catenin accumulation in neoplastic hepatocytes. These results indicate that intervention with TGF-β signaling is highly promising in liver cancer therapy. PMID:16883581

Stiffness of hyaluronic acid gels containing liver extracellular matrix supports human hepatocyte function and alters cell morphology.

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Deegan, Daniel B; Zimmerman, Cynthia; Skardal, Aleksander; Atala, Anthony; Shupe, Thomas D

2015-03-01

Tissue engineering and cell based liver therapies have utilized primary hepatocytes with limited success due to the failure of hepatocytes to maintain their phenotype in vitro. In order to overcome this challenge, hyaluronic acid (HA) cell culture substrates were formulated to closely mimic the composition and stiffness of the normal liver cellular microenvironment. The stiffness of the substrate was modulated by adjusting HA hydrogel crosslinking. Additionally, the repertoire of bioactive molecules within the HA substrate was bolstered by supplementation with normal liver extracellular matrix (ECM). Primary human hepatocyte viability and phenotype were determined over a narrow physiologically relevant range of substrate stiffnesses from 600 to 4600Pa in both the presence and absence of liver ECM. Cell attachment, viability, and organization of the actin cytoskeleton improved with increased stiffness up to 4600Pa. These differences were not evident in earlier time points or substrates containing only HA. However, gene expression for the hepatocyte markers hepatocyte nuclear factor 4 alpha (HNF4α) and albumin significantly decreased on the 4600Pa stiffness at day 7 indicating that cells may not have maintained their phenotype long-term at this stiffness. Function, as measured by albumin secretion, varied with both stiffness and time in culture and peaked at day 7 at the 1200Pa stiffness, slightly below the stiffness of normal liver ECM at 3000Pa. Overall, gel stiffness affected primary human hepatocyte cell adhesion, functional marker expression, and morphological characteristics dependent on both the presence of liver ECM in gel substrates and time in culture. Copyright © 2015 Elsevier Ltd. All rights reserved.

Evaluating the uptake and intracellular fate of polystyrene nanoparticles by primary and hepatocyte cell lines in vitro

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Johnston, Helinor J.; Semmler-Behnke, Manuela; Brown, David M.; Kreyling, Wolfgang; Tran, Lang; Stone, Vicki

2010-01-01

Nanoparticles (NPs) are being used within diverse applications such as medicines, clothing, cosmetics and food. In order to promote the safe development of such nanotechnologies it is essential to assess the potential adverse health consequences associated with human exposure. The liver is recognised as a target site for NP toxicity, due to NP accumulation within this organ subsequent to injection, inhalation or instillation. The uptake of fluorescent polystyrene carboxylated particles (20 nm or 200 nm diameter) by hepatocytes was determined using confocal microscopy; with cells imaged 'live' during particle exposure or after exposure within fixed cells. Comparisons between the uptake of polystyrene particles by primary rat hepatocytes, and human hepatocyte cell lines (C3A and HepG2) were made. Uptake of particles by hepatocytes was size, time, and serum dependent. Specifically, the uptake of 200 nm particles was limited, but 20 nm NPs were internalised by all cell types from 10 min onwards. At 10 min, 20 nm NP fluorescence co-localised with the tubulin cytoskeleton staining; after 30 min NP fluorescence compartmentalised into structures located within and/or between cells. The fate of internalised NPs was considered and they were not contained within early endosomes or lysosomes, but within mitochondria of cell lines. NPs accumulated within bile canaliculi to a limited extent, which suggests that NPs can be eliminated within bile. This is in keeping with the finding that gold NPs were eliminated in bile following intravenous injection into rats. The findings were, in the main, comparable between primary rat hepatocytes and the different human hepatocyte cell lines.

Influence of flow conditions and matrix coatings on growth and differentiation of three-dimensionally cultured rat hepatocytes.

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Fiegel, Henning C; Havers, Joerg; Kneser, Ulrich; Smith, Molly K; Moeller, Tim; Kluth, Dietrich; Mooney, David J; Rogiers, Xavier; Kaufmann, Peter M

2004-01-01

Maintenance of liver-specific function of hepatocytes in culture is still difficult. Improved culture conditions may enhance the cell growth and function of cultured cells. We investigated the effect of three-dimensional culture under flow conditions, and the influence of surface modifications in hepatocyte cultures. Hepatocytes were harvested from Lewis rats. Cells were cultured on three-dimensional polymeric poly-lactic-co-glycolic acid (PLGA) matrices in static culture, or in a pulsatile flow-bioreactor system. Different surface modifications of matrices were investigated: coating with collagen I, collagen IV, laminin, or fibronectin; or uncoated matrix. Hepatocyte numbers, DNA content, and albumin secretion rate were assessed over the observation period. Culture under flow condition significantly enhanced cell numbers. An additional improvement of this effect was observed, when matrix coating was used. Cellular function also showed a significant increase (4- to 5-fold) under flow conditions when compared with static culture. Our data showed that culture under flow conditions improves cell number, and strongly enhances cellular function. Matrix modification by coating with extracellular matrix showed overall an additive stimulatory effect. Our conclusion is that combining three-dimensional culture under flow conditions and using matrix modification significantly improves culture conditions and is therefore attractive for the development of successful culture systems for hepatocytes.

The Hepatitis B Virus (HBV) HBx Protein Activates AKT To Simultaneously Regulate HBV Replication and Hepatocyte Survival

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Rawat, Siddhartha

2014-01-01

ABSTRACT Chronic infection with hepatitis B virus (HBV) is a risk factor for developing liver diseases such as hepatocellular carcinoma (HCC). HBx is a multifunctional protein encoded by the HBV genome; HBx stimulates HBV replication and is thought to play an important role in the development of HBV-associated HCC. HBx can activate the phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway in some cell lines; however, whether HBx regulates PI3K/AKT signaling in normal hepatocytes has not been evaluated. In studies described here, we assessed HBx activation of PI3K/AKT signaling in an ex vivo model of cultured primary hepatocytes and determined how this HBx activity affects HBV replication. We report that HBx activates AKT in primary hepatocytes and that the activation of AKT decreases HBV replication and HBV mRNA and core protein levels. We show that the transcription factor hepatocyte nuclear factor 4α (HNF4α) is a target of HBx-regulated AKT, and we link HNF4α to HBx-regulated AKT modulation of HBV transcription and replication. Although we and others have shown that HBx stimulates and is likely required for HBV replication, we now report that HBx also activates signals that can diminish the overall level of HBV replication. While this may seem counterintuitive, we show that an important effect of HBx activation of AKT is inhibition of apoptosis. Consequently, our studies suggest that HBx balances HBV replication and cell survival by stimulating signaling pathways that enhance hepatocyte survival at the expense of higher levels of HBV replication. IMPORTANCE Chronic hepatitis B virus (HBV) infection is a common cause of the development of liver cancer. Regulation of cell signaling pathways by the HBV HBx protein is thought to influence the development of HBV-associated liver cancer. HBx stimulates, and may be essential for, HBV replication. We show that HBx activates AKT in hepatocytes to reduce HBV replication. While this seems contradictory to an

A transgenic rat hepatocyte - Kupffer cell co-culture model for evaluation of direct and macrophage-related effect of poly(amidoamine) dendrimers.

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Jemnitz, Katalin; Bátai-Konczos, Attila; Szabó, Mónika; Ioja, Enikő; Kolacsek, Orsolya; Orbán, Tamás I; Török, György; Homolya, László; Kovács, Eszter; Jablonkai, István; Veres, Zsuzsa

2017-02-01

Increasing number of papers demonstrate that Kupffer cells (KCs) play a role in the development of drug induced liver injury (DILI). Furthermore, elevated intracellular Ca 2+ level of hepatocytes is considered as a common marker of DILI. Here we applied an in vitro model based on hepatocyte mono- and hepatocyte/KC co-cultures (H/KC) isolated from transgenic rats stably expressing the GCaMP2 fluorescent Ca 2+ sensor protein to investigate the effects of polycationic (G5), polyanionic (G4.5) and polyethylene-glycol coated neutral (G5 Peg) dendrimers known to accumulate in the liver, primarily in KCs. Following dendrimer exposure, hepatocyte homeostasis was measured by MTT cytotoxicity assay and by Ca 2+ imaging, while hepatocyte functions were studied by CYP2B1/2 inducibility, and bilirubin and taurocholate transport. G5 was significantly more cytotoxic than G4.5 for hepatocytes and induced Ca 2+ oscillation and sustained Ca 2+ signals at 1μM and10 μM, respectively both in hepatocytes and KCs. Dendrimer-induced Ca 2+ signals in hepatocytes were attenuated by macrophages. Activation of KCs by lipopolysaccharide and G5 decreased the inducibility of CYP2B1/2, which was restored by depleting the KCs with gadolinium-chloride and pentoxyphylline, suggesting a role of macrophages in the hindrance of CYP2B1/2 induction by G5 and lipopolysaccharide. In the H/KC, but not in the hepatocyte mono-culture, G5 reduced the canalicular efflux of bilirubin and stimulated the uptake and canalicular efflux of taurocholate. In conclusion, H/KC provides a good model for the prediction of hepatotoxic potential of drugs, especially of nanomaterials known to be trapped by macrophages, activation of which presumably contributes to DILI. Copyright © 2016 Elsevier B.V. All rights reserved.

Temperature shift and host cell contact up-regulate sporozoite expression of Plasmodium falciparum genes involved in hepatocyte infection.

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Anthony Siau

Full Text Available Plasmodium sporozoites are deposited in the skin by Anopheles mosquitoes. They then find their way to the liver, where they specifically invade hepatocytes in which they develop to yield merozoites infective to red blood cells. Relatively little is known of the molecular interactions during these initial obligatory phases of the infection. Recent data suggested that many of the inoculated sporozoites invade hepatocytes an hour or more after the infective bite. We hypothesised that this pre-invasive period in the mammalian host prepares sporozoites for successful hepatocyte infection. Therefore, the genes whose expression becomes modified prior to hepatocyte invasion would be those likely to code for proteins implicated in the subsequent events of invasion and development. We have used P. falciparum sporozoites and their natural host cells, primary human hepatocytes, in in vitro co-culture system as a model for the pre-invasive period. We first established that under co-culture conditions, sporozoites maintain infectivity for an hour or more, in contrast to a drastic loss in infectivity when hepatocytes were not included. Thus, a differential transcriptome of salivary gland sporozoites versus sporozoites co-cultured with hepatocytes was established using a pan-genomic P. falciparum microarray. The expression of 532 genes was found to have been up-regulated following co-culture. A fifth of these genes had no orthologues in the genomes of Plasmodium species used in rodent models of malaria. Quantitative RT-PCR analysis of a selection of 21 genes confirmed the reliability of the microarray data. Time-course analysis further indicated two patterns of up-regulation following sporozoite co-culture, one transient and the other sustained, suggesting roles in hepatocyte invasion and liver stage development, respectively. This was supported by functional studies of four hitherto uncharacterized proteins of which two were shown to be sporozoite surface

3D-printed gelatin scaffolds of differing pore geometry modulate hepatocyte function and gene expression.

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Lewis, Phillip L; Green, Richard M; Shah, Ramille N

2018-03-15

Three dimensional (3D) printing is highly amenable to the fabrication of tissue-engineered organs of a repetitive microstructure such as the liver. The creation of uniform and geometrically repetitive tissue scaffolds can also allow for the control over cellular aggregation and nutrient diffusion. However, the effect of differing geometries, while controlling for pore size, has yet to be investigated in the context of hepatocyte function. In this study, we show the ability to precisely control pore geometry of 3D-printed gelatin scaffolds. An undifferentiated hepatocyte cell line (HUH7) demonstrated high viability and proliferation when seeded on 3D-printed scaffolds of two different geometries. However, hepatocyte specific functions (albumin secretion, CYP activity, and bile transport) increases in more interconnected 3D-printed gelatin cultures compared to a less interconnected geometry and to 2D controls. Additionally, we also illustrate the disparity between gene expression and protein function in simple 2D culture modes, and that recreation of a physiologically mimetic 3D environment is necessary to induce both expression and function of cultured hepatocytes. Three dimensional (3D) printing provides tissue engineers the ability spatially pattern cells and materials in precise geometries, however the biological effects of scaffold geometry on soft tissues such as the liver have not been rigorously investigated. In this manuscript, we describe a method to 3D print gelatin into well-defined repetitive geometries that show clear differences in biological effects on seeded hepatocytes. We show that a relatively simple and widely used biomaterial, such as gelatin, can significantly modulate biological processes when fabricated into specific 3D geometries. Furthermore, this study expands upon past research into hepatocyte aggregation by demonstrating how it can be manipulated to enhance protein function, and how function and expression may not precisely correlate in

R-spondin3-LGR4 signaling protects hepatocytes against DMOG-induced hypoxia/reoxygenation injury through activating β-catenin.

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Liu, Shiying; Yin, Yue; Yu, Ruili; Li, Yin; Zhang, Weizhen

2018-04-30

Leucine-rich repeat G-protein-coupled receptor 4 (LGR4) and its ligands R-spondin1-4 (Rspos) have been vastly investigated in embryonic development. The biological functions of Rspos-LGR4 system in liver remains largely unknown. Here, we explored whether it protects hepatocytes against hypoxia/reoxygenation (H/R) induced damage. H/R injury was induced by dimethyloxalylglycine (DMOG) in AML12 cells and the effects of Rspo3 on cell proliferation and apoptosis were assessed. Specific shRNAs were used to interfere LGR4 or β-catenin. DMOG caused hepatocytes damage evidenced by increase in HIF-1α, cell death and apoptosis genes p27 and Bax, with concurrent decrease of cell proliferation genes PCNA and CyclinD1. Of all the Rspos, Rspo3 is predominantly expressed in AML12 hepatocytes. Importantly, Rspo3 demonstrated an alteration in a manner similar to proliferation-related genes during H/R injury. Rspo3 pretreatment rendered hepatocytes less vulnerable to DMOG induced H/R injury. Ablation of LGR4 using shRNA attenuated the protective effects of Rspo3. Wnt3a also protected AML12 cells from damages caused by H/R, showing enhanced proliferation activity. Notably, knockdown of β-catenin in hepatocytes completely abolished the effect of Rspo3 pretreatment on the expression levels of PCNA and CyclinD1. Rspo3-LGR4 axis protects hepatocytes from H/R injury via activating β-catenin. Copyright © 2018. Published by Elsevier Inc.

A survey of consumer attitudes to the supply and use of human hepatocytes in the United Kingdom.

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Lloyd, Tom D R; Orr, Samantha; Dennison, Ashley R

2003-11-01

Human hepatocytes are the model of choice for pharmacotoxicological studies, but their acquisition is often problematic due to ethical and logistical difficulties. The UK Human Tissue Bank is a not-for-profit organisation that acquires and processes human tissue, with a specialist interest in the isolation of human hepatocytes. A recent in-house survey of the processing of liver tissue over 1 year revealed that freshly isolated hepatocytes were underutilised due to mismatched consumer demand, despite the published need for them. We present the results of a telephone survey to investigate the reasons behind this paradox. This survey highlighted some problem areas, including "out of hours" availability of cells and personnel difficulties, but overall, demonstrated the value of such a service, with numerous researchers taking advantage of available good quality human hepatocytes. Although further work is required in optimising long-term storage protocols through cryopreservation, we have demonstrated that tissue handling of this type can be successful and beneficial to the pharmaceutical and biotechnology industries.

Interleukin-1 inhibition facilitates recovery from liver injury and promotes regeneration of hepatocytes in alcoholic hepatitis in mice.

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Iracheta-Vellve, Arvin; Petrasek, Jan; Gyogyosi, Benedek; Bala, Shashi; Csak, Timea; Kodys, Karen; Szabo, Gyongyi

2017-07-01

Inflammation and impaired hepatocyte regeneration contribute to liver failure in alcoholic hepatitis (AH). Interleukin (IL)-1 is a key inflammatory cytokine in the pathobiology of AH. The role of IL-1 in liver regeneration in the recovery phase of alcohol-induced liver injury is unknown. In this study, we tested IL-1 receptor antagonist to block IL-1 signalling in a mouse model of acute-on-chronic liver injury on liver inflammation and hepatocyte regeneration in AH. We observed that inhibition of IL-1 signalling decreased liver inflammation and neutrophil infiltration, and resulted in enhanced regeneration of hepatocytes and increased rate of recovery from liver injury in AH. Our novel findings suggest that IL-1 drives sustained liver inflammation and impaired hepatocyte regeneration even after cessation of ethanol exposure. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Hepatic iron overload is associated with hepatocyte apoptosis during Clonorchis sinensis infection.

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Han, Su; Tang, Qiaoran; Chen, Rui; Li, Yihong; Shu, Jing; Zhang, Xiaoli

2017-08-01

Hepatic iron overload has been implicated in many liver diseases; however, whether it is involved in clonorchiasis remains unknown. The purpose of this study is to investigate whether Clonorchis sinensis (C. sinensis) infection causes hepatic iron overload, analyze the relationship between the iron overload and associated cell apoptosis, so as to determine the role of excess iron plays in C. sinensis-induced liver injury. The Perls' Prussian staining and atomic absorption spectrometry methods were used to investigate the iron overload in hepatic sections of wistar rats and patients infected with C. sinensis. The hepatic apoptosis was detected by transferase uridyl nick end labeling (TUNEL) methods. Spearman analysis was used for determining the correlation of the histological hepatic iron index and the apoptotic index. Blue iron particles were deposited mainly in the hepatocytes, Kupffer cells and endothelial cells, around the liver portal and central vein area of both patients and rats. The total iron score was found to be higher in the infected groups than the respective control from 8 weeks. The hepatic iron concentration was also significantly higher in treatment groups than in control rats from 8 weeks. The hepatocyte apoptosis was found to be significantly higher in the portal area of the liver tissue and around the central vein. However, spearman's rank correlation coefficient revealed that there was a mildly negative correlation between the iron index and hepatocyte apoptosis. This present study confirmed that hepatic iron overload was found during C. sinensis infection. This suggests that iron overload may be associated with hepatocyte apoptosis and involved in liver injury during C. sinensis infection. Further studies are needed to investigate the molecular mechanism involved here.

Effect of honokiol on the induction of drug-metabolizing enzymes in human hepatocytes

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Cho YY

2014-11-01

Full Text Available Yong-Yeon Cho,1 Hyeon-Uk Jeong,1 Jeong-Han Kim,2 Hye Suk Lee1 1College of Pharmacy, The Catholic University of Korea, Bucheon, Korea; 2Department of Agricultural Biotechnology, Seoul National University, Seoul, Korea Abstract: Honokiol, 2-(4-hydroxy-3-prop-2-enyl-phenyl-4-prop-2-enyl-phenol, an active component of Magnolia officinalis and Magnolia grandiflora, exerts various pharmacological activities such as antitumorigenic, antioxidative, anti-inflammatory, neurotrophic, and antithrombotic effects. To investigate whether honokiol acts as a perpetrator in drug interactions, messenger ribonucleic acid (mRNA levels of phase I and II drug-metabolizing enzymes, including cytochrome P450 (CYP, UDP-glucuronosyltransferase (UGT, and sulfotransferase 2A1 (SULT2A1, were analyzed by real-time reverse transcription polymerase chain reaction following 48-hour honokiol exposure in three independent cryopreserved human hepatocyte cultures. Honokiol treatment at the highest concentration tested (50 µM increased the CYP2B6 mRNA level and CYP2B6-catalyzed bupropion hydroxylase activity more than two-fold in three different hepatocyte cultures, indicating that honokiol induces CYP2B6 at higher concentrations. However, honokiol treatment (0.5–50 µM did not significantly alter the mRNA levels of phase I enzymes (CYP1A2, CYP3A4, CYP2C8, CYP2C9, and CYP2C19 or phase II enzymes (UGT1A1, UGT1A4, UGT1A9, UGT2B7, and SULT2A1 in cryopreserved human hepatocyte cultures. CYP1A2-catalyzed phenacetin O-deethylase and CYP3A4-catalyzed midazolam 1'-hydroxylase activities were not affected by 48-hour honokiol treatment in cryopreserved human hepatocytes. These results indicate that honokiol is a weak CYP2B6 inducer and is unlikely to increase the metabolism of concomitant CYP2B6 substrates and cause pharmacokinetic-based drug interactions in humans. Keywords: honokiol, human hepatocytes, drug interactions, cytochrome P450, UDP-glucuronosyltransferases

Study on radioprotection of alliin and damage mechanism in hepatocyte after irradiation

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Ji, Tae Jeong; Kim, Won Tae [Dept, of Radiological Science, Kaya University, Kimhae (Korea, Republic of)

2016-12-15

Liver tissue damage by a radiation exposure caused a jaundice and ascitic fluid e form harden atrophy. The reason for this lies in morphological damage of a liver cells. This study tried that observe damage mechanism of the cell organelles. It was especially observed mitochondria, endoplasmic reticulum and nuclear membrane associated with energy metabolizable. also, This study had with a radio-protector development research at the same time. Radio-protector was used to alliin that has an blood flow increase. Cell observation make used of transmission electron microscope(TEM). The result of an experiment, 7Gy of whole body irradiation was caused an inflammation in cell organelles and hypertrophy of the nucleus membrane. After 20 days, The hepatocyte has been observed in a damaged membrane on peroxisome, mitochondria and vacuole of the cell organelles. After 30 days, The hepatocyte has been observed in disconnected ribosomes on a rough endoplasmic reticulum. There was looked a giant lipoblast. There was clearly normal observed a mitochondria and nucleus membrane in the hepatocyte after alliin injection. aslo, It was no damaged the nucleus membrane. Therefore, It was identified portion a radio-protector effect from alliin.

Cholangiocarcinoma in Cirrhosis: Value of Hepatocyte Specific Magnetic Resonance Imaging.

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Piscaglia, Fabio; Iavarone, Massimo; Galassi, Marzia; Vavassori, Sara; Renzulli, Matteo; Forzenigo, Laura Virginia; Granito, Alessandro; Salvatore, Veronica; Sangiovanni, Angelo; Golfieri, Rita; Colombo, Massimo; Bolondi, Luigi

2015-10-01

The diagnosis of intrahepatic cholangiocellular carcinoma (ICC) remains elusive at imaging, which is a critical issue in cirrhotic patients in whom a diagnosis of hepatocellular carcinoma (HCC) can be established only by imaging. The aim of the study was to evaluate the potential of MRI in the diagnosis of ICC in cirrhosis using 'hepatocyte-specific' Gadolinium (Gd)-based contrast agents. Sixteen histologically proven and retrospectively identified ICCs on cirrhosis were investigated with hepatocyte-specific magnetic resonance contrast agents (6 in Bologna with Gd-EOB-DTPA and 10 in Milan with Gd-BOPTA). The control group consisted of 41 consecutively and prospectively collected nodules (31 HCCs) imaged with Gd-EOB-DTPA. Fifteen ICC nodules (94%) displayed hypointensity in the hepatobiliary phase, suggesting malignancy. Thirteen cholangiocarcinomas (81%) showed hyperenhancement in the venous phase. Only 2 cholangiocarcinoma nodules showed hypoenhancement in the venous phase, corresponding to washout, in both cases preceded by rim enhancement in arterial phase. All the hepatocarcinomas showed hypointensity in hepatobiliary phase, but was always preceded by hypointensity in the venous phase; arterial rim enhancement was never observed in any hepatocarcinoma or regenerative nodule. MRI with hepatocyte-specific Gd-based contrast agents showed a pattern of malignancy in almost all the ICCs, concurrently avoiding misdiagnosis with hepatocarcinoma. These findings suggest a greater diagnostic capacity for this technique compared with the results of MRI with conventional contrast agents reported in the literature in this setting. © 2015 S. Karger AG, Basel.

Developing a New Two-Step Protocol to Generate Functional Hepatocytes from Wharton’s Jelly-Derived Mesenchymal Stem Cells under Hypoxic Condition

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Patcharee Prasajak

2013-01-01

Full Text Available The shortage of donor livers and hepatocytes is a major limitation of liver transplantation. Thus, generation of hepatocyte-like cells may provide alternative choice for therapeutic applications. In this study, we developed a new method to establish hepatocytes from Wharton’s jelly-derived mesenchymal stem cells (WJ-MSCs cell lines named WJMSCs-SUT1 and WJMSCs-SUT2 under hypoxic condition. This new method could rapidly drive both WJ-MSCs cell lines into hepatic lineage within 18 days. The achievement of hepatogenic differentiation was confirmed by the characterization of both phenotypes and functions. More than 80% MSCs-derived hepatocyte-like cells (MSCDHCs achieved functional hepatocytes including hepatic marker expressions both at gene and protein levels, glycogen storage, low-density lipoprotein uptake, urea production, and albumin secretion. This study highlights the establishment of new hepatogenic induction protocol under hypoxic condition in order to mimic hypoxic microenvironment in typical cell physiology. In conclusion, we present a simple, high-efficiency, and time saving protocol for the generation of functional hepatocyte-like cells from WJ-MSCs in hypoxic condition. The achievement of this method may overcome the limitation of donor hepatocytes and provides a new avenue for therapeutic value in cell-based therapy for life-threatening liver diseases, regenerative medicine, toxicity testing for pharmacological drug screening, and other medical related applications.

MCD-induced steatohepatitis is associated with hepatic adiponectin resistance and adipogenic transformation of hepatocytes.

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Larter, Claire Z; Yeh, Matthew M; Williams, Jacqueline; Bell-Anderson, Kim S; Farrell, Geoffrey C

2008-09-01

In these studies, we tested the hypothesis that increased lipid intake would exacerbate the severity of nutritional steatohepatitis. C57Bl/6J mice were fed methionine-and-choline deficient (MCD) diets containing 20% (high) or 5% (low) fat by weight for 3 weeks and compared to lipid-matched controls. MCD feeding increased serum ALT levels and induced hepatic steatosis, lobular inflammation and ballooning degeneration of hepatocytes, irrespective of dietary fat content. Hepatic triglyceride accumulation was similar between high and low-fat MCD-fed mice, but lipoperoxide levels were approximately 3-fold higher in the high-fat MCD-fed animals. Serum adiponectin levels increased in MCD-fed mice, although to a lesser extent in high-fat fed animals. AMPK phosphorylation was correspondingly increased in muscle of MCD-fed mice, but hepatic AMPK phosphorylation decreased, and there was little evidence of PPAR alpha activation, suggesting impaired adiponectin action in the livers of MCD-fed animals. Hepatocyte PPAR gamma mRNA levels increased in MCD-fed mice, and were associated with increased aP2 expression, indicating adipogenic transformation of hepatocytes. Increased dietary lipid intake did not alter steatohepatitis severity in MCD-fed mice despite increased lipoperoxide accumulation. Instead, steatohepatitis was associated with impaired hepatic adiponectin action, and adipogenic transformation of hepatocytes in both low and high-fat MCD-fed mice.

Modulation of protein synthesis and secretion by substratum in primary cultures of rat hepatocytes

International Nuclear Information System (INIS)

Sudhakaran, P.R.; Stamatoglou, S.C.; Hughes, R.C.

1986-01-01

Hepatocytes isolated by perfusion of adult rat liver and cultured on substrata consisting of one or more of the major components of the liver biomatrix (fibronectin, laminin, type IV collagen) have been examined for the synthesis of defined proteins. Under these conditions, tyrosine amino transferase, a marker of hepatocyte function, is maintained at similar levels in response to dexamethasone over 5 days in culture on each substratum, and total cellular protein synthesis remains constant. By contrast, there is a rapid decrease in synthesis and secretion of albumin and a 3-7-fold increase in synthesis and section of α-fetoprotein which are most marked on a laminin substratum, but least evident on type IV collagen, and an increased synthesis of fibronectin and type IV collagen. The newly synthesized matrix proteins are present in the cell layer as well as in cell secretions. The enhanced synthesis of fibronectin is less in cells seeded onto a fibronectin substratum than on laminin or type IV collagen substrata. These results indicate that hepatocytes cultured in serum-free medium on substrata composed of components of the liver biomatrix maintain certain functions of the differentiated state (tyrosine amino transferase), lose others (albumin secretion) and switch to increased synthesis of matrix components as well as fetal markers such as α-fetoprotein. The magnitude of these effects depends on the substratum on which the hepatocytes are cultured

Manganese Transport and Toxicity in Polarized WIF-B Hepatocytes.

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Thompson, Khristy J; Hein, Jennifer; Baez, Andrew; Sosa, Jose Carlo; Wessling-Resnick, Marianne

2018-05-24

Mn toxicity arises from nutritional problems, community and occupational exposures, and genetic risks. Mn blood levels are controlled by hepatobiliary clearance. The goals of this study were to determine the cellular distribution of Mn transporters in polarized hepatocytes, to establish an in vitro assay for hepatocyte Mn efflux, and to examine possible roles the Mn transporters would play in metal import and export. For these experiments, hepatocytoma WIF-B cells were grown for 12-14 days to achieve maximal polarity. Immunoblots showed that Mn transporters ZIP8, ZnT10, ferroportin (Fpn), and ZIP14 were present. Indirect immunofluorescence microscopy localized Fpn and ZIP14 to WIF-B cell basolateral domains while ZnT10 and ZIP8 associated with intracellular vesicular compartments. ZIP8-positive structures were distributed uniformly throughout the cytoplasm, but ZnT10-positive vesicles were adjacent to apical bile compartments. WIF-B cells were sensitive to Mn toxicity, showing decreased viability after 16 h exposure to > 250 M MnCl2. However, the hepatocytes were resistant to 4 h exposures of up to 500 M MnCl2 despite 50-fold increased Mn content. Washout experiments showed time-dependent efflux with 80% Mn released after a 4 h chase period. Hepcidin reduced levels of Fpn in WIF-B cells, clearing Fpn from the cell surface, but Mn efflux was unaffected. The secretory inhibitor brefeldin A did block release of Mn from WIF-B cells, suggesting vesicle fusion may be involved in export. These results point to a possible role of ZnT10 to import Mn into vesicles that subsequently fuse with the apical membrane and empty their contents into bile.

Ontogenetic changes in the ultrastructure of rat hepatocyte organelles after prenatal x irradiation

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Chedid, A.; Nair, V.

1975-01-01

The effects of prenatal x irradiation on the development of hepatocyte organelles have been studied in Sprague-Dawley rats. Pregnant rats received 50 R to the pelvic region on the 13th gestation day (g.d.). Animals were sacrificed on g.d.'s 15 and 20, day of birth, and 5th postnatal day. The fetal and neonatal livers were obtained and processed for electron-microscopic examination. The most striking discernible change after irradiation involves the appearance of cytoplasmic ''polyribosomal aggregates'' in the hepatocyte specimens of 15th and 20th g.d.'s. In the control rat, smooth endoplasmic reticulum (SER) appears for the first time on 20th g.d., while no SER could be detected in the hepatocytes from the irradiated animals at this period nor on day of birth. In the irradiated animals, SER was observed on the 5th postnatal day. Our results are consistent with the hypothesis that lipid peroxidation membrane alteration, delayed appearance of SER, and ''polyribosomal aggregation'' may be sequentially linked events after prenatal x irradiation. (U.S.)

Knockdown of autophagy enhances innate immune response in hepatitis C virus infected hepatocytes

Science.gov (United States)

Shrivastava, Shubham; Raychoudhuri, Amit; Steele, Robert; Ray, Ranjit; Ray, Ratna B.

2010-01-01

The role of autophagy in disease pathogenesis following viral infection is beginning to be elucidated. We have previously reported that hepatitis C virus (HCV) infection in hepatocytes induces autophagy. However, the biological significance of HCV induced autophagy has not been clarified. Autophagy has recently been identified as a novel component of innate immune system against viral infection. In the present study, we have shown that knockdown of autophagy related protein Beclin1 or ATG7 in immortalized human hepatocytes (IHH) inhibited HCV growth. Beclin1 or ATG7 knockdown IHH when infected with HCV exhibited an increased expression of IFN-β, OAS-1, IFN-α and IFI27 mRNAs of the interferon signaling pathways as compared to infection of control IHH. Subsequent study demonstrated that HCV infection in autophagy impaired IHH displayed caspase activation, PARP cleavage and apoptotic cell death. Conclusion The disruption of autophagy machinery in HCV infected hepatocytes activated IFN signaling pathway, and induced apoptosis. Together, these results suggest that HCV induced autophagy impairs innate immune response. PMID:21274862

An improved microphotometry system for measurement of cytochrome P-450 in hepatocyte cytoplasm.

Science.gov (United States)

Watanabe, J; Kanamura, S

1991-05-01

To measure cytochrome P-450 (P-450) content in hepatocyte cytoplasm, we developed a dual monochromator-equipped microphotometry system (KWSP-1). Simultaneous measurements of absorbance at 450 and 490 nm with narrow band width (0.5 nm) and small spot size (2 microns) were accomplished by this system. Corresponding fields in serial sections could be easily and rapidly identified under the Nomarski imaging mode of KWSP-1. Photometric accuracy and repeatability of wavelength setting of KWSP-1 were also satisfactory for measurement of P-450. With this system, it is thus possible to measure the extinction of P-450 from many small measuring areas and to precisely determine P-450 content in the cytoplasm of rat hepatocytes. A microphotometric method was developed using cuvette slides and two serial 10-microns thick sections (mapping method). The intracellular distribution of P-450 in individual hepatocytes could be visualized by the mapping method with KWSP-1. However, this method was not applicable to tissue sections containing hemoglobin larger than 4 microM.

Frequency of micronuclei in hepatocytes following X and fast-neutron irradiations--an analysis by a linear-quadratic model

International Nuclear Information System (INIS)

Ono, K.; Nagata, Y.; Akuta, K.; Abe, M.; Ando, K.; Koike, S.

1990-01-01

The usefulness of the micronucleus assay for investigating the radiation response of hepatocytes was examined. The frequency was defined as the ratio of the total number of micronuclei to the number of hepatocytes examined. The dose-response curves were curvilinear after X rays and linear after neutrons. These dose-response curves were analyzed by a linear-quadratic model, frequency = aD + bD2 + c. The a/b ratio was 3.03 +/- 1.26 Gy following X irradiation. This value is within the range of the alpha/beta ratios reported by others using the clonogenic assay of hepatocytes. While the a/b value for neutrons was 24.3 +/- 11.7 Gy, the maximum relative biological effectiveness of neutrons was 6.30 +/- 2.53. Since the micronucleus assay is simple and rapid, it may be a good tool for evaluating the radiation response of hepatocytes in vivo

Acetic acid activates the AMP-activated protein kinase signaling pathway to regulate lipid metabolism in bovine hepatocytes.

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Xinwei Li

Full Text Available The effect of acetic acid on hepatic lipid metabolism in ruminants differs significantly from that in monogastric animals. Therefore, the aim of this study was to investigate the regulation mechanism of acetic acid on the hepatic lipid metabolism in dairy cows. The AMP-activated protein kinase (AMPK signaling pathway plays a key role in regulating hepatic lipid metabolism. In vitro, bovine hepatocytes were cultured and treated with different concentrations of sodium acetate (neutralized acetic acid and BML-275 (an AMPKα inhibitor. Acetic acid consumed a large amount of ATP, resulting in an increase in AMPKα phosphorylation. The increase in AMPKα phosphorylation increased the expression and transcriptional activity of peroxisome proliferator-activated receptor α, which upregulated the expression of lipid oxidation genes, thereby increasing lipid oxidation in bovine hepatocytes. Furthermore, elevated AMPKα phosphorylation reduced the expression and transcriptional activity of the sterol regulatory element-binding protein 1c and the carbohydrate responsive element-binding protein, which reduced the expression of lipogenic genes, thereby decreasing lipid biosynthesis in bovine hepatocytes. In addition, activated AMPKα inhibited the activity of acetyl-CoA carboxylase. Consequently, the triglyceride content in the acetate-treated hepatocytes was significantly decreased. These results indicate that acetic acid activates the AMPKα signaling pathway to increase lipid oxidation and decrease lipid synthesis in bovine hepatocytes, thereby reducing liver fat accumulation in dairy cows.

Metformin protects primary rat hepatocytes against oxidative stress-induced apoptosis

NARCIS (Netherlands)

Conde de la Rosa, Laura; Vrenken, Titia E; Buist-Homan, Manon; Faber, Klaas Nico; Moshage, Han

The majority of chronic liver diseases are accompanied by oxidative stress, which induces apoptosis in hepatocytes and liver injury. Recent studies suggest that oxidative stress and insulin resistance are important in the pathogenesis of nonalcoholic fatty liver disease (NAFLD) and the

Hepatocyte heterogeneity in the metabolism of amino acids and ammonia

NARCIS (Netherlands)

Häussinger, D.; Lamers, W. H.; Moorman, A. F.

1992-01-01

With respect to hepatocyte heterogeneity in ammonia and amino acid metabolism, two different patterns of sublobular gene expression are distinguished: 'gradient-type' and 'strict- or compartment-type' zonation. An example for strict-type zonation is the reciprocal distribution of carbamoylphosphate

Choline or methionine reverses impaired secretion of VLDL by hepatocytes from choline-deficient rats

International Nuclear Information System (INIS)

Yao, Z.; Vance, D.E.

1987-01-01

Male rats fed a choline-deficient (CD) diet for three days accumulated triacylglycerol (TG) in the liver. Hepatocytes from these rats were cultured and maintained in a medium + choline. The rate of secretion of TG was reduced by 50% in the CD cells. Correspondingly, [ 3 H]oleate and [ 3 H]glycerol were incorporated at a 2-fold higher rate into TG secreted by choline-supplemented cells compared to CD cells. Isolation of lipoprotein fractions by ultracentrifugation showed that the reduced secretion of TG by CD hepatocytes was mainly due to an impaired secretion of very low density lipoprotein (VLDL). Incorporation of [ 3 H]leucine into secreted apoB/sub H/, apoB/sub L/ and apoE was markedly reduced in CD cells compared to choline-supplemented cells. Secretion of high density lipoprotein was not reduced in the CD hepatocytes. Normal secretion of VLDL was resumed upon addition of methionine to the CD cells

Glucose metabolism and recycling by hepatocytes of OB/OB and ob/ob mice

International Nuclear Information System (INIS)

Lahtela, J.T.; Wals, P.A.; Katz, J.

1990-01-01

Hepatocytes were prepared from livers of ob/ob (obese diabetic) mice and their lean (OB/OB) siblings that had been fasted for 24 h. The hepatocytes were incubated with [U-14C, 2-3H]-, [U-14C, 3-3H]-, and [U-14C, 6-3H]glucose at concentrations from 20 to 120 mM. 14C was recovered mainly in CO2, glycogen, and lactate. Tritium was recovered in water and glycogen. The yield in labeled products from [2-3H]glucose ranged from two to three times that from [U-14C]glucose. The yields from [3-3H]- and [6-3H]glucose were similar, and 1.3-1.7 times that from [U-14C]glucose. At 40 mM, total utilization of glucose by obese mice was about twice that for lean mice, but there was little difference at 120 mM. The rate of recycling between glucose and glucose 6-phosphate was calculated. An equation to calculate the rate of recycling of glucose from the 2-3H/U-14C ratio in glycogen is derived in the APPENDIX. Our results show that (1) the utilization of glucose by hepatocytes from obese diabetic mice exceeds that of their lean controls, (2) the rate of glucose phosphorylation in both groups greatly exceeds glucose uptake and the rate of glycogen synthesis, (3) glucose phosphorylation represents a difference between a high glucokinase rate and hydrolysis of glucose 6-phosphate, and (4) recycling of glucose carbon between glucose 6-phosphate and pyruvate occurs within mouse hepatocytes

Prolyl hydroxylase-1 regulates hepatocyte apoptosis in an NF-κB-dependent manner

Energy Technology Data Exchange (ETDEWEB)

Fitzpatrick, Susan F.; Fábián, Zsolt; Schaible, Bettina; Lenihan, Colin R.; Schwarzl, Thomas [School of Medicine and Medical Science, The Conway Institute, University College Dublin, Belfield, Dublin 4 Ireland (Ireland); Rodriguez, Javier [Systems Biology Ireland, University College Dublin, Dublin 4 (Ireland); Zheng, Xingnan; Li, Zongwei [Lineberger Comprehensive Cancer Center, University of North Carolina School of Medicine, Chapel Hill, NC (United States); Tambuwala, Murtaza M. [School of Pharmacy and Pharmaceutical Sciences, Ulster University, Coleraine, BT52 1SA, Northern Ireland (United Kingdom); Higgins, Desmond G.; O' Meara, Yvonne [School of Medicine and Medical Science, The Conway Institute, University College Dublin, Belfield, Dublin 4 Ireland (Ireland); Slattery, Craig [School of Biomolecular and Biomedical Science, The Conway Institute, University College Dublin, Belfield, Dublin 4 Ireland (Ireland); Manresa, Mario C. [School of Medicine and Medical Science, The Conway Institute, University College Dublin, Belfield, Dublin 4 Ireland (Ireland); Fraisl, Peter; Bruning, Ulrike [Laboratory of Angiogenesis and Vascular Metabolism, Department of Oncology, University of Leuven, Vesalius Research Center, VIB, B-3000 (Belgium); Baes, Myriam [Laboratory for Cell Metabolism, Department of Pharmaceutical and Pharmacological Sciences, KU Leuven (Belgium); Carmeliet, Peter; Doherty, Glen [Laboratory of Angiogenesis and Vascular Metabolism, Department of Oncology, University of Leuven, Vesalius Research Center, VIB, B-3000 (Belgium); Kriegsheim, Alex von [Systems Biology Ireland, University College Dublin, Dublin 4 (Ireland); Cummins, Eoin P. [School of Medicine and Medical Science, The Conway Institute, University College Dublin, Belfield, Dublin 4 Ireland (Ireland); and others

2016-06-03

Hepatocyte death is an important contributing factor in a number of diseases of the liver. PHD1 confers hypoxic sensitivity upon transcription factors including the hypoxia inducible factor (HIF) and nuclear factor-kappaB (NF-κB). Reduced PHD1 activity is linked to decreased apoptosis. Here, we investigated the underlying mechanism(s) in hepatocytes. Basal NF-κB activity was elevated in PHD1{sup −/−} hepatocytes compared to wild type controls. ChIP-seq analysis confirmed enhanced binding of NF-κB to chromatin in regions proximal to the promoters of genes involved in the regulation of apoptosis. Inhibition of NF-κB (but not knock-out of HIF-1 or HIF-2) reversed the anti-apoptotic effects of pharmacologic hydroxylase inhibition. We hypothesize that PHD1 inhibition leads to altered expression of NF-κB-dependent genes resulting in reduced apoptosis. This study provides new information relating to the possible mechanism of therapeutic action of hydroxylase inhibitors that has been reported in pre-clinical models of intestinal and hepatic disease. -- Highlights: •Genetic ablation of PHD1 upregulates NF-kappaB (NF-κB) in hepatocytes. •Activation of NF-κB leads to differential DNA-binding of p50/p65 and results in differential regulation of apoptotic genes. •We identified proline 191 in the beta subunit of the I-kappaB kinase as a target for PHD1-mediated hydroxylation. •Blockade of prolyl-4-hydroxylases has been found cytoprotective in liver cells.

Hepatocyte Hyperproliferation upon Liver-Specific Co-disruption of Thioredoxin-1, Thioredoxin Reductase-1, and Glutathione Reductase

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Justin R. Prigge

2017-06-01

Full Text Available Energetic nutrients are oxidized to sustain high intracellular NADPH/NADP+ ratios. NADPH-dependent reduction of thioredoxin-1 (Trx1 disulfide and glutathione disulfide by thioredoxin reductase-1 (TrxR1 and glutathione reductase (Gsr, respectively, fuels antioxidant systems and deoxyribonucleotide synthesis. Mouse livers lacking both TrxR1 and Gsr sustain these essential activities using an NADPH-independent methionine-consuming pathway; however, it remains unclear how this reducing power is distributed. Here, we show that liver-specific co-disruption of the genes encoding Trx1, TrxR1, and Gsr (triple-null causes dramatic hepatocyte hyperproliferation. Thus, even in the absence of Trx1, methionine-fueled glutathione production supports hepatocyte S phase deoxyribonucleotide production. Also, Trx1 in the absence of TrxR1 provides a survival advantage to cells under hyperglycemic stress, suggesting that glutathione, likely via glutaredoxins, can reduce Trx1 disulfide in vivo. In triple-null livers like in many cancers, deoxyribonucleotide synthesis places a critical yet relatively low-volume demand on these reductase systems, thereby favoring high hepatocyte turnover over sustained hepatocyte integrity.

Inhibition of Drp1 protects against senecionine-induced mitochondria-mediated apoptosis in primary hepatocytes and in mice

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Xiao Yang

2017-08-01

Full Text Available Pyrrolizidine alkaloids (PAs are a group of compounds found in various plants and some of them are widely consumed in the world as herbal medicines and food supplements. PAs are potent hepatotoxins that cause irreversible liver injury in animals and humans. However, the mechanisms by which PAs induce liver injury are not clear. In the present study, we determined the hepatotoxicity and molecular mechanisms of senecionine, one of the most common toxic PAs, in primary cultured mouse and human hepatocytes as well as in mice. We found that senecionine administration increased serum alanine aminotransferase levels in mice. H&E and TUNEL staining of liver tissues revealed increased hemorrhage and hepatocyte apoptosis in liver zone 2 areas. Mechanistically, senecionine induced loss of mitochondrial membrane potential, release of mitochondrial cytochrome c as well as mitochondrial JNK translocation and activation prior to the increased DNA fragmentation and caspase-3 activation in primary cultured mouse and human hepatocytes. SP600125, a specific JNK inhibitor, and ZVAD-fmk, a general caspase inhibitor, alleviated senecionine-induced apoptosis in primary hepatocytes. Interestingly, senecionine also caused marked mitochondria fragmentation in hepatocytes. Pharmacological inhibition of dynamin-related protein1 (Drp1, a protein that is critical to regulate mitochondrial fission, blocked senecionine-induced mitochondrial fragmentation and mitochondrial release of cytochrome c and apoptosis. More importantly, hepatocyte-specific Drp1 knockout mice were resistant to senecionine-induced liver injury due to decreased mitochondrial damage and apoptosis. In conclusion, our results uncovered a novel mechanism of Drp1-mediated mitochondrial fragmentation in senecionine-induced liver injury. Targeting Drp1-mediated mitochondrial fragmentation and apoptosis may be a potential avenue to prevent and treat hepatotoxicity induced by PAs. Keywords: Senecionine, Drp1

The metabolism of aflatoxin B1 by hepatocytes isolated from rats following the in vivo administration of some xenobiotics

International Nuclear Information System (INIS)

Metcalfe, S.A.; Neal, G.E.

1983-01-01

Isolated rat hepatocytes, an intact cellular system capable of performing phase I and phase II metabolism, have been used to investigate metabolism of aflatoxin B1. These cells were found to metabolise [ 14 C]aflatoxin B1 to aflatoxins M1 and Q1, and to radiolabelled polar material, presumably conjugates, as analysed by h.p.l.c., t.l.c. and radioactive determination. In vivo administration of the mixed function oxidase inducers, phenobarbitone and 3-methylcholanthrene, resulted in enhanced hepatocyte phase I (microsomal) metabolism of aflatoxin B1. In contrast to metabolism of AFB1 by in vitro subcellular systems increased production of polar material (conjugated metabolites) derived from [ 14 C]aflatoxin B1 was also detected in hepatocytes isolated from these pretreated animals. Formation of aflatoxin Q1 by isolated hepatocytes appeared to be mediated by cytochrome P450-linked enzymes whereas cytochrome P448-linked enzymes were apparently involved in aflatoxin M1 production. Chronic feeding of aflatoxin B1 to rats enhanced hepatocyte production of conjugated material only and did not elevate cellular cytochrome P450 levels, thus suggesting that aflatoxin B1 is not an inducer of its own primary metabolism

Protective effect of kombucha tea against tertiary butyl hydroperoxide induced cytotoxicity and cell death in murine hepatocytes.

Science.gov (United States)

Bhattacharya, Semantee; Manna, Prasenjit; Gachhui, Ratan; Sil, Parames C

2011-07-01

Kombucha (KT), a fermented black tea (BT), is known to have many beneficial properties. In the present study, antioxidant property of KT has been investigated against tertiary butyl hydroperoxide (TBHP) induced cytotoxicity using murine hepatocytes. TBHP, a reactive oxygen species inducer, causes oxidative stress resulting in organ pathophysiology. Exposure to TBHP caused a reduction in cell viability, increased membrane leakage and disturbed the intra-cellular antioxidant machineries in hepatocytes. TBHP exposure disrupted mitochondrial membrane potential and induced apoptosis as evidenced by flow cytometric analyses. KT treatment, however, counteracted the changes in mitochondrial membrane potential and prevented apoptotic cell death of the hepatocytes. BT treatment also reverted TBHP induced hepatotoxicity, however KT was found to be more efficient. This may be due to the formation of antioxidant molecules like D-saccharic acid-1,4-lactone (DSL) during fermentation process and are absent in BT. Moreover, the radical scavenging activities of KT were found to be higher than BT. Results of the study showed that KT has the potential to ameliorate TBHP induced oxidative insult and cell death in murine hepatocytes more effectively than BT.

Radiation-induced PKC signaling system in cultured rat hepatocytes

International Nuclear Information System (INIS)

Nakajima, Tetsuo; Yukawa, Osami

1998-01-01

Radiation effects on living organisms are mainly caused through reactive oxygen species (ROS) on living cells. It is known that ROS damages various membranes and the bio membranes play an important role in cellular signal transduction pathways. The effects of radiation on cellular signal transduction pathways in cultured rat hepatocytes have been studied

Differential Impacts of Soybean and Fish Oils on Hepatocyte Lipid Droplet Accumulation and Endoplasmic Reticulum Stress in Primary Rabbit Hepatocytes

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Zhu, Xueping; Xiao, Zhihui; Xu, Yumin; Zhao, Xingli; Cheng, Ping; Cui, Ningxun; Cui, Mingling; Li, Jie; Zhu, Xiaoli

2016-01-01

Parenteral nutrition-associated liver disease (PNALD) is a severe ailment associated with long-term parenteral nutrition. Soybean oil-based lipid emulsions (SOLE) are thought to promote PNALD development, whereas fish oil-based lipid emulsions (FOLE) are thought to protect against PNALD. This study aimed to investigate the effects of SOLE and FOLE on primary rabbit hepatocytes. The results reveal that SOLE caused significant endoplasmic reticulum (ER) and mitochondrial damage, ultimately resu...

Spatiotemporal multiple coherence resonances and calcium waves in a coupled hepatocyte system

International Nuclear Information System (INIS)

Bao-Hua, Wang; Qi-Shao, Lu; Shu-Juan, Lü; Xiu-Feng, Lang

2009-01-01

Spatiotemporal multiple coherence resonances for calcium activities induced by weak Gaussian white noise in coupled hepatocytes are studied. It is shown that bi-resonances in hepatocytes are induced by the interplay and competition between noise and coupling of cells, in other words, the cell in network can be excited either by noise or by its neighbour via gap junction which can transfer calcium ions between cells. Furthermore, the intercellular annular calcium waves induced by noise are observed, in which the wave length decreases with noise intensity augmenting but increases monotonically with coupling strength increasing. And for a fixed noise level, there is an optimal coupling strength that makes the coherence resonance reach maximum. (general)

Calcium-mediated signaling and calmodulin-dependent kinase regulate hepatocyte-inducible nitric oxide synthase expression.

Science.gov (United States)

Zhang, Baochun; Crankshaw, Will; Nesemeier, Ryan; Patel, Jay; Nweze, Ikenna; Lakshmanan, Jaganathan; Harbrecht, Brian G

2015-02-01

Induced nitric oxide synthase (iNOS) is induced in hepatocytes by shock and inflammatory stimuli. Excessive NO from iNOS mediates shock-induced hepatic injury and death, so understanding the regulation of iNOS will help elucidate the pathophysiology of septic shock. In vitro, cytokines induce iNOS expression through activation of signaling pathways including mitogen-activated protein kinases and nuclear factor κB. Cytokines also induce calcium (Ca(2+)) mobilization and activate calcium-mediated intracellular signaling pathways, typically through activation of calmodulin-dependent kinases (CaMK). Calcium regulates NO production in macrophages but the role of calcium and calcium-mediated signaling in hepatocyte iNOS expression has not been defined. Primary rat hepatocytes were isolated, cultured, and induced to produce NO with proinflammatory cytokines. Calcium mobilization and Ca(2+)-mediated signaling were altered with ionophore, Ca(2+) channel blockers, and inhibitors of CaMK. The Ca(2+) ionophore A23187 suppressed cytokine-stimulated NO production, whereas Ethylene glycol tetraacetic acid and nifedipine increased NO production, iNOS messenger RNA, and iNOS protein expression. Inhibition of CaMK with KN93 and CBD increased NO production but the calcineurin inhibitor FK 506 decreased iNOS expression. These data demonstrate that calcium-mediated signaling regulates hepatocyte iNOS expression and does so through a mechanism independent of calcineurin. Changes in intracellular calcium levels may regulate iNOS expression during hepatic inflammation induced by proinflammatory cytokines. Copyright © 2015 Elsevier Inc. All rights reserved.

A preliminary study for constructing a bioartificial liver device with induced pluripotent stem cell-derived hepatocytes

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Iwamuro Masaya

2012-12-01

Full Text Available Abstract Background Bioartificial liver systems, designed to support patients with liver failure, are composed of bioreactors and functional hepatocytes. Immunological rejection of the embedded hepatocytes by the host immune system is a serious concern that crucially degrades the performance of the device. Induced pluripotent stem (iPS cells are considered a desirable source for bioartificial liver systems, because patient-derived iPS cells are free from immunological rejection. The purpose of this paper was to test the feasibility of a bioartificial liver system with iPS cell-derived hepatocyte-like cells. Methods Mouse iPS cells were differentiated into hepatocyte-like cells by a multi-step differentiation protocol via embryoid bodies and definitive endoderm. Differentiation of iPS cells was evaluated by morphology, PCR assay, and functional assays. iPS cell-derived hepatocyte-like cells were cultured in a bioreactor module with a pore size of 0.2 μm for 7 days. The amount of albumin secreted into the circulating medium was analyzed by ELISA. Additionally, after a 7-day culture in a bioreactor module, cells were observed by a scanning electron microscope. Results At the final stage of the differentiation program, iPS cells changed their morphology to a polygonal shape with two nucleoli and enriched cytoplasmic granules. Transmission electron microscope analysis revealed their polygonal shape, glycogen deposition in the cytoplasm, microvilli on their surfaces, and a duct-like arrangement. PCR analysis showed increased expression of albumin mRNA over the course of the differentiation program. Albumin and urea production was also observed. iPS-Heps culture in bioreactor modules showed the accumulation of albumin in the medium for up to 7 days. Scanning electron microscopy revealed the attachment of cell clusters to the hollow fibers of the module. These results indicated that iPS cells were differentiated into hepatocyte-like cells after culture

Utilization of supplemental methionine sources by primary cultures of chick hepatocytes

International Nuclear Information System (INIS)

Dibner, J.J.

1983-01-01

Utilization of 2-hydroxy-4-(methylthio) butanoic acid (HMB) as a substrate for protein synthesis was studied by using primary cultures of chick liver cells. Cultures were prepared by enzymatic dissociation of livers from week old Hubbard broiler chicks and were maintained for 4 days under nonproliferative conditions. Hepatocyte differentiation was verified by using dexamethasone induction of tyrosine aminotransferase activity. Conversion of [14C]HMB to L-methionine was shown by chromatographic analysis of hepatocyte protein hydrolysate and incorporation into protein was proven by cycloheximide inhibition of synthesis. When incorporation of HMB was compared to that of DL-methionine (DLM) equimolar quantities of the two sources were found in liver cell protein. These results support, at a cellular level, the conclusion that HMB and DLM are biochemically equivalent sources of methionine for protein synthesis

Curcumin inhibits activation of TRPM2 channels in rat hepatocytes

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E. Kheradpezhouh

2016-04-01

Full Text Available Oxidative stress is a hallmark of many liver diseases including viral and drug-induced hepatitis, ischemia-reperfusion injury, and non-alcoholic steatohepatitis. One of the consequences of oxidative stress in the liver is deregulation of Ca2+ homeostasis, resulting in a sustained elevation of the free cytosolic Ca2+ concentration ([Ca2+]c in hepatocytes, which leads to irreversible cellular damage. Recently it has been shown that liver damage induced by paracetamol and subsequent oxidative stress is, in large part, mediated by Ca2+ entry through Transient Receptor Potential Melastatin 2 (TRPM2 channels. Involvement of TRPM2 channels in hepatocellular damage induced by oxidative stress makes TRPM2 a potential therapeutic target for treatment of a range of oxidative stress-related liver diseases. We report here the identification of curcumin ((1E,6E-1,7-bis(4-hydroxy-3-methoxyphenyl-1,6-heptadiene-3,5-dione, a natural plant-derived polyphenol in turmeric spice, as a novel inhibitor of TRPM2 channel. Presence of 5 µM curcumin in the incubation medium prevented the H2O2- and paracetamol-induced [Ca2+]c rise in rat hepatocytes. Furthermore, in patch clamping experiments incubation of hepatocytes with curcumin inhibited activation of TRPM2 current by intracellular ADPR with IC50 of approximately 50 nM. These findings enhance understanding of the actions of curcumin and suggest that the known hepatoprotective properties of curcumin are, at least in part, mediated through inhibition of TRPM2 channels.

Direct induction of hepatocyte-like cells from immortalized human bone marrow mesenchymal stem cells by overexpression of HNF4α

International Nuclear Information System (INIS)

Hu, Xiaojun; Xie, Peiyi; Li, Weiqiang; Li, Zhengran; Shan, Hong

2016-01-01

Hepatocytes from human bone marrow-derived mesenchymal stem cells (hBM-MSCs) are expected to be a useful source for cell transplantation. However, relatively low efficiency and repeatability of hepatic differentiation of human BM-MSCs remains an obstacle for clinical translation. Hepatocyte nuclear factor 4 alpha (HNF4α), a critical transcription factor, plays an essential role in the entire process of liver development. In this study, immortalized hBM-MSCs, UE7T-13 cells were transduced with a lentiviral vector containing HNF4α. The typical fibroblast-like morphology of the MSCs changed, and polygonal, epithelioid cells grew out after HNF4α transduction. In hepatocyte culture medium, HNF4α-transduced MSCs (E7-hHNF4α cells) strongly expressed the albumin (ALB), CYP2B6, alpha-1 antitrypsin (AAT), and FOXA2 mRNA and exhibited morphology markedly similar to that of mature hepatocytes. The E7-hHNF4α cells showed hepatic functions such as Indocyanine green (ICG) uptake and release, glycogen storage, urea production and ALB secretion. Approximately 28% of E7-hHNF4α cells expressed both ALB and AAT. Furthermore, these E7-hHNF4α cells via superior mesenteric vein (SMV) injection expressed human ALB in mouse chronic injured liver. In conclusion, this study represents a novel strategy by directly inducing hepatocyte-like cells from MSCs. - Highlights: • We overexpressed HNF4α in immortalized BM-MSCs by lentiviral transduction. • HNF4α-transduced MSCs transdifferentiated into hepatocytes with mature hepatic metabolic functions. • Our study represents a novel strategy by direct induction of hepatocyte-like cells from MSCs.

Aluminum Exposure from Parenteral Nutrition: Early Bile Canaliculus Changes of the Hepatocyte

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Amanda R. Hall

2018-06-01

Full Text Available Background: Neonates on long-term parenteral nutrition (PN may develop parenteral nutrition-associated liver disease (PNALD. Aluminum (Al is a known contaminant of infant PN, and we hypothesize that it substantially contributes to PNALD. In this study, we aim to assess the impact of Al on hepatocytes in a piglet model. Methods: We conducted a randomized control trial using a Yucatan piglet PN model. Piglets, aged 3–6 days, were placed into two groups. The high Al group (n = 8 received PN with 63 µg/kg/day of Al, while the low Al group (n = 7 received PN with 24 µg/kg/day of Al. Serum samples for total bile acids (TBA were collected over two weeks, and liver tissue was obtained at the end of the experiment. Bile canaliculus morphometry were studied by transmission electron microscopy (TEM and ImageJ software analysis. Results: The canalicular space was smaller and the microvilli were shorter in the high Al group than in the low Al group. There was no difference in the TBA between the groups. Conclusions: Al causes structural changes in the hepatocytes despite unaltered serum bile acids. High Al in PN is associated with short microvilli, which could decrease the functional excretion area of the hepatocytes and impair bile flow.

Higher protein kinase C ζ in fatty rat liver and its effect on insulin actions in primary hepatocytes.

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Wei Chen

Full Text Available We previously showed the impairment of insulin-regulated gene expression in the primary hepatocytes from Zucker fatty (ZF rats, and its association with alterations of hepatic glucose and lipid metabolism. However, the molecular mechanism is unknown. A preliminary experiment shows that the expression level of protein kinase C ζ (PKCζ, a member of atypical PKC family, is higher in the liver and hepatocytes of ZF rats than that of Zucker lean (ZL rats. Herein, we intend to investigate the roles of atypical protein kinase C in the regulation of hepatic gene expression. The insulin-regulated hepatic gene expression was evaluated in ZL primary hepatocytes treated with atypical PKC recombinant adenoviruses. Recombinant adenovirus-mediated overexpression of PKCζ, or the other atypical PKC member PKCι/λ, alters the basal and impairs the insulin-regulated expressions of glucokinase, sterol regulatory element-binding protein 1c, the cytosolic form of phosphoenolpyruvate carboxykinase, the catalytic subunit of glucose 6-phosphatase, and insulin like growth factor-binding protein 1 in ZL primary hepatocytes. PKCζ or PKCι/λ overexpression also reduces the protein level of insulin receptor substrate 1, and the insulin-induced phosphorylation of AKT at Ser473 and Thr308. Additionally, PKCι/λ overexpression impairs the insulin-induced Prckz expression, indicating the crosstalk between PKCζ and PKCι/λ. We conclude that the PKCζ expression is elevated in hepatocytes of insulin resistant ZF rats. Overexpressions of aPKCs in primary hepatocytes impair insulin signal transduction, and in turn, the down-stream insulin-regulated gene expression. These data suggest that elevation of aPKC expression may contribute to the hepatic insulin resistance at gene expression level.

Modulation of Mitochondrial DNA Copy Number to Induce Hepatocytic Differentiation of Human Amniotic Epithelial Cells.

Science.gov (United States)

Vaghjiani, Vijesh; Cain, Jason E; Lee, William; Vaithilingam, Vijayaganapathy; Tuch, Bernard E; St John, Justin C

2017-10-15

Mitochondrial deoxyribonucleic acid (mtDNA) copy number is tightly regulated during pluripotency and differentiation. There is increased demand of cellular adenosine triphosphate (ATP) during differentiation for energy-intensive cell types such as hepatocytes and neurons to meet the cell's functional requirements. During hepatocyte differentiation, mtDNA copy number should be synchronously increased to generate sufficient ATP through oxidative phosphorylation. Unlike bone marrow mesenchymal cells, mtDNA copy number failed to increase by 28 days of differentiation of human amniotic epithelial cells (hAEC) into hepatocyte-like cells (HLC) despite their expression of some end-stage hepatic markers. This was due to higher levels of DNA methylation at exon 2 of POLGA, the mtDNA-specific replication factor. Treatment with a DNA demethylation agent, 5-azacytidine, resulted in increased mtDNA copy number, reduced DNA methylation at exon 2 of POLGA, and reduced hepatic gene expression. Depletion of mtDNA followed by subsequent differentiation did not increase mtDNA copy number, but reduced DNA methylation at exon 2 of POLGA and increased expression of hepatic and pluripotency genes. We encapsulated hAEC in barium alginate microcapsules and subsequently differentiated them into HLC. Encapsulation resulted in no net increase of mtDNA copy number but a significant reduction in DNA methylation of POLGA. RNAseq analysis showed that differentiated HLC express hepatocyte-specific genes but also increased expression of inflammatory interferon genes. Differentiation in encapsulated cells showed suppression of inflammatory genes as well as increased expression of genes associated with hepatocyte function pathways and networks. This study demonstrates that an increase in classical hepatic gene expression can be achieved in HLC through encapsulation, although they fail to effectively regulate mtDNA copy number.

Cadmium-induced apoptosis through the mitochondrial pathway in rainbow trout hepatocytes: involvement of oxidative stress

International Nuclear Information System (INIS)

Risso-de Faverney, C.; Orsini, N.; Sousa, G. de; Rahmani, R.

2004-01-01

Cadmium (Cd) induces oxidative stress and apoptosis in trout hepatocytes. We therefore investigated the involvement of the mitochondrial pathway in the initiation of apoptosis and the possible role of oxidative stress in that process. This study demonstrates that hepatocyte exposure to Cd (2, 5 and 10 μM) triggers significant caspase-3, but also caspase-8 and -9 activation in a dose-dependent manner. Western-blot analysis of hepatocyte mitochondrial and cytosolic fractions revealed that cytochrome c (Cyt c) was released in the cytosol in a dose-dependent manner, whereas the pro-apoptotic protein Bax was redistributed to mitochondria after 24 and 48 h exposure. We also found that the expression of anti-apoptotic protein Bcl-xL, known to be regulated under mild oxidative stress to protect cells from apoptosis, did not change after 3 and 6 h exposure to Cd, then increased after 24 and 48 h exposure to 10 μM Cd. In the second part of this work, two antioxidant agents, 2,2,6,6-tetramethylpiperidinyl-1-oxyl (TEMPO) (100 μM) and N-acetylcysteine (NAC, 100 μM) were used to determine the involvement of reactive oxygen species (ROS) in Cd-induced apoptosis. Simultaneously exposing trout hepatocytes to Cd and TEMPO or NAC significantly reduced caspase-3 activation after 48 h and had a suppressive effect on caspase-8 and -9 also, mostly after 24 h. Lastly, the presence of either one of these antioxidants in the treatment medium also attenuated Cd-induced Cyt c release in cytosol and the level of Bax in the mitochondria after 24 and 48 h, while high Bcl-xL expression was observed. Taken together, these data clearly evidenced the key role of mitochondria in the cascade of events leading to trout hepatocyte apoptosis in response to Cd and the relationship that exists between oxidative stress and cell death

Efficient Generation of Functional Hepatocytes From Human Embryonic Stem Cells and Induced Pluripotent Stem Cells by HNF4α Transduction

OpenAIRE

Takayama, Kazuo; Inamura, Mitsuru; Kawabata, Kenji; Katayama, Kazufumi; Higuchi, Maiko; Tashiro, Katsuhisa; Nonaka, Aki; Sakurai, Fuminori; Hayakawa, Takao; Kusuda Furue, Miho; Mizuguchi, Hiroyuki

2012-01-01

Hepatocyte-like cells from human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) are expected to be a useful source of cells drug discovery. Although we recently reported that hepatic commitment is promoted by transduction of SOX17 and HEX into human ESC- and iPSC-derived cells, these hepatocyte-like cells were not sufficiently mature for drug screening. To promote hepatic maturation, we utilized transduction of the hepatocyte nuclear factor 4α (HNF4α) gene, which is kn...

Liver fatty acid binding protein is the mitosis-associated polypeptide target of a carcinogen in rat hepatocytes

International Nuclear Information System (INIS)

Bassuk, J.A.; Tsichlis, P.N.; Sorof, S.

1987-01-01

Hepatocytes in normal rat liver were found previously to contain a cytoplasmic 14,000-dalton polypeptide (p14) that is associated with mitosis and is the principal early covalent target of activated metabolites of the carcinogen N-2-fluorenylacetamide (2-acetylaminofluorene). The level of immunohistochemically detected p14 was low when growth activity of hepatocytes was low, was markedly elevated during mitosis in normal and regenerating livers, but was very high throughout interphase during proliferation of hyperplastic and malignant hepatocytes induced in rat liver by a carcinogen (N-2-fluorenylacetamide or 3'-methyl-4-dimethylaminoazobenzene). The authors report here that p14 is the liver fatty acid binding protein. The nucleotide sequence of p14 cDNA clones, isolated by screening a rat liver cDNA library in bacteriophage λgt11 using p14 antiserum, was completely identical to part of the sequence reported for liver fatty acid binding protein. Furthermore, the two proteins shared the following properties: size of mRNA, amino acid composition, molecular size according to NaDodSO 4 gel electrophoresis, and electrophoretic mobilities in a Triton X-100/acetic acid/urea gel. The two polypeptides bound oleic acid similarly. Finally, identical elevations of cytoplasmic immunostain were detected specifically in mitotic hepatocytes with either antiserum. The collected findings are suggestive that liver fatty acid binding protein may carry ligands that promote hepatocyte division and may transport certain activated chemical carcinogens

Study of Valproic Acid-Enhanced Hepatocyte Steatosis

Science.gov (United States)

Chang, Renin; Chou, Mei-Chia; Hung, Li-Ying; Wang, Mu-En; Hsu, Meng-Chieh; Chiu, Chih-Hsien

2016-01-01

Valproic acid (VPA) is one of the most widely used antiepilepsy drugs. However, several side effects, including weight gain and fatty liver, have been reported in patients following VPA treatment. In this study, we explored the molecular mechanisms of VPA-induced hepatic steatosis using FL83B cell line-based in vitro model. Using fluorescent lipid staining technique, we found that VPA enhanced oleic acid- (OLA-) induced lipid accumulation in a dose-dependent manner in hepatocytes; this may be due to upregulated lipid uptake, triacylglycerol (TAG) synthesis, and lipid droplet formation. Real-time PCR results showed that, following VPA treatment, the expression levels of genes encoding cluster of differentiation 36 (Cd36), low-density lipoprotein receptor-related protein 1 (Lrp1), diacylglycerol acyltransferase 2 (Dgat2), and perilipin 2 (Plin2) were increased, that of carnitine palmitoyltransferase I a (Cpt1a) was not affected, and those of acetyl-Co A carboxylase α (Acca) and fatty acid synthase (Fasn) were decreased. Furthermore, using immunofluorescence staining and flow cytometry analyses, we found that VPA also induced peroxisome proliferator-activated receptor γ (PPARγ) nuclear translocation and increased levels of cell-surface CD36. Based on these results, we propose that VPA may enhance OLA-induced hepatocyte steatosis through the upregulation of PPARγ- and CD36-dependent lipid uptake, TAG synthesis, and lipid droplet formation. PMID:27034954

Study of Valproic Acid-Enhanced Hepatocyte Steatosis

Directory of Open Access Journals (Sweden)

Renin Chang

2016-01-01

Full Text Available Valproic acid (VPA is one of the most widely used antiepilepsy drugs. However, several side effects, including weight gain and fatty liver, have been reported in patients following VPA treatment. In this study, we explored the molecular mechanisms of VPA-induced hepatic steatosis using FL83B cell line-based in vitro model. Using fluorescent lipid staining technique, we found that VPA enhanced oleic acid- (OLA- induced lipid accumulation in a dose-dependent manner in hepatocytes; this may be due to upregulated lipid uptake, triacylglycerol (TAG synthesis, and lipid droplet formation. Real-time PCR results showed that, following VPA treatment, the expression levels of genes encoding cluster of differentiation 36 (Cd36, low-density lipoprotein receptor-related protein 1 (Lrp1, diacylglycerol acyltransferase 2 (Dgat2, and perilipin 2 (Plin2 were increased, that of carnitine palmitoyltransferase I a (Cpt1a was not affected, and those of acetyl-Co A carboxylase α (Acca and fatty acid synthase (Fasn were decreased. Furthermore, using immunofluorescence staining and flow cytometry analyses, we found that VPA also induced peroxisome proliferator-activated receptor γ (PPARγ nuclear translocation and increased levels of cell-surface CD36. Based on these results, we propose that VPA may enhance OLA-induced hepatocyte steatosis through the upregulation of PPARγ- and CD36-dependent lipid uptake, TAG synthesis, and lipid droplet formation.

Ketose induced respiratory inhibition in isolated hepatocytes.

Science.gov (United States)

Martínez, P; Carrascosa, J M; Núñez de Castro, I

1987-06-01

The addition of 10 mM fructose or 10 mM tagatose to a suspension of hepatocytes caused respiratory inhibition, whereas no change in oxygen uptake was observed following the addition of glucose. However, incubations in the presence of fructose showed a high, aerobic glycolytic activity. Tagatose is phosphorylated to tagatose 1-phosphate but is not further metabolized by cell free liver extract. Moreover, the addition of fructose to glucagon treated cells also caused the Crabtree-like effect. The concentration of adenine nucleotides and inorganic phosphate (Pi) in the mitochondrial and cytosolic compartments during incubation (time 30 min) was determined by the digitonin fractionation procedure. In the presence of 10 mM fructose or tagatose, the total adenine nucleotide pools decreased by 40%; however, glucose produced no change. The addition of ketoses diminished the asymmetric distribution of extramitochondrial (ATP/ADP)e ratio and intramitochondrial (ATP/ADP)i ratio. At the same time the total mitochondrial Pi fell from 17 mM to 6-7 mM. The mitochondrial membrane potential (-161 mV) in the presence of fructose showed no changes during the 30 min experimental period. An increase in the NADH/NAD+ ratio was observed. These results suggest that in hepatocytes the inhibition of respiration is not necessarily linked with the enhanced aerobic glycolysis, by competition for common substrates.

Protein profiling as early detection biomarkers for TiO2 nanoparticle toxicity in Daphnia magna.