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Sample records for human hepatocytes egcg

  1. Influence of Aluminium and EGCG on Fibrillation and Aggregation of Human Islet Amyloid Polypeptide

    Science.gov (United States)

    Xu, Zhi-Xue; Zhang, Qiang; Ma, Gong-Li; Chen, Cong-Heng; He, Yan-Ming; Xu, Li-Hui; Zhang, Yuan; Zhou, Guang-Rong; Li, Zhen-Hua

    2016-01-01

    The abnormal fibrillation of human islet amyloid polypeptide (hIAPP) has been implicated in the development of type II diabetes. Aluminum is known to trigger the structural transformation of many amyloid proteins and induce the formation of toxic aggregate species. The (−)-epigallocatechin gallate (EGCG) is considered capable of binding both metal ions and amyloid proteins with inhibitory effect on the fibrillation of amyloid proteins. However, the effect of Al(III)/EGCG complex on hIAPP fibrillation is unclear. In the present work, we sought to view insight into the structures and properties of Al(III) and EGCG complex by using spectroscopic experiments and quantum chemical calculations and also investigated the influence of Al(III) and EGCG on hIAPP fibrillation and aggregation as well as their combined interference on this process. Our studies demonstrated that Al(III) could promote fibrillation and aggregation of hIAPP, while EGCG could inhibit the fibrillation of hIAPP and lead to the formation of hIAPP amorphous aggregates instead of the ordered fibrils. Furthermore, we proved that the Al(III)/EGCG complex in molar ratio of 1 : 1 as Al(EGCG)(H2O)2 could inhibit the hIAPP fibrillation more effectively than EGCG alone. The results provide the invaluable reference for the new drug development to treat type II diabetes. PMID:28074190

  2. Influence of Aluminium and EGCG on Fibrillation and Aggregation of Human Islet Amyloid Polypeptide

    Directory of Open Access Journals (Sweden)

    Zhi-Xue Xu

    2016-01-01

    Full Text Available The abnormal fibrillation of human islet amyloid polypeptide (hIAPP has been implicated in the development of type II diabetes. Aluminum is known to trigger the structural transformation of many amyloid proteins and induce the formation of toxic aggregate species. The (−-epigallocatechin gallate (EGCG is considered capable of binding both metal ions and amyloid proteins with inhibitory effect on the fibrillation of amyloid proteins. However, the effect of Al(III/EGCG complex on hIAPP fibrillation is unclear. In the present work, we sought to view insight into the structures and properties of Al(III and EGCG complex by using spectroscopic experiments and quantum chemical calculations and also investigated the influence of Al(III and EGCG on hIAPP fibrillation and aggregation as well as their combined interference on this process. Our studies demonstrated that Al(III could promote fibrillation and aggregation of hIAPP, while EGCG could inhibit the fibrillation of hIAPP and lead to the formation of hIAPP amorphous aggregates instead of the ordered fibrils. Furthermore, we proved that the Al(III/EGCG complex in molar ratio of 1 : 1 as Al(EGCG(H2O2 could inhibit the hIAPP fibrillation more effectively than EGCG alone. The results provide the invaluable reference for the new drug development to treat type II diabetes.

  3. In vitro drug metabolism of green tea catechins in human, monkey, dog, rat and mouse hepatocytes.

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    Chen, Wendy W; Qin, Geng-Yao; Zhang, Ting; Feng, Wan-Yong

    2012-06-01

    The metabolic fate of green tea catechins [(-)-epicatechin ((-)-EC), (-)-epicatechin-3-gallate (ECG) (-)- epigallocatechin (EGC) and (-)-epigallocatechin-3-gallate (EGCG)] in cryopreserved human, monkey, dog, rat and mouse hepatocytes was studied. Methylation, glucuronidation, sulfation and isomerization pathways of (-)-EC in all five species were found. Methylation, glucuronidation, sulfation, hydrolysis, isomerization and glucosidation pathways of ECG were found. Species differences in metabolism of (-)-EC or ECG were observed. Surprisingly, no metabolites of EGC or EGCG were detected, but chemical oxidation and polymerization were observed under these experimental conditions. It appeared that enzymatic reactions and chemical reactions were differentiated by an additional hydroxyl group on the B-ring between (-)-EC/ECG and EGC/EGCG. For (-)-EC, thirty-five metabolites including isomerized (M6. M10 and M25), glucuronidated (M3, M5 and M11), sulfated (M7, M15, M16, M18, M20, M23, M26), methylated (M2, M9, M12, M17, M19, M21, M27, M30, M32), glucuronated/methylated (M4, M8, M13, M14), sulfated/methylated (M22, M24, M28, M29, M31, M33, M34, M35) and diglucuronidate (M1), were detected and characterized. M11, M18, M19 and M23 were major metabolites in human hepatocytes; M11, M26 and M31 were major metabolites in monkey hepatocytes; M10, M20, M22, M26 and M31 were major metabolites in dog hepatocytes; M5, M6 and M10 were major metabolites in rat hepatocytes; and M5, M6 and M13 were major metabolites in mouse hepatocytes. For ECG, twelve metabolites including isomerized (M1), hydrolyzed (M3), glucosidated (M2), glucuronidated (M4 and M6), sulfated (M9, M11 and M12), methylated (M7), sulfated/glucuronidated/methylated (M8 and M10) and diglucuronidated (M5), were detected and characterized. M4, M11 and M12 were major metabolites in human hepatocytes; M11 and M12 were major metabolites in monkey hepatocytes; M3 and M11 were major metabolites in dog hepatocytes; M4, M6 and

  4. Human cathelicidin LL-37 enhance the antibiofilm effect of EGCG on Streptococcus mutans

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    Guo, Yi-jie; Zhang, Bo; Feng, Xue-song; Ren, Hui-xun; Xu, Ji-ru

    2016-01-01

    Background Streptococcus mutans forms biofilms as a resistance mechanism against antimicrobial agents in the human oral cavity. We recently showed that human cathelicidin LL-37 exhibits inhibitory effects on biofilm formation of S. mutans through interaction with lipoteichoic acid (LTA), but without antibacterial or biofilm dispersal abilities. (−)-Epigallocatechin gallate (EGCG) is the most abundant constituent of tea catechins that has the greatest anti-infective potential to inhibit the gr...

  5. Bile acid formation in primary human hepatocytes

    Institute of Scientific and Technical Information of China (English)

    Curt Einarsson; Ewa Ellis; Anna Abrahamsson; Bo-G6ran Ericzon; Ingemar Bj rkhem; Magnus Axelson

    2000-01-01

    AIM To evaluate a culture system for bile acid formation in primary human hepatocytes in comparison with HepG2 cells. METHODS Hepatocytes were isolated from normal human liver tissue and were cultured in serum-free William's E medium. The medium was collected and renewed every 24 h. Bile acids and their precursors in media were finally analysed by gas chromatography-mass spectrometry. RESULTS Cholic acid ( CA ) andchenodeoxycholic acid (CDCA) conjugated with glycine or taurine accounted for 70% and 25% of total steroids. A third of CDCA was also conjugated with sulphuric acid. Dexamathasone and thyroid hormorm alone or in combination did not significantly effect bile acid formation. The addition of cyclosporin A (10 μmol/L) inhibited the synthesis of CA and CDCA by about 13% and 30%, respectively. CONCLUSION Isolated human hepatocytes in primary culture behave as in the intact liver by converting cholesterol to conjugated CA and CDCA. This is in contrast to cultured HepG2 cells, which release large amounts of bile acid precursors and unconjugated bile acids into the medium.

  6. Bile acid formation in primary human hepatocytes

    Institute of Scientific and Technical Information of China (English)

    Curt Einarsson; Ewa Ellis; Anna Abrahamsson; Bo-G ran Ericzon; Ingemar Bj rkhem; Magnus Axelson

    2000-01-01

    AIM To evaluate a system for bile acid formation in human hepatocytes in comparison with HepG2 cells.METHODS Hepatocytes were isolated from normal human liver tissue and were cultured in serum-freeWilliam's E medium. The medium was collected and renewed every 24 h. Bile acids and their precursors inmedia were finally analysed by gas chromatography-mass spectrometry.RESULTS Cholic acid (CA) and chenodeoxycholic acid (CDCA) conjugated with glycine or taurineaccounted for 70% and 25% of total steroids. One third of CDCA was also conjugated with sulphuric acid.Dexamethasone and thyroid hormone alone or in combination did not significantly affect bile acid formation.The addition of cyclosporin A (10 tm) inhibited the synthesis of CA and CDCA by about 13% and 30%,respectively.CONCLUSION Isolated human hepatocytes in primary culture behave as in the intact liver by convertingalmost quantitatively cholesterol to conjugated CA and CDCA. This is in contrast to cultured HepG2 cells,which release large amounts of bile acid precursors and unconjugated bile acids into the medium.

  7. 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

  8. 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 expressi

  9. Epigallocatechin-3-gallate (EGCG) as a pro-osteogenic agent to enhance osteogenic differentiation of mesenchymal stem cells from human bone marrow: an in vitro study.

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    Jin, Pan; Wu, Huayu; Xu, Guojie; Zheng, Li; Zhao, Jinmin

    2014-05-01

    The proliferation and osteogenic capacity of mesenchymal stem cells (MSCs) needs to be improved for their use in cell-based therapy for osteoporosis. (-)-Epigallocatechin-3-gallate (EGCG), one of the green tea catechins, has been widely investigated in studies of osteoblasts and osteoclasts. However, no consensus on its role as an osteogenic inducer has been reached, possibly because of the various types of cell lines examined and the range of concentrations of EGCG used. In this study, the osteogenic effects of EGCG are studied in primary human bone-marrow-derived MSCs (hBMSCs) by detecting cell proliferation, alkaline phosphatase (ALP) activity and the expression of relevant osteogenic markers. Our results show that EGCG has a strong stimulatory effect on hBMSCs developing towards the osteogenic lineage, especially at a concentration of 5 μM, as evidenced by an increased ALP activity, the up-regulated expression of osteogenic genes and the formation of bone-like nodules. Further exploration has indicated that EGCG directes osteogenic differentiation via the continuous up-regulation of Runx2. The underlying mechanism might involve EGCG affects on osteogenic differentiation through the modulation of bone morphogenetic protein-2 expression. EGCG has also been found to promote the proliferation of hBMSCs in a dose-dependent manner. This might be associated with its antioxidative effect leading to favorable amounts of reactive oxygen species in the cellular environment. Our study thus indicates that EGCG can be used as a pro-osteogenic agent for the stem-cell-based therapy of osteoporosis.

  10. Synthesis and Biological Testing of Novel Glucosylated Epigallocatechin Gallate (EGCG Derivatives

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    Xin Zhang

    2016-05-01

    Full Text Available Epigallocatechin gallate (EGCG is the most abundant component of green tea catechins and has strong physiological activities. In this study, two novel EGCG glycosides (EGCG-G1 and EGCG-G2 were chemoselectively synthesized by a chemical modification strategy. Each of these EGCG glycosides underwent structure identification, and the structures were assigned as follows: epigallocatechin gallate-4′′-O-β-d-glucopyranoside (EGCG-G1, 2 and epigallocatechin gallate-4′,4′′-O-β-d-gluco-pyranoside (EGCG-G2, 3. The EGCG glycosides were evaluated for their anticancer activity in vitro against two human breast cell lines (MCF-7 and MDA-MB-231 using MTT assays. The inhibition rate of EGCG glycosides (EGCG-G1 and EGCG-G2 is not obvious. The EGCG glycosides are more stable than EGCG in aqueous solutions, but exhibited decreasing antioxidant activity in the DPPH radical-scavenging assay (EGCG > EGCG-G2 > EGCG-G1. Additionally, the EGCG glycosides exhibited increased water solubility: EGCG-G2 and EGCG-G1 were 15 and 31 times as soluble EGCG, respectively. The EGCG glycosides appear to be useful, and further studies regarding their biological activity are in progress.

  11. Effects of green tea and its epigallocatechin (EGCG) content on body weight and fat mass in humans: a systematic review

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    Vázquez Cisneros, Lucía Cristina; López-Uriarte, Patricia; López-Espinoza, Antonio; Navarro Meza, Mónica; Espinoza-Gallardo, Ana Cristina; Guzmán Aburto, Martha Beatriz

    2017-06-05

    The prevalence and incidence of overweight and obesity worldwide continues to increase, as well as diseases related to these conditions. This is attributed to an increase in energy intake and a decrease in energy expenditure. Consumption of green tea has been linked to a reduction in body fat and body weight. However, research on green tea has been very diverse. This review assesses the investigations that have been made with green tea and its epigallocatechin gallato (EGCG) content, evaluating its effect on body fat and body weight in humans. A search was made in the PubMed and Web of the Science databases that gave a first total result of 424 potential articles; 409 were excluded and 15 articles were used for this systematic review. Research has been very varied, however, daily consumption of green tea with doses of EGCG between 100 and 460 mg/day has shown greater effectiveness on body fat and body weight reduction in intervention periods of 12 weeks or more. In addition, the use of caffeine doses between 80 and 300 mg/day has been shown to be an important factor for this effects, when the participants did not have a high caffeine intake (> 300 mg/day) prior to the intervention.

  12. EGCG inhibits growth of human pancreatic tumors orthotopically implanted in Balb C nude mice through modulation of FKHRL1/FOXO3a and neuropilin.

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    Shankar, Sharmila; Marsh, Luke; Srivastava, Rakesh K

    2013-01-01

    Human pancreatic cancer is currently one of the fourth leading causes of cancer-related mortality with a 5-year survival rate of less than 5 %. Since pancreatic carcinoma is largely refractory to conventional therapies, there is a strong medical need for the development of novel and innovative cancer preventive strategies. The forkhead transcription factors of the O class (FOXO) play a major role in cell proliferation, angiogenesis, metastasis, and tumorigenesis. The objectives of this study were to examine whether FKHRL1/FOXO3a modulates antitumor activity of (-)-epigallocatechin-3-gallate (EGCG), an active ingredient in green tea, in pancreatic cancer model in vivo. PANC-1 cells were orthotopically implanted into Balb c nude mice and gavaged with EGCG after tumor formation. Cell proliferation and apoptosis were measured by Ki67 and TUNEL staining, respectively. The expression of PI3K, AKT, ERK, and FOXO3a/FKHRL1 and its target genes were measured by the western blot analysis and/or q-RT-PCR. FOXO-DNA binding was measured by gel shift assay. EGCG-treated mice showed significant inhibition in tumor growth which was associated with reduced phosphorylation of ERK, PI3K, AKT, and FKHRL1/FOXO3a, and modulation of FOXO target genes. EGCG induced apoptosis by upregulating Bim and activating caspase-3. EGCG modulated markers of cell cycle (p27/KIP1), angiogenesis (CD31, VEGF, IL-6, IL-8, SEMA3F, and HIF1α), and metastasis (MMP2 and MMP7). The inhibition of VEGF by EGCG was associated with suppression of neuropilin. EGCG inhibited epithelial-mesenchymal transition by upregulating the expression of E-cadherin and inhibiting the expression of N-cadherin and Zeb1. These data suggest that EGCG inhibits pancreatic cancer orthotopic tumor growth, angiogenesis, and metastasis which are associated with inhibition of PI3K/AKT and ERK pathways and activation of FKHRL1/FOXO3a. As a conclusion, EGCG can be used for the prevention and/or treatment of pancreatic cancer.

  13. 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.

  14. Transplantation of human hepatocytes into tolerized genetically immunocompetent rats

    Institute of Scientific and Technical Information of China (English)

    Edwin C. Ouyang; Catherine H. Wu; Cherie Walton; Kittichai Promrat; George Y. Wu

    2001-01-01

    AIM To determine whether normal geneticallyirnmunocornpetent rodent hosts could bemanipulated to accept human hepatocytetransplants with long term survival withoutirnrnunosuppression.METHODS Tolerance towards humanhepatocytes was established by injection ofprimary human hepatocytes or Huh7 humanhepatoma cells into the peritoneal cavities offetal rats. Corresponding cells weresubsequently transplanted into newborn rats viaintrasplenic injection within 24 h after birth.RESULTS Mixed lymphocyte assays showedthat spleen cells from non-tolerized rats werestimulated to proliferate when exposed to humanhepatocytes, while cells from tolerized ratswere not. Injections made between 15 d and 17 dof gestation produced optimal tolerizaton.Transplanted human hepatocytes in rat liverswere visualized by immunohistochemicalstaining of human albumin. By dot blotting ofgenomic DNA in livers of tolerized rats 16 weeksafter hepatocyte transplantation, it was foundthat approximately 2.5 × 105 human hepatocytessurvived per rat liver. Human albumin mRNA wasdetected in rat livers by RT-PCR for 15 wk, andhuman albumin protein was also detectable in ratserum.CONCLUSION Tolerization of an immuno-competent rat can permit transplantation, andsurvival of functional human hepatocytes.

  15. Caffeine induces CYP1A2 expression in rat hepatocytes but not in human hepatocytes.

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    Vaynshteyn, David; Jeong, Hyunyoung

    2012-06-01

    Caffeine is the active constituent in coffee. Continual consumption of caffeine can lead to an attenuated response also known as tolerance. Results from rat studies have shown that caffeine is an inducer of CYP1A2, the enzyme responsible for caffeine's metabolism. This suggests that CYP1A2 induction by caffeine may be in part responsible for caffeine tolerance. However, whether caffeine induces CYP1A2 expression in humans remains unknown. Our results from luciferase assays performed in HepG2 cells showed that caffeine is not an activator of the aromatic hydrocarbon receptor (AhR), a major transcription factor involved in upregulation of CYP1A2. Furthermore, caffeine did not induce CYP1A2 expression in primary human hepatocytes at a concentration attained by ordinary coffee drinking. On the other hand, caffeine enhanced CYP1A2 expression by 9-fold in rat hepatocytes. Our results suggest that caffeine from ordinary coffee drinking does not induce CYP1A2 expression in humans and that factors other than CYP1A2 induction by caffeine likely contribute to development of caffeine tolerance in humans.

  16. Effect of matrine on primary human hepatocytes in vitro.

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    Gong, Xiaobing; Gao, Yuan; Guo, Guoqing; Vondran, Florian W R; Schwartlander, Ruth; Efimova, Ekaterina; Pless, Gesine; Sauera, Igor M; Neuhaus, Peter

    2015-03-01

    Matrine is a bioactive component of the traditional Chinese medical herb Sophora flavescens that has been used in China to treat various kinds of diseases including virus hepatitis. However, the molecular mechanisms underlying its hepatoprotective effects remains elusive. In the present study, primary human hepatocytes were employed to elucidate the protective effects and molecular mechanisms of matrine. We observed that low concentrations of matrine had no significant impact on albumin secretion, but high concentrations (>140 mg/L) of matrine decreased the albumin secretion in hepatocytes. Western blot data indicated that matrine at 140 mg/L at 72 h induced protein expression of CYP2A6, CYP2B6 and CYP3A4. Furthermore, high concentrations of matrine reduced LDH and AST levels and were cytotoxic to hepatocytes, leading to a decreased cell viability and total protein amount. Moreover, low concentrations of matrine, enhanced the ECOD activity and decreased the level of NO2 (-) induced by cytokines in human hepatocytes. Taken together, the present study sheds novel light on the molecular mechanisms of matrine and potential application of matrine in hepatic diseases.

  17. 3D Cultivation Techniques for Primary Human Hepatocytes

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    Anastasia Bachmann

    2015-02-01

    Full Text Available One of the main challenges in drug development is the prediction of in vivo toxicity based on in vitro data. The standard cultivation system for primary human hepatocytes is based on monolayer cultures, even if it is known that these conditions result in a loss of hepatocyte morphology and of liver-specific functions, such as drug-metabolizing enzymes and transporters. As it has been demonstrated that hepatocytes embedded between two sheets of collagen maintain their function, various hydrogels and scaffolds for the 3D cultivation of hepatocytes have been developed. To further improve or maintain hepatic functions, 3D cultivation has been combined with perfusion. In this manuscript, we discuss the benefits and drawbacks of different 3D microfluidic devices. For most systems that are currently available, the main issues are the requirement of large cell numbers, the low throughput, and expensive equipment, which render these devices unattractive for research and the drug-developing industry. A higher acceptance of these devices could be achieved by their simplification and their compatibility with high-throughput, as both aspects are of major importance for a user-friendly device.

  18. The effects of human umbilical cord perivascular cells on rat hepatocyte structure and functional polarity.

    Science.gov (United States)

    Gómez-Aristizábal, Alejandro; Davies, John Edward

    2013-06-01

    Hepatocyte culture is a useful tool for the study of their biology and the development of bioartificial livers. However, many challenges have to be overcome since hepatocytes rapidly lose their normal phenotype in vitro. We have recently demonstrated that human umbilical cord perivascular cells (HUCPVCs) are able to provide support to hepatocytes. In the present study we go further into exploring the effects that HUCPVCs have in the functional polarization, and both the internal and external organization, of hepatocytes. Also, we investigate HUCPVC-hepatocyte crosstalk by tracking both the effects of HUCPVCs on hepatocyte transcription factors and those of hepatocytes on the expression of hepatotrophic factors in HUCPVCs. Our results show that HUCPVCs maintain the functional polarity of hepatocytes ex vivo, as judged by the secretion of fluorescein into bile canaliculi, for at least 40 days. Transmission electron microscopy revealed that hepatocytes in coculture organize in an organoid-like structure embedded in extracellular matrix surrounded by HUCPVCs. In coculture, hepatocytes displayed a higher expression of C/EBPα, implicated in maintenance of the mature hepatocyte phenotype, and HUCPVCs upregulated hepatocyte growth factor and Jagged1 indicating that these genes may play important roles in HUCPVC-hepatocyte interactions.

  19. Transplantation of human stem cell-derived hepatocytes in an animal model of acute liver failure.

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    Ramanathan, Rajesh; Pettinato, Giuseppe; Beeston, John T; Lee, David D; Wen, Xuejun; Mangino, Martin J; Fisher, Robert A

    2015-08-01

    Hepatocyte cell transplantation can be life-saving in patients with acute liver failure (ALF); however, primary human hepatocyte transplantation is limited by the scarcity of donor hepatocytes. We investigated the effect of stem cell-derived, hepatocyte-like cells in an animal xenotransplant model of ALF. Intraperitoneal d-galactosamine was used to develop a lethal model of ALF in the rat. Human induced pluripotent stem cells (iPSC), human mesenchymal stem cells, and human iPSC combined with human endothelial cells (iPSC + EC) were differentiated into hepatocyte-like cells and transplanted into the spleens of athymic nude rats with ALF. A reproducible lethal model of ALF was achieved with nearly 90% death within 3 days. Compared with negative controls, rats transplanted with stem cell-derived, hepatocyte-like cells were associated with increased survival. Human albumin was detected in the rat serum 3 days after transplantation in more than one-half the animals transplanted with hepatocyte-like cells. Only animals transplanted with iPSC + EC-derived hepatocytes had serum human albumin at 14 days posttransplant. Transplanted hepatocyte-like cells homed to the injured rat liver, whereas the ECs were only detected in the spleen. Transplantation of stem cell-derived, hepatocyte-like cells improved survival with evidence of in vivo human albumin production. Combining ECs may prolong cell function after transplantation. Copyright © 2015 Elsevier Inc. All rights reserved.

  20. A novel mouse model for stable engraftment of a human immune system and human hepatocytes.

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    Helene Strick-Marchand

    Full Text Available Hepatic infections by hepatitis B virus (HBV, hepatitis C virus (HCV and Plasmodium parasites leading to acute or chronic diseases constitute a global health challenge. The species tropism of these hepatotropic pathogens is restricted to chimpanzees and humans, thus model systems to study their pathological mechanisms are severely limited. Although these pathogens infect hepatocytes, disease pathology is intimately related to the degree and quality of the immune response. As a first step to decipher the immune response to infected hepatocytes, we developed an animal model harboring both a human immune system (HIS and human hepatocytes (HUHEP in BALB/c Rag2-/- IL-2Rγc-/- NOD.sirpa uPAtg/tg mice. The extent and kinetics of human hepatocyte engraftment were similar between HUHEP and HIS-HUHEP mice. Transplanted human hepatocytes were polarized and mature in vivo, resulting in 20-50% liver chimerism in these models. Human myeloid and lymphoid cell lineages developed at similar frequencies in HIS and HIS-HUHEP mice, and splenic and hepatic compartments were humanized with mature B cells, NK cells and naïve T cells, as well as monocytes and dendritic cells. Taken together, these results demonstrate that HIS-HUHEP mice can be stably (> 5 months and robustly engrafted with a humanized immune system and chimeric human liver. This novel HIS-HUHEP model provides a platform to investigate human immune responses against hepatotropic pathogens and to test novel drug strategies or vaccine candidates.

  1. A novel mouse model for stable engraftment of a human immune system and human hepatocytes.

    Science.gov (United States)

    Strick-Marchand, Helene; Dusséaux, Mathilde; Darche, Sylvie; Huntington, Nicholas D; Legrand, Nicolas; Masse-Ranson, Guillemette; Corcuff, Erwan; Ahodantin, James; Weijer, Kees; Spits, Hergen; Kremsdorf, Dina; Di Santo, James P

    2015-01-01

    Hepatic infections by hepatitis B virus (HBV), hepatitis C virus (HCV) and Plasmodium parasites leading to acute or chronic diseases constitute a global health challenge. The species tropism of these hepatotropic pathogens is restricted to chimpanzees and humans, thus model systems to study their pathological mechanisms are severely limited. Although these pathogens infect hepatocytes, disease pathology is intimately related to the degree and quality of the immune response. As a first step to decipher the immune response to infected hepatocytes, we developed an animal model harboring both a human immune system (HIS) and human hepatocytes (HUHEP) in BALB/c Rag2-/- IL-2Rγc-/- NOD.sirpa uPAtg/tg mice. The extent and kinetics of human hepatocyte engraftment were similar between HUHEP and HIS-HUHEP mice. Transplanted human hepatocytes were polarized and mature in vivo, resulting in 20-50% liver chimerism in these models. Human myeloid and lymphoid cell lineages developed at similar frequencies in HIS and HIS-HUHEP mice, and splenic and hepatic compartments were humanized with mature B cells, NK cells and naïve T cells, as well as monocytes and dendritic cells. Taken together, these results demonstrate that HIS-HUHEP mice can be stably (> 5 months) and robustly engrafted with a humanized immune system and chimeric human liver. This novel HIS-HUHEP model provides a platform to investigate human immune responses against hepatotropic pathogens and to test novel drug strategies or vaccine candidates.

  2. Fipronil induces CYP isoforms and cytotoxicity in human hepatocytes.

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    Das, Parikshit C; Cao, Yan; Cherrington, Nathan; Hodgson, Ernest; Rose, Randy L

    2006-12-15

    Recent studies have demonstrated the potential of pesticides to either inhibit or induce xenobiotic metabolizing enzymes in humans. Exposure of human hepatocytes to doses of fipronil (5-amino-1-[2,6-dichloro-4-(trifluoromethyl)phenyl]-4-[(trifluoromethyl) sulfinyl]-1H-pyrazole-3-carbonitrile) ranging from 0.1 to 25 microM resulted in a dose dependent increase in CYP1A1 mRNA expression (3.5 to approximately 55-fold) as measured by the branched DNA assay. In a similar manner, CYP3A4 mRNA expression was also induced (10-30-fold), although at the higher doses induction returned to near control levels. CYP2B6 and 3A5 were also induced by fipronil, although at lower levels (2-3-fold). Confirmation of bDNA results were sought through western blotting and/or enzyme activity assays. Western blots using CYP3A4 antibody demonstrated a dose responsive increase from 0.5 to 1 microM followed by decreasing responses at higher concentrations. Similar increases and decreases were observed in CYP3A4-specific activity levels as measured using 6beta-hydroxytestosterone formation following incubation with testosterone. Likewise, activity levels for a CYP1A1-specific substrate, luciferin CEE, demonstrated that CYP1A1 enzyme activities were maximally induced by 1 microM fipronil followed by dramatically declining activity measurements at 10 and 25 microM. Cytotoxic effects of fipronil and fipronil sulfone were examined using the adenylate kinase and the trypan blue exclusion assays in HepG2 cells and human hepatocytes. The results indicate both that HepG2 cells and primary human hepatocytes are sensitive to the cytotoxic effects of fipronil. The maximum induction of adenylate kinase was ca. 3-fold greater than the respective controls in HepG2 and 6-10-fold in the case of primary hepatocytes. A significant time- and dose-dependent induction of adenylate kinase activity in HepG2 cells was noted from 0.1 to 12.5 microM fipronil followed by decreasing activities at 25 and 50 microM. For

  3. Species-specific toxicity of troglitazone on rats and human by gel entrapped hepatocytes

    Energy Technology Data Exchange (ETDEWEB)

    Shen, Chong [College of Materials Science and Chemical Engineering, Zhejiang University, Zhejiang 310027 (China); Meng, Qin, E-mail: mengq@zju.edu.cn [College of Materials Science and Chemical Engineering, Zhejiang University, Zhejiang 310027 (China); Zhang, Guoliang [Institute of Biological and Environmental Engineering, Zhejiang University of Technology, Zhejiang 310012 (China)

    2012-01-01

    Troglitazone, despite passing preclinical trials on animals, was shortly withdrawn from market due to its severe hepatotoxicity in clinic. As rat hepatocyte monolayer consistently showed sensitive troglitazone toxicity as human hepatocyte monolayer in contrast to the species-specific toxicity in vivo, this paper utilized both hepatocytes in three-dimensional culture of gel entrapment to reflect the species difference on hepatotoxicity. Rat hepatocytes in gel entrapment did not show obvious cellular damage even under a long-term exposure for 21 days while gel entrapped human hepatocytes significantly displayed oxidative stress, steatosis, mitochondrial damage and cell death at a short exposure for 4 days. As a result, the detected species-specific toxicity of troglitazone between gel entrapped rat and human hepatocytes consisted well with the situation in vivo but was in a sharp contrast to the performance of two hepatocytes by monolayer culture. Such contradictory toxicity of rat hepatocytes between monolayer and gel entrapment culture could be explained by the fact that troglitazone was cleared more rapidly in gel entrapment than in monolayer culture. Similarly, the differential clearance of troglitazone in rat and human might also explain its species-specific toxicity. Therefore, gel entrapment of hepatocytes might serve as a platform for evaluation of drug toxicity at early stage of drug development by reducing costs, increasing the likelihood of clinical success and limiting human exposure to unsafe drugs. -- Highlights: ► Species-specific toxicity of troglitazone reflected by rat/human hepatocytes ► 3D hepatocytes in 21 days’ long-term culture used for drug hepatotoxicity ► Oversensitive toxicity in hepatocyte monolayer by slow troglitazone clearance.

  4. Hepatitis B virus infection and replication in primarily cultured human fetal hepatocytes

    Institute of Scientific and Technical Information of China (English)

    Min Lin; Qun Chen; Li-Ye Yang; Wen-Yu Li; Xi-Biao Cao; Jiao-Ren Wu; You-Peng Peng; Mo-Rui Chen

    2007-01-01

    AIM:To investigate the infection and replication of hepatitis B virus(HBV)in primarily cultured human fetal hepatocytes(HFHs).METHODS:The human fetal hepatocytes were cultured in serum-free medium,HBV-positive serum was added into the medium to study the susceptibility of hepatocytes to HBV infection.The supernatant was collected for ELISA assay of HBsAg and HBeAg,and quantitative fluorescence PCR for HBV-DNA assay daily.Albumin and HBcAg,CK8 and CK18 expressions were detected by immunohistochemistry in cultured hepatocytes.Content of lactate dehydrogenate(LDH)was measured to find out the integrity of the cell membrane.RESULTS:A stable hepatocyte culture system was established.HBV could infect the hepatocytes and replicate,and HBcAg expression could be detected by immunohistochemistry in hepatocyte-like cells.HBV-DNA in the supernatant could be detected from d 2 to d 18 and HBsAg and HBeAg were positive on d 3-d 18 after HBV infection.HBV in medium increased from d 0 to d 6 and subsequently decreased as the cells were progressively loosing their hepatocyte phenotypes.CONCLUSION:HBV could infect human fetal hepatocytes and replicate.This in vitro model allowed a detailed Study on early events associated with human HBV entry into cells and subsequent replication.

  5. Evaluation of the Bitter-Masking Potential of Food Proteins for EGCG by a Cell-Based Human Bitter Taste Receptor Assay and Binding Studies

    NARCIS (Netherlands)

    Bohin, M.C.; Roland, W.S.U.; Gruppen, H.; Gouka, R.J.; Hijden, H.T.W.M.; Dekker, P.; Smit, G.; Vincken, J.P.

    2013-01-01

    Epigallocatechin gallate (EGCG) has been ascribed to several health benefits, but its bitter taste influences the liking of products with high concentrations of this compound. ß-Casein, in particular, and several gelatins are known as strong binders of EGCG, contrary to ß-lactoglobulin. The current

  6. Maintenance of Hepatic Functions in Primary Human Hepatocytes Cultured on Xeno-Free and Chemical Defined Human Recombinant Laminins.

    Science.gov (United States)

    Watanabe, Masaaki; Zemack, Helen; Johansson, Helene; Hagbard, Louise; Jorns, Carl; Li, Meng; Ellis, Ewa

    2016-01-01

    Refined methods for maintaining specific functions of isolated hepatocytes under xeno-free and chemical defined conditions is of great importance for the development of hepatocyte research and regenerative therapy. Laminins, a large family of heterotrimeric basement membrane adhesion proteins, are highly cell and tissue type specific components of the extracellular matrix and strongly influence the behavior and function of associated cells and/or tissues. However, detailed biological functions of many laminin isoforms are still to be evaluated. In this study, we determined the distribution of laminin isoforms in human liver tissue and isolated primary human hepatocytes by western blot analysis, and investigated the efficacy of different human recombinant laminin isoforms on hepatic functions during culture. Protein expressions of laminin-chain α2, α3, α4, β1, β3, γ1, and γ2 were detected in both isolated human hepatocytes and liver tissue. No α1 and α5 expression could be detected in liver tissue or hepatocytes. Hepatocytes were isolated from five different individual livers, and cultured on human recombinant laminin isoforms -111, -211, -221, -332, -411, -421, -511, and -521 (Biolamina AB), matrigel (extracted from Engelbreth-Holm-Swarm sarcoma), or collagen type IV (Collagen). Hepatocytes cultured on laminin showed characteristic hexagonal shape in a flat cell monolayer. Viability, double stranded DNA concentration, and Ki67 expression for hepatocytes cultured for six days on laminin were comparable to those cultured on EHS and Collagen. Hepatocytes cultured on laminin also displayed production of human albumin, alpha-1-antitrypsin, bile acids, and gene expression of liver-enriched factors, such as hepatocyte nuclear factor 4 alpha, glucose-6-phosphate, cytochrome P450 3A4, and multidrug resistance-associated protein 2. We conclude that all forms of human recombinant laminin tested maintain cell viability and liver-specific functions of primary human

  7. Proliferation of L02 human hepatocytes in tolerized geneticall yimmunocompetent rats

    Institute of Scientific and Technical Information of China (English)

    Hu Lin; Qing Mao; Yu-Ming Wang; Li Jiang

    2008-01-01

    AIM: To investigate whether human hepatocytes could proliferate after transplantation to normal immunocompetent rats treated with 2-acetaminofluorene or Retrorsine and partial hepatectomy.METHODS: L02 hepatocyte-tolerant Sprague-Dawley rats were injected with Retrorsine, 2-acetaminofluorene or normal saline. L02 hepatocytes were then transplanted via the spleen. Human albumin and its mRNA, specific proliferating cell nuclear antigen (PCNA), L02 hepatocyte dynamic distribution, number density and area density of PCNA-positive cells in the liver were determined.RESULTS: All the examined indicators were not significantly different between the rats treated with 2-acetaminofluorene and normal saline, which was not the case with rats treated with Retrorsine. A dynamic distribution of L02 hepatocytes in the rat liver was detected from wk 1 to mo 6 after transplantation in the Retrorsine group and from wk 1 to 10 in the 2-acetaminofluorene group. Human albumin and its mRNA were detected from wk 2 to mo 6 in the Retrorsine group and from wk 1 to 8 in the 2-acetaminofiuorene group.Specific human PCNA was detected in the rat liver from wk 2 to mo 6 in the Retrorsine group and from wk 2 to 6 in the 2-acetaminofluorene group. Human albumin and its mRNA contents as well as the number of PCNA positive cells reached a peak at wk 4.CONCLUSION: L02 human hepatocytes could not proliferate significiantly after transplantation to the normal,immunocompetent rats treated with 2-acetaminofluorene.L02 human hepatocytes can survive for 10 wk after transplantation and express human albumin for 8 wk. L02human hepatocytes can proliferate and express human albumin for 6 mo after transplantation to the rats treated with Retrorsine. The chimeric L02 human hepatocytes,which then underwent transplantation into tolerant rats,were normal in morphogenesis, biochemistry and function.

  8. Epigallocatechin-gallate (EGCG) regulates autophagy in human retinal pigment epithelial cells: A potential role for reducing UVB light-induced retinal damage

    Energy Technology Data Exchange (ETDEWEB)

    Li, Chao-Peng; Yao, Jin; Tao, Zhi-Fu; Li, Xiu-Miao; Jiang, Qin, E-mail: jqin710@vip.sina.com; Yan, Biao, E-mail: yanbiao1982@hotmail.com

    2013-09-06

    Highlights: •UVB irradiation induces RPE autophagy. •EGCG treatment represses UVB-mediated autophagy. •EGCG regulates UVB-mediated autophagy through mTOR signaling pathway. •EGCG sensitizes RPE cells to UVB-induced damage in an autophagy-dependent manner. -- Abstract: Autophagy is an intracellular catabolic process involved in protein and organelle degradation via the lysosomal pathway that has been linked in the pathogenesis of age-related macular degeneration (AMD). UVB irradiation-mediated degeneration of the macular retinal pigment epithelial (RPE) cells is an important hallmark of AMD, which is along with the change in RPE autophagy. Thus, pharmacological manipulation of RPE autophagy may offer an alternative therapeutic target in AMD. Here, we found that epigallocatechin-3-gallate (EGCG), a polyphenolic compound from green tea, plays a regulatory role in UVB irradiation-induced autophagy in RPE cells. UVB irradiation results in a marked increase in the amount of LC3-II protein in a dose-dependent manner. EGCG administration leads to a significant reduction in the formation of LC3-II and autophagosomes. mTOR signaling activation is required for EGCG-induced LC3-II formation, as evidenced by the fact that EGCG-induced LC3-II formation is significantly impaired by rapamycin administration. Moreover, EGCG significantly alleviates the toxic effects of UVB irradiation on RPE cells in an autophagy-dependent manner. Collectively, our study reveals a novel role of EGCG in RPE autophagy. EGCG may be exploited as a potential therapeutic reagent for the treatment of pathological conditions associated with abnormal autophagy.

  9. Chemically induced hepatotoxicity in human stem cell-induced hepatocytes compared with primary hepatocytes and HepG2.

    Science.gov (United States)

    Kang, Seok-Jin; Lee, Hyuk-Mi; Park, Young-Il; Yi, Hee; Lee, Hunjoo; So, ByungJae; Song, Jae-Young; Kang, Hwan-Goo

    2016-10-01

    Stem cell-induced hepatocytes (SC-iHeps) have been suggested as a valuable model for evaluating drug toxicology. Here, human-induced pluripotent stem cells (QIA7) and embryonic stem cells (WA01) were differentiated into hepatocytes, and the hepatotoxic effects of acetaminophen (AAP) and aflatoxin B1 (AFB1) were compared with primary hepatocytes (p-Heps) and HepG2. In a cytotoxicity assay, the IC50 of SC-iHeps was similar to that in p-Heps and HepG2 in the AAP groups but different from that in p-Heps of the AFB1 groups. In a multi-parameter assay, phenotypic changes in mitochondrial membrane potential, calcium influx and oxidative stress were similar between QIA7-iHeps and p-Heps following AAP and AFB1 treatment but relatively low in WA01-iHeps and HepG2. Most hepatic functional markers (hepatocyte-specific genes, albumin/urea secretion, and the CYP450 enzyme activity) were decreased in a dose-dependent manner following AAP and AFB1 treatment in SC-iHeps and p-Heps but not in HepG2. Regarding CYP450 inhibition, the cell viability of SC-iHeps and p-Heps was increased by ketoconazole, a CYP3A4 inhibitor. Collectively, SC-iHeps and p-Heps showed similar cytotoxicity and hepatocyte functional effects for AAP and AFB1 compared with HepG2. Therefore, SC-iHeps have phenotypic characteristics and sensitivity to cytotoxic chemicals that are more similar to p-Heps than to HepG2 cells.

  10. DIFFERENTIAL-EFFECTS OF EICOSAPENTAENOIC ACID ON GLYCEROLIPID AND APOLIPOPROTEIN-B METABOLISM IN PRIMARY HUMAN HEPATOCYTES COMPARED TO HEPG2 CELLS AND PRIMARY RAT HEPATOCYTES

    NARCIS (Netherlands)

    LIN, YG; SMIT, MJ; HAVINGA, R; VERKADE, HJ; VONK, RJ; KUIPERS, F

    1995-01-01

    We compared the effects of eicosapentaenoic acid (EPA) and oleic acid (OA) on glycerolipid and apolipoprotein B (apoB) metabolism in primary human hepatocytes, HepG2 cells and primary rat hepatocytes. Cells were incubated for 1 to 5 h with 0.25 mM bovine serum albumin in the absence (control) or

  11. Preparation and characterization of (−)-Epigallocatechin-3-gallate (EGCG)-loaded nanoparticles and their inhibitory effects on Human breast cancer MCF-7 cells

    Science.gov (United States)

    Zeng, Liang; Yan, Jingna; Luo, Liyong; Ma, Mengjun; Zhu, Huiqun

    2017-01-01

    We were employing nanotechnology to improve the targeting ability of (−)-Epigallocatechin-3-gallate (EGCG) towards MCF-7 cells, and two kinds of EGCG nanoparticles (FA-NPS-PEG and FA-PEG-NPS) were obtained, besides, their characteristics and effects on MCF-7 cells were studied. The results indicated that (i) both FA-NPS-PEG and FA-PEG-NPS have high stabilities; (ii) their particles sizes were 185.0 ± 13.5 nm and 142.7 ± 7.2 nm, respectively; (iii) their encapsulation efficiencies of EGCG were 90.36 ± 2.20% and 39.79 ± 7.54%, respectively. (iv) there was no cytotoxicity observed in EGCG, FA-NPS-PEG and FA-PEG-NPS toward MCF-7 cells over all concentrations (0~400 μg/mL) tested; (v) EGCG, FA-NPS-PEG and FA-PEG-NPS inhibited MCF-7 cells proliferation in dose-dependent manners, with the average IC50 of 470.5 ± 33.0, 65.9 ± 0.4 and 66.6 ± 0.6 μg/mL; (vi) EGCG, FA-NPS-PEG and FA-PEG-NPS could modulated the expressions of several key regulatory proteins in PI3K-Akt pathway such as up-regulation of PTEN, p21 and Bax, and down-regulation of p-PDK1, p-AKT, CyclinD1 and Bcl-2, which gave an illustration about the mechanism by which EGCG nanoparticles inhibited MCF-7 cells proliferation. In this study, EGCG nanoparticles can significantly enhance the targeting ability and efficacy of EGCG, which is considered to an experimental foundation for further research on its activity, targeting ability and metabolism in vivo.

  12. CYP2E1-dependent hepatotoxicity and oxidative damage after ethanol administration in human primary hepatocytes

    Institute of Scientific and Technical Information of China (English)

    Lie-Gang Liu; Hong Yan; Ping Yao; Wen Zhang; Li-Jun Zou; Fang-Fang Song; Ke Li; Xiu-Fa Sun

    2005-01-01

    AIM: To observe the relationship between ethanol-induced oxidative damage in human primary cultured hepatocytes and cytochrome P450 2E1 (CYP2E1) activity, in order to address if inhibition of CYP2E1 could attenuate ethanol-induced cellular damage.METHODS: The dose-dependent (25-100 mmol/L) and time-dependent (0-24 h) exposures of primary human cultured hepatocytes to ethanol were carried out. CYP2E1 activity and protein expression were detected by spectrophotometer and Western blot analysis respectively.Hepatotoxicity was investigated by determination of lactate dehydrogenase (LDH) and aspartate transaminase (AST) level in hepatocyte culture supernatants, as well as the intracellular formation of malondialdehyde (MDA).RESULTS: A dose-and time-dependent response between ethanol exposure and CYP2E1 activity in human hepatocytes was demonstrated. Moreover, there was a time-dependent increase of CYP2E1 protein after 100 mmol/L ethanol exposure. Meanwhile, ethanol exposure of hepatocytes caused a time-dependent increase of ceilular MDA level, LDH, and AST activities in supernatants.Furthermore, the inhibitor of CYP2E1, diallyl sulfide (DAS) could partly attenuate the increases of MDA, LDH, and AST in human hepatocytes.CONCLUSION: A positive relationship between ethanol-induced oxidative aamage in human primary cultured hepatocytes and CYP2E1 activity was exhibited, and the inhibition of CYP2E1 could partly attenuate ethanol-induced oxidative damage.

  13. Discovering multiple transcripts of human hepatocytes using massively parallel signature sequencing (MPSS

    Directory of Open Access Journals (Sweden)

    Li Yi-Xue

    2007-07-01

    Full Text Available Abstract Background The liver is the largest human internal organ – it is composed of multiple cell types and plays a vital role in fulfilling the body's metabolic needs and maintaining homeostasis. Of these cell types the hepatocytes, which account for three-quarters of the liver's volume, perform its main functions. To discover the molecular basis of hepatocyte function, we employed Massively Parallel Signature Sequencing (MPSS to determine the transcriptomic profile of adult human hepatocytes obtained by laser capture microdissection (LCM. Results 10,279 UniGene clusters, representing 7,475 known genes, were detected in human hepatocytes. In addition, 1,819 unique MPSS signatures matching the antisense strand of 1,605 non-redundant UniGene clusters (such as APOC1, APOC2, APOB and APOH were highly expressed in hepatocytes. Conclusion Apart from a large number of protein-coding genes, some of the antisense transcripts expressed in hepatocytes could play important roles in transcriptional interference via a cis-/trans-regulation mechanism. Our result provided a comprehensively transcriptomic atlas of human hepatocytes using MPSS technique, which could be served as an available resource for an in-depth understanding of human liver biology and diseases.

  14. Physiological effects of epigallocatechin-3-gallate (EGCG) on energy expenditure for prospective fat oxidation in humans: A systematic review and meta-analysis.

    Science.gov (United States)

    Kapoor, Mahendra P; Sugita, Masaaki; Fukuzawa, Yoshitaka; Okubo, Tsutomu

    2017-05-01

    Green tea catechins (GTCs) are known to improve fat oxidation (FOX) during fasted, rested and exercise conditions wherein epigallocatechin-3-gallate (EGCG) is thought to be the most pharmacologically active and has been studied extensively. From the available data of randomized controlled trials (RCTs) on EGCG, we carried out a systematic review and meta-analysis to elucidate whether EGCG consumption indeed increase energy expenditure (EE) and promote FOX. A systematic review of the literature was conducted using electronic databases (PubMed, Embase, Cochrane Library, CINAHL, JICST, JSTPLUS, and JMEDPLUS and others) and eight RCTs were included. RCTs were reviewed using Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines and methodological quality was assessed. After data extraction, results were aggregated using fixed- and random-effect approaches and expressed to quantify the relationship between the dose of EGCG for respiratory quotient (RQ), EE and rate of FOX to compare the EGCG and placebo treatments. The meta-analysis results of verities of studies in terms of dose and length of duration revealed that EGCG supplementation provided significant mean difference (MD) when compared with placebo for RQ [MD: -0.02; 95% confidence intervals (95% CI), -0.04 to 0.00; I(2)=67%; P=.01] and EE [MD: 158.05 kJ/day; 95% CI, 4.72 to 311.38; I(2)=0%; P=.04] in fixed-effect approach. Changes in FOX did not reach the level of statistical significance. Meta-analyses of EGCG influence on the body mass index, waist circumference and total body fat mass (TBFM) were also examined and their impact on the promotion of FOX is reported. Effect of EGCG doses was also systematically reviewed. Finding showed that EGCG intake moderately accelerates EE and reduces RQ. The analyses revealed that the EGCG resulted in difference in RQ and EE but the effect on the other measures of energy metabolism was relatively mild. Possibly, EGCG alone has the potential to increase

  15. Differentiation of human embryonic stem cells along a hepatocyte lineage and its application in liver regeneration

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    Hepatocyte transplantation and bioartificial liver(BAL)as alternatives to liver transplantation offer the possibility of effective treatment for many inherited and acquired hepatic disorders.Unfortunately,the limited availability of donated livers and the variability of their derived hepatocytes make it difficult to obtain enough viable human hepatocytes for the hepatocyte-based therapies.Embryonic stem cells (ESCs),which could be isolated directly from the blastocyst inner cell mass,have permanent self-renewal capability and developmental pluripotency and therefore might be an ideal cell source in the treatment of hepatic discords.However,differentiation of hESCS into hepatocytes with significant numbers remains a challenge.This review updates our current understanding of differentiation of ESCs into hepatic lineage cells,their future therapeutic uses and problems in liver regeneration.

  16. 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

  17. Implication of Akt, ERK1/2 and alternative p38MAPK signalling pathways in human colon cancer cell apoptosis induced by green tea EGCG.

    Science.gov (United States)

    Cerezo-Guisado, María Isabel; Zur, Rafal; Lorenzo, María Jesús; Risco, Ana; Martín-Serrano, Miguel A; Alvarez-Barrientos, Alberto; Cuenda, Ana; Centeno, Francisco

    2015-10-01

    We investigated apoptosis induced by the green tea component the epigallocatechin-3-gallate (EGCG) and the pathways underlying its activity in a colon cancer cell line. A complete understanding of the mechanism(s) and molecules targeted by green tea polyphenols could be useful in developing novel therapeutic approaches for cancer treatment. EGCG, which is the major polyphenol in green tea, has cytotoxic effects and induced cell death in HT-29 cell death. In this study, we evaluated the effect EGCG on mitogen-activated protein kinase (MAPK) and Akt pathways. EGCG treatment increased phospho-ERK1/2, -JNK1/2 and -p38α, -p38γ and -p38δ, as well as phospho-Akt levels. Using a combination of kinase inhibitors, we found that EGCG-induced cell death is partially blocked by inhibiting Akt, ERK1/2 or alternative p38MAPK activity. Our data suggest that these kinase pathways are involved in the anti-cancer effects of EGCG and indicate potential use of this compound as chemotherapeutic agent for colon cancer treatment.

  18. In vitro induction of human embryonic stem cells into hepatocyte-like cells

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Human embryonic stem cells (hES cells) are pluripotent and provide a unique, unlimited resource for human hepatocytes, which can serve as a novel cell source for cell transplantation and bioartificial liver (BAL). Here, we have developed a procedure by which hES cells can differentiate into hepatocyte-like cells. After being cultured in suspension in bacteriological petri dishes for 7 d, hES cells developed into cystic embryoid bodies (EBs). The EBs were then cultured in conditional medium containing dexamethasone and insulin in collagen type I-coated tissue culture dishes for two weeks. The hES cell-derived hepatocyte-like cells (HLCs) displayed some morphologic characteristics of hepatocytes. Reverse-transcription polymerase chain reaction (RT-PCR) and immunofluorescence cell staining proved that the induced HLCs expressed several hepatocyte specific genes including AFP, ALB, CYP1B1 and cytokeratins CK18 and CK19. Furthermore, the induced cells executed a range of hepatocyte functions, such as ICG uptake/excretion, glycogen deposits, albumin production and ammonium metabolism. Taken together, our results show that HLCs exhibit similar morphologic, phenotypic, and functional characteristics to hepatocytes.

  19. Activated human neutrophils release hepatocyte growth factor/scatter factor.

    LENUS (Irish Health Repository)

    McCourt, M

    2012-02-03

    BACKGROUND: Hepatocyte growth factor or scatter factor (HGF\\/SF) is a pleiotropic cytokine that has potent angiogenic properties. We have previously demonstrated that neutrophils (PMN) are directly angiogenic by releasing vascular endothelial growth factor (VEGF). We hypothesized that the acute inflammatory response can stimulate PMN to release HGF. AIMS: To examine the effects of inflammatory mediators on PMN HGF release and the effect of recombinant human HGF (rhHGF) on PMN adhesion receptor expression and PMN VEGF release. METHODS: In the first experiment, PMN were isolated from healthy volunteers and stimulated with tumour necrosis factor-alpha (TNF-alpha), lipopolysaccharide (LPS), interleukin-8 (IL-8), and formyl methionyl-leucyl-phenylalanine (fMLP). Culture supernatants were assayed for HGF using ELISA. In the second experiment, PMN were lysed to measure total HGF release and HGF expression in the PMN was detected by Western immunoblotting. Finally, PMN were stimulated with rhHGF. PMN CD 11a, CD 11b, and CD 18 receptor expression and VEGF release was measured using flow cytometry and ELISA respectively. RESULTS: TNF-alpha, LPS and fMLP stimulation resulted in significantly increased release of PMN HGF (755+\\/-216, 484+\\/-221 and 565+\\/-278 pg\\/ml, respectively) compared to controls (118+\\/-42 pg\\/ml). IL-8 had no effect. Total HGF release following cell lysis and Western blot suggests that HGF is released from intracellular stores. Recombinant human HGF did not alter PMN adhesion receptor expression and had no effect on PMN VEGF release. CONCLUSIONS: This study demonstrates that pro-inflammatory mediators can stimulate HGF release from a PMN intracellular store and that activated PMN in addition to secreting VEGF have further angiogenic potential by releasing HGF.

  20. Generation of human pluripotent stem cell-derived hepatocyte-like cells for drug toxicity screening.

    Science.gov (United States)

    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.

  1. Characterization of rat or human hepatocytes cultured in microphysiological systems (MPS) to identify hepatotoxicity.

    Science.gov (United States)

    Chang, Shih-Yu; Voellinger, Jenna L; Van Ness, Kirk P; Chapron, Brian; Shaffer, Rachel M; Neumann, Thomas; White, Collin C; Kavanagh, Terrance J; Kelly, Edward J; Eaton, David L

    2017-04-01

    The liver is the main site for drug and xenobiotics metabolism, including inactivation or bioactivation. In order to improve the predictability of drug safety and efficacy in clinical development, and to facilitate the evaluation of the potential human health effects from exposure to environmental contaminants, there is a critical need to accurately model human organ systems such as the liver in vitro. We are developing a microphysiological system (MPS) based on a new commercial microfluidic platform (Nortis, Inc.) that can utilize primary liver cells from multiple species (e.g., rat and human). Compared to conventional monolayer cell culture, which typically survives for 5-7days or less, primary rat or human hepatocytes in an MPS exhibited higher viability and improved hepatic functions, such as albumin production, expression of hepatocyte marker HNF4α and canaliculi structure, for up to 14days. Additionally, induction of Cytochrome P450 (CYP) 1A and 3A4 in cryopreserved human hepatocytes was observed in the MPS. The acute cytotoxicity of the potent hepatotoxic and hepatocarcinogen, aflatoxin B1, was evaluated in human hepatocytes cultured in an MPS, demonstrating the utility of this model for acute hepatotoxicity assessment. These results indicate that MPS-cultured hepatocytes provide a promising approach for evaluating chemical toxicity in vitro. Copyright © 2017. Published by Elsevier Ltd.

  2. Human Upcyte Hepatocytes: Characterization of the Hepatic Phenotype and Evaluation for Acute and Long-Term Hepatotoxicity Routine Testing.

    Science.gov (United States)

    Tolosa, Laia; Gómez-Lechón, M José; López, Silvia; Guzmán, Carla; Castell, José V; Donato, M Teresa; Jover, Ramiro

    2016-07-01

    The capacity of human hepatic cell-based models to predict hepatotoxicity depends on the functional performance of cells. The major limitations of human hepatocytes include the scarce availability and rapid loss of the hepatic phenotype. Hepatoma cells are readily available and easy to handle, but are metabolically poor compared with hepatocytes. Recently developed human upcyte hepatocytes offer the advantage of combining many features of primary hepatocytes with the unlimited availability of hepatoma cells. We analyzed the phenotype of upcyte hepatocytes comparatively with HepG2 cells and adult primary human hepatocytes to characterize their functional features as a differentiated hepatic cell model. The transcriptomic analysis of liver characteristic genes confirmed that the upcyte hepatocytes expression profile comes closer to human hepatocytes than HepG2 cells. CYP activities were measurable and showed a similar response to prototypical CYP inducers than primary human hepatocytes. Upcyte hepatocytes also retained conjugating activities and key hepatic functions, e.g. albumin, urea, lipid and glycogen synthesis, at levels close to hepatocytes. We also investigated the suitability of this cell model for preclinical hepatotoxicity risk assessments using multiparametric high-content screening, as well as transcriptomics and targeted metabolomic analysis. Compounds with well-documented in vivo hepatotoxicity were screened after acute and repeated doses up to 1 week. The evaluation of complex mechanisms of cell toxicity, drug-induced steatosis and oxidative stress biomarkers demonstrated that, by combining the phenotype of primary human hepatocytes and the ease of handling of HepG2 cells, upcyte hepatocytes offer suitable properties to be potentially used for toxicological assessments during drug development.

  3. In Vitro Toxicity of Epigallocatechin Gallate in Rat Liver Mitochondria and Hepatocytes

    Directory of Open Access Journals (Sweden)

    Otto Kucera

    2015-01-01

    Full Text Available Epigallocatechin-3-gallate (EGCG is the main compound of green tea with well-described antioxidant, anti-inflammatory, and tumor-suppressing properties. However, EGCG at high doses was reported to cause liver injury. In this study, we evaluated the effect of EGCG on primary culture of rat hepatocytes and on rat liver mitochondria in permeabilized hepatocytes. The 24-hour incubation with EGCG in concentrations of 10 μmol/L and higher led to signs of cellular injury and to a decrease in hepatocyte functions. The effect of EGCG on the formation of reactive oxygen species (ROS was biphasic. While low doses of EGCG decreased ROS production, the highest tested dose induced a significant increase in ROS formation. Furthermore, we observed a decline in mitochondrial membrane potential in cells exposed to EGCG when compared to control cells. In permeabilized hepatocytes, EGCG caused damage of the outer mitochondrial membrane and an uncoupling of oxidative phosphorylation. EGCG in concentrations lower than 10 μmol/L was recognized as safe for hepatocytes in vitro.

  4. Gene expression analysis of primary normal human hepatocytes infected with human hepatitis B virus

    Institute of Scientific and Technical Information of China (English)

    Hyun Mi Ryu; Sung Gyoo Park; Sung Su Yea; Won Hee Jang; Young-Il Yang; Guhung Jung

    2006-01-01

    AIM: To find the relationship between hepatitis B virus (HBV) and hepatocytes during the initial state of infection by cDNA microarray.METHODS: Primary normal human hepatocytes (PNHHs)were isolated and infected with HBV. From the PNHHs,RNA was isolated and inverted into complement DNA (cDNA) with Cy3- or Cy5- labeled dUTP for microarray analysis. The labeled cDNA was hybridized with microarray chip, including 4224 cDNAs. From the image of the microarray, expression profiles were produced and some of them were confirmed by RT-PCR, immunoblot analysis, and NF-κB luciferase reporter assay.RESULTS: From the cDNA microarray, we obtained 98differentially regulated genes. Of the 98 genes, 53 were up regulated and 45 down regulated. Interestingly, in the up regulated genes, we found the TNF signaling pathway-related genes: LT-α, TRAF2, and NIK. By using RT-PCR, we confirmed the up-regulation of these genes in HepG2, Huh7, and Chang liver cells, which were transfected with pHBV1.2x, a plasmid encoding all HBV messages. Moreover, these three genes participated in HBVmediated NF-κB activation.CONCLUSION: During the initial state of HBV infection,hepatocytes facilitate the activation of NF-κB through up regulation of LT-α, TRAF2, and NIK.

  5. SIRT1 Disruption in Human Fetal Hepatocytes Leads to Increased Accumulation of Glucose and Lipids.

    Directory of Open Access Journals (Sweden)

    Takamasa Tobita

    Full Text Available There are unprecedented epidemics of obesity, such as type II diabetes and non-alcoholic fatty liver diseases (NAFLD in developed countries. A concerning percentage of American children are being affected by obesity and NAFLD. Studies have suggested that the maternal environment in utero might play a role in the development of these diseases later in life. In this study, we documented that inhibiting SIRT1 signaling in human fetal hepatocytes rapidly led to an increase in intracellular glucose and lipids levels. More importantly, both de novo lipogenesis and gluconeogenesis related genes were upregulated upon SIRT1 inhibition. The AKT/FOXO1 pathway, a major negative regulator of gluconeogenesis, was decreased in the human fetal hepatocytes inhibited for SIRT1, consistent with the higher level of gluconeogenesis. These results indicate that SIRT1 is an important regulator of lipid and carbohydrate metabolisms within human fetal hepatocytes, acting as an adaptive transcriptional response to environmental changes.

  6. Effect of butylated hydroxytoluene, curcumin, propyl gallate and thiabendazole on cytochrome P450 forms in cultured human hepatocytes

    NARCIS (Netherlands)

    Price, R.J.; Scott, M.P.; Giddings, A.M.; Walters, D.G.; Stierum, R.H.; Meredith, C.; Lake, B.G.

    2008-01-01

    1. The objective of this study was to investigate the effects of four food chemicals, namely butylated hydroxytoluene (BHT), curcumin (CC), propyl gallate (PG) and thiabendazole (TB), on cytochrome P450 (CYP) forms in cultured human hepatocytes. 2. Treatment of human hepatocytes for 72 h with 2-200

  7. Effect of butylated hydroxytoluene, curcumin, propyl gallate and thiabendazole on cytochrome P450 forms in cultured human hepatocytes

    NARCIS (Netherlands)

    Price, R.J.; Scott, M.P.; Giddings, A.M.; Walters, D.G.; Stierum, R.H.; Meredith, C.; Lake, B.G.

    2008-01-01

    1. The objective of this study was to investigate the effects of four food chemicals, namely butylated hydroxytoluene (BHT), curcumin (CC), propyl gallate (PG) and thiabendazole (TB), on cytochrome P450 (CYP) forms in cultured human hepatocytes. 2. Treatment of human hepatocytes for 72 h with 2-200

  8. 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 participants (10 women/12 men, BMI 20.6-34.5 kg/m(2), age 18-49 years) underwent a hyperinsulinemic euglycemic clamp with measurement of HGF at baseline and steady state. Relationships between baseline HGF, anthropometrics, triglycerides, liver enzymes, c-reactive protein and adiponectin were also evaluated...

  9. EGCG attenuates atherosclerosis through the Jagged-1/Notch pathway.

    Science.gov (United States)

    Yin, Jianguo; Huang, Fang; Yi, Yuhong; Yin, Liang; Peng, Daoquan

    2016-02-01

    Atherosclerosis is the most common cause of cardiovascular diseases worldwide. Oxidized low-density lipoprotein (ox-LDL) is a particularly important risk factor in the pathogenesis of atherosclerosis. Accumulating evidence has indicated that epigallocatechin-3-gallate (EGCG; a catechin found in the popular beverage, greent tea) protects against ox-LDL-induced atherosclerosis. However, the underlying mechanisms remain unclear. In the present study, ox-LDL (100 mg/l) induced damage to, and the apoptosis of human umbilical vein endothelial cells (HUVECs) by reducing endothelial nitric oxide synthase (eNOS) expression and promoting inducible nitric oxide synthase (iNOS) expression; these effects were abrogated by the addition of 50 µM EGCG. Furthermore, ox-LDL rapidly activated the membrane translocation of p22phox, and altered the protein expression of Jagged-1 and Notch pathway-related proteins [Math1, hairy and enhancer of split (HES)1 and HES5]; these effects were also prevented by pre-treatment with 50 µM EGCG. In addition, Jagged-1 played a significant role in the EGCG-mediated protection against ox-LDL-induced apoptosis and ox-LDL‑diminished cell adhesion in the HUVECs. Finally, EGCG inhibited high-fat diet (HFD)-induced atherosclerosis in apolipoprotein E (ApoE) knockout (ApoE-KO) mice through the Jagged-1/Notch pathway. Taken together, these findings demonstrate that 50 µM EGCG protects against ox-LDL-induced endothelial dysfunction through the Jagged-1/Notch signaling pathway. Moreover, our data provide insight into the possible molecular mechanisms through which EGCG attenuates ox-LDL‑induced vascular endothelial dysfunction.

  10. 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.

  11. Potential barriers to human hepatocyte transplantation in MUP-uPAtg(⁺/⁺)Rag2⁻/⁻γC⁻/⁻ mice.

    Science.gov (United States)

    Komori, Junji; DeWard, Aaron D; Gramignoli, Roberto; Strom, Stephen C; Fontes, Paulo; Lagasse, Eric

    2014-01-01

    Primary human fetal and adult hepatocytes have been considered feasible donor cell sources for cell transplantation. We compared the engraftment efficiencies between adult human, fetal human, and adult porcine hepatocytes after transplantation into MUP-uPA(tg(+/+))Rag2(-/-)γC(-/-)mice. Transplantation of adult human hepatocytes yielded a 1,000-fold higher serum albumin level compared to transplantation of fetal human hepatocytes, while transplantation of adult porcine hepatocytes resulted in a 100-fold higher serum albumin level than adult human hepatocytes. These results suggest that adult liver cells are superior to fetal liver cells for transplantation, and caution should be applied if porcine hepatocytes are used for preclinical studies as a proof of concept for human hepatocytes.

  12. Hepatic Stellate Cells Improve Engraftment of Human Primary Hepatocytes: A Preclinical Transplantation Study in an Animal Model.

    Science.gov (United States)

    Dusabineza, Ange-Clarisse; Najimi, Mustapha; van Hul, Noémi; Legry, Vanessa; Khuu, Dung Ngoc; van Grunsven, Leo A; Sokal, Etienne; Leclercq, Isabelle A

    2015-01-01

    Human hepatocytes are used for liver cell therapy, but the small number of engrafting cells limits the benefit of cell transplantation. We tested whether cotransplantation of hepatocytes with hepatic stellate cells (HSCs) could improve hepatocyte engraftment in vivo. Human primary hepatocytes were transplanted into SCID mice either alone or in a mixture with HSCs (quiescent or after culture activation) or LX-2 cells (ratio 20:1). Four weeks after transplantation into mouse livers, human albumin-positive (huAlb(+)) hepatocytes were found scattered. When cotransplanted in a mixture with HSCs or LX-2 cells, huAlb(+) hepatocytes formed clusters and were more numerous occupying 2- to 5.9-fold more surface on the tissue section than in livers transplanted with hepatocytes alone. Increased huAlb mRNA expression in livers transplanted with the cell mixtures confirmed those results. The presence of HSCs increased the number of hepatocytes entrapped in the host liver at an early time point posttransplantation but not their proliferation in situ as assessed by cumulative incorporation of BrdU. Importantly, 4 weeks posttransplantation, we found no accumulation of αSMA(+)-activated HSCs or collagen deposition. To follow the fate of transplanted HSCs, HSCs derived from GFP(+) mice were injected into GFP(-) littermates: 17 h posttransplant, GFP(+) HSCs were found in the sinusoids, without proliferating or actively producing ECM; they were undetectable at later time points. Coculture with HSCs improved the number of adherent hepatocytes, with best attachment obtained when hepatocytes were seeded in contact with activated HSCs. In vivo, cotransplantation of hepatocytes with HSCs into a healthy liver recipient does not generate fibrosis, but significantly improves the engraftment of hepatocytes, probably by ameliorating cell homing.

  13. Hepatocyte differentiation of human fibroblasts from cirrhotic liver in vitro and in vivo

    Institute of Scientific and Technical Information of China (English)

    Yu-Ling Sun; Sheng-Yong Yin; Lin Zhou; Hai-Yang Xie; Feng Zhang; Li-Ming Wu; Shu-Sen Zheng

    2011-01-01

    BACKGROUND: Mesenchymal stem cells (MSCs) and fibro-blasts have intimate relationships, and the phenotypic homology between fibroblasts and MSCs has been recently described. The aim of this study was to investigate the hepatic differentiating potentialofhumanfibroblastsincirrhoticliver. METHODS: The phenotypes of fibroblasts in cirrhotic liver were labeled by biological methods. After that, the differentiation potential of these fibroblasts in vitro was characterized in terms of liver-specific gene and protein expression. Finally, an animal model of hepatocyte regeneration in severe combined immunodeficient (SCID) mice was created by retrorsine injection and partial hepatectomy, and the expression of human hepatocyte proteins in SCID mouse livers was checked by immunohistochemicalanalysisafterfibroblastadministration. RESULTS: Surface immunophenotyping revealed that a minority of fibroblasts expressed markers of MSCs and hepatic epithelial cytokeratins as well as alpha-smooth muscle actin, but homogeneously expressed vimentin, desmin, prolyl 4-hydroxylase and fibronectin. These fibroblasts presented the characteristics of hepatocytes in vitro and differentiated directly into functional hepatocytes in the liver of hepatecto-mized SCID mice. CONCLUSIONS: This study demonstrated that fibroblasts in cirrhotic liver have the potential to differentiate into hepatocyte-like cells in vitro and in vivo. Our findings infer that hepatic differentiation of fibroblasts may serve as a new target for reversion of liver fibrosis and a cell source for tissue engineering.

  14. Caffeine induces CYP1A2 expression in rat hepatocytes but not in human hepatocytes

    OpenAIRE

    Vaynshteyn, David; Jeong, Hyunyoung

    2012-01-01

    Caffeine is the active constituent in coffee. Continual consumption of caffeine can lead to an attenuated response also known as tolerance. Results from rat studies have shown that caffeine is an inducer of CYP1A2, the enzyme responsible for caffeine’s metabolism. This suggests that CYP1A2 induction by caffeine may be in part responsible for caffeine tolerance. However, whether caffeine induces CYP1A2 expression in humans remains unknown. Our results from luciferase assays performed in HepG2 ...

  15. Effects of human pharmaceuticals on cytotoxicity, EROD activity and ROS production in fish hepatocytes.

    OpenAIRE

    2004-01-01

    Pharmaceuticals are found in the aquatic environment but their potential effects on non-target species like fish remain unknown. This in vitro study is a first approach in the toxicity assessment of human drugs on fish. Nine pharmaceuticals were tested on two fish hepatocyte models: primary cultures of rainbow trout hepatocytes (PRTH) and PLHC-1 fish cell line. Cell viability, interaction with cytochrome P450 1A (CYP1A) enzyme and oxidative stress were assessed by using 3-(4,5-dimethylthiazol...

  16. Organochlorine pesticides induce epithelial to mesenchymal transition of human primary cultured hepatocytes.

    Science.gov (United States)

    Zucchini-Pascal, Nathalie; Peyre, Ludovic; de Sousa, Georges; Rahmani, Roger

    2012-11-01

    Persistent organic pollutants (POPs) are a group of organic or chemicals that adversely affect human health and are persistent in the environment. These highly toxic compounds include industrial chemicals, pesticides such as organochlorines, and unwanted wastes such as dioxins. Although studies have described the general toxicity effects of organochlorine pesticides, the mechanisms underlying its potential carcinogenic effects in the liver are not well understood. In this study, we analyzed the effect of three organochlorine pesticides (dichlorodiphenyltrichloroethane, heptachlore and endosulfan) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the epithelial to mesenchymal transition (EMT) in primary cultured human hepatocytes. We found that these compounds modified the hepatocyte phenotype, inducing cell spread, formation of lamellipodia structures and reorganization of the actin cytoskeleton in stress fibers. These morphological alterations were accompanied by disruption of cell-cell junctions, E-cadherin repression and albumin down-regulation. Interestingly, these characteristic features of dedifferentiating hepatocytes were correlated with the gain of expression of various mesenchymal genes, including vimentin, fibronectin and its receptor ITGA5. These various results show that organochlorines and TCDD accelerate cultured human hepatocyte dedifferentiation and EMT processes. These events could account, at least in part, for the carcionogenic and/or fibrogenic activities of these POPs. Copyright © 2012 Elsevier Ltd. All rights reserved.

  17. Farnesoid X Receptor Inhibits the Transcriptional Activity of Carbohydrate Response Element Binding Protein in Human Hepatocytes

    Science.gov (United States)

    Caron, Sandrine; Huaman Samanez, Carolina; Dehondt, Hélène; Ploton, Maheul; Briand, Olivier; Lien, Fleur; Dorchies, Emilie; Dumont, Julie; Postic, Catherine; Cariou, Bertrand; Lefebvre, Philippe

    2013-01-01

    The glucose-activated transcription factor carbohydrate response element binding protein (ChREBP) induces the expression of hepatic glycolytic and lipogenic genes. The farnesoid X receptor (FXR) is a nuclear bile acid receptor controlling bile acid, lipid, and glucose homeostasis. FXR negatively regulates hepatic glycolysis and lipogenesis in mouse liver. The aim of this study was to determine whether FXR regulates the transcriptional activity of ChREBP in human hepatocytes and to unravel the underlying molecular mechanisms. Agonist-activated FXR inhibits glucose-induced transcription of several glycolytic genes, including the liver-type pyruvate kinase gene (L-PK), in the immortalized human hepatocyte (IHH) and HepaRG cell lines. This inhibition requires the L4L3 region of the L-PK promoter, known to bind the transcription factors ChREBP and hepatocyte nuclear factor 4α (HNF4α). FXR interacts directly with ChREBP and HNF4α proteins. Analysis of the protein complex bound to the L4L3 region reveals the presence of ChREBP, HNF4α, FXR, and the transcriptional coactivators p300 and CBP at high glucose concentrations. FXR activation does not affect either FXR or HNF4α binding to the L4L3 region but does result in the concomitant release of ChREBP, p300, and CBP and in the recruitment of the transcriptional corepressor SMRT. Thus, FXR transrepresses the expression of genes involved in glycolysis in human hepatocytes. PMID:23530060

  18. Generation of functional hepatocyte-like cells from human deciduous periodontal ligament stem cells

    Science.gov (United States)

    Vasanthan, Punitha; Jayaraman, Pukana; Kunasekaran, Wijenthiran; Lawrence, Anthony; Gnanasegaran, Nareshwaran; Govindasamy, Vijayendran; Musa, Sabri; Kasim, Noor Hayaty Abu

    2016-08-01

    Human deciduous periodontal ligament stem cells have been introduced for as an easily accessible source of stem cells from dental origin. Although recent studies have revealed the ability of these stem cells in multipotential attribute, their efficiency of hepatic lineage differentiation has not been addressed so far. The aim of this study is to investigate hepatic lineage fate competence of periodontal ligament stem cells through direct media induction. Differentiation of periodontal ligament stem cells into hepatocyte-like cells was conducted by the exposure of two phase media induction. First phase was performed in the presence of hepatocyte growth factors to induce a definitive endoderm formation. In the subsequent phase, the cells were treated with oncostatin M and dexamethosone followed by insulin and transferrin to generate hepatocyte-like cells. Hepatic-related characters of the generated hepatocyte-like cells were determined at both mRNA and protein level followed by functional assays. Foremost changes observed in the generation of hepatocyte-like cells were the morphological features in which these cells were transformed from fibroblastic shape to polygonal shape. Temporal expression of hepatic markers ranging from early endodermal up to late markers were detected in the hepatocyte-like cells. Crucial hepatic markers such as glycogen storage, albumin, and urea secretion were also shown. These findings exhibited the ability of periodontal ligament stem cells of dental origin to be directed into hepatic lineage fate. These cells can be regarded as an alternative autologous source in the usage of stem cell-based treatment for liver diseases.

  19. 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.

  20. 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

  1. Generation of functional hepatocyte-like cells from human deciduous periodontal ligament stem cells.

    Science.gov (United States)

    Vasanthan, Punitha; Jayaraman, Pukana; Kunasekaran, Wijenthiran; Lawrence, Anthony; Gnanasegaran, Nareshwaran; Govindasamy, Vijayendran; Musa, Sabri; Kasim, Noor Hayaty Abu

    2016-08-01

    Human deciduous periodontal ligament stem cells have been introduced for as an easily accessible source of stem cells from dental origin. Although recent studies have revealed the ability of these stem cells in multipotential attribute, their efficiency of hepatic lineage differentiation has not been addressed so far. The aim of this study is to investigate hepatic lineage fate competence of periodontal ligament stem cells through direct media induction. Differentiation of periodontal ligament stem cells into hepatocyte-like cells was conducted by the exposure of two phase media induction. First phase was performed in the presence of hepatocyte growth factors to induce a definitive endoderm formation. In the subsequent phase, the cells were treated with oncostatin M and dexamethosone followed by insulin and transferrin to generate hepatocyte-like cells. Hepatic-related characters of the generated hepatocyte-like cells were determined at both mRNA and protein level followed by functional assays. Foremost changes observed in the generation of hepatocyte-like cells were the morphological features in which these cells were transformed from fibroblastic shape to polygonal shape. Temporal expression of hepatic markers ranging from early endodermal up to late markers were detected in the hepatocyte-like cells. Crucial hepatic markers such as glycogen storage, albumin, and urea secretion were also shown. These findings exhibited the ability of periodontal ligament stem cells of dental origin to be directed into hepatic lineage fate. These cells can be regarded as an alternative autologous source in the usage of stem cell-based treatment for liver diseases.

  2. Rifampicin does not significantly affect the expression of Small heterodimer partner (SHP in primary human hepatocytes

    Directory of Open Access Journals (Sweden)

    Petr ePavek

    2012-01-01

    Full Text Available The small/short heterodimer partner (SHP, NR0B2 is a nuclear receptor corepressor lacking a DNA binding domain. SHP is induced by bile acid-activated farnesoid X receptor (FXR resulting in CYP7A1 gene suppression. In contrast, Pregnane X receptor (PXR activation by its ligands was recently suggested to inhibit SHP gene transactivation to maximize the induction of PXR target genes. However, there are also conflicting reports in literature whether PXR or rodent Pxr activation down-regulates SHP/Shp expression. Moreover, the PXR-mediated regulation of the SHP gene has been studied only at the SHP mRNA and transactivation (gene reporter assay levels.In this study, we studied the effect of rifampicin, a prototype PXR ligand, on SHP mRNA and protein expression in three primary human hepatocyte cultures.We found that SHP mRNA is not systematically down-regulated in hepatocyte in culture after 24 h treatment with rifampicin. Consistently, we did not observe down-regulation of SHP protein in primary human hepatocytes after 24 and 48 h of incubation with rifampicin.We can conclude that although we observed slight down-regulation of SHP mRNA and protein in several hepatocyte preparations, the phenomenon is unlikely critical for PXR-mediated induction of its target genes.

  3. Comparative Proteomic Analysis of Human Liver Tissue and Isolated Hepatocytes with a Focus on Proteins Determining Drug Exposure.

    Science.gov (United States)

    Vildhede, Anna; Wiśniewski, Jacek R; Norén, Agneta; Karlgren, Maria; Artursson, Per

    2015-08-07

    Freshly isolated human hepatocytes are considered the gold standard for in vitro studies of liver functions, including drug transport, metabolism, and toxicity. For accurate predictions of the in vivo outcome, the isolated hepatocytes should reflect the phenotype of their in vivo counterpart, i.e., hepatocytes in human liver tissue. Here, we quantified and compared the membrane proteomes of freshly isolated hepatocytes and human liver tissue using a label-free shotgun proteomics approach. A total of 5144 unique proteins were identified, spanning over 6 orders of magnitude in abundance. There was a good global correlation in protein abundance. However, the expression of many plasma membrane proteins was lower in the isolated hepatocytes than in the liver tissue. This included transport proteins that determine hepatocyte exposure to many drugs and endogenous compounds. Pathway analysis of the differentially expressed proteins confirmed that hepatocytes are exposed to oxidative stress during isolation and suggested that plasma membrane proteins were degraded via the protein ubiquitination pathway. Finally, using pitavastatin as an example, we show how protein quantifications can improve in vitro predictions of in vivo liver clearance. We tentatively conclude that our data set will be a useful resource for improved hepatocyte predictions of the in vivo outcome.

  4. The therapeutic effects of EGCG on vitiligo.

    Science.gov (United States)

    Zhu, Yiping; Wang, Suiquan; Lin, Fuquan; Li, Qing; Xu, Aie

    2014-12-01

    Epigallocatechin-3-gallate (EGCG) is one of the main chemical constituents of green tea, which has been used as an important traditional Chinese medicine. Green tea has anti-inflammatory, anti-oxidant, and immunomodulatory properties. However, the effects of EGCG on vitiligo are not known. We assessed the role of EGCG in vitiligo induced by monobenzone in mice. We demonstrated that EGCG: delayed the time of depigmentation; reduced the prevalence of depigmentation; and decreased the area of depigmentation. Examination of depigmented skin treated with EGCG by reflectance confocal microscopy suggested increased numbers of epidermal melanocytes and histologic examination showed decreased perilesional accumulation of CD8(+) T cells. To further investigate the mechanism of the anti-inflammatory effects of EGCG, levels of inflammatory mediator tumor necrosis factor (TNF)-α, interferon (IFN)-γ and interleukin (IL)-6 were tested by enzyme-linked immunosorbent assay. Serum cytokine levels were significantly decreased after administration of EGCG compared with the model group. These results suggested that EGCG may have protective effects against vitiligo, and that it could contribute to suppression of activation of CD8(+) T cells and inflammatory mediators. Based on these results, 5% EGCG was considered to be the most suitable concentration for treating vitiligo, and was used for further study. In addition, we investigated the gene-expression profile of this model in relation to EGCG. Using a 4×44K whole genome oligo microarray assay, 1264 down-regulated genes and 1332 up-regulated genes were recorded in the 5% EGCG group compared with the model group, and selected genes were validated by real-time polymerase chain reaction. Our study demonstrated that EGCG administration was significantly associated with a decreased risk of vitiligo. EGCG could be a new preventive agent against vitiligo in the clinical setting.

  5. Nuclear lactate dehydrogenase modulates histone modification in human hepatocytes

    Energy Technology Data Exchange (ETDEWEB)

    Castonguay, Zachary; Auger, Christopher; Thomas, Sean C.; Chahma, M’hamed; Appanna, Vasu D., E-mail: vappanna@laurentian.ca

    2014-11-07

    Highlights: • Nuclear LDH is up-regulated under oxidative stress. • SIRT1 is co-immunoprecipitated bound to nuclear LDH. • Nuclear LDH is involved in histone deacetylation and epigenetics. - Abstract: It is becoming increasingly apparent that the nucleus harbors metabolic enzymes that affect genetic transforming events. Here, we describe a nuclear isoform of lactate dehydrogenase (nLDH) and its ability to orchestrate histone deacetylation by controlling the availability of nicotinamide adenine dinucleotide (NAD{sup +}), a key ingredient of the sirtuin-1 (SIRT1) deacetylase system. There was an increase in the expression of nLDH concomitant with the presence of hydrogen peroxide (H{sub 2}O{sub 2}) in the culture medium. Under oxidative stress, the NAD{sup +} generated by nLDH resulted in the enhanced deacetylation of histones compared to the control hepatocytes despite no discernable change in the levels of SIRT1. There appeared to be an intimate association between nLDH and SIRT1 as these two enzymes co-immunoprecipitated. The ability of nLDH to regulate epigenetic modifications by manipulating NAD{sup +} reveals an intricate link between metabolism and the processing of genetic information.

  6. Optimizing human hepatocyte models for metabolic phenotype and function: effects of treatment with dimethyl sulfoxide (DMSO).

    Science.gov (United States)

    Nikolaou, Nikolaos; Green, Charlotte J; Gunn, Pippa J; Hodson, Leanne; Tomlinson, Jeremy W

    2016-11-01

    Primary human hepatocytes are considered to be the "gold standard" cellular model for studying hepatic fatty acid and glucose metabolism; however, they come with limitations. Although the HepG2 cell line retains many of the primary hepatocyte metabolic functions they have a malignant origin and low rates of triglyceride secretion. The aim of this study was to investigate whether dimethyl sulfoxide supplementation in the media of HepG2 cells would enhance metabolic functionality leading to the development of an improved in vitro cell model that closely recapitulates primary human hepatocyte metabolism. HepG2 cells were cultured in media containing 1% dimethyl sulfoxide for 2, 4, 7, 14, and 21 days. Gene expression, protein levels, intracellular triglyceride, and media concentrations of triglyceride, urea, and 3-hydroxybutyrate concentrations were measured. Dimethyl sulfoxide treatment altered the expression of genes involved in lipid (FAS, ACC1, ACC2, DGAT1, DGAT2, SCD) and glucose (PEPCK, G6Pase) metabolism as well as liver functionality (albumin, alpha-1-antitrypsin, AFP). mRNA changes were paralleled by alterations at the protein level. DMSO treatment decreased intracellular triglyceride content and lactate production and increased triglyceride and 3-hydroxybutyrate concentrations in the media in a time-dependent manner. We have demonstrated that the addition of 1% dimethyl sulfoxide to culture media changes the metabolic phenotype of HepG2 cells toward a more primary human hepatocyte phenotype. This will enhance the currently available in vitro model systems for the study of hepatocyte biology related to pathological processes that contribute to disease and their response to specific therapeutic interventions. © 2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.

  7. Benzylpenicillin, acetylcysteine and silibinin as antidotes in human hepatocytes intoxicated with alpha-amanitin.

    Science.gov (United States)

    Magdalan, Jan; Ostrowska, Alina; Piotrowska, Aleksandra; Gomułkiewicz, Agnieszka; Podhorska-Okołów, Marzena; Patrzałek, Dariusz; Szelag, Adam; Dziegiel, Piotr

    2010-07-01

    Fatalities due to mushroom poisonings are increasing worldwide, with high mortality rate resulting from ingestion of amanitin-producing species. Intoxications caused by amanitin-containing mushrooms represent an unresolved problem in clinical toxicology since no specific and fully efficient antidote is available. The objective of this study was a comparative evaluation of benzylpenicillin (BPCN), acetylcysteine (ACC) and silibinin (SIL) as an antidotes in human hepatocytes intoxicated with alpha-amanitin (alpha-AMA). All experiments were performed on cultured human hepatocytes. Cytotoxicity evaluation of cultured cells using MTT assay and measurement of lactate dehydrogenase (LDH) activity was performed at 12, 24 and 48h of exposure to alpha-AMA and/or antidotes. The significant decline of cell viability and significant increase of LDH activity were observed in all experimental hepatocyte cultures after 12, 24 and 36h exposure to alpha-AMA at concentration 2microM. Exposure of the cells to alpha-AMA resulted also in significant reduction of cell spreading and attachment. However, addition of tested antidotes to experimental cultures significantly stimulated cell proliferation and attachment. In cell cultures exposed simultaneously to alpha-AMA and tested antidotes cytotoxic parameters (MTT and LDH) were not significantly different from control incidences. The cytoprotective effect of all antidotes was not dose-related, which reflects a high efficacy of all these substances. Administration of studied antidotes was not associated with any adverse effects in hepatocytes. The administration of ACC, BPCN or SIL to human hepatocyte cultures showed a similar strong protective effect against cell damage in alpha-AMA toxicity.

  8. Evaluation of the metabolism of propranolol by linear ion trap technology in mouse, rat, dog, monkey, and human cryopreserved hepatocytes.

    Science.gov (United States)

    Baughman, Todd M; Talarico, Christine L; Soglia, John R

    2009-07-01

    Propranolol is a widely used quality control and validation compound for liver microsome and hepatocyte metabolism studies. A multitude of literature reports describing the identification of propranolol metabolites exists today. However, no literature reports currently exist showing hepatocyte metabolism across the five species commonly used during pre-clinical drug discovery, namely mouse, rat, dog, monkey, and human. Herein, we present full metabolic profiles of propranolol in mouse, rat, dog, monkey and human hepatocytes. As expected, extensive phase I and phase II metabolism was observed across all five species and species-specific metabolites were detected in monkey and dog hepatocytes. Of particular interest was the detection of an N-hydroxylamine glucuronide metabolite in monkey and dog hepatocytes.

  9. Conversion of mononuclear cells from human umbilical cord blood into hepatocyte-like cells

    Institute of Scientific and Technical Information of China (English)

    ZHANG Fang-ting; FANG Jia-zhi; YU Jie; WAN Hui-juan; YE Jing; LONG Xia; YIN Mei-jun; HUANG Chun-qiao

    2006-01-01

    Objective:To evaluate the differentiation of human umbilical cord blood cells into hepatocyte-like cells. Methods: Mononuclear cells (MNCs) derived from human umbilical cord blood were isolated using Ficoll. The experiment was derived into 3 categories: (1) MNCs co-cultured with 50 mg minced liver tissue separated by a trans-well membrane and then collected at 0 h,24 h,48 h and 72 h; (2) MNCs cultured along supplemented with 100 ml/L FBS, 100 μ/ml penicillin, 100 μg/ml streptomycin, 4. 7 μg/ml linoleic acid, 1×ITS, 10-4 mol/L L-Ascorbic acid 2-P and a combination of FGF4 (100 ng/ml) and HGF (20 ng/Ml). Cells were then collected at 0 d and 16 d to examine the expression profile of hepatocyte correlating markers; (3) 0.2-0.3 ml of MNCs with a cell density of 2×107/ml were transplanted into prepared recipient mice [n= 12, injected with 0.4 ml/kg (20%) CCl4 and 150 ng/kg 5-fluorouracil (5-Fu) prior the transplant 24 h and 48 h, respectively] via injection through tail vein. Mice were sacrificed 4 weeks after transplantation. The hepatocyte correlating mRNAs and proteins were determined by RTPCR, immunohistochemical analysis and immunoflurence technique. Results: (1) After 72 h, a number of glycogen positive stained cells were observed with MNCs co-cultured with damaged mouse liver tissues.The expression of hepatocyte markers, human albumin (ALB), α-fetal protein (AFP) and human GATA4 Mrna and proteins were detected by RT-PCR and immunohistochemistry as well. For the confirmation,the DNA sequencing of PCR products was performed. In control groups, MNCs co-cultured with normal mouse hepatocytes or MNCs cultured alone, all markers remained negative. (2) In growth factor supplemented culture system, MNCs developed into larger volume with richer cytoplasm and binucleation after 16 d. Positive expression of ALB, AFP, CK18 and CK19 Mrna were detected with RT-PCR, and ALB positive staining was observed by immunocytochemistry as well. In contrast, MNCs cultured without

  10. Spheroid culture for enhanced differentiation of human embryonic stem cells to hepatocyte-like cells.

    Science.gov (United States)

    Subramanian, Kartik; Owens, Derek Jason; Raju, Ravali; Firpo, Meri; O'Brien, Timothy D; Verfaillie, Catherine M; Hu, Wei-Shou

    2014-01-15

    Stem cell-derived hepatocyte-like cells hold great potential for the treatment of liver disease and for drug toxicity screening. The success of these applications hinges on the generation of differentiated cells with high liver specific activities. Many protocols have been developed to guide human embryonic stem cells (hESCs) to differentiate to the hepatic lineage. Here we report cultivation of hESCs as three-dimensional aggregates that enhances their differentiation to hepatocyte-like cells. Differentiation was first carried out in monolayer culture for 20 days. Subsequently cells were allowed to self-aggregate into spheroids. Significantly higher expression of liver-specific transcripts and proteins, including Albumin, phosphoenolpyruvate carboxykinase, and asialoglycoprotein receptor 1 was observed. The differentiated phenotype was sustained for more than 2 weeks in the three-dimensional spheroid culture system, significantly longer than in monolayer culture. Cells in spheroids exhibit morphological and ultrastructural characteristics of primary hepatocytes by scanning and transmission electron microscopy in addition to mature functions, such as biliary excretion of metabolic products and cytochrome P450 activities. This three-dimensional spheroid culture system may be appropriate for generating high quality, functional hepatocyte-like cells from ESCs.

  11. Assessment of a micropatterned hepatocyte coculture system to generate major human excretory and circulating drug metabolites.

    Science.gov (United States)

    Wang, Wendy WeiWei; Khetani, Salman R; Krzyzewski, Stacy; Duignan, David B; Obach, R Scott

    2010-10-01

    Metabolism is one of the important determinants of the overall disposition of drugs, and the profile of metabolites can have an impact on efficacy and safety. Predicting which drug metabolites will be quantitatively predominant in humans has become increasingly important in the research and development of new drugs. In this study, a novel micropatterned hepatocyte coculture system was evaluated for its ability to generate human in vivo metabolites. Twenty-seven compounds of diverse chemical structure and subject to a range of drug biotransformation reactions were assessed for metabolite profiles in the micropatterned coculture system using pooled cryopreserved human hepatocytes. The ability of this system to generate metabolites that are >10% of dose in excreta or >10% of total drug-related material in circulation was assessed and compared to previously reported data obtained in human hepatocyte suspensions, liver S-9 fraction, and liver microsomes. The micropatterned coculture system was incubated for up to 7 days without a change in medium, which offered an ability to generate metabolites for slowly metabolized compounds. The micropatterned coculture system generated 82% of the excretory metabolites that exceed 10% of dose and 75% of the circulating metabolites that exceed 10% of total circulating drug-related material, exceeds the performance of hepatocyte suspension incubations and other in vitro systems. Phase 1 and phase 2 metabolites were generated, as well as metabolites that arise via two or more sequential reactions. These results suggest that this in vitro system offers the highest performance among in vitro metabolism systems to predict major human in vivo metabolites.

  12. Relationship between the biological activities of methylated derivatives of (-)-epigallocatechin-3-O-gallate (EGCG) and their cell surface binding activities.

    Science.gov (United States)

    Yano, Satomi; Fujimura, Yoshinori; Umeda, Daisuke; Miyase, Toshio; Yamada, Koji; Tachibana, Hirofumi

    2007-08-22

    It was previously reported that (-)-epigallocatechin-3-O-gallate (EGCG) suppresses the expression of the high-affinity IgE receptor FcepsilonRI in human basophilic cells and that this suppressive effect is associated with EGCG binding to the cell surface. This study examined the effects of five methylated derivatives of EGCG, (-)-epigallocatechin-3-O-(3-O-methyl)gallate (EGCG 3' 'Me), (-)-epigallocatechin-3-O-(4-O-methyl)gallate (EGCG 4' 'Me), (-)-4'-O-methyl-epigallocatechin-3-O-gallate (EGCG 4'Me), (-)-epigallocatechin-3-O-(3,4-O-methyl)gallate (EGCG 3' '4' 'diMe), and (-)-4'-O-methyl-epigallocatechin-3-O-(4-O-methyl)gallate (EGCG 4'4' 'diMe) on FcepsilonRI expression and ERK1/2 phosphorylation, and each of their cell surface binding activities was measured. Of these five methylated derivatives, three that are methylated at the 3' '- and/or 4' '-position, EGCG 3' 'Me, EGCG 4' 'Me, and EGCG 3' '4' 'diMe, suppressed FcepsilonRI expression and ERK1/2 phosphorylation, although the suppressive effects were lower than that of EGCG. EGCG 4'Me and EGCG 4'4' 'diMe, both of which are methylated at the 4'-position, did not demonstrate a suppressive effect. Furthermore, it was found that EGCG 3' 'Me, EGCG 4' 'Me, EGCG 3' '4' 'diMe, and EGCG 4'Me, which are methylated at the 3' '- and/or 4' '-positions or the 4'-position, could bind to the cell surface even though their binding activities were lower than that of EGCG. Only EGCG 4'4' 'diMe, which is methylated at both the 4'- and 4' '-positions, could not bind. These results suggest that the trihydroxyl structure of the B ring is essential for EGCG to exert the suppressive effects and that the hydroxyl groups on both the 4'-position in the B ring and the 4' '-position in the gallate are crucial for the cell surface binding activity of EGCG.

  13. 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

    2017-09-05

    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.

  14. FcRn expression, ligands binding properties and its regulation in human immune cells and hepatocytes

    OpenAIRE

    2007-01-01

    ABSTRACT Expression and diverse functions of MHC class I related neonatal Fc receptor in different tissues is continually reported. To contribute to the understanding of how the receptor functions according to cell type, we investigated the expression and ligands binding properties of FcRn in human immune cells and hepatocytes. Here, we report that heterodimeric FcRn is expressed in these cells as evidenced by RT-PCR, Western immunoblottting and flow cytometry. The receptor expression i...

  15. Effect of honokiol on the induction of drug-metabolizing enzymes in human hepatocytes.

    Science.gov (United States)

    Cho, Yong-Yeon; Jeong, Hyeon-Uk; Kim, Jeong-Han; Lee, Hye Suk

    2014-01-01

    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.

  16. Genome-wide binding and transcriptome analysis of human farnesoid X receptor in primary human hepatocytes.

    Directory of Open Access Journals (Sweden)

    Le Zhan

    Full Text Available Farnesoid X receptor (FXR, NR1H4 is a ligand-activated transcription factor, belonging to the nuclear receptor superfamily. FXR is highly expressed in the liver and is essential in regulating bile acid homeostasis. FXR deficiency is implicated in numerous liver diseases and mice with modulation of FXR have been used as animal models to study liver physiology and pathology. We have reported genome-wide binding of FXR in mice by chromatin immunoprecipitation - deep sequencing (ChIP-seq, with results indicating that FXR may be involved in regulating diverse pathways in liver. However, limited information exists for the functions of human FXR and the suitability of using murine models to study human FXR functions.In the current study, we performed ChIP-seq in primary human hepatocytes (PHHs treated with a synthetic FXR agonist, GW4064 or DMSO control. In parallel, RNA deep sequencing (RNA-seq and RNA microarray were performed for GW4064 or control treated PHHs and wild type mouse livers, respectively.ChIP-seq showed similar profiles of genome-wide FXR binding in humans and mice in terms of motif analysis and pathway prediction. However, RNA-seq and microarray showed more different transcriptome profiles between PHHs and mouse livers upon GW4064 treatment.In summary, we have established genome-wide human FXR binding and transcriptome profiles. These results will aid in determining the human FXR functions, as well as judging to what level the mouse models could be used to study human FXR functions.

  17. Effect of honokiol on the induction of drug-metabolizing enzymes in human hepatocytes

    Directory of Open Access Journals (Sweden)

    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

  18. Induction of Heme Oxygenase-1 in Human Hepatocytes to Protect Them From Ethanol-induced Cytotoxicity

    Institute of Scientific and Technical Information of China (English)

    LIE-GANG LIU; HONG YAN; WEN ZHANG; PING YAO; XI-PING ZHANG; XIU-FA SUN; ANDREAS K. NUSSLER

    2004-01-01

    We investigated the relationship between ethanol exposure and heme oxygenase (HO-1) in human hepatocytes in order to ascertain if induction of HO-1 can prevent ethanol induced cellular damage. Methods Dose-dependent (25-100 mmol/L) and time-dependent (0-24 h) ethanol exposure were used in the present study. HO-1 mRNA and protein expression were detected by PT-PCR and Western blot respectively. HO-1 activity was indicated by bilirubin and Fe2+ formation. Cytotoxicity was investigated by means of lactate dehydrogenate (LDH) and aspartate transaminase (AST) level in culture supernatants, as well as the intracellular formation of malondialdehyde (MDA), cellular glutathione (GSH) status and CYP 2E1 activity. Results We first demonstrated a dose-dependent response between ethanol exposure and HO-1 mRNA and protein expression in human hepatocytes. We further observed a time-dependent increase of HO-1 mRNA expression using 100 mmol/L ethanol starting 30 minutes after ethanol exposure, reaching its maximum between 3 h and 9 h. Being similar to what had been demonstrated with the mRNA level, increased protein expression started at 6 h after ethanol exposure, and kept continuous elevated over 18 h. In addition, we found that ethanol exposure to hepatocytes markedly increased HO-1 enzyme activity in a time-dependent manner measured as bilirubin and Fe2+ formation in human hepatocytes. Our results clearly showed that ethanol exposure caused a significant increase of LDH, AST, and MDA levels, while the antioxidant GSH was time-dependently reduced. Furthermore, we demonstrated that pre-administration of cobalt protoporphyrin (CoPP) induced HO-1 in human hepatocytes, and prevented an increase of MDA and a decrease of GSH. These effects could be partially reversed by zinc protoporphyrin (ZnPP), an antagonist of HO-1 induction. Conclusion HO-1 expression in cells or organs could lead to new strategies for better prevention and treatment of ethanol-induced oxidative damage in human

  19. Serotonin receptor agonist quipazine promotes proliferation and apoptosis of human hepatocyte strain of L-02 strain

    Institute of Scientific and Technical Information of China (English)

    Yang Liu; Zhi-Yong Zhang

    2009-01-01

    BACKGROUND:Liver disease is commonly seen in the clinic and its pathological characteristic is combined hepatocellular death and apoptosis. Promoting hepatocyte regeneration is one of the main methods of treating liver disease. Serotonin (5-HT) is an important compound which participates in various life process, and 95% of it is carried by platelets in the blood. A recent ifnding showed that platelet-derived serotonin is the key factor in liver regeneration, which fails without serotonin. This study aimed to investigate the effects of quipazine, a selective 5-HT receptor agonist, on proliferation and apoptosis in the human hepatocyte strain L-02. METHODS:L-02 cells were cultured in medium with 5-HT and quipazine, and samples were collected at 24, 48, and 72 hours. The methyl thiazolyl tetrazolium (MTT) method was used to test viability, lfow cytometry to assess the cell cycle, the Annexin-V/PI method to evaluate apoptosis, and immunohistochemistry to detect proliferating cell nuclear antigen (PCNA). RESULTS:Compared with the control group, the viability of L-02 cells was improved in the 10, 50, and 250 μg/ml quipazine groups (P0.05); and no difference in the percentage of apoptotic cells was found between the 50μg/ml quipazine and control groups (P>0.05).CONCLUSION:Quipazine improves proliferation of a human hepatocyte strainin vitro, and this is not based on the inhibition of apoptosis.

  20. The effect of human milk on DNA synthesis of neonatal rat hepatocytes in primary culture.

    Science.gov (United States)

    Kohno, Y; Shiraki, K; Mura, T

    1991-03-01

    We studied the effect of human milk on DNA synthesis of neonatal hepatocytes to elucidate the physiologic role of human milk in growth of the liver. Neonatal hepatocytes were isolated from 5-d-old rats and cultured in serum-free medium. Human milk stimulated DNA synthesis of these hepatocytes in a concentration-dependent manner. The stimulatory activity of 7.5% (vol/vol) human milk plus 0.1 mumol/L insulin was five times that of control and was almost the same as that of 20 micrograms/L human epidermal growth factor (hEGF) plus insulin. The effect of human milk was additive with treatment with hEGF and insulin. The milk associated with prolonged jaundice of infants was significantly more active than the milk that was not associated with jaundice, although the concentration of hEGF was not different between the two types of milk. The mitogenic activity of milk was heat-labile, inactivated by DTT and stable after treatment with trypsin. Three peaks of the activity were detected in milk by gel filtration and the fraction containing proteins of molecular weight between 36,000 and 76,000 showed the highest activity. Anti-hEGF antibody did not inhibit this activity completely. These results suggested the presence of mitogens other than hEGF or a more active form of hEGF in human milk. The milk associated with breast-milk jaundice exerts a different influence on cell growth and may affect maturation of the liver function related to bilirubin metabolism. The mitogenic activity of milk might be important for growth and development of the liver in infants.

  1. Learning and memory changes in rats following exogenous human hepatocyte growth factor gene injection into cerebral ischemic penumbra

    Institute of Scientific and Technical Information of China (English)

    Zhijun You; Yong Liu; Jianye Yang; Qingping Jiang

    2011-01-01

    Human hepatocyte growth factor can be used to treat cerebral infarction, administered by lateral ventricular, cerebellomedullary cistern or subarachnoid injections. However, the target gene expression product is scarcely found in the ischemic penumbra, but extensively distributes in other regions, increasing the risks of gene therapy. The present study directly transfected hepatocyte growth factor gene into the ischemic penumbra of rats with transient middle cerebral artery occlusion. Immunohistochemical analysis revealed that infarct volume was significantly decreased, hepatocyte growth factor protein expression level and vessel quantity in the ischemic penumbra were significantly increased, and learning and memory were significantly improved.

  2. Pathway Analysis and Modeling of the Differentiation of Human Embryonic Stem Cells into Hepatocyte-like Cells

    Science.gov (United States)

    Daskalaki, Andriani; Jozefczuk, Justyna; Lehrach, Hans; Adjaye, James; Wierling, Christoph

    2011-06-01

    A more detailed understanding of the differentiation of human embryonic and induced pluripotent stem cells into hepatocyte-like cells can help to improve therapies for liver diseases, like steatohepatitis. In this work we used microarray-based expression data to analyze the in vitro differentiation of human embryonic stem cells into hepatocytes. Pathway analysis has been carried out on gene expression data of different stages of the differentiation process from embryonic stem cells into hepatocyte-like cells via definitive endoderm and hepatic endoderm. Based on pathway analysis we identified signaling pathways, like the GPCR signaling pathway as well as FOXA2 regulatory networks. Based on these highly enriched pathways we constructed a model prototype to better understand and study the differentiation of stem cells into hepatocytes.

  3. Mechanistic insights from comparing intrinsic clearance values between human liver microsomes and hepatocytes to guide drug design.

    Science.gov (United States)

    Di, Li; Keefer, Christopher; Scott, Dennis O; Strelevitz, Timothy J; Chang, George; Bi, Yi-An; Lai, Yurong; Duckworth, Jonathon; Fenner, Katherine; Troutman, Matthew D; Obach, R Scott

    2012-11-01

    Metabolic stability of drug candidates are often determined in both liver microsome and hepatocyte assays. Comparison of intrinsic clearance values between the two assays provides additional information to guide drug design. Intrinsic clearance values from human liver microsomes and hepatocytes were compared for a set of commercial drugs with known metabolic pathways and transporter characteristics. The results showed that for compounds that were predominately metabolized by CYP mediated mechanisms, the intrinsic clearance values from the two assays were comparable. For compounds with non-CYP pathways, such as UGT and AO, intrinsic clearance was faster in hepatocytes than in microsomes. Substrates of uptake or efflux transporters in this study did not have significant differences of intrinsic clearance between microsomes and hepatocytes, when uptake into the hepatocytes was not the rate-limiting step. When hepatic uptake was rate limiting, intrinsic clearance in microsomes was faster than that in hepatocytes, which was more prevalent for compounds with rapid metabolism. Low passive permeability can limit the exposure to drug molecules to the metabolizing enzymes in the hepatocytes in relationship to the rate of metabolism. The faster the rate of metabolism, the higher permeability is needed for molecule to enter the cells and not becoming rate-limiting. The findings are very useful for drug discovery programs to gain additional insights on mechanistic information to help drug design without added experiments. Follow-up studies can then be designed to address specific questions.

  4. Metabolism of Oxycodone in Human Hepatocytes from Different Age Groups and Prediction of Hepatic Plasma Clearance

    Science.gov (United States)

    Korjamo, Timo; Tolonen, Ari; Ranta, Veli-Pekka; Turpeinen, Miia; Kokki, Hannu

    2012-01-01

    Oxycodone is commonly used to treat severe pain in adults and children. It is extensively metabolized in the liver in adults, but the maturation of metabolism is not well understood. Our aim was to study the metabolism of oxycodone in cryopreserved human hepatocytes from different age groups (3 days, 2 and 5 months, 4 years, adult pool) and predict hepatic plasma clearance of oxycodone using these data. Oxycodone (0.1, 1, and 10 μM) was incubated with hepatocytes for 4 h, and 1 μM oxycodone also with CYP3A inhibitor ketoconazole (1 μM). Oxycodone and noroxycodone concentrations were determined at several time points with liquid chromatography–mass spectrometry. In vitro clearance of oxycodone was used to predict hepatic plasma clearance, using the well-stirred model and published physiological parameters. Noroxycodone was the major metabolite in all batches and ketoconazole inhibited the metabolism markedly in most cases. A clear correlation between in vitro oxycodone clearance and CYP3A4 activity was observed. The predicted hepatic plasma clearances were typically much lower than the published median total plasma clearance from pharmacokinetic studies. The data suggests that there are no children-specific metabolites of oxycodone. Moreover, CYP3A activity seems to be the major determinant in metabolic clearance of oxycodone regardless of age group or individual variability in hepatocyte batches. PMID:22291644

  5. Sulindac and its metabolites inhibit multiple transport proteins in rat and human hepatocytes.

    Science.gov (United States)

    Lee, Jin Kyung; Paine, Mary F; Brouwer, Kim L R

    2010-08-01

    Sulindac is a commonly used nonsteroidal anti-inflammatory drug. This study tested the hypothesis that sulindac-mediated drug-drug interactions and/or hepatotoxicity may be caused, in part, by inhibition of proteins responsible for the hepatic transport of drugs and/or bile acids by sulindac and/or sulindac metabolites [sulindac sulfone (S-sulfone) and sulindac sulfide (S-sulfide)]. The uptake and excretion of model substrates, [(3)H]taurocholate (TC), [(3)H]estradiol 17-beta-glucuronide (E217G), and nitrofurantoin (NF), were investigated in rat and human suspended and sandwich-cultured hepatocytes (SCH). In suspended rat hepatocytes, S-sulfone and S-sulfide inhibited Na(+)-dependent TC initial uptake (IC(50) of 24.9 +/- 6.4 and 12.5 +/- 1.8 microM, respectively) and Na(+)-independent E217G initial uptake (IC(50) of 12.1 +/- 1.6 and 6.3 +/- 0.3 microM, respectively). In rat SCH, sulindac metabolites (100 microM) decreased the in vitro biliary clearance (Cl(biliary)) of TC, E217G, and NF by 38 to 83%, 81 to 97%, and 33 to 57%, respectively; S-sulfone and S-sulfide also decreased the TC and NF biliary excretion index by 39 to 55%. In suspended human hepatocytes, S-sulfone and S-sulfide inhibited Na(+)-dependent TC initial uptake (IC(50) of 42.2 and 3.1 microM, respectively); S-sulfide also inhibited the TC Cl(biliary) in human SCH. Sulindac/metabolites markedly inhibited hepatic uptake and biliary excretion of E217G by 51 to 100% in human SCH. In conclusion, sulindac and metabolites are potent inhibitors of the uptake and biliary clearance of bile acids in rat and human hepatocytes and also inhibit substrates of rat breast cancer resistance protein, rat and human organic anion-transporting polypeptides, and human multidrug resistance-associated protein 2. Inhibition of multiple hepatic transport proteins by sulindac/metabolites may play an important role in clinically significant sulindac-mediated drug-drug interactions and/or liver injury.

  6. Certain (-)-epigallocatechin-3-gallate (EGCG) auto-oxidation products (EAOPs) retain the cytotoxic activities of EGCG.

    Science.gov (United States)

    Wei, Yaqing; Chen, Pingping; Ling, Tiejun; Wang, Yijun; Dong, Ruixia; Zhang, Chen; Zhang, Longjie; Han, Manman; Wang, Dongxu; Wan, Xiaochun; Zhang, Jinsong

    2016-08-01

    (-)-Epigallocatechin-3-gallate (EGCG) from green tea has anti-cancer effect. The cytotoxic actions of EGCG are associated with its auto-oxidation, leading to the production of hydrogen peroxide and formation of numerous EGCG auto-oxidation products (EAOPs), the structures and bioactivities of them remain largely unclear. In the present study, we compared several fundamental properties of EGCG and EAOPs, which were prepared using 5mg/mL EGCG dissolved in 200mM phosphate buffered saline (pH 8.0 at 37°C) and normal oxygen partial pressure for different periods of time. Despite the complete disappearance of EGCG after the 4-h auto-oxidation, 4-h EAOPs gained an enhanced capacity to deplete cysteine thiol groups, and retained the cytotoxic effects of EGCG as well as the capacity to produce hydrogen peroxide and inhibit thioredoxin reductase, a putative target for cancer prevention and treatment. The results indicate that certain EAOPs possess equivalent cytotoxic activities to EGCG, while exhibiting simultaneously enhanced capacity for cysteine depletion. These results imply that EGCG and EAOPs formed extracellularly function in concert to exhibit cytotoxic effects, which previously have been ascribed to EGCG alone.

  7. Metabolism of bromopride in mouse, rat, rabbit, dog, monkey, and human hepatocytes.

    Science.gov (United States)

    Dunne, Christine E; Bushee, Jennifer L; Argikar, Upendra A

    2013-01-01

    Bromopride (BRP) has been utilized clinically for treatment of nausea, vomiting and gastro-intestinal motility disorders. The pharmacokinetics of BRP have been characterized in dogs and humans; however, the metabolic profile of BRP has not been well studied. The present study was aimed at better understanding BRP metabolism across species. We investigated biotransformation of BRP in mouse, rat, rabbit, dog, monkey, and human hepatocytes with the help of LC-MS(n) and accurate mass measurement. Mice, rats, dogs, and monkeys are relevant in drug discovery and development as pre-clinical species to be compared with humans, whereas rabbits were efficacy models for BRP. Overall, twenty metabolites of BRP were identified across hepatocytes from the six species. Monkeys offered the most coverage for humans, in terms of number of metabolites identified. Interestingly, M14, an N-sulfate metabolite of BRP, was identified as a human-specific metabolite. BRP metabolism had only been reported in dog plasma and urine, historically. Our investigation is the first documentation of in vitro metabolism of BRP in the six species reported here. Metabolites M1, M2, M4-M10, M12, M13, and M15-M20 have not been previously reported. In summary, this report documents seventeen metabolites of BRP for the first time, thus providing a deeper insight into the biotransformation of BRP.

  8. Perfused human hepatocyte microtissues identify reactive metabolite-forming and mitochondria-perturbing hepatotoxins.

    Science.gov (United States)

    Rowe, Cliff; Shaeri, Mohsen; Large, Emma; Cornforth, Terri; Robinson, Angela; Kostrzewski, Tomasz; Sison-Young, Rowena; Goldring, Christopher; Park, Kevin; Hughes, David

    2017-09-15

    Hepatotoxins cause liver damage via many mechanisms but the formation of reactive metabolites and/or damage to liver mitochondria are commonly implicated. We assess 3D human primary hepatocyte microtissues as a platform for hepatotoxicity studies with reactive metabolite-forming and mitochondria-perturbing compounds. We show that microtissues formed from cryopreserved human hepatocytes had bile canaliculi, transcribed mRNA from genes associated with xenobiotic metabolism and expressed functional cytochrome P450 enzymes. Hierarchical clustering was used to distinguish dose-dependent hepatotoxicity elicited by clozapine, fialuridine and acetaminophen (APAP) from control cultures and less liver-damaging compounds, olanzapine and entecavir. The regio-isomer of acetaminophen, N-acetyl-meta-aminophenol (AMAP) clustered with the hepatotoxic compounds. The principal metabolites of APAP were formed and dose-dependent changes in metabolite profile similar to those seen in patient overdose was observed. The toxicological profile of APAP was indistinguishable from that of AMAP, confirming AMAP as a human hepatotoxin. Tissue oxygen consumption rate was significantly decreased within 2h of exposure to APAP or AMAP, concomitant with glutathione depletion. These data highlight the potential utility of perfused metabolically functional human liver microtissues in drug development and mechanistic toxicology. Copyright © 2017. Published by Elsevier Ltd.

  9. Stiffness of hyaluronic acid gels containing liver extracellular matrix supports human hepatocyte function and alters cell morphology.

    Science.gov (United States)

    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.

  10. Genetic heterogeneity among slow acetylator N-acetyltransferase 2 phenotypes in cryopreserved human hepatocytes.

    Science.gov (United States)

    Doll, Mark A; Hein, David W

    2017-07-01

    Genetic polymorphisms in human N-acetyltransferase 2 (NAT2) modify the metabolism of numerous drugs and carcinogens. These genetic polymorphisms modify both drug efficacy and toxicity and cancer risk associated with carcinogen exposure. Previous studies have suggested phenotypic heterogeneity among different NAT2 slow acetylator genotypes. NAT2 phenotype was investigated in vitro and in situ in samples of human hepatocytes obtained from various NAT2 slow and intermediate NAT2 acetylator genotypes. NAT2 gene dose response (NAT2*5B/*5B > NAT2*5B/*6A > NAT2*6A/*6A) was observed towards the N-acetylation of the NAT2-specific drug sulfamethazine by human hepatocytes both in vitro and in situ. N-acetylation of 4-aminobiphenyl, an arylamine carcinogen substrate for both N-acetyltransferase 1 and NAT2, showed the same trend both in vitro and in situ although the differences were not significant (p > 0.05). The N-acetylation of the N-acetyltransferase 1-specific substrate p-aminobenzoic acid did not follow this trend. In comparisons of NAT2 intermediate acetylator genotypes, differences in N-acetylation between NAT2*4/*5B and NAT2*4/*6B hepatocytes were not observed in vitro or in situ towards any of these substrates. These results further support phenotypic heterogeneity among NAT2 slow acetylator genotypes, consistent with differential risks of drug failure or toxicity and cancer associated with carcinogen exposure.

  11. Suppression of bile acid synthesis by thyroid hormone in primary human hepatocytes

    Institute of Scientific and Technical Information of China (English)

    Ewa Cristine Siljevik Ellis

    2006-01-01

    AIM: It is known that thyroid hormones alter the bile acid metabolism in humans, however the effect on individual enzymes has been difficult to elucidate.This is mainly due to the lack of human liver cell lines producing bile acids. We used cultures of primary human hepatocytes to study the effects of triiodothyronine (T3) on bile acid synthesis.METHODS: Primary hepatocytes were isolated from liver tissue obtained from three different patients undergoing liver resection due to underlying malignancy. The hepatocytes were cultured under serum-free conditions and treated from d 1 to d 5 with culture containing 0.1-1000 nmol/L of T3. Bile acid formation and mRNA levels of key enzymes were analysed.RESULTS: The lowest concentration of T3 decreased cholic acid (CA) formation to 43%-53% of controls and chenodeoxycholic acid (CDCA) to 52%-75% of controls on d 5. The highest dose further decreased CA formation to 16%-48% of controls while CDCA formation remained at 50%-117% of controls. Expression of mRNA levels of cholesterol 7α-hydroxylase (CYP7A1) and sterol 12α-hydroxylase (CYP8B1) dose-dependently decreased.Sterol 27-hydroxylase (CYP27A1) levels also decreased,but not to the same extent.CONCLUSION: T3 dose-dependently decreased total bile acid formation in parallel with decreased expression of CYP7A1 and CYP8B1. CA formation is inhibited to a higher degree than CDCA, resulting in a marked decrease in the CA/CDCA ratio.

  12. Transplantation of human thioredoxin gene-modified hepatocytes for treatment of acute liver failure in rat model

    Institute of Scientific and Technical Information of China (English)

    LI Hua; JIANG Nan; ZHANG Jian; WANG Gen-shu; YANG Yang; CHEN Gui-hua

    2009-01-01

    Background Mostly because of the limited number and proliferative ability of the transplanted hepatocytes,hepatocyte transplantation offers only temporary support to the hepatic function with rather poor functional replacement of the damaged liver parenchyma.This study aimed to observe the therapeutic effect of human thioredoxin(hTrx)gene-modified hepatocytes on experimental acute liver failure in rats.Methods hTrx cDNA was obtained by reverse transcription-polymerase chain reaction(RT-PCR)from human osteosercoma 143(TK-)cells to construct the recombinant retrovirus vector pLEGFP/hTrx,which was packaged into PA317 cells to collect the recombinant retrovirus containing hTrx gene.After titration and characterization,the recombinant retrovirus was applied to primary cultured rat hepatocyte for infection to generate hTrx gene-modified rat hepatocytes,whose viability and antioxidative capacity were examined by immunohistochemistry and MIF assay,respectively.In a Sprague-Dawley(SD)rat model of acute liver failure,the modified hepatocytes were injected into the spleen,and the hepatic function and survival rate of the recipient rats were evaluated at different time points after the transplantation.Results NIH3T3 cells infected by the recombinant retrovirus were capable of expressing bioactive hTrx in the form of fusion proteins.Immunohistochemistry demonstrated normal function of the hTrx gene-modified hepatocytes,which possessed strong antioxidative capacity as shown by MTT assay.Transplantation of the modified hepatocytes in rats with acute liver failure resulted in significantly lowered serum alanine aminotransferase(ALT)and total bilirubin(TBIL)levels(P<0.05).The hepatocytes exhibited long-term survival and efficient proliferation after transplantation.Fourteen days after the operation,the rat models receiving hTrx gene-modified hepatocytes had significantly higher survival rate than those without the transplantation.Conclusion hTrx gene-modifled hepatocyte

  13. Scalable spheroid model of human hepatocytes for hepatitis C infection and replication.

    Science.gov (United States)

    Ananthanarayanan, Abhishek; Nugraha, Bramasta; Triyatni, Miriam; Hart, Stefan; Sankuratri, Suryanarayana; Yu, Hanry

    2014-07-07

    Developing effective new drugs against hepatitis C (HCV) virus has been challenging due to the lack of appropriate small animal and in vitro models recapitulating the entire life cycle of the virus. Current in vitro models fail to recapitulate the complexity of human liver physiology. Here we present a method to study HCV infection and replication on spheroid cultures of Huh 7.5 cells and primary human hepatocytes. Spheroid cultures are constructed using a galactosylated cellulosic sponge with homogeneous macroporosity, enabling the formation and maintenance of uniformly sized spheroids. This facilitates easy handling of the tissue-engineered constructs and overcomes limitations inherent of traditional spheroid cultures. Spheroids formed in the galactosylated cellulosic sponge show enhanced hepatic functions in Huh 7.5 cells and maintain liver-specific functions of primary human hepatocytes for 2 weeks in culture. Establishment of apical and basolateral polarity along with the expression and localization of all HCV specific entry proteins allow for a 9-fold increase in viral entry in spheroid cultures over conventional monolayer cultures. Huh 7.5 cells cultured in the galactosylated cellulosic sponge also support replication of the HCV clone, JFH (Japanese fulminant hepatitis)-1 at higher levels than in monolayer cultures. The advantages of our system in maintaining liver-specific functions and allowing HCV infection together with its ease of handling make it suitable for the study of HCV biology in basic research and pharmaceutical R&D.

  14. A Nonhuman Primate Model of Human Radiation-Induced Venocclusive Liver Disease and Hepatocyte Injury

    Energy Technology Data Exchange (ETDEWEB)

    Yannam, Govardhana Rao [Department of Surgery, University of Nebraska Medical Center, Omaha, Nebraska (United States); Han, Bing [Department of Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania (United States); Department of Hepatobiliary Surgery, First Affiliated Hospital of Xi' an Jiaotong University, Xi' an, Shaanxi (China); Setoyama, Kentaro [Department of Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania (United States); Yamamoto, Toshiyuki [Department of Surgery, University of Nebraska Medical Center, Omaha, Nebraska (United States); Ito, Ryotaro; Brooks, Jenna M. [Department of Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania (United States); Guzman-Lepe, Jorge [Department of Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania (United States); Department of Pathology, Children' s Hospital of Pittsburgh, Pittsburgh, Pennsylvania (United States); Galambos, Csaba [Department of Pathology, Children' s Hospital of Pittsburgh, Pittsburgh, Pennsylvania (United States); Fong, Jason V. [Department of Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania (United States); Deutsch, Melvin; Quader, Mubina A. [Department of Radiation Oncology, Children' s Hospital of Pittsburgh, Pittsburgh, Pennsylvania (United States); Yamanouchi, Kosho [Department of Radiation Oncology, Albert Einstein College of Medicine, Bronx, New York (United States); Marion Bessin Liver Research Center, Albert Einstein College of Medicine, Bronx, New York (United States); Kabarriti, Rafi; Mehta, Keyur [Department of Radiation Oncology, Albert Einstein College of Medicine, Bronx, New York (United States); Soto-Gutierrez, Alejandro [Department of Pathology, Children' s Hospital of Pittsburgh, Pittsburgh, Pennsylvania (United States); McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania (United States); and others

    2014-02-01

    Background: Human liver has an unusual sensitivity to radiation that limits its use in cancer therapy or in preconditioning for hepatocyte transplantation. Because the characteristic veno-occlusive lesions of radiation-induced liver disease do not occur in rodents, there has been no experimental model to investigate the limits of safe radiation therapy or explore the pathogenesis of hepatic veno-occlusive disease. Methods and Materials: We performed a dose-escalation study in a primate, the cynomolgus monkey, using hypofractionated stereotactic body radiotherapy in 13 animals. Results: At doses ≥40 Gy, animals developed a systemic syndrome resembling human radiation-induced liver disease, consisting of decreased albumin, elevated alkaline phosphatase, loss of appetite, ascites, and normal bilirubin. Higher radiation doses were lethal, causing severe disease that required euthanasia approximately 10 weeks after radiation. Even at lower doses in which radiation-induced liver disease was mild or nonexistent, latent and significant injury to hepatocytes was demonstrated by asialoglycoprotein-mediated functional imaging. These monkeys developed hepatic failure with encephalopathy when they received parenteral nutrition containing high concentrations of glucose. Histologically, livers showed central obstruction via an unusual intimal swelling that progressed to central fibrosis. Conclusions: The cynomolgus monkey, as the first animal model of human veno-occlusive radiation-induced liver disease, provides a resource for characterizing the early changes and pathogenesis of venocclusion, for establishing nonlethal therapeutic dosages, and for examining experimental therapies to minimize radiation injury.

  15. Influence of commonly used clinical antidotes on antioxidant systems in human hepatocyte culture intoxicated with alpha-amanitin.

    Science.gov (United States)

    Magdalan, Jan; Piotrowska, Aleksandra; Gomułkiewicz, Agnieszka; Sozański, Tomasz; Szeląg, Adam; Dziegięl, Piotr

    2011-01-01

    α-Amanitin (α-AMA) is the main toxin of Amanita phalloides and its subspecies (A. virosa and A. verna). The primary mechanism of α-AMA toxicity is associated with protein synthesis blocking in hepatocytes. Additionally, α-AMA exhibits prooxidant properties that may contribute to its severe hepatotoxicity. The aim of the present study was to assess the effect of α-AMA on lipid peroxidation and the activities of superoxide dismutase (SOD) and catalase (CAT) in human hepatocyte culture. The effects of benzylpenicillin (BPCN), N-acetyl-L-cysteine (ACC), and silibinin (SIL) on SOD and CAT activities and on lipid peroxidation in human hepatocyte culture intoxicated with α-AMA were also examined. In human hepatocyte culture, 48-hour exposure to α-AMA at a 2-μM concentration caused an increase in SOD activity, a reduction of CAT activity, and a significant increase in lipid peroxidation. Changes in SOD and CAT activity caused by α-AMA could probably enhance lipid peroxidation by increased generation of hydrogen peroxide combined with reduced detoxification of that oxygen radical. The addition of antidotes (ACC or SIL) to the culture medium provided more effective protection against lipid peroxidation in human hepatocytes intoxicated with α-AMA than the addition of BPCN, possessing no antioxidant properties.

  16. Differences in the Epigenetic Regulation of Cytochrome P450 Genes between Human Embryonic Stem Cell-Derived Hepatocytes and Primary Hepatocytes.

    Science.gov (United States)

    Park, Han-Jin; Choi, Young-Jun; Kim, Ji Woo; Chun, Hang-Suk; Im, Ilkyun; Yoon, Seokjoo; Han, Yong-Mahn; Song, Chang-Woo; Kim, Hyemin

    2015-01-01

    Human pluripotent stem cell-derived hepatocytes have the potential to provide in vitro model systems for drug discovery and hepatotoxicity testing. However, these cells are currently unsuitable for drug toxicity and efficacy testing because of their limited expression of genes encoding drug-metabolizing enzymes, especially cytochrome P450 (CYP) enzymes. Transcript levels of major CYP genes were much lower in human embryonic stem cell-derived hepatocytes (hESC-Hep) than in human primary hepatocytes (hPH). To verify the mechanism underlying this reduced expression of CYP genes, including CYP1A1, CYP1A2, CYP1B1, CYP2D6, and CYP2E1, we investigated their epigenetic regulation in terms of DNA methylation and histone modifications in hESC-Hep and hPH. CpG islands of CYP genes were hypermethylated in hESC-Hep, whereas they had an open chromatin structure, as represented by hypomethylation of CpG sites and permissive histone modifications, in hPH. Inhibition of DNA methyltransferases (DNMTs) during hepatic maturation induced demethylation of the CpG sites of CYP1A1 and CYP1A2, leading to the up-regulation of their transcription. Combinatorial inhibition of DNMTs and histone deacetylases (HDACs) increased the transcript levels of CYP1A1, CYP1A2, CYP1B1, and CYP2D6. Our findings suggest that limited expression of CYP genes in hESC-Hep is modulated by epigenetic regulatory factors such as DNMTs and HDACs.

  17. Metabolism of lysergic acid diethylamide (LSD) to 2-oxo-3-hydroxy LSD (O-H-LSD) in human liver microsomes and cryopreserved human hepatocytes.

    Science.gov (United States)

    Klette, K L; Anderson, C J; Poch, G K; Nimrod, A C; ElSohly, M A

    2000-10-01

    The metabolism of lysergic acid diethylamide (LSD) to 2-oxo-3-hydroxy lysergic acid diethylamide (O-H-LSD) was investigated in liver microsomes and cyropreserved hepatocytes from humans. Previous studies have demonstrated that O-H-LSD is present in human urine at concentrations 16-43 times greater than LSD, the parent compound. Additionally, these studies have determined that O-H-LSD is not generated during the specimen extraction and analytical processes or due to parent compound degradation in aqueous urine samples. However, these studies have not been conclusive in demonstrating that O-H-LSD is uniquely produced during in vivo metabolism. Phase I drug metabolism was investigated by incubating human liver microsomes and cryopreserved human hepatocytes with LSD. The reaction was quenched at various time points, and the aliquots were extracted using liquid partitioning and analyzed by liquid chromatography-mass spectrometry. O-H-LSD was positively identified in all human liver microsomal and human hepatocyte fractions incubated with LSD. In addition, O-H-LSD was not detected in any microsomal or hepatocyte fraction not treated with LSD nor in LSD specimens devoid of microsomes or hepatocytes. This study provides definitive evidence that O-H-LSD is produced as a metabolic product following incubation of human liver microsomes and hepatocytes with LSD.

  18. Therapeutic angiogenesis induced by human hepatocyte growth factor (HGF) gene in rat myocardial ischemia models

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    In order to investigate the feasibility of myocardial ischemia gene therapy, we cloned human hepatocyte growth factor gene from human placenta cDNA library by the RT-PCR method. Recombination adenovirus Ad-HGF was constructed by the method of co-transfection and homologous recombination of plasmids in 293 cells. Ad-HGF was amplified in 293 cells and purified through CsCl density gradient centrifugation. Ad-HGF could be expressed in rat primary myocardial cells and HGF secreted into the culture media, which was tested by ELISA. The distribution and persistence of adenovirus in rat were investigated by green fluorescence protein as a report gene. In vivo we found that intramyocardial administration of Ad-HGF could induce angiogenesis in rat myocardium after ligation of coronary artery. The results suggested that Ad-HGF was effective in vitro and in vivo, and the data for designing human trial of gene therapy-- mediated cardiac angiogenesis were provided.

  19. Effect of EGCG-induced Autophagy on Human Ovarian Cancer Cell Line SKOV3 and the Underlying Mechanism%茶多酚对卵巢癌SKOV3细胞自噬水平的影响及相关机制研究

    Institute of Scientific and Technical Information of China (English)

    詹欣; 张玲云; 王志华

    2015-01-01

    Objective To investigate the effect of EGCG-induced autophagy on human ovarian cancer cell line SKOV3 and to explore the mechanism. Methods Human ovarian cancer cell SKOV3 were treated with EGCG, then autophage-associated protein LC3-II and AKT protein expression on the cells was detected by Western blot. Results After EGCG treatment, the level of LC3-II was up-regulated in a dose dependent manner, while the level of phospho-AKT was decreased. Pretreatment with AKT activator insulin-like growth factor 1, the EGCG-induced LC3-II expression was suppressed. Conclusion EGCG can induce autophagy in huaman ovarian cancer cell SKOV3 through AKT signaling pathway.%目的:研究茶多酚(EGCG)对人卵巢癌SKOV3细胞自噬水平的影响及相关机制。方法用EGCG处理SKOV3细胞,Western blot检测自噬相关蛋白LC3-II及蛋白激酶B(PKB)信号通路相应蛋白表达变化。结果 EGCG处理SKOV3细胞后,自噬相关蛋白LC3-II表达上调,并呈一定的时间浓度依赖性。EGCG处理SKOV3细胞后,AKT的磷酸化水平下调,AKT激活剂胰岛素样生长因子1(IGF-1)预处理后,自噬相关蛋白LC3-II高表达被抑制。结论 EGCG通过AKT介导的信号通路诱导SKOV3细胞自噬水平升高。

  20. Comparative metabolism of honokiol in mouse, rat, dog, monkey, and human hepatocytes.

    Science.gov (United States)

    Jeong, Hyeon-Uk; Kim, Ju-Hyun; Kong, Tae Yeon; Choi, Won Gu; Lee, Hye Suk

    2016-04-01

    Honokiol has antitumor, antioxidative, anti-inflammatory, and antithrombotic effects. Here we aimed to identify the metabolic profile of honokiol in mouse, rat, dog, monkey, and human hepatocytes and to characterize the enzymes responsible for the glucuronidation and sulfation of honokiol. Honokiol had a high hepatic extraction ratio in all five species, indicating that it was extensively metabolized. A total of 32 metabolites, including 17 common and 15 different metabolites, produced via glucuronidation, sulfation, and oxidation of honokiol allyl groups were tentatively identified using liquid chromatography-high resolution quadrupole Orbitrap mass spectrometry. Glucuronidation of honokiol to M8 (honokiol-4-glucuronide) and M9 (honokiol-2'-glucuronide) was the predominant metabolic pathway in hepatocytes of all five species; however, interspecies differences between 4- and 2'-glucuronidation of honokiol were observed. UGT1A1, 1A8, 1A9, 2B15, and 2B17 played major roles in M8 formation, whereas UGT1A7 and 1A9 played major roles in M9 formation. Human cDNA-expressed SULT1C4 played a major role in M10 formation (honokiol-2'-sulfate), whereas SULT1A1*1, 1A1*2, and 1A2 played major roles in M11 formation (honokiol-4-sulfate). In conclusion, honokiol metabolism showed interspecies differences.

  1. Downregulation of IGF-1 receptor occurs after hepatic linage commitment during hepatocyte differentiation from human embryonic stem cells.

    Science.gov (United States)

    Waraky, Ahmed; Aleem, Eiman; Larsson, Olle

    2016-09-30

    The insulin-like growth factor 1 receptor (IGF-1R) has been suggested to be involved in hepatocyte differentiation. Human hepatocyte cancer cells and stem cells are known to express IGF-1R whereas normal hepatocytes do not. In the present study we optimized a differentiation protocol and verified the different stages by established markers. The expression levels of IGF-1R and major downstream signaling proteins during differentiation from human embryonic stem cells (hESC) to mature hepatocytes were investigated. We could only demonstrate a minor decrease in IGF-1R expression during endodermal differentiation compared to hESC, but declined substantially (>50%) after hepatic lineage commitment during the hepatocyte specification and maturation stages. This downregulation was paralleled by an upregulation of ERK 1/2, AKT and insulin substrate-1. Neither inhibition nor activation of IGF-1R had any essential effect on endoderm differentiation of human embryonic stem cells. Therefore, our data suggest that IGF-1R downregulation may have a regulatory impact after initiation of hepatic lineage commitment. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  2. Potential inhibition of cytochrome P450 3A4 by propofol in human primary hepatocytes

    Institute of Scientific and Technical Information of China (English)

    Li-Qun Yang; Wei-Feng Yu; Yun-Fei Cao; Bin Gong; Qing Chang; Guang-Shun Yang

    2003-01-01

    AIM: Hepatic cytochrome P450 isoenzymes constitute a superfamily of hemoproteins that play a major role in the metabolism of endogenous compounds and in the detoxification of xenobiotic molecules. P450 3A4 is one of the most important forms in human being, and mediates the metabolism of around 70% of therapeutic drugs and endogenous compounds. Propofol, a widely used intravenous anesthetic drug, is known to inhibit cytochrome P450activities in isolated rat hepatocytes. The goal of this study was to evaluate the potential efficacy of propofol on P4503A4 in a dose-dependent manner to understand its drugdrug interaction.METHODS: Hepatocytes were isolated from liver specimens from hepatic angioma patients undergone hepatic surgery.Primary incubated hepatocytes were treated with 0, 0.01,0.05, 0.1, 0.5, and 1.0 mM propofol for 24 hours. P450 3A4activity was measured with Nash′s colorimetry. The protein expression was assessed by Western blot analysis.RESULTS: A dose-dependent inhibitory effect of propofol was observed in cytochrome P450 3A4 activity. A minimal dosage of propofol (0.01 mM) induced a significant inhibition of P450 3A4 activity, although its regular dosages (0.01-0.1mM) showed no inhibitory effect on the cellular protein expression of P450 3A4.CONCLUSION: Propofol may be a potential CYP3A4 inhibitor as this anesthetic can inhibit isoenzyme activity significantly and reduce the metabolic rate of CYP3A4 substrates. This inhibition occurs at post-expression level, and concentration of propofol used clinically does not affect CYP3A4 protein expression. propofol may thus induce drug interaction of cytochrome P450 3A4 activity at the dosage used clinically.

  3. (-)-Epigallocatechin-3-O-gallate (EGCG) attenuates the hemodynamics stimulated by caffeine through decrease of catecholamines release.

    Science.gov (United States)

    Han, Jin-Yi; Moon, Yong-Jin; Han, Jong-Hyun; Kim, Jong-Hoon; Woo, Jae-Hoon; Yoo, Hwan-Soo; Hong, Jin Tae; Ahn, Hee-Yul; Hong, Jong-Myeon; Oh, Ki-Wan

    2016-09-01

    A human study of the effects on hemodynamics of caffeine and epigallocatechin-3-O-gallate (EGCG) was performed. Caffeine tablets (200 mg) were orally administered to healthy males aged between 25 and 35 years 30 min after oral administration of EGCG tablets (100 and 200 mg). The increase in BP induced by caffeine was inhibited when co-administrated with EGCG. We found that caffeine slightly decreased heart rate (HR) in the volunteers. Although EGCG enhanced HR reduction, the effect was not significant. In addition, caffeine increased blood catecholamine levels, but EGCG inhibited the increase in noradrenaline, adrenaline and dopamine levels induced by caffeine. Whether EGCG decreases the elevated HR and systolic perfusion pressure, and ventricular contractility induced by adrenergic agonists in the isolated rat heart was investigated. The modified Krebs-Henseleit solution was perfused through a Langendorff apparatus to the isolated hearts of rats. HR, systolic perfusion pressure, and developed maximal rates of contraction (+dP/dtmax) and relaxation (-dP/dtmax) were increased by epinephrine (EP) and isoproterenol (IP). In contrast, EGCG decreased the elevated HR, systolic perfusion pressure, and left ventricular ±dp/dtmax induced by EP and/or IP. In conclusion, EGCG could attenuate the hemodynamics stimulated by caffeine through decreasing catecholamine release.

  4. Mechanisms of acetaminophen-induced cell death in primary human hepatocytes

    Energy Technology Data Exchange (ETDEWEB)

    Xie, Yuchao; McGill, Mitchell R.; Dorko, Kenneth [Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, KS 66160 (United States); Kumer, Sean C.; Schmitt, Timothy M.; Forster, Jameson [Department of Surgery, University of Kansas Medical Center, Kansas City, KS 66160 (United States); Jaeschke, Hartmut, E-mail: hjaeschke@kumc.edu [Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, KS 66160 (United States)

    2014-09-15

    Acetaminophen (APAP) overdose is the most prevalent cause of drug-induced liver injury in western countries. Numerous studies have been conducted to investigate the mechanisms of injury after APAP overdose in various animal models; however, the importance of these mechanisms for humans remains unclear. Here we investigated APAP hepatotoxicity using freshly isolated primary human hepatocytes (PHH) from either donor livers or liver resections. PHH were exposed to 5 mM, 10 mM or 20 mM APAP over a period of 48 h and multiple parameters were assessed. APAP dose-dependently induced significant hepatocyte necrosis starting from 24 h, which correlated with the clinical onset of human liver injury after APAP overdose. Interestingly, cellular glutathione was depleted rapidly during the first 3 h. APAP also resulted in early formation of APAP-protein adducts (measured in whole cell lysate and in mitochondria) and mitochondrial dysfunction, indicated by the loss of mitochondrial membrane potential after 12 h. Furthermore, APAP time-dependently triggered c-Jun N-terminal kinase (JNK) activation in the cytosol and translocation of phospho-JNK to the mitochondria. Both co-treatment and post-treatment (3 h) with the JNK inhibitor SP600125 reduced JNK activation and significantly attenuated cell death at 24 h and 48 h after APAP. The clinical antidote N-acetylcysteine offered almost complete protection even if administered 6 h after APAP and a partial protection when given at 15 h. Conclusion: These data highlight important mechanistic events in APAP toxicity in PHH and indicate a critical role of JNK in the progression of injury after APAP in humans. The JNK pathway may represent a therapeutic target in the clinic. - Highlights: • APAP reproducibly causes cell death in freshly isolated primary human hepatocytes. • APAP induces adduct formation, JNK activation and mitochondrial dysfunction in PHH. • Mitochondrial adducts and JNK translocation are delayed in PHH compared to

  5. Human Hepatocyte-Derived Induced Pluripotent Stem Cells: MYC Expression, Similarities to Human Germ Cell Tumors, and Safety Issues

    Directory of Open Access Journals (Sweden)

    Carmen Unzu

    2016-01-01

    Full Text Available Induced pluripotent stem cells (iPSC are a most promising approach to the development of a hepatocyte transplantable mass sufficient to induce long-term correction of inherited liver metabolic diseases, thus avoiding liver transplantation. Their intrinsic self-renewal ability and potential to differentiate into any of the three germ layers identify iPSC as the most promising cell-based therapeutics, but also as drivers of tumor development. Teratoma development currently represents the gold standard to assess iPSC pluripotency. We analyzed the tumorigenic potential of iPSC generated from human hepatocytes (HEP-iPSC and compared their immunohistochemical profiles to that of tumors developed from fibroblast and hematopoietic stem cell-derived iPSC. HEP-iPSC generated tumors significantly presented more malignant morphological features than reprogrammed fibroblasts or CD34+ iPSC. Moreover, the protooncogene myc showed the strongest expression in HEP-iPSC, compared to only faint expression in the other cell subsets. Random integration of transgenes and the use of potent protooncogenes such as myc might be a risk factor for malignant tumor development if hepatocytes are used for reprogramming. Nonviral vector delivery systems or reprogramming of cells obtained from less invasive harvesting methods would represent interesting options for future developments in stem cell-based approaches for liver metabolic diseases.

  6. METABOLISM OF 3 PHARMACOLOGICALLY ACTIVE-DRUGS IN ISOLATED HUMAN AND RAT HEPATOCYTES - ANALYSIS OF INTERSPECIES VARIABILITY AND COMPARISON WITH METABOLISM IN-VIVO

    NARCIS (Netherlands)

    SANDKER, GW; VOS, RME; DELBRESSINE, LPC; SLOOFF, MJH; MEIJER, DKF; GROOTHUIS, GMM

    1994-01-01

    1. The metabolism of the three drugs (Org GB 94, Org 3770 and Org OD 14) was studied in isolated human and rat hepatocytes. The metabolic profiles in rat and human hepatocytes were compared with the available in vivo data in both species. 2. All three drugs were metabolized extensively under the con

  7. PRIMARY STUDY ON MECHANISM OF COLLAGEN SYNTHESIS INHIBITION IN CULTURED HUMAN FETAL HEPATOCYTES PRETREATED BY SALVIA MILTIORRHIZA BUNGE

    Institute of Scientific and Technical Information of China (English)

    赵英仁; 朱风群; 和水祥; 苌新明; 乔文

    2003-01-01

    Objective The effects of salvia miltiorrhiza bunge (SMB) on collagen synthesis in the human fetal hepatocytes culture were studied. Methods The collagen synthesis of hepatocytes were stimulated by the addition of carbon tetrachloride (CCl4) to the culture medium, the concentration of type procollagen (PC) in the culture medium and the hydroxyproline (Hyp) in hepatocytes were determined, as well as the activity of se-dependent glutathione peroxidase (Se-GSH-Px) and the concentration of malondiadehyde (MDA) in the culture medium. Results A significant decrease in PC, Hyp and MDA production, and the significant increase in Se-GSH-Px activity were observed in the cultures pretreated with 1 g L-1 SMB for 4 hours compared with the untreated cultures. Analysis of the Se-GSH-Px/MDA ratio in SMB pretreated group showed more marked increase compared to that of the untreated group (P<0.01). There was a significant negative correlation between the ratio of Se-GSH-Px/MDA and the concentration of PC in SMB pretreated group (r=-0.9017, P<0.01). Conclusion Our results indicate that SMB may suppress the collagen synthesis of cultured human fetal hepatocytes stimulated by CCl4, and its mechanism may be related to the increase in Se-GSH-Px/MDA ratio and the enhancement of hepatocytes antioxidation capability.

  8. Amelioration of Hyperbilirubinemia in Gunn Rats after Transplantation of Human Induced Pluripotent Stem Cell-Derived Hepatocytes

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    Yong Chen

    2015-07-01

    Full Text Available Hepatocyte transplantation has the potential to cure inherited liver diseases, but its application is impeded by a scarcity of donor livers. Therefore, we explored whether transplantation of hepatocyte-like cells (iHeps differentiated from human induced pluripotent stem cells (iPSCs could ameliorate inherited liver diseases. iPSCs reprogrammed from human skin fibroblasts were differentiated to iHeps, which were transplanted into livers of uridinediphosphoglucuronate glucuronosyltransferase-1 (UGT1A1-deficient Gunn rats, a model of Crigler-Najjar syndrome 1 (CN1, where elevated unconjugated bilirubin causes brain injury and death. To promote iHep proliferation, 30% of the recipient liver was X-irradiated before transplantation, and hepatocyte growth factor was expressed. After transplantation, UGT1A1+ iHep clusters constituted 2.5%–7.5% of the preconditioned liver lobe. A decline of serum bilirubin by 30%–60% and biliary excretion of bilirubin glucuronides indicated that transplanted iHeps expressed UGT1A1 activity, a postnatal function of hepatocytes. Therefore, iHeps warrant further exploration as a renewable source of hepatocytes for treating inherited liver diseases.

  9. Fibroblast growth factor-4 and hepatocyte growth factor induce differentiation of human umbilical cord blood-derived mesenchymal stem cells into hepatocytes

    Institute of Scientific and Technical Information of China (English)

    Xin-Qin Kang; Wei-Jin Zang; Li-Jun Bao; Dong-Ling Li; Tu-Sheng Song; Xiao-Li Xu; Xiao-Jiang Yu

    2005-01-01

    AIM: To investigate the differentiation of human umbilical cord blood (HUCB)-derived mesenchymal stem cells (MSCs) into hepatocytes by induction of fibroblast growth factor-4 (FGF-4) and hepatocyte growth factor (HGF), and to find a new source of cell types for therapies of hepatic diseases.METHODS: vSCs were isolated by combining gradient density centrifugation with plastic adherence. When HUCB-derived MSCs reached 70% confluence, they were cultured in Iscove modified Dulbecco medium (IMDM) supplemented with 10 mL/L FBS, 20 ng/mL HGF and 10 ng/mL FGF-4. The medium was changed every 4 d and stored for albumin, alpha-fetoprotein (AFP) and urea assay. Expression of CK-18 was detected by immunocytochemistry. Glycogen storage in hepatocytes was determined by PAS staining.RESULTS: By combining gradient density centrifugation with plastic adherence, we could isolate MSCs from 25.6% of human umbilical cord blood. When MSCs were cultured with FGF-4 and HGF, approximately 63.6% of cells became small, round and epithelioid on d 28 by morphology. Compared with the control, the level of AFP increased significantly from d 12 to 18.20±1.16 μg/L (t = 2.884, P<0.05) in MSCs cultured with FGF-4 and HGF, and was higher (54.28±3.11 μg/L) on d 28 (t = 13.493, P<0.01). Albumin increased significantly on d 16 (t = 6.68, P<0.01) to 1.02±0.15 μg/mL, and to 3.63±0.30 μg/mL on d 28 (t = 11.748, P<0.01). Urea(4.72±1.03 μmol/L) was detected on d 20 (t = 4.272,P<0.01), and continued to increase to 10.28±1.06 μmol/L on d 28 (t = 9.276, P<0.01). Cells expressed CK-18 on d 16. Glycogen storage was observed on d 24. CONCLUSION: HUCB-derived MSCs can differentiate into hepatocytes by induction of FGF-4 and HGF. HUCBderived MSCs are a new source of cell types for cell transplantation therapy of hepatic diseases.

  10. Human hepatocyte growth factor promotes functional recovery in primates after spinal cord injury.

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    Kazuya Kitamura

    Full Text Available Many therapeutic interventions for spinal cord injury (SCI using neurotrophic factors have focused on reducing the area damaged by secondary, post-injury degeneration, to promote functional recovery. Hepatocyte growth factor (HGF, which is a potent mitogen for mature hepatocytes and a mediator of the inflammatory responses to tissue injury, was recently highlighted as a potent neurotrophic factor in the central nervous system. We previously reported that introducing exogenous HGF into the injured rodent spinal cord using a herpes simplex virus-1 vector significantly reduces the area of damaged tissue and promotes functional recovery. However, that study did not examine the therapeutic effects of administering HGF after injury, which is the most critical issue for clinical application. To translate this strategy to human treatment, we induced a contusive cervical SCI in the common marmoset, a primate, and then administered recombinant human HGF (rhHGF intrathecally. Motor function was assessed using an original open field scoring system focusing on manual function, including reach-and-grasp performance and hand placement in walking. The intrathecal rhHGF preserved the corticospinal fibers and myelinated areas, thereby promoting functional recovery. In vivo magnetic resonance imaging showed significant preservation of the intact spinal cord parenchyma. rhHGF-treatment did not give rise to an abnormal outgrowth of calcitonin gene related peptide positive fibers compared to the control group, indicating that this treatment did not induce or exacerbate allodynia. This is the first study to report the efficacy of rhHGF for treating SCI in non-human primates. In addition, this is the first presentation of a novel scale for assessing neurological motor performance in non-human primates after contusive cervical SCI.

  11. Differentiation of human umbilical cord blood stem cells into hepatocytes in vivo and in vitro

    Institute of Scientific and Technical Information of China (English)

    Xiao-Peng Tang; Min Zhang; Xu Yang; Li-Min Chen; Yang Zeng

    2006-01-01

    AIM: To study the condition and potentiality of human umbilical cord blood stem cells (HUCBSC) to differentiate into hepatocytes in vivo or in vitro.METHODS: In a cell culture study of human umbilical cord blood stem cell (HUCBSC) differentiation, human umbilical cord blood mononuclear cells (HUCBMNC) were separated by density gradient centrifugation.Fibroblast growth factor (FGF) and hepatocyte growth factor (HGF) and the supernatant of fetal liver were added in the inducing groups. Only FGF was added in the control group. The expansion and differentiation of HUCBMNC in each group were observed. Human alpha fetoprotein (AFP) and albumin (ALB) were detected by immunohistochemistry. In the animal experiments, the survival SD rats with acute hepatic injury after carbon tetrachloride (CCL4) injection 48 h were randomly divided into three groups. The rats in group A were treated with human umbilical cord blood serum. The rats in group B were treated with HUCBMNC transplantation. The rats in group C were treated with HUCBMNC transplantation followed by intraperitoneal cyclophosphamide for 7 d.The rats were killed at different time points after the treatment and the liver tissue was histopathologically studied and human AFP and ALB detected by immunohistochemistry. The human X inactive-specific transcript gene fragment in the liver tissue was amplified by PCR to find human DNA.RESULTS: The results of cell culture showed that adherent cells were stained negative for AFP or ALB in control group. However, the adherent cells in the inducing groups stained positive for AFP or ALB. The result of animal experiment showed that no human AFP or ALB positive cells present in the liver tissue of group A (control group). However, many human AFP or ALB positive cells were scattered around sinus hepaticus and the central veins of hepatic lobules and in the portal area in group B and group C after one month. The fragment of human X chromagene could be detected in the liver tissue of

  12. Green tea polyphenol EGCG suppresses lung cancer cell growth through upregulating miR-210 expression caused by stabilizing HIF-1α.

    Science.gov (United States)

    Wang, Hong; Bian, Shengjie; Yang, Chung S

    2011-12-01

    (-)-Epigallocatechin-3-gallate (EGCG) has been reported to affect many cellular regulatory pathways. This study aims to determine whether EGCG could target microRNA (miRNA), one of the mechanisms for cells to achieve subtle change in multiple targets. We found that, in both human and mouse lung cancer cells in culture, EGCG specifically upregulated the expression of miR-210, a major miRNA regulated by HIF-1α. Furthermore, we found that overexpression of miR-210 led to reduced cell proliferation rate and anchorage-independent growth as well as reduced sensitivity to EGCG. On the mechanisms of miR-210 regulation by EGCG, we demonstrated that the regulation was mediated through the hypoxia-response element in miR-210 promoter. Consistently, the upregulation of miR-210 was found to be correlated with the stabilized HIF-1α in lung cancer cell lines after EGCG treatment. This EGCG-induced stabilization of HIF-1α was further shown by the stabilization of HA-tagged HIF-1α but not the P402A/P564A-mutated HIF-1α by EGCG, suggesting that EGCG targets the oxygen-dependent degradation (ODD) domain. Direct evidence was obtained by affinity binding assay showing that EGCG specifically binds HIF-1α with a K(d) = 3.47 μM. This result suggests that EGCG binding interferes with the hydroxylation of key Pro residues in the ODD domain, preventing HIF-1α from the Pro hydroxylation-dependent ubiquitination and subsequent proteosome-mediated degradation. In summary, our results demonstrated, for the first time, the elevation of miR-210 by EGCG in lung cancer cell lines and this is mediated by the stabilization of HIF-1α. This event contributes to the anticancer activity of EGCG.

  13. Adenoviral-mediated correction of methylmalonyl-CoA mutase deficiency in murine fibroblasts and human hepatocytes

    Directory of Open Access Journals (Sweden)

    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.

  14. Hypoxia and proinflammatory factors upregulate apelin receptor expression in human stellate cells and hepatocytes.

    Science.gov (United States)

    Melgar-Lesmes, Pedro; Pauta, Montserrat; Reichenbach, Vedrana; Casals, Gregori; Ros, Josefa; Bataller, Ramon; Morales-Ruiz, Manuel; Jiménez, Wladimiro

    2011-10-01

    The activation of the apelin receptor (APJ) plays a major role in both angiogenic and fibrogenic response to chronic liver injury. However, the mechanisms that govern the induction of APJ expression have not been clarified so far. The regulation and the role of APJ in cultured human liver cells were investigated. Tissular expression of APJ and α-smooth muscle actin was analysed by immunocolocalisation in human cirrhotic liver and in control samples. mRNA and protein expression of APJ were analysed in two cell lines, LX-2 (as hepatic stellate cells, HSCs) and HepG2 (as hepatocytes), under hypoxic conditions or after exposure to proinflammatory or profibrogenic factors. Additionally, both hepatic cell lines were stimulated with apelin to assess cell survival and the expression of angiogenic factors. The APJ-positive signal was negligible in control livers. In contrast, APJ was highly expressed in HSCs and slightly expressed in hepatocytes of human cirrhotic liver. Sustained hypoxia and lipopolysaccharide stimulated the expression of APJ in LX-2 cells. Moreover, hypoxia, tumour necrosis factor α and angiotensin II induced the expression of APJ in HepG2 cells. Activation of APJ stimulated angiopoietin-1 expression and cell survival in LX-2 cells and, in turn, triggered the synthesis of vascular endothelial growth factor type A and platelet-derived growth factor-BB in HepG2 cells. These results suggest that hypoxia and inflammatory factors could play a major role in the activation of the hepatic apelin system leading to angiogenic and fibroproliferative response occurring in chronic liver disease.

  15. Endogenous bile acid disposition in rat and human sandwich-cultured hepatocytes

    Energy Technology Data Exchange (ETDEWEB)

    Marion, Tracy L., E-mail: tracylmarion@qualyst.com [Curriculum in Toxicology, UNC School of Medicine, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7270 (United States); Perry, Cassandra H., E-mail: cassandraperry@qualyst.com [Qualyst, Inc., Durham, NC 27713 (United States); St Claire, Robert L., E-mail: bobstclaire@qualyst.com [Qualyst, Inc., Durham, NC 27713 (United States); Brouwer, Kim L.R., E-mail: kbrouwer@unc.edu [Division of Pharmacotherapy and Experimental Therapeutics, UNC Eshelman School of Pharmacy, The University of North Carolina at Chapel Hill, CB 7569 Kerr Hall, Chapel Hill, NC 27599-7569 (United States)

    2012-05-15

    Sandwich-cultured hepatocytes (SCH) are used commonly to investigate hepatic transport protein-mediated uptake and biliary excretion of substrates. However, little is known about the disposition of endogenous bile acids (BAs) in SCH. In this study, four endogenous conjugated BAs common to rats and humans [taurocholic acid (TCA), glycocholic acid (GCA), taurochenodeoxycholic acid (TCDCA), and glycochenodeoxycholic acid (GCDCA)], as well as two BA species specific to rodents (α- and β-tauromuricholic acid; α/β TMCA), were profiled in primary rat and human SCH. Using B-CLEAR{sup ®} technology, BAs were measured in cells + bile canaliculi, cells, and medium of SCH by LC-MS/MS. Results indicated that, just as in vivo, taurine-conjugated BA species were predominant in rat SCH, while glycine-conjugated BAs were predominant in human SCH. Total intracellular BAs remained relatively constant over days in culture in rat SCH. Total BAs in control (CTL) cells + bile, cells, and medium were approximately 3.4, 2.9, and 8.3-fold greater in human than in rat. The estimated intracellular concentrations of the measured total BAs were 64.3 ± 5.9 μM in CTL rat and 183 ± 56 μM in CTL human SCH, while medium concentrations of the total BAs measured were 1.16 ± 0.21 μM in CTL rat SCH and 9.61 ± 6.36 μM in CTL human SCH. Treatment of cells for 24 h with 10 μM troglitazone (TRO), an inhibitor of the bile salt export pump (BSEP) and the Na{sup +}-taurocholate cotransporting polypeptide (NTCP), had no significant effect on endogenous BAs measured at the end of the 24-h culture period, potentially due to compensatory mechanisms that maintain BA homeostasis. These data demonstrate that BAs in SCH are similar to in vivo, and that SCH may be a useful in vitro model to study alterations in BA disposition if species differences are taken into account. -- Highlights: ► Bile acids (BAs) were measured in rat and human sandwich-cultured hepatocytes (SCH). ► Cell and medium BA

  16. Epigallocatechin gallate (EGCG), a major component of green tea, is a dual phosphoinositide-3-kinase/mTOR inhibitor

    Energy Technology Data Exchange (ETDEWEB)

    Van Aller, Glenn S., E-mail: glenn.s.van.aller@gsk.com [Department of Cancer Research, GlaxoSmithKline, Collegeville, PA 19426 (United States); Carson, Jeff D. [Department of Cancer Research, GlaxoSmithKline, Collegeville, PA 19426 (United States); Tang, Wei; Peng, Hao; Zhao, Lin [Discovery Biology, BioDuro, No. 29 Life Science Park Road, Changping, Beijing (China); Copeland, Robert A.; Tummino, Peter J. [Department of Cancer Research, GlaxoSmithKline, Collegeville, PA 19426 (United States); Luo, Lusong [Discovery Biology, BioDuro, No. 29 Life Science Park Road, Changping, Beijing (China)

    2011-03-11

    Research highlights: {yields} Epigallocatechin-3-gallate (EGCG) is an ATP-competitive inhibitor of PI3K and mTOR with Ki values around 300 nM. {yields} EGCG inhibits cell proliferation and AKT phosphorylation at Ser473 in MDA-MB-231and A549 cells. {yields} Molecular docking studies show that EGCG binds well to the PI3K kinase domain active site. {yields} These results suggest another important molecular mechanism for the anticancer activities of EGCG. -- Abstract: The PI3K signaling pathway is activated in a broad spectrum of human cancers, either directly by genetic mutation or indirectly via activation of receptor tyrosine kinases or inactivation of the PTEN tumor suppressor. The key nodes of this pathway have emerged as important therapeutic targets for the treatment of cancer. In this study, we show that (-)-epigallocatechin-3-gallate (EGCG), a major component of green tea, is an ATP-competitive inhibitor of both phosphoinositide-3-kinase (PI3K) and mammalian target of rapamycin (mTOR) with K{sub i} values of 380 and 320 nM respectively. The potency of EGCG against PI3K and mTOR is within physiologically relevant concentrations. In addition, EGCG inhibits cell proliferation and AKT phosphorylation at Ser473 in MDA-MB-231 and A549 cells. Molecular docking studies show that EGCG binds well to the PI3K kinase domain active site, agreeing with the finding that EGCG competes for ATP binding. Our results suggest another important molecular mechanism for the anticancer activities of EGCG.

  17. Effect of cold and warm ischaemia on drug metabolism in isolated hepatocytes and slices from human and monkey liver

    NARCIS (Netherlands)

    Olinga, Peter; Hof, I.H; de Jager, M.H; de Jong, Kurt; Slooff, M.JH; Meijer, D.K F; Groothuis, Geny; Merema, M.T.

    1998-01-01

    1. The influence of short-term cold storage in University of Wisconsin organ preservation solution (UW) on the ability to metabolize lidocaine, testosterone and 7-ethoxycoumarin in isolated human and cynomolgus monkey (Macaca fascicularis) hepatocytes and liver slices has been investigated. 2. The h

  18. An algorithm that predicts the viability and the yield of human hepatocytes isolated from remnant liver pieces obtained from liver resections.

    Science.gov (United States)

    Lee, Serene M L; Schelcher, Celine; Laubender, Rüdiger P; Fröse, Natalja; Thasler, Reinhard M K; Schiergens, Tobias S; Mansmann, Ulrich; Thasler, Wolfgang E

    2014-01-01

    Isolated human primary hepatocytes are an essential in vitro model for basic and clinical research. For successful application as a model, isolated hepatocytes need to have a good viability and be available in sufficient yield. Therefore, this study aims to identify donor characteristics, intra-operative factors, tissue processing and cell isolation parameters that affect the viability and yield of human hepatocytes. Remnant liver pieces from tissue designated as surgical waste were collected from 1034 donors with informed consent. Human hepatocytes were isolated by a two-step collagenase perfusion technique with modifications and hepatocyte yield and viability were subsequently determined. The accompanying patient data was collected and entered into a database. Univariate analyses found that the viability and the yield of hepatocytes were affected by many of the variables examined. Multivariate analyses were then carried out to confirm the factors that have a significant relationship with the viability and the yield. It was found that the viability of hepatocytes was significantly decreased by the presence of fibrosis, liver fat and with increasing gamma-glutamyltranspeptidase activity and bilirubin content. Yield was significantly decreased by the presence of liver fat, septal fibrosis, with increasing aspartate aminotransferase activity, cold ischemia times and weight of perfused liver. However, yield was significantly increased by chemotherapy treatment. In conclusion, this study determined the variables that have a significant effect on the viability and the yield of isolated human hepatocytes. These variables have been used to generate an algorithm that can calculate projected viability and yield of isolated human hepatocytes. In this way, projected viability can be determined even before isolation of hepatocytes, so that donors that result in high viability and yield can be identified. Further, if the viability and yield of the isolated hepatocytes is lower

  19. Current status of human hepatocyte transplantation and its potential for Wilson's disease.

    Science.gov (United States)

    Filippi, Celine; Dhawan, Anil

    2014-05-01

    Wilson's disease (WD) is a genetic disorder of liver copper excretion leading to its accumulation in various vital organs like the liver, brain, and kidneys. Drugs such as penicillamine, trientine, and zinc salts are the mainstay of treatment, with good outcomes; but nonresponders or a lack of compliance to the drug treatment can result in disease progression and acute liver failure (ALF). Current treatment for WD with ALF is an emergency liver transplantation and lifelong immunosuppression. Human hepatocyte transplantation (HTx) is increasingly used as treatment for liver-based metabolic defects. HTx may benefit WD patients with ALF, either as transient support until chelation treatment shows its effect or as a definitive cure through liver repopulation by healthy donor cells, as shown in animal models of WD. Although clinical trials of HTx have already proven safety and efficacy in different ALF etiologies, it remains to be demonstrated similarly in cases of WD.

  20. Targeting Nuclear Receptors with Lentivirus-Delivered Small RNAs in Primary Human Hepatocytes

    Directory of Open Access Journals (Sweden)

    Maria Thomas

    2014-07-01

    Full Text Available Background: RNA interference (RNAi has tremendous potential for investigating gene function and for developing new therapies. Primary human hepatocytes (PHH are the “gold standard” for studying the regulation of hepatic metabolism in vitro. However, application of RNAi in PHH has some technical hurdles. The objective of this study was to develop effective and robust protocol for transduction of PHH with lentiviral vectors. Methods: We used lentiviral vectors to transduce PHH for introduction of short hairpin RNAs (shRNAs targeting constitutive androstane receptor (CAR, peroxisome proliferator activated receptor alpha (PPARα, and microRNA, miR-143. Infection efficiency was quantitatively analyzed by flow cytometry and microscopy. Target gene expression was assessed using quantitative real-time (qRT-PCR method. Results: Lentiviral vector transduction resulted in ≥95% of infected cells at low multiplicity of infection (MOI of 3, which did not impair cellular viability. We demonstrated the feasibility of this technique in studies on targeting nuclear receptors, PPARα and CAR, with shRNAs as well as in lentivirus-mediated overexpression and knock-down of miRNA-143 experiments. Conclusions: We developed an efficient and robust protocol with standardized procedures for virus production, method of titer determination, and infection procedure for RNAi in primary human hepatocytes based on delivery of shRNAs, microRNAs or anti-microRNAs in different laboratory settings. This approach should be useful to study not only the regulation via nuclear receptors but also other biological, pharmacological, and toxicological aspects of drug metabolism.

  1. Metabolic Activation of Cholestatic Drug-Induced Bile Acid-Dependent Toxicity in Human Sandwich-Cultured Hepatocytes.

    Science.gov (United States)

    Ogimura, Eiichiro; Tokizono, Mayuko; Sekine, Shuichi; Nakagawa, Tetsuya; Bando, Kiyoko; Ito, Kousei

    2017-09-01

    We previously reported a cell-based toxicity assay using sandwich-cultured hepatocytes in combination with a titrated amount of human bile acid (BA) species. In this assay, test compound-induced inhibition of BA efflux from sandwich-cultured hepatocytes leads to BA-dependent cell toxicity (BAtox, i.e., cell death due to the accumulation of BAs). Using this assay, we investigated whether 1-aminobenzotriazole (1-ABT; a nonselective cytochrome P450 inhibitor) enhanced or suppressed test compound-induced BAtox. There was a tendency that BAtox of many compounds was enhanced by 1-ABT in human hepatocytes; in contrast, such a tendency was not observed in rat hepatocytes. In particular, 1-ABT tended to enhance BAtox of several compounds (clopidogrel, ticlopidine, everolimus, etc.) in human, whereas 1-ABT tended to enhance BAtox of only ticlopidine in rat. These results indicate that this system can be used to evaluate BAtox while taking into account drug metabolism and the existence of an interspecies difference in the effect of 1-ABT treatment on BAtox. Copyright © 2017 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

  2. CHARACTERIZATION OF TRANSPORT IN ISOLATED HUMAN HEPATOCYTES - A STUDY WITH THE BILE-ACID TAUROCHOLIC ACID, THE UNCHARGED OUABAIN AND THE ORGANIC CATIONS VECURONIUM AND ROCURONIUM

    NARCIS (Netherlands)

    SANDKER, GW; WEERT, B; OLINGA, P; WOLTERS, H; SLOOFF, MJH; MEIJER, DKF; GROOTHUIS, GMM

    1994-01-01

    The uptake and efflux of three categories of substrates were measured in isolated human hepatocytes and compared to those in rat hepatocytes. In addition, the extent to which the in vitro experiments quantitatively reflect liver function in vivo in both species was investigated. The anionic bile aci

  3. Association of filamin A and vimentin with hepatitis C virus proteins in infected human hepatocytes.

    Science.gov (United States)

    Ghosh, S; Ahrens, W A; Phatak, S U; Hwang, S; Schrum, L W; Bonkovsky, H L

    2011-10-01

    Chronic hepatitis C (CHC) infection caused by hepatitis C virus (HCV) is a major cause of liver disease and remains a major therapeutic challenge. A variety of host proteins interact with HCV proteins. The definitive role of cytoskeletal (CS) proteins in HCV infection remains to be determined. In this study, our aim was to determine the expression profile of differentially regulated and expressed selected CS proteins and their association with HCV proteins in infected hepatocytes as possible therapeutic targets. Using proteomics, qRT-PCR, Western blot and immunofluorescence techniques, we revealed that filamin A (fila) and vimentin (vim) were prominently increased proteins in HCV-expressing human hepatoma cells compared with parental cells and in liver biopsies from patients with CHC vs controls. HCV nonstructural (NS) 3 and NS5A proteins were associated with fila, while core protein partially with fila and vim. Immunoprecipitation showed interactions among fila and NS3 and NS5A proteins. Cells treated with interferon-α showed a dose- and time-dependent decrease in CS and HCV proteins. NS proteins clustered at the perinuclear region following cytochalasin b treatment, whereas disperse cytoplasmic and perinuclear distribution was observed in the no-treatment group. This study demonstrates and signifies that changes occur in the expression of CS proteins in HCV-infected hepatocytes and, for the first time, shows the up-regulation and interaction of fila with HCV proteins. Association between CS and HCV proteins may have implications in future design of CS protein-targeted therapy for the treatment for HCV infection.

  4. Identification of New Synthetic Cannabinoid ADB-CHMINACA (MAB-CHMINACA) Metabolites in Human Hepatocytes.

    Science.gov (United States)

    Carlier, Jeremy; Diao, Xingxing; Sempio, Cristina; Huestis, Marilyn A

    2017-03-01

    ADB-CHMINACA (MAB-CHMINACA) is a new synthetic cannabinoid with high potency and many reported adverse events and fatalities. The drug is currently scheduled in several countries in Europe and the USA. Analytical methods need to be developed to confirm ADB-CHMINACA intake for clinical and forensic programs. For many synthetic cannabinoids, parent compound is not detectable in biological samples after intake, making the detection of metabolites the only way to prove consumption. Therefore, detection of ADB-CHMINACA metabolites in biological specimens is critical. Since there are currently no published data on ADB-CHMINACA metabolism, we aimed to identify its major metabolites. Cryopreserved human hepatocytes were incubated with 10 μmol/L ADB-CHMINACA for 3 h. Incubations were analyzed with liquid chromatography on a biphenyl column, high resolution tandem mass spectrometry (orbitrap), and metabolite identification software. A reference standard of six commercially available potential metabolites was simultaneously analyzed under the same conditions to allow correct assignment of isomers. We detected ten major metabolites. Biotransformations mainly occurred at the cyclohexylmethyl tail of the compound, as also observed with structural analogs' metabolism. Minor reactions also occurred at the tert-butyl chain. Only two analytical standards of potential metabolites matched an actual metabolite detected in hepatocyte incubations. We recommend A9 (ADB-CHMINACA hydroxycyclohexylmethyl), A4 (ADB-CHMINACA 4″-hydroxycyclohexyl), and A6 (ADB-CHMINACA hydroxycyclohexylmethyl) as metabolite targets to document ADB-CHMINACA intake in clinical and forensic cases. Additionally, these results will guide analytical standard manufacturers to better provide suitable references for further studies on ADB-CHMINACA metabolism.

  5. Human hepatocyte growth factor (hHGF-modified hepatic oval cells improve liver transplant survival.

    Directory of Open Access Journals (Sweden)

    Zhu Li

    Full Text Available Despite progress in the field of immunosuppression, acute rejection is still a common postoperative complication following liver transplantation. This study aims to investigate the capacity of the human hepatocyte growth factor (hHGF in modifying hepatic oval cells (HOCs administered simultaneously with orthotopic liver transplantation as a means of improving graft survival. HOCs were activated and isolated using a modified 2-acetylaminofluorene/partial hepatectomy (2-AAF/PH model in male Lewis rats. A HOC line stably expressing the HGF gene was established following stable transfection of the pBLAST2-hHGF plasmid. Our results demonstrated that hHGF-modified HOCs could efficiently differentiate into hepatocytes and bile duct epithelial cells in vitro. Administration of HOCs at the time of liver transplantation induced a wider distribution of SRY-positive donor cells in liver tissues. Administration of hHGF-HOC at the time of transplantation remarkably prolonged the median survival time and improved liver function for recipients compared to these parameters in the other treatment groups (P<0.05. Moreover, hHGF-HOC administration at the time of liver transplantation significantly suppressed elevation of interleukin-2 (IL-2, tumor necrosis factor-α (TNF-α and interferon-γ (IFN-γ levels while increasing the production of IL-10 and TGF-β1 (P<0.05. HOC or hHGF-HOC administration promoted cell proliferation, reduced cell apoptosis, and decreased liver allograft rejection rates. Furthermore, hHGF-modified HOCs more efficiently reduced acute allograft rejection (P<0.05 versus HOC transplantation only. Our results indicate that the combination of hHGF-modified HOCs with liver transplantation decreased host anti-graft immune responses resulting in a reduction of allograft rejection rates and prolonging graft survival in recipient rats. This suggests that HOC-based cell transplantation therapies can be developed as a means of treating severe liver

  6. Application of three-dimensional culture conditions to human embryonic stem cell-derived definitive endoderm cells enhances hepatocyte differentiation and functionality.

    Science.gov (United States)

    Ramasamy, Thamil Selvee; Yu, Jason S L; Selden, Clare; Hodgson, Humphery; Cui, Wei

    2013-02-01

    Human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) provide an unlimited source for the generation of human hepatocytes, owing to their indefinite self-renewal and pluripotent properties. Both hESC-/iPSC-derived hepatocytes hold great promise in treating liver diseases as potential candidates for cell replacement therapies or as an in vitro platform to conduct new drug trials. It has been previously demonstrated that the initiation of hESC differentiation in monolayer cultures increases the generation of definitive endoderm (DE) and subsequently of hepatocyte differentiation. However, monolayer culture may hinder the maturation of hESC-derived hepatocytes, since such two-dimensional (2D) conditions do not accurately reflect the complex nature of three-dimensional (3D) hepatocyte specification in vivo. Here, we report the sequential application of 2D and 3D culture systems to differentiate hESCs to hepatocytes. Human ESCs were initially differentiated in a monolayer culture to DE cells, which were then inoculated into Algimatrix scaffolds. Treatments of hESC-DE cells with a ROCK inhibitor before and after inoculation dramatically enhanced their survival and the formation of spheroids, which are distinct from HepG2 carcinoma cells. In comparison with monolayer culture alone, sequential 2D and 3D cultures significantly improved hepatocyte differentiation and function. Our results demonstrate that hESC-DE cells can be incorporated into Algimatrix 3D culture systems to enhance hepatocyte differentiation and function.

  7. Measurement of Blood Coagulation Factor Synthesis in Cultures of Human Hepatocytes.

    Science.gov (United States)

    Heinz, Stefan; Braspenning, Joris

    2015-01-01

    An important function of the liver is the synthesis and secretion of blood coagulation factors. Within the liver, hepatocytes are involved in the synthesis of most blood coagulation factors, such as fibrinogen, prothrombin, factor V, VII, IX, X, XI, XII, as well as protein C and S, and antithrombin, whereas liver sinusoidal endothelial cells produce factor VIII and von Willebrand factor. Here, we describe methods for the detection and quantification of most blood coagulation factors in hepatocytes in vitro. Hepatocyte cultures indeed provide a valuable tool to study blood coagulation factors. In addition, the generation and expansion of hepatocytes or hepatocyte-like cells may be used in future for cell-based therapies of liver diseases, including blood coagulation factor deficiencies.

  8. Does prostaglandin-E1 modulate d-galactosamine induced cell death in primary culture of human hepatocytes?

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    Sameh S. Tawfik

    2015-12-01

    Full Text Available Background: Cell death pathway can occur under physiological or pathological conditions. In vitro and in vivo studies have shown that d-galactosamine (DGA induces hepatocyte damage. Objective: The present study aims to evaluate the protective effect of prostaglandin E1 (PGE1 on DGA-induced apoptosis, necrosis and oxidative stress in primary culture of human hepatocytes. Methods: Normal human hepatocytes were obtained from the safety margin of liver specimens, removed during hepatectomy operation to liver cancer patients, and isolated using the classical collagenase perfusion method. After culture stabilization, PGE1 (1 μM was added 2 h before DGA (5 mM. Cultures were maintained for 24 h before the parameters for apoptosis, necrosis and oxidative stress were measured. Apoptosis was studied by DNA-fragmentation, neutral (nSMase and acid (aSMase sphingomyelinase and caspase-3 activity. Necrosis was investigated by lactate dehydrogenase (LDH and transaminases (ALT & AST enzymes. The oxidative stress was assessed by malondialdehyde (MDA, glutathione (GSH, oxidized glutathione (GSSH, glutathione S-transferase (GST, glutathione peroxidase (GSPx, catalase (CAT, superoxide dismutase (SOD and nitric oxide (NO. Results: The hepatotoxin DGA induced apoptosis and enhanced all parameters related to necrosis and intracellular oxidative stress. On the other hand, PGE1 reduced the measured values for the parameters indicative for the DAG induced apoptosis, necrosis and oxidative stress. In addition, PGE1 proved also to prevent GSH depletion. The obtained results provided evidences for the biochemical hepatotoxic effects of DGA (5 mM especially through the induction of apoptosis, necrosis and oxidative stress alterations in the cultured human hepatocytes. Conclusion: PGE1 could be a useful protective treatment against DGA-induced hepatocyte cell death.

  9. Histone deacetylase inhibitor valproic acid promotes the differentiation of human induced pluripotent stem cells into hepatocyte-like cells.

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    Yuki Kondo

    Full Text Available In this study, we aimed to elucidate the effects and mechanism of action of valproic acid on hepatic differentiation from human induced pluripotent stem cell-derived hepatic progenitor cells. Human induced pluripotent stem cells were differentiated into endodermal cells in the presence of activin A and then into hepatic progenitor cells using dimethyl sulfoxide. Hepatic progenitor cells were matured in the presence of hepatocyte growth factor, oncostatin M, and dexamethasone with valproic acid that was added during the maturation process. After 25 days of differentiation, cells expressed hepatic marker genes and drug-metabolizing enzymes and exhibited drug-metabolizing enzyme activities. These expression levels and activities were increased by treatment with valproic acid, the timing and duration of which were important parameters to promote differentiation from human induced pluripotent stem cell-derived hepatic progenitor cells into hepatocytes. Valproic acid inhibited histone deacetylase activity during differentiation of human induced pluripotent stem cells, and other histone deacetylase inhibitors also enhanced differentiation into hepatocytes. In conclusion, histone deacetylase inhibitors such as valproic acid can be used to promote hepatic differentiation from human induced pluripotent stem cell-derived hepatic progenitor cells.

  10. Troglitazone, but not rosiglitazone, damages mitochondrial DNA and induces mitochondrial dysfunction and cell death in human hepatocytes.

    Science.gov (United States)

    Rachek, Lyudmila I; Yuzefovych, Larysa V; Ledoux, Susan P; Julie, Neil L; Wilson, Glenn L

    2009-11-01

    Thiazolidinediones (TZDs), such as troglitazone (TRO) and rosiglitazone (ROSI), improve insulin resistance by acting as ligands for the nuclear receptor peroxisome proliferator-activated receptor-gamma (PPARgamma). TRO was withdrawn from the market because of reports of serious hepatotoxicity. A growing body of evidence suggests that TRO caused mitochondrial dysfunction and induction of apoptosis in human hepatocytes but its mechanisms of action remain unclear. We hypothesized that damage to mitochondrial DNA (mtDNA) is an initiating event involved in TRO-induced mitochondrial dysfunction and hepatotoxicity. Primary human hepatocytes were exposed to TRO and ROSI. The results obtained revealed that TRO, but not ROSI at equimolar concentrations, caused a substantial increase in mtDNA damage and decreased ATP production and cellular viability. The reactive oxygen species (ROS) scavenger, N-acetyl cystein (NAC), significantly diminished the TRO-induced cytotoxicity, suggesting involvement of ROS in TRO-induced hepatocyte cytotoxicity. The PPARgamma antagonist (GW9662) did not block the TRO-induced decrease in cell viability, indicating that the TRO-induced hepatotoxicity is PPARgamma-independent. Furthermore, TRO induced hepatocyte apoptosis, caspase-3 cleavage and cytochrome c release. Targeting of a DNA repair protein to mitochondria by protein transduction using a fusion protein containing the DNA repair enzyme Endonuclease III (EndoIII) from Escherichia coli, a mitochondrial translocation sequence (MTS) and the protein transduction domain (PTD) from HIV-1 TAT protein protected hepatocytes against TRO-induced toxicity. Overall, our results indicate that significant mtDNA damage caused by TRO is a prime initiator of the hepatoxicity caused by this drug.

  11. Inhibition of preS1-hepatocyte interaction by an array of recombinant human antibodies from naturally recovered individuals

    DEFF Research Database (Denmark)

    Sankhyan, Anurag; Sharma, Chandresh; Dutta, Durgashree;

    2016-01-01

    binding signature, interacting with different amino acids within the preS1-peptide region. Ability to prevent binding of the preS1 protein (N-terminus 60a.a.) to HepG2 cells stably expressing hNTCP (HepG2-hNTCP-C4 cells), the HBV receptor on human hepatocytes was taken as a surrogate marker...

  12. Mapping the Cell-Surface N-Glycoproteome of Human Hepatocytes Reveals Markers for Selecting a Homogeneous Population of iPSC-Derived Hepatocytes.

    Science.gov (United States)

    Mallanna, Sunil K; Cayo, Max A; Twaroski, Kirk; Gundry, Rebekah L; Duncan, Stephen A

    2016-09-13

    When comparing hepatic phenotypes between iPSC-derived hepatocyte-like cells from different liver disease patients, cell heterogeneity can confound interpretation. We proposed that homogeneous cell populations could be generated by fluorescence-activated cell sorting (FACS). Using cell-surface capture proteomics, we identified a total of 300 glycoproteins on hepatocytes. Analyses of the expression profiles during the differentiation of iPSCs revealed that SLC10A1, CLRN3, and AADAC were highly enriched during the final stages of hepatocyte differentiation. FACS purification of hepatocyte-like cells expressing SLC10A1, CLRN3, or AADAC demonstrated enrichment of cells with hepatocyte characteristics. Moreover, transcriptome analyses revealed that cells expressing the liver gene regulatory network were enriched while cells expressing a pluripotent stem cell network were depleted. In conclusion, we report an extensive catalog of cell-surface N-linked glycoproteins expressed in primary hepatocytes and identify cell-surface proteins that facilitate the purification of homogeneous populations of iPSC-derived hepatocyte-like cells.

  13. Mapping the Cell-Surface N-Glycoproteome of Human Hepatocytes Reveals Markers for Selecting a Homogeneous Population of iPSC-Derived Hepatocytes

    Directory of Open Access Journals (Sweden)

    Sunil K. Mallanna

    2016-09-01

    Full Text Available When comparing hepatic phenotypes between iPSC-derived hepatocyte-like cells from different liver disease patients, cell heterogeneity can confound interpretation. We proposed that homogeneous cell populations could be generated by fluorescence-activated cell sorting (FACS. Using cell-surface capture proteomics, we identified a total of 300 glycoproteins on hepatocytes. Analyses of the expression profiles during the differentiation of iPSCs revealed that SLC10A1, CLRN3, and AADAC were highly enriched during the final stages of hepatocyte differentiation. FACS purification of hepatocyte-like cells expressing SLC10A1, CLRN3, or AADAC demonstrated enrichment of cells with hepatocyte characteristics. Moreover, transcriptome analyses revealed that cells expressing the liver gene regulatory network were enriched while cells expressing a pluripotent stem cell network were depleted. In conclusion, we report an extensive catalog of cell-surface N-linked glycoproteins expressed in primary hepatocytes and identify cell-surface proteins that facilitate the purification of homogeneous populations of iPSC-derived hepatocyte-like cells.

  14. Survival of primary human hepatocytes and death of induced pluripotent stem cells in media lacking glucose and arginine.

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    Minoru Tomizawa

    Full Text Available BACKGROUND: Tumorigenicity is an associated risk for transplantation of hepatocytes differentiated from human induced pluripotent stem (hiPS cells. Hepatocytes express the enzymes galactokinase and ornithine transcarbamylase (OTC to aid in their own survival. However, hiPS cells do not express these enzymes, and therefore, are not be expected to survive in a medium containing galactose and ornithine and lacking glucose and arginine. MATERIALS AND METHODS: Real-time quantitative polymerase chain reaction (PCR was performed to analyze the expression of galactokinase 1 (GALK11 and GALK2, ornithine carbamyltransferase, and phenylalanine hydroxylase (PAH. The hiPS cell line 201B7 was cultured in hepatocyte selection medium (HSM, which lacks glucose and arginine but contains galactose and ornithine. Furthermore, microscopic analysis of the cultured cells was performed after hematoxylin and eosin (H&E staining, terminal deoxynucleotidyl transferase (TdT-mediated dUTP nick end labeling (TUNEL. The hiPS cells were immunostained to assess their pluripotency in HSM. In addition, the primary human hepatocytes were cultured with or without hiPS cells in HSM. RESULTS: The expression levels of GALK1, GALK2, OTC, and PAH in 201B7 were 22.2±5.0 (average ± standard deviation, 14.2% ±1.1%, 1.2% ±0.2%, and 8.4% ±0.7% respectively, compared with those in the adult liver. The hiPS cell population diminished when cultured in HSM and completely disappeared after 3 days. The cultured cells showed condensation or fragmentation of their nuclei, thereby suggesting apoptosis. TUNEL staining confirmed that the cells had undergone apoptosis. The 201B7 cells were positive for Nanog, SSEA-4, and TRA-1-60. The primary human hepatocytes survived when cultured alone in HSM and when co-cultured with hiPS cells. CONCLUSION: Therefore, HSM is and ideal medium for eliminating hiPS cells and purifying hepatocytes without inducing any damage.

  15. Effects of Fluoride on Lipid Peroxidation, DNA Damage and Apoptosis in Human Embryo Hepatocytes

    Institute of Scientific and Technical Information of China (English)

    AI-GUO WANG; TAO XIA; QI-LONG CHU; MING ZHANG; FANG LIU; XUE-MIN CHEN; KE-DI YANG

    2004-01-01

    Objective To investigate the effects of fluoride on lipid peroxidation, DNA damage and apoptosis in human embryo hepatocyte L-02 cells. Methods Lipid peroxide (LPO) level, reduced glutathione (GSH) content, DNA damage, apoptosis, and cell cycle analysis were measured after in vitro cultured L-02 cells were exposed to sodium fluoride at different doses (40 μg/mL, 80 μg/mL, and 160 μg/mL) for 24 hours. Results Fluoride caused an increase of LPO levels and a decrease of GSH content in L-02 cells. There appeared to be an obvious dose-effect relationship between the fluoride concentration and the observed changes. Fluoride also caused DNA damage and apoptosis and increased the cell number in S phase of cell cycle in the cells tested. There was a statistically significant difference in DNA damage and apoptosis when comparing the high dose of fluoride treated cells with the low dose of fluoride treated cells. Conclusion Fluoride can cause lipid peroxidation, DNA damage, and apoptosis in the L-02 cell experimental model and there is a significant positive correlation between fluoride concentration and these pathological changes.

  16. Evidence of a DHA Signature in the Lipidome and Metabolome of Human Hepatocytes

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    Veronica Ghini

    2017-02-01

    Full Text Available Cell supplementation with bioactive molecules often causes a perturbation in the whole intracellular environment. Omics techniques can be applied for the assessment of this perturbation. In this study, the overall effect of docosahexaenoic acid (DHA supplementation on cultured human hepatocyte lipidome and metabolome has been investigated using nuclear magnetic resonance (NMR in combination with traditional techniques. The effect of two additional bioactives sharing with DHA the lipid-lowering effect—propionic acid (PRO and protocatechuic acid (PCA—has also been evaluated in the context of possible synergism. NMR analysis of the cell lipid extracts showed that DHA supplementation, alone or in combination with PCA or PRO, strongly altered the cell lipid profile. The perfect discrimination between cells receiving DHA (alone or in combination and the other cells reinforced the idea of a global rearrangement of the lipid environment induced by DHA. Notably, gas chromatography and fluorimetric analyses confirmed the strong discrimination obtained by NMR. The DHA signature was evidenced not only in the cell lipidome, but also in the metabolome. Results reported herein indicate that NMR, combined with other techniques, represents a fundamental approach to studying the effect of bioactive supplementation, particularly in the case of molecules with a broad spectrum of mechanisms of action.

  17. Evidence of a DHA Signature in the Lipidome and Metabolome of Human Hepatocytes

    Science.gov (United States)

    Ghini, Veronica; Di Nunzio, Mattia; Tenori, Leonardo; Valli, Veronica; Danesi, Francesca; Capozzi, Francesco; Luchinat, Claudio; Bordoni, Alessandra

    2017-01-01

    Cell supplementation with bioactive molecules often causes a perturbation in the whole intracellular environment. Omics techniques can be applied for the assessment of this perturbation. In this study, the overall effect of docosahexaenoic acid (DHA) supplementation on cultured human hepatocyte lipidome and metabolome has been investigated using nuclear magnetic resonance (NMR) in combination with traditional techniques. The effect of two additional bioactives sharing with DHA the lipid-lowering effect—propionic acid (PRO) and protocatechuic acid (PCA)—has also been evaluated in the context of possible synergism. NMR analysis of the cell lipid extracts showed that DHA supplementation, alone or in combination with PCA or PRO, strongly altered the cell lipid profile. The perfect discrimination between cells receiving DHA (alone or in combination) and the other cells reinforced the idea of a global rearrangement of the lipid environment induced by DHA. Notably, gas chromatography and fluorimetric analyses confirmed the strong discrimination obtained by NMR. The DHA signature was evidenced not only in the cell lipidome, but also in the metabolome. Results reported herein indicate that NMR, combined with other techniques, represents a fundamental approach to studying the effect of bioactive supplementation, particularly in the case of molecules with a broad spectrum of mechanisms of action. PMID:28208746

  18. Similarities in the immunoglobulin response and VH gene usage in rhesus monkeys and humans exposed to porcine hepatocytes

    Directory of Open Access Journals (Sweden)

    Borie Dominic C

    2006-03-01

    Full Text Available Abstract Background The use of porcine cells and organs as a source of xenografts for human patients would vastly increase the donor pool; however, both humans and Old World primates vigorously reject pig tissues due to xenoantibodies that react with the polysaccharide galactose α (1,3 galactose (αGal present on the surface of many porcine cells. We previously examined the xenoantibody response in patients exposed to porcine hepatocytes via treatment(s with bioartficial liver devices (BALs, composed of porcine cells in a support matrix. We determined that xenoantibodies in BAL-treated patients are predominantly directed at porcine αGal carbohydrate epitopes, and are encoded by a small number of germline heavy chain variable region (VH immunoglobulin genes. The studies described in this manuscript were designed to identify whether the xenoantibody responses and the IgVH genes encoding antibodies to porcine hepatocytes in non-human primates used as preclinical models are similar to those in humans. Adult non-immunosuppressed rhesus monkeys (Macaca mulatta were injected intra-portally with porcine hepatocytes or heterotopically transplanted with a porcine liver lobe. Peripheral blood leukocytes and serum were obtained prior to and at multiple time points after exposure, and the immune response was characterized, using ELISA to evaluate the levels and specificities of circulating xenoantibodies, and the production of cDNA libraries to determine the genes used by B cells to encode those antibodies. Results Xenoantibodies produced following exposure to isolated hepatocytes and solid organ liver grafts were predominantly encoded by genes in the VH3 family, with a minor contribution from the VH4 family. Immunoglobulin heavy-chain gene (VH cDNA library screening and gene sequencing of IgM libraries identified the genes as most closely-related to the IGHV3-11 and IGHV4-59 germline progenitors. One of the genes most similar to IGHV3-11, VH3-11cyno, has

  19. Featured Article: Isolation, characterization, and cultivation of human hepatocytes and non-parenchymal liver cells

    Science.gov (United States)

    Pfeiffer, Elisa; Kegel, Victoria; Zeilinger, Katrin; Hengstler, Jan G; Nüssler, Andreas K; Seehofer, Daniel

    2015-01-01

    Primary human hepatocytes (PHH) are considered to be the gold standard for in vitro testing of xenobiotic metabolism and hepatotoxicity. However, PHH cultivation in 2D mono-cultures leads to dedifferentiation and a loss of function. It is well known that hepatic non-parenchymal cells (NPC), such as Kupffer cells (KC), liver endothelial cells (LEC), and hepatic stellate cells (HSC), play a central role in the maintenance of PHH functions. The aims of the present study were to establish a protocol for the simultaneous isolation of human PHH and NPC from the same tissue specimen and to test their suitability for in vitro co-culture. Human PHH and NPC were isolated from tissue obtained by partial liver resection by a two-step EDTA/collagenase perfusion technique. The obtained cell fractions were purified by Percoll density gradient centrifugation. KC, LEC, and HSC contained in the NPC fraction were separated using specific adherence properties and magnetic activated cell sorting (MACS®). Identified NPC revealed a yield of 1.9 × 106 KC, 2.7 × 105 LEC and 4.7 × 105 HSC per gram liver tissue, showing viabilities >90%. Characterization of these NPC showed that all populations went through an activation process, which influenced the cell fate. The activation of KC strongly depended on the tissue quality and donor anamnesis. KC became activated in culture in association with a loss of viability within 4–5 days. LEC lost specific features during culture, while HSC went through a transformation process into myofibroblasts. The testing of different culture conditions for HSC demonstrated that they can attenuate, but not prevent dedifferentiation in vitro. In conclusion, the method described allows the isolation and separation of PHH and NPC in high quality and quantity from the same donor. PMID:25394621

  20. Inducing effects of hepatocyte growth factor on the expression of vascular endothelial growth factor in human colorectal carcinoma cells through MEK and PI3K signaling pathways

    Institute of Scientific and Technical Information of China (English)

    ZHANG Yu-hua; WEI Wei; XU Hao; WANG Yan-yan; WU Wen-xi

    2007-01-01

    Background Vascular endothelial growth factor plays a key role in human colorectal carcinoma invasion and metastasis. However, the regulation mechanism remains unknown. Recent studies have shown that several cytokines can regulate the expression of vascular endothelial growth factor in tumor cells. In this study, we investigated whether hepatocyte growth factor can regulate the expression of vascular endothelial growth factor in colorectal carcinoma cells.Methods Hepatocyte growth factor and vascular endothelial growth factor in human serum were measured by ELISA.The mRNA level of vascular endothelial growth factor was analyzed by reverse transcription-PCR. Western blot assay was performed to evaluate levels of c-Met and several other proteins involved in the MAPK and PI3K signaling pathways in colorectal carcinoma cells.Results Serum hepatocyte growth factor and vascular endothelial growth factor were significantly increased in colorectal carcinoma subjects. In vitro extraneous hepatocyte growth factor markedly increased protein and mRNA levels of vascular endothelial growth factor in colorectal carcinoma cells. Hepatocyte growth factor induced phosphorylation of c-Met, ERK1/2 and AKT in a dose-dependent manner. Specific inhibitors on MEK and PI3K inhibited the hepatocyte growth factor-induced expression of vascular endothelial growth factor in colorectal carcinoma cells.Conclusion This present study indicates that hepatocyte growth factor upregulates the expression of vascular endothelial growth factor in colorectal carcinoma cells via the MEK/ERK and PI3K/AKT signaling pathways.

  1. Differential Expression Profile of lncRNAs from Primary Human Hepatocytes Following DEET and Fipronil Exposure.

    Science.gov (United States)

    Mitchell Iii, Robert D; Wallace, Andrew D; Hodgson, Ernest; Roe, R Michael

    2017-10-07

    While the synthesis and use of new chemical compounds is at an all-time high, the study of their potential impact on human health is quickly falling behind, and new methods are needed to assess their impact. We chose to examine the effects of two common environmental chemicals, the insect repellent N,N-diethyl-m-toluamide (DEET) and the insecticide fluocyanobenpyrazole (fipronil), on transcript levels of long non-protein coding RNAs (lncRNAs) in primary human hepatocytes using a global RNA-Seq approach. While lncRNAs are believed to play a critical role in numerous important biological processes, many still remain uncharacterized, and their functions and modes of action remain largely unclear, especially in relation to environmental chemicals. RNA-Seq showed that 100 µM DEET significantly increased transcript levels for 2 lncRNAs and lowered transcript levels for 18 lncRNAs, while fipronil at 10 µM increased transcript levels for 76 lncRNAs and decreased levels for 193 lncRNAs. A mixture of 100 µM DEET and 10 µM fipronil increased transcript levels for 75 lncRNAs and lowered transcript levels for 258 lncRNAs. This indicates a more-than-additive effect on lncRNA transcript expression when the two chemicals were presented in combination versus each chemical alone. Differentially expressed lncRNA genes were mapped to chromosomes, analyzed by proximity to neighboring protein-coding genes, and functionally characterized via gene ontology and molecular mapping algorithms. While further testing is required to assess the organismal impact of changes in transcript levels, this initial analysis links several of the dysregulated lncRNAs to processes and pathways critical to proper cellular function, such as the innate and adaptive immune response and the p53 signaling pathway.

  2. Effect of remifentanil on mitochondrial oxygen consumption of cultured human hepatocytes.

    Directory of Open Access Journals (Sweden)

    Siamak Djafarzadeh

    remifentanil increases cellular respiration of human hepatocytes and prevents TNF-α-induced mitochondrial dysfunction. The results were not explained by uncoupling of mitochondrial respiration.

  3. HCV core protein represses the apoptosis and improves the autophagy of human hepatocytes

    Science.gov (United States)

    Liu, Changhong; Qu, Aihua; Han, Xiaochun; Wang, Yiguo

    2015-01-01

    Objectives: This study aims to investigate the influence on human hepatocytes apoptosis and autophagy by the hepatitis C virus (HCV) core protein. Methods: QSG-7701, a human-derived non-neoplastic liver cell line, was transfected with PIRES-core vector that was a eukaryotic vector to express HCV core protein. Fluorescence microscope was used to observe the changes of nuclei in apoptosis cells by Annex in V-FITC/PI double staining. Flow cytometry was applied to detect the rate of cell apoptosis. Western blotting was used to detect the expression of HCV core protein, transcription factor nuclear factor-kappa B (NF-κB), autophagic biomarker microtubule associated protein 1 light chain 3 (LC3), and Beclin-1. Results: The apoptosis rate was significantly lower (P < 0.05) in QSG7701/core group (transfected with PIRES-core vector, (1.34±0.07)%) than in QSG7701 group (no transfection, (2.35±0.11)%) and in QSG7701 QSG7701/pcDNA3.1 group (transfected with pcDNA3.1 vector, (2.58±0.1)%). NF-κB expression was up-expressed in QSG7701/core group than in QSG7701/pcDNA3.1 group and QSG7701 group (P < 0.05). LC3-II expression and Beclin-1 expression was significant higher in QSG7701/core group than in the QSG7701/pcDNA3.1 group and QSG7701 group (P < 0.05). Conclusion: HCV core protein can repress the apoptosis and improve the autophagy of QSG7701 through up-regulating NF-κB and Beclin-1 expression. PMID:26629077

  4. Effect of Remifentanil on Mitochondrial Oxygen Consumption of Cultured Human Hepatocytes

    Science.gov (United States)

    Djafarzadeh, Siamak; Vuda, Madhusudanarao; Takala, Jukka; Jakob, Stephan M.

    2012-01-01

    increases cellular respiration of human hepatocytes and prevents TNF-α-induced mitochondrial dysfunction. The results were not explained by uncoupling of mitochondrial respiration. PMID:23028840

  5. Pharmacokinetics and effects on serum cholinesterase activities of organophosphorus pesticides acephate and chlorpyrifos in chimeric mice transplanted with human hepatocytes.

    Science.gov (United States)

    Suemizu, Hiroshi; Sota, Shigeto; Kuronuma, Miyuki; Shimizu, Makiko; Yamazaki, Hiroshi

    2014-11-01

    Organophosphorus pesticides acephate and chlorpyrifos in foods have potential to impact human health. The aim of the current study was to investigate the pharmacokinetics of acephate and chlorpyrifos orally administered at lowest-observed-adverse-effect-level doses in chimeric mice transplanted with human hepatocytes. Absorbed acephate and its metabolite methamidophos were detected in serum from wild type mice and chimeric mice orally administered 150mg/kg. Approximately 70% inhibition of cholinesterase was evident in plasma of chimeric mice with humanized liver (which have higher serum cholinesterase activities than wild type mice) 1day after oral administrations of acephate. Adjusted animal biomonitoring equivalents from chimeric mice studies were scaled to human biomonitoring equivalents using known species allometric scaling factors and in vitro metabolic clearance data with a simple physiologically based pharmacokinetic (PBPK) model. Estimated plasma concentrations of acephate and chlorpyrifos in humans were consistent with reported concentrations. Acephate cleared similarly in humans and chimeric mice but accidental/incidental overdose levels of chlorpyrifos cleared (dependent on liver metabolism) more slowly from plasma in humans than it did in mice. The data presented here illustrate how chimeric mice transplanted with human hepatocytes in combination with a simple PBPK model can assist evaluations of toxicological potential of organophosphorus pesticides.

  6. Human recombinant vascular endothelial growth factor reduces necrosis and enhances hepatocyte regeneration in a mouse model of acetaminophen toxicity.

    Science.gov (United States)

    Donahower, Brian C; McCullough, Sandra S; Hennings, Leah; Simpson, Pippa M; Stowe, Cindy D; Saad, Ali G; Kurten, Richard C; Hinson, Jack A; James, Laura P

    2010-07-01

    We reported previously that vascular endothelial growth factor (VEGF) was increased in acetaminophen (APAP) toxicity in mice and treatment with a VEGF receptor inhibitor reduced hepatocyte regeneration. The effect of human recombinant VEGF (hrVEGF) on APAP toxicity in the mouse was examined. In early toxicity studies, B6C3F1 mice received hrVEGF (50 microg s.c.) or vehicle 30 min before receiving APAP (200 mg/kg i.p.) and were sacrificed at 2, 4, and 8 h. Toxicity was comparable at 2 and 4 h, but reduced in the APAP/hrVEGF mice at 8 h (p toxicity and increased hepatocyte regeneration in APAP toxicity in the mouse. Attenuation of sinusoidal cell endothelial dysfunction and changes in neutrophil dynamics may be operant mechanisms in the hepatoprotection mediated by hrVEGF in APAP toxicity.

  7. Expression kinetics of hepatic progenitor markers in cellular models of human liver development recapitulating hepatocyte and biliary cell fate commitment.

    Science.gov (United States)

    Chaudhari, Pooja; Tian, Lipeng; Deshmukh, Abhijeet; Jang, Yoon-Young

    2016-09-01

    Due to the limitations of research using human embryos and the lack of a biological model of human liver development, the roles of the various markers associated with liver stem or progenitor cell potential in humans are largely speculative, and based on studies utilizing animal models and certain patient tissues. Human pluripotent stem cell-based in vitro multistage hepatic differentiation systems may serve as good surrogate models for mimicking normal human liver development, pathogenesis and injury/regeneration studies. Here, we describe the implications of various liver stem or progenitor cell markers and their bipotency (i.e. hepatocytic- and biliary-epithelial cell differentiation), based on the pluripotent stem cell-derived model of human liver development. Future studies using the human cellular model(s) of liver and biliary development will provide more human relevant biological and/or pathological roles of distinct markers expressed in heterogeneous liver stem/progenitor cell populations.

  8. Inhibition of bile salt transport by drugs associated with liver injury in primary hepatocytes from human, monkey, dog, rat, and mouse.

    Science.gov (United States)

    Zhang, Jie; He, Kan; Cai, Lining; Chen, Yu-Chuan; Yang, Yifan; Shi, Qin; Woolf, Thomas F; Ge, Weigong; Guo, Lei; Borlak, Jürgen; Tong, Weida

    2016-08-05

    Interference of bile salt transport is one of the underlying mechanisms for drug-induced liver injury (DILI). We developed a novel bile salt transport activity assay involving in situ biosynthesis of bile salts from their precursors in primary human, monkey, dog, rat, and mouse hepatocytes in suspension as well as LC-MS/MS determination of extracellular bile salts transported out of hepatocytes. Glycine- and taurine-conjugated bile acids were rapidly formed in hepatocytes and effectively transported into the extracellular medium. The bile salt formation and transport activities were time‒ and bile-acid-concentration‒dependent in primary human hepatocytes. The transport activity was inhibited by the bile salt export pump (BSEP) inhibitors ketoconazole, saquinavir, cyclosporine, and troglitazone. The assay was used to test 86 drugs for their potential to inhibit bile salt transport activity in human hepatocytes, which included 35 drugs associated with severe DILI (sDILI) and 51 with non-severe DILI (non-sDILI). Approximately 60% of the sDILI drugs showed potent inhibition (with IC50 values drugs showed this strength of inhibition in primary human hepatocytes and these drugs are associated only with cholestatic and mixed hepatocellular cholestatic (mixed) injuries. The sDILI drugs, which did not show substantial inhibition of bile salt transport activity, are likely to be associated with immune-mediated liver injury. Twenty-four drugs were also tested in monkey, dog, rat and mouse hepatocytes. Species differences in potency were observed with mouse being less sensitive than other species to inhibition of bile salt transport. In summary, a novel assay has been developed using hepatocytes in suspension from human and animal species that can be used to assess the potential for drugs and/or drug-derived metabolites to inhibit bile salt transport and/or formation activity. Drugs causing sDILI, except those by immune-mediated mechanism, are highly associated with potent

  9. Transcriptional regulation of the human Liver X Receptor α gene by Hepatocyte Nuclear Factor 4α

    Energy Technology Data Exchange (ETDEWEB)

    Theofilatos, Dimitris; Anestis, Aristomenis [University of Crete Medical School and Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology of Hellas, Heraklion, 71003, Crete (Greece); Hashimoto, Koshi [Department of Preemptive Medicine and Metabolism, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-city, Tokyo, 113-8510 (Japan); Kardassis, Dimitris, E-mail: kardasis@imbb.forth.gr [University of Crete Medical School and Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology of Hellas, Heraklion, 71003, Crete (Greece)

    2016-01-15

    Liver X Receptors (LXRs) are sterol-activated transcription factors that play major roles in cellular cholesterol homeostasis, HDL biogenesis and reverse cholesterol transport. The aim of the present study was to investigate the mechanisms that control the expression of the human LXRα gene in hepatic cells. A series of reporter plasmids containing consecutive 5′ deletions of the hLXRα promoter upstream of the luciferase gene were constructed and the activity of each construct was measured in HepG2 cells. This analysis showed that the activity of the human LXRα promoter was significantly reduced by deleting the −111 to −42 region suggesting the presence of positive regulatory elements in this short proximal fragment. Bioinformatics data including motif search and ChIP-Seq revealed the presence of a potential binding motif for Hepatocyte Nuclear Factor 4 α (HNF-4α) in this area. Overexpression of HNF-4α in HEK 293T cells increased the expression of all LXRα promoter constructs except −42/+384. In line, silencing the expression of endogenous HNF-4α in HepG2 cells was associated with reduced LXRα protein levels and reduced activity of the −111/+384 LXRα promoter but not of the −42/+384 promoter. Using ChiP assays in HepG2 cells combined with DNAP assays we mapped the novel HNF-4α specific binding motif (H4-SBM) in the −50 to −40 region of the human LXRα promoter. A triple mutation in this H4-SBM abolished HNF-4α binding and reduced the activity of the promoter to 65% relative to the wild type. Furthermore, the mutant promoter could not be transactivated by HNF-4α. In conclusion, our data indicate that HNF-4α may have a wider role in cell and plasma cholesterol homeostasis by controlling the expression of LXRα in hepatic cells. - Highlights: • The human LXRα promoter contains a HNF-4α specific binding motif in the proximal −50/−40 region. • Mutations in this motif abolished HNF4α binding and transactivation of the h

  10. A putative role of micro RNA in regulation of cholesterol 7α-hydroxylase expression in human hepatocytes[S

    OpenAIRE

    Song, Kwang-Hoon; Li, Tiangang; Owsley, Erika; Chiang, John Y. L.

    2010-01-01

    Cholesterol 7α-hydroxylase (CYP7A1) plays a critical role in regulation of bile acid synthesis in the liver. CYP7A1 mRNAs have very short half-lives, and bile acids destabilize CYP7A1 mRNA via the 3′-untranslated region (3′-UTR). However, the underlying mechanism of translational regulation of CYP7A1 mRNA remains unknown. Screening of a human micro RNA (miRNA) microarray has identified five differentially expressed miRNAs in human primary hepatocytes treated with chenodeoxycholic acid, GW4064...

  11. Co-culture of primary human tumor hepatocytes from patients with hepatocellular carcinoma with autologous peripheral blood mononuclear cells: study of their in vitro immunological interactions

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    Doumba Polyxeni P

    2013-01-01

    Full Text Available Abstract Background Many studies have suggested that the immune response may play a crucial role in the progression of hepatocellular carcinoma (HCC. Therefore, our aim was to establish a (i functional culture of primary human tumor hepatocytes and non-tumor from patients with hepatocellular carcinoma (HCC and (ii a co-culture system of HCC and non-HCC hepatocytes with autologous peripheral blood mononuclear cells (PBMCs in order to study in vitro cell-to-cell interactions. Methods Tumor (HCC and non-tumor (non-HCC hepatocytes were isolated from the liver resection specimens of 11 patients operated for HCC, while PBMCs were retrieved immediately prior to surgery. Four biopsies were obtained from patients with no liver disease who had surgery for non malignant tumor (normal hepatocytes. Hepatocytes were either cultured alone (monoculture or co-cultured with PBMCs. Flow cytometry measurements for MHC class II expression, apoptosis, necrosis and viability (7AAD were performed 24 h, 48 h and 72 h in co-culture and monocultures. Results HCC and non-HCC hepatocytes exhibited increased MHC-II expression at 48h and 72h in co-culture with PBMCs as compared to monoculture, with MHC II-expressing HCC hepatocytes showing increased viability at 72 h. PBMCs showed increased MHC-II expression (activation in co-culture with HCC as compared to non-HCC hepatocytes at all time points. Moreover, CD8+ T cells had significantly increased apoptosis and necrosis at 48h in co-culture with HCC hepatocytes as compared to monocultures. Interestingly, MHC-II expression on both HCC and non-HCC hepatocytes in co-culture was positively correlated with the respective activated CD8+ T cells. Conclusions We have established an in vitro co-culture model to study interactions between autologous PBMCs and primary HCC and non-HCC hepatocytes. This direct interaction leads to increased antigen presenting ability of HCC hepatocytes, activation of PBMCs with a concomitant apoptosis of

  12. Effect of recombinant human growth hormone on age-related hepatocyte changes in old male and female Wistar rats.

    Science.gov (United States)

    Castillo, Carmen; Salazar, Veronica; Ariznavarreta, Carmen; Vara, Elena; Tresguerres, Jesus A F

    2004-10-01

    Aging induces changes in several organs, such as the liver, and this process might be due to damage caused by free radicals and inflammatory mediators. The growth hormone (GH)/insulin-like growth factor-1 (IGF-1) axis shows a reduction with age, and this fact could be associated with some age-related changes. The aim of this study was to investigate the effect of GH administration on age-induced alterations in hepatocytes. Two and twenty two month-old male and female Wistar rats were used. Old rats were treated with human recombinant GH for 10 wk. At the end of the treatment, hepatocytes were isolated from the liver and cultured, and different parameters were measured in cells and medium. Plasma IGF-1 was also measured. Aging significantly decreased plasma IGF-1 in males. In females, plasma IGF-1 was also reduced, but not significantly. GH treatment restored plasma IGF-1 levels to values similar to young males. Aging was associated with a significant increase in lipid peroxidation (LPO), nitric oxide (NO), carbon monoxide (CO) and cyclic guanosyl-monophosphate (cGMP), as well as a reduction in adenosyl triphosphate (ATP) and phosphatidylcholine (PC) synthesis. GH administration partially prevented all these changes in males. In females, some of the parameters were significantly improved by GH (ATP, CO, cGMP), while others showed a tendency to improvement, although differences did not reach significance. In conclusion, GH administration could exert beneficial effects against age-related changes in hepatocytes, mainly in males.

  13. Use of Sandwich-Cultured Human Hepatocytes to Predict Biliary Clearance of Angiotensin II Receptor Blockers and HMG-CoA Reductase Inhibitors

    Science.gov (United States)

    Abe, Koji; Bridges, Arlene S.; Brouwer, Kim L. R.

    2009-01-01

    Previous reports have indicated that in vitro biliary clearance (Clbiliary) determined in sandwich-cultured hepatocytes correlates well with in vivo Clbiliary for limited sets of compounds. The purpose of this study was 1) to determine the in vitro Clbiliary in sandwich-cultured human hepatocytes of angiotensin II receptor blockers and HMG-CoA reductase inhibitors that undergo limited metabolism and 2) to compare the predicted Clbiliary values with estimated in vivo hepatic clearance data in humans. The average biliary excretion index and in vitro intrinsic Clbiliary values of olmesartan, valsartan, pravastatin, rosuvastatin, and pitavastatin in sandwich-cultured human hepatocytes were 35, 23, 31, 25, and 16%, respectively, and 0.943, 1.20, 0.484, 3.39, and 5.48 ml/min/kg, respectively. Clbiliary values predicted from sandwich-cultured human hepatocytes correlated with estimated in vivo hepatic clearance values based on published data (no in vivo data in humans was available for pitavastatin), and the rank order was also consistent. In conclusion, in vitro Clbiliary determined in sandwich-cultured human hepatocytes can be used to predict in vivo Clbiliary of compounds in humans. PMID:19074974

  14. Hepatitis C virus infection suppresses the interferon response in the liver of the human hepatocyte chimeric mouse.

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    Masataka Tsuge

    Full Text Available BACKGROUND AND AIMS: Recent studies indicate that hepatitis C virus (HCV can modulate the expression of various genes including those involved in interferon signaling, and up-regulation of interferon-stimulated genes by HCV was reported to be strongly associated with treatment outcome. To expand our understanding of the molecular mechanism underlying treatment resistance, we analyzed the direct effects of interferon and/or HCV infection under immunodeficient conditions using cDNA microarray analysis of human hepatocyte chimeric mice. METHODS: Human serum containing HCV genotype 1b was injected into human hepatocyte chimeric mice. IFN-α was administered 8 weeks after inoculation, and 6 hours later human hepatocytes in the mouse livers were collected for microarray analysis. RESULTS: HCV infection induced a more than 3-fold change in the expression of 181 genes, especially genes related to Organismal Injury and Abnormalities, such as fibrosis or injury of the liver (P = 5.90E-16∼3.66E-03. IFN administration induced more than 3-fold up-regulation in the expression of 152 genes. Marked induction was observed in the anti-fibrotic chemokines such as CXCL9, suggesting that IFN treatment might lead not only to HCV eradication but also prevention and repair of liver fibrosis. HCV infection appeared to suppress interferon signaling via significant reduction in interferon-induced gene expression in several genes of the IFN signaling pathway, including Mx1, STAT1, and several members of the CXCL and IFI families (P = 6.0E-12. Genes associated with Antimicrobial Response and Inflammatory Response were also significantly repressed (P = 5.22×10(-10∼1.95×10(-2. CONCLUSIONS: These results provide molecular insights into possible mechanisms used by HCV to evade innate immune responses, as well as novel therapeutic targets and a potential new indication for interferon therapy.

  15. Gene disruption of Plasmodium falciparum p52 results in attenuation of malaria liver stage development in cultured primary human hepatocytes.

    Directory of Open Access Journals (Sweden)

    Ben C L van Schaijk

    Full Text Available Difficulties with inducing sterile and long lasting protective immunity against malaria with subunit vaccines has renewed interest in vaccinations with attenuated Plasmodium parasites. Immunizations with sporozoites that are attenuated by radiation (RAS can induce strong protective immunity both in humans and rodent models of malaria. Recently, in rodent parasites it has been shown that through the deletion of a single gene, sporozoites can also become attenuated in liver stage development and, importantly, immunization with these sporozoites results in immune responses identical to RAS. The promise of vaccination using these genetically attenuated sporozoites (GAS depends on translating the results in rodent malaria models to human malaria. In this study, we perform the first essential step in this transition by disrupting, p52, in P. falciparum an ortholog of the rodent parasite gene, p36p, which we had previously shown can confer long lasting protective immunity in mice. These P. falciparum P52 deficient sporozoites demonstrate gliding motility, cell traversal and an invasion rate into primary human hepatocytes in vitro that is comparable to wild type sporozoites. However, inside the host hepatocyte development is arrested very soon after invasion. This study reveals, for the first time, that disrupting the equivalent gene in both P. falciparum and rodent malaria Plasmodium species generates parasites that become similarly arrested during liver stage development and these results pave the way for further development of GAS for human use.

  16. Predictivity of dog co-culture model, primary human hepatocytes and HepG2 cells for the detection of hepatotoxic drugs in humans

    Energy Technology Data Exchange (ETDEWEB)

    Atienzar, Franck A., E-mail: franck.atienzar@ucb.com [UCB Pharma SA, Non-Clinical Development, Chemin du Foriest, 1420 Braine-l' Alleud (Belgium); Novik, Eric I. [H mu rel Corporation, 675 U.S. Highway 1, North Brunswick, NJ 08902 (United States); Gerets, Helga H. [UCB Pharma SA, Non-Clinical Development, Chemin du Foriest, 1420 Braine-l' Alleud (Belgium); Parekh, Amit [H mu rel Corporation, 675 U.S. Highway 1, North Brunswick, NJ 08902 (United States); Delatour, Claude; Cardenas, Alvaro [UCB Pharma SA, Non-Clinical Development, Chemin du Foriest, 1420 Braine-l' Alleud (Belgium); MacDonald, James [Chrysalis Pharma Consulting, LLC, 385 Route 24, Suite 1G, Chester, NJ 07930 (United States); Yarmush, Martin L. [Department of Biomedical Engineering, Rutgers University, Piscataway, NJ 08854 (United States); Dhalluin, Stéphane [UCB Pharma SA, Non-Clinical Development, Chemin du Foriest, 1420 Braine-l' Alleud (Belgium)

    2014-02-15

    Drug Induced Liver Injury (DILI) is a major cause of attrition during early and late stage drug development. Consequently, there is a need to develop better in vitro primary hepatocyte models from different species for predicting hepatotoxicity in both animals and humans early in drug development. Dog is often chosen as the non-rodent species for toxicology studies. Unfortunately, dog in vitro models allowing long term cultures are not available. The objective of the present manuscript is to describe the development of a co-culture dog model for predicting hepatotoxic drugs in humans and to compare the predictivity of the canine model along with primary human hepatocytes and HepG2 cells. After rigorous optimization, the dog co-culture model displayed metabolic capacities that were maintained up to 2 weeks which indicates that such model could be also used for long term metabolism studies. Most of the human hepatotoxic drugs were detected with a sensitivity of approximately 80% (n = 40) for the three cellular models. Nevertheless, the specificity was low approximately 40% for the HepG2 cells and hepatocytes compared to 72.7% for the canine model (n = 11). Furthermore, the dog co-culture model showed a higher superiority for the classification of 5 pairs of close structural analogs with different DILI concerns in comparison to both human cellular models. Finally, the reproducibility of the canine system was also satisfactory with a coefficient of correlation of 75.2% (n = 14). Overall, the present manuscript indicates that the dog co-culture model may represent a relevant tool to perform chronic hepatotoxicity and metabolism studies. - Highlights: • Importance of species differences in drug development. • Relevance of dog co-culture model for metabolism and toxicology studies. • Hepatotoxicity: higher predictivity of dog co-culture vs HepG2 and human hepatocytes.

  17. Comparison of the effects of the synthetic pyrethroid Metofluthrin and phenobarbital on CYP2B form induction and replicative DNA synthesis in cultured rat and human hepatocytes.

    Science.gov (United States)

    Hirose, Yukihiro; Nagahori, Hirohisa; Yamada, Tomoya; Deguchi, Yoshihito; Tomigahara, Yoshitaka; Nishioka, Kazuhiko; Uwagawa, Satoshi; Kawamura, Satoshi; Isobe, Naohiko; Lake, Brian G; Okuno, Yasuyoshi

    2009-04-05

    High doses of Metofluthrin (MTF) have been shown to produce liver tumours in rats by a mode of action (MOA) involving activation of the constitutive androstane receptor leading to liver hypertrophy, induction of cytochrome P450 (CYP) forms and increased cell proliferation. The aim of this study was to compare the effects of MTF with those of the known rodent liver tumour promoter phenobarbital (PB) on the induction CYP2B forms and replicative DNA synthesis in cultured rat and human hepatocytes. Treatment with 50 microM MTF and 50 microM PB for 72 h increased CYP2B1 mRNA levels in male Wistar rat hepatocytes and CYP2B6 mRNA levels in human hepatocytes. Replicative DNA synthesis was determined by incorporation of 5-bromo-2'-deoxyuridine over the last 24 h of a 48 h treatment period. Treatment with 10-1000 microM MTF and 100-500 microM PB resulted in significant increases in replicative DNA synthesis in rat hepatocytes. While replicative DNA synthesis was increased in human hepatocytes treated with 5-50 ng/ml epidermal growth factor or 5-100 ng/ml hepatocyte growth factor, treatment with MTF and PB had no effect. These results demonstrate that while both MTF and PB induce CYP2B forms in both species, MTF and PB only induced replicative DNA synthesis in rat and not in human hepatocytes. These results provide further evidence that the MOA for MTF-induced rat liver tumour formation is similar to that of PB and some other non-genotoxic CYP2B form inducers and that the key event of increased cell proliferation would not occur in human liver.

  18. Explanted diseased livers - a possible source of metabolic competent primary human hepatocytes.

    Science.gov (United States)

    Kleine, Moritz; Riemer, Marc; Krech, Till; DeTemple, Daphne; Jäger, Mark D; Lehner, Frank; Manns, Michael P; Klempnauer, Jürgen; Borlak, Jürgen; Bektas, Hueseyin; Vondran, Florian W R

    2014-01-01

    Being an integral part of basic, translational and clinical research, the demand for primary human hepatocytes (PHH) is continuously growing while the availability of tissue resection material for the isolation of metabolically competent PHH remains limited. To overcome current shortcomings, this study evaluated the use of explanted diseased organs from liver transplantation patients as a potential source of PHH. Therefore, PHH were isolated from resected surgical specimens (Rx-group; n = 60) and explanted diseased livers obtained from graft recipients with low labMELD-score (Ex-group; n = 5). Using established protocols PHH were subsequently cultured for a period of 7 days. The viability and metabolic competence of cultured PHH was assessed by the following parameters: morphology and cell count (CyQuant assay), albumin synthesis, urea production, AST-leakage, and phase I and II metabolism. Both groups were compared in terms of cell yield and metabolic function, and results were correlated with clinical parameters of tissue donors. Notably, cellular yields and viabilities were comparable between the Rx- and Ex-group and were 5.3±0.5 and 2.9±0.7×106 cells/g liver tissue with 84.3±1.3 and 76.0±8.6% viability, respectively. Moreover, PHH isolated from the Rx- or Ex-group did not differ in regards to loss of cell number in culture, albumin synthesis, urea production, AST-leakage, and phase I and II metabolism (measured by the 7-ethoxycoumarin-O-deethylase and uracil-5'-diphosphate-glucuronyltransferase activity). Likewise, basal transcript expressions of the CYP monooxygenases 1A1, 2C8 and 3A4 were comparable as was their induction when treated with a cocktail that consisted of 3-methylcholantren, rifampicin and phenobarbital, with increased expression of CYP 1A1 and 3A4 mRNA while transcript expression of CYP 2C8 was only marginally changed. In conclusion, the use of explanted diseased livers obtained from recipients with low labMELD-score might represent

  19. Investigation of Orlistat effects on PXR activation and CYP3A4 expression in primary human hepatocytes and human intestinal LS174T cells.

    Science.gov (United States)

    Novotna, Aneta; Doricakova, Aneta; Vrzal, Radim; Maurel, Patrick; Pavek, Petr; Dvorak, Zdenek

    2010-10-09

    Drugs for weight loss have been in use for nearly hundred years. Orlistat (Xenical) is a non-centrally acting anti-obesity drug that inactivates gastric and intestinal lipases, thus, preventing absorption of dietary triglycerides. There are reports indicating that Orlistat reduces bioavailability of Cyclosporin to a clinically relevant degree. Since Cyclosporin is metabolized by cytochrome P450 CYP3A4, we examined whether interaction between Orlistat and Cyclosporin involves induction of CYP3A4. Human Caucasian colon adenocarcinoma cells LS174T and primary cultures of human hepatocytes were used, as in vitro models of intestinal and hepatic cells, respectively. Treatment of LS174T cells for 24h with Orlistat (1-100mg/L) did not cause induction of CYP3A4 mRNA levels as compared to control cells while Orlistat (100mg/L) slightly induced CYP3A4 mRNA in human hepatocytes. Rifampicin, a model CYP3A4 inducer, significantly induced CYP3A4 mRNA in both types of cells. The level of CYP3A4 protein in human hepatocytes was increased by Orlistat after 48h, while rifampicin strongly induced CYP3A4 protein level. In addition, Orlistat moderately dose-independently activated pregnane X receptor (PXR) in LS174T cells transiently transfected with p3A4-luc reporter construct containing the basal promoter (-362/+53) with proximal PXR response element and the distal xenobiotic responsive enhancer module (-7836/-7208) of the CYP3A4 gene 5'-flanking region. In conclusion, we report here that Orlistat is weak PXR activator and CYP3A4 inducer in human hepatocytes, but it has no effect on CYP3A4 in intestinal cells, implying no role of CYP3A4 induction in the interaction between Orlistat and Cyclosporin in absorption process.

  20. Formation of GSH-trapped reactive metabolites in human liver microsomes, S9 fraction, HepaRG-cells, and human hepatocytes.

    Science.gov (United States)

    Lassila, Toni; Rousu, Timo; Mattila, Sampo; Chesné, Christophe; Pelkonen, Olavi; Turpeinen, Miia; Tolonen, Ari

    2015-11-10

    The objective was to compare several in vitro human liver-derived subcellular and cellular incubation systems for the formation of GSH-trapped reactive metabolites. Incubations of pooled human liver microsomes, human liver S9 fractions, HepaRG-cells, and human hepatocytes were performed with glutathione as a trapping agent. Experiments with liver S9 were performed under two conditions, using only NADPH and using a full set of cofactors enabling also conjugative metabolism. Ten structurally different compounds were used as a test set, chosen as either "positive" (ciprofloxacin, clozapine, diclofenac, ethinyl estradiol, pulegone, and ticlopidine) or "negative" (caffeine, citalopram, losartan, montelukast) compounds, based on their known adverse reactions on liver or bone marrow. GSH conjugates were observed for seven of the ten compounds; while no conjugates were observed for caffeine, citalopram, or ciprofloxacin. Hepatocyte and HepaRG assays produced a clearly lower number and lower relative abundance of GSH conjugates compared to assays with microsomes and S9 fractions. The major GSH conjugates were different for many compounds in cellular subfractions and cell-based systems. Hepatocytes generally produced a higher number of GSH conjugates than HepaRG cells, although the differences were minor. The results show that the hepatic enzyme system used for screening of GSH-trapped reactive metabolites do have a high impact on the results, and results between different systems are comparable only qualitatively. Copyright © 2015 Elsevier B.V. All rights reserved.

  1. Metabolism of RCS-8, a synthetic cannabinoid with cyclohexyl structure, in human hepatocytes by high-resolution MS

    Science.gov (United States)

    Wohlfarth, Ariane; Pang, Shaokun; Zhu, Mingshe; Gandhi, Adarsh S; Scheidweiler, Karl B; Huestis, Marilyn A

    2015-01-01

    Background Since 2008, synthetic cannabinoids are major new designer drugs of abuse. They are extensively metabolized and excreted in urine, but limited human metabolism data are available. As there are no reports on the metabolism of RCS-8, a scheduled phenylacetylindole synthetic cannabinoid with an N-cyclohexylethyl moiety, we investigated metabolism of this new designer drug by human hepatocytes and high resolution MS. Methods After human hepatocyte incubation with RCS-8, samples were analyzed on a TripleTOF 5600+ mass spectrometer with time-of-flight survey scan and information-dependent acquisition triggered product ion scans. Data mining of the accurate mass full scan and product ion spectra employed different data processing algorithms. Results and Conclusion More than 20 RCS-8 metabolites were identified, products of oxidation, demethylation, and glucuronidation. Major metabolites and targets for analytical methods were hydroxyphenyl RCS - 8 glucuronide, a variety of hydroxycyclohexyl-hydroxyphenyl RCS-8 glucuronides, hydroxyphenyl RCS-8, as well as the demethyl-hydroxycyclohexyl RCS-8 glucuronide. PMID:24946920

  2. Evaluation of UGT protein interactions in human hepatocytes: effect of siRNA down regulation of UGT1A9 and UGT2B7 on propofol glucuronidation in human hepatocytes.

    Science.gov (United States)

    Konopnicki, Camille M; Dickmann, Leslie J; Tracy, Jeffrey M; Tukey, Robert H; Wienkers, Larry C; Foti, Robert S

    2013-07-15

    Previous experiments performed in recombinant systems have suggested that protein-protein interactions occur between the UGTs and may play a significant role in modulating enzyme activity. However, evidence of UGT protein-protein interactions either in vivo or in more physiologically relevant in vitro systems has yet to be demonstrated. In this study, we examined oligomerization and its ability to affect glucuronidation in plated human hepatocytes. siRNA down regulation experiments and activity studies were used to examine changes in metabolite formation of one UGT isoform due to down regulation of a second UGT isoform. Selective siRNA directed towards UGT1A9 or UGT2B7 resulted in significant and selective decreases in their respective mRNA levels. As expected, the metabolism of the UGT1A9 substrate propofol decreased with UGT1A9 down regulation. Interestingly, UGT1A9 activity, but not UGT1A9 mRNA expression, was also diminished when UGT2B7 expression was selectively inhibited, implying potential interactions between the two isoforms. Minor changes to UGT1A4, UGT2B4 and UGT2B7 activity were also observed when UGT1A9 expression was selectively down regulated. To our knowledge, this represents the first piece of evidence that UGT protein-protein interactions occur in human hepatocytes and suggests that expression levels of UGT2B7 may directly impact the glucuronidation activity of selective UGT1A9 substrates.

  3. Xanthohumol induces phase II enzymes via Nrf2 in human hepatocytes in vitro.

    Science.gov (United States)

    Krajka-Kuźniak, Violetta; Paluszczak, Jarosław; Baer-Dubowska, Wanda

    2013-02-01

    The aim of this study was to investigate whether xanthohumol may exert chemoprotective activity through the modulation of the nuclear factor erythroid-2-related factor 2 (Nrf2) pathway in immortalized normal THLE-2 hepatocytes and a hepatocellular carcinoma HepG2 cell line. Cells were incubated in the presence of xanthohumol and the activation of Nrf2 and expression of genes controlled by this transcription factor were evaluated. Additionally, p53 level was assessed. Xanthohumol increased the expression and led to the activation of Nrf2 in both cell lines. However, in contrast to normal cells the expression of genes controlled by this transcription factor was not affected in HepG2 cells, except for GSTA and GSTP. Xanthohumol, beside the induction of GSTs and HO-1, significantly elevated NQO1 expression in concert with p53 level in normal hepatocytes. The activation of Nrf2 pathway and subsequently phase II enzymes in concert with p53 induction in normal hepatocytes may account for the molecular mechanism of the chemopreventive activity of xanthohumol. On the other hand its cytotoxicity towards HCC cells shown in this study indicates that it may also be considered as potentially chemotherapeutic.

  4. Arsenite Effects on Mitochondrial Bioenergetics in Human and Mouse Primary Hepatocytes Follow a Nonlinear Dose Response

    Directory of Open Access Journals (Sweden)

    Hemantkumar Chavan

    2017-01-01

    Full Text Available Arsenite is a known carcinogen and its exposure has been implicated in a variety of noncarcinogenic health concerns. Increased oxidative stress is thought to be the primary cause of arsenite toxicity and the toxic effect is thought to be linear with detrimental effects reported at all concentrations of arsenite. But the paradigm of linear dose response in arsenite toxicity is shifting. In the present study we demonstrate that arsenite effects on mitochondrial respiration in primary hepatocytes follow a nonlinear dose response. In vitro exposure of primary hepatocytes to an environmentally relevant, moderate level of arsenite results in increased oxidant production that appears to arise from changes in the expression and activity of respiratory Complex I of the mitochondrial proton circuit. In primary hepatocytes the excess oxidant production appears to elicit adaptive responses that promote resistance to oxidative stress and a propensity to increased proliferation. Taken together, these results suggest a nonlinear dose-response characteristic of arsenite with low-dose arsenite promoting adaptive responses in a process known as mitohormesis, with transient increase in ROS levels acting as transducers of arsenite-induced mitohormesis.

  5. Excretory/secretory products of Fasciola hepatica but not recombinant phosphoglycerate kinase induce death of human hepatocyte cells.

    Science.gov (United States)

    Bąska, Piotr; Norbury, Luke J; Wiśniewski, Marcin; Januszkiewicz, Kamil; Wędrychowicz, Halina

    2013-06-01

    The liver fluke Fasciola hepatica infects a wide range of hosts, and has a considerable impact on the agriculture industry, mainly through infections of sheep and cattle. Further, human infection is now considered of public health importance and is hyperendemic in some regions. The fluke infection causes considerable damage to the hosts' liver. However, the mechanisms of liver destruction have not yet been completely elucidated. In the present report we incubated a human liver cell line in the presence of either F. hepatica excretory/secretory material (FhES) or recombinant phosphoglycerate kinase (FhPGK). Dosedependent cytotoxicity in the presence of FhES was observed, indicating that FhES is capable of killing human hepatocytes, supporting a role for FhES in damaging host liver cells during infection; while treatment with a recombinant intracellular protein - FhPGK, had no impact on cell survival.

  6. Arsenic induces mitochondria-dependent apoptosis by reactive oxygen species generation rather than glutathione depletion in Chang human hepatocytes

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Yi; Xu, Yuanyuan; Wang, Huihui; Xue, Peng; Li, Xin; Li, Bing; Zheng, Quanmei; Sun, Guifan [China Medical University, Department of Occupational and Environmental Health, College of Public Health, Shenyang (China)

    2009-10-15

    This study was conducted to evaluate the possible involvement of mitochondrial pathway in NaAsO{sub 2}-induced apoptosis and the role of reactive oxygen species (ROS) and reduced glutathione (GSH) in the apoptotic effect in Chang human hepatocytes. The MTT assay demonstrated that sodium arsenite (NaAsO{sub 2}) treatment for 24 h caused a dose-dependent decrease of cell viability. NaAsO{sub 2} treatment (0-30{mu}M) was also found to induce phosphatidylserine externalization, a hallmark of apoptosis; to disrupt the mitochondrial membrane potential ({delta}{psi}{sub m}); to cause the release of cytochrome c into the cytosol, and to trigger cleavage of caspase-3 and poly (ADP-ribose) polymerase (PARP) in a dose-dependent manner. All these changes were accompanied with the enhanced generation of intracellular ROS and malondialdehyde (MDA). Increase of intracellular GSH also coincided unexpectedly. Moreover, the extracellular addition of N-acetyl-l-cysteine (NAC, 5 mM) effectively reduced the generation of ROS and MDA, and rescued the cells from NaAsO{sub 2} induced apoptosis and related alteration of mitochondria. These data suggest that the arsenic-induced cell apoptosis occurs though the mitochondrial pathway, and is mostly dependent on generation of ROS rather than GSH depletion in Chang human hepatocytes. (orig.)

  7. Metabolite profiling of RCS-4, a novel synthetic cannabinoid designer drug, using human hepatocyte metabolism and TOF-MS

    Science.gov (United States)

    Gandhi, Adarsh S; Zhu, Mingshe; Pang, Shaokun; Wohlfarth, Ariane; Scheidweiler, Karl B; Huestis, Marilyn A

    2014-01-01

    Background Since 2009, scheduling legislation of synthetic cannabinoids prompted new compound emergence to circumvent legal restrictions. 2-(4-methoxyphenyl)-1-(1-pentyl-indol-3-yl)methanone (RCS-4) is a potent cannabinoid receptor agonist sold in herbal smoking blends. Absence of parent synthetic cannabinoids in urine suggests the importance of metabolite identification for detecting RCS-4 consumption in clinical and forensic investigations. Materials & methods & Results With 1 h human hepatocyte incubation and TOF high-resolution MS, we identified 18 RCS-4 metabolites, many not yet reported. Most metabolites were hydroxylated with or without demethylation, carboxylation and dealkylation followed by glucuronidation. One additional sulfated metabolite was also observed. O-demethylation was the most common biotransformation and generated the major metabolite. Conclusion For the first time, we present a metabolic scheme of RCS-4 obtained from human hepatocytes, including Phase I and II metabolites. Metabolite structural information and associated high-resolution mass spectra can be employed for developing clinical and forensic laboratory RCS-4 urine screening methods. PMID:25046048

  8. First characterization of AKB-48 metabolism, a novel synthetic cannabinoid, using human hepatocytes and high-resolution mass spectrometry.

    Science.gov (United States)

    Gandhi, Adarsh S; Zhu, Mingshe; Pang, Shaokun; Wohlfarth, Ariane; Scheidweiler, Karl B; Liu, Hua-Fen; Huestis, Marilyn A

    2013-10-01

    Since the federal authorities scheduled the first synthetic cannabinoids, JWH-018 and JWH-073, new synthetic cannabinoids were robustly marketed. N-(1-Adamantyl)-1-pentylindazole-3-carboxamide (AKB-48), also known as APINACA, was recently observed in Japanese herbal smoking blends. The National Forensic Laboratory Information System registered 443 reports of AKB-48 cases in the USA from March 2010 to January 2013. In May 2013, the Drug Enforcement Administration listed AKB-48 as a Schedule I drug. Recently, AKB-48 was shown to have twice the CB1 receptor binding affinity than CB2. These pharmacological effects and the difficulty in detecting the parent compound in urine highlight the importance of metabolite identification for developing analytical methods for clinical and forensic investigations. Using human hepatocytes and TripleTOF mass spectrometry, we identified 17 novel phase I and II AKB-48 metabolites, products of monohydroxylation, dihydroxylation, or trihydroxylation on the aliphatic adamantane ring or N-pentyl side chain. Glucuronide conjugation of some mono- and dihydroxylated metabolites also occurred. Oxidation and dihydroxylation on the adamantane ring and N-pentyl side chain formed a ketone. More metabolites were identified after 3 h of incubation than at 1 h. For the first time, we present a AKB-48 metabolic scheme obtained from human hepatocytes and high-resolution mass spectrometry. These data are needed to develop analytical methods to identify AKB-48 consumption in clinical and forensic testing.

  9. A human hepatoma cell line FLC4 cultured on the radial flow bioreactor as a model for human hepatocytes

    Institute of Scientific and Technical Information of China (English)

    LiYW; BabuE

    2002-01-01

    Hepatocytes play central roles in the metabolism and excretion of drugs and xenobiovics.For this purpose,hepatocytes were endowed with high levels of enzyme activity for the phase I and phase Ⅱ metabolism as well as high levels of transmembrane transport activity which enables the entrance and the exit of drugs and xenobiotics and their metabolites through the plasma membrane of the hepatocytes.They include the transporters in the canalicular and sinusoidal membrane.Although a lot of cell lines were established from hepatoma cells or normal hepatocytes,none of them are fully satisfactory in the expression of the enzymes and transportens.We have established and characterized a hepatoma cell line designated FLC4 and found that this cell line exhibits properties quite similar to those of the normal hepatocytes in the light of enzymes and transporters for drug metabolism and transkport when they are cultured on the radial flow bioreactors.Using FLC4 cells cultured on the radial flow bioreactors,we are developing in vitro systems to evaluate the interaction of drugs with liver transporters and drug-drug interaction through the hepa tocyte transporters.

  10. Laminin Receptor-Avid Nanotherapeutic EGCg-AuNPs as a Potential Alternative Therapeutic Approach to Prevent Restenosis

    Directory of Open Access Journals (Sweden)

    Menka Khoobchandani

    2016-03-01

    Full Text Available In our efforts to develop new approaches to treat and prevent human vascular diseases, we report herein our results on the proliferation and migration of human smooth muscles cells (SMCs and endothelial cells (ECs using epigallocatechin-3-gallate conjugated gold nanoparticles (EGCg-AuNPs as possible alternatives to drug coated stents. Detailed in vitro stability studies of EGCg-AuNPs in various biological fluids, affinity and selectivity towards SMCs and ECs have been investigated. The EGCg-AuNPs showed selective inhibitory efficacy toward the migration of SMCs. However, the endothelial cells remained unaffected under similar experimental conditions. The cellular internalization studies have indicated that EGCg-AuNPs internalize into the SMCs and ECs within short periods of time through laminin receptor mediated endocytosis mode. Favorable toxicity profiles and selective affinity toward SMCs and ECs suggest that EGCg-AuNPs may provide attractive alternatives to drug coated stents and therefore offer new therapeutic approaches in treating cardiovascular diseases.

  11. Protocol for Isolation of Primary Human Hepatocytes and Corresponding Major Populations of Non-parenchymal Liver Cells.

    Science.gov (United States)

    Kegel, Victoria; Deharde, Daniela; Pfeiffer, Elisa; Zeilinger, Katrin; Seehofer, Daniel; Damm, Georg

    2016-03-30

    Beside parenchymal hepatocytes, the liver consists of non-parenchymal cells (NPC) namely Kupffer cells (KC), liver endothelial cells (LEC) and hepatic Stellate cells (HSC). Two-dimensional (2D) culture of primary human hepatocyte (PHH) is still considered as the "gold standard" for in vitro testing of drug metabolism and hepatotoxicity. It is well-known that the 2D monoculture of PHH suffers from dedifferentiation and loss of function. Recently it was shown that hepatic NPC play a central role in liver (patho-) physiology and the maintenance of PHH functions. Current research focuses on the reconstruction of in vivo tissue architecture by 3D- and co-culture models to overcome the limitations of 2D monocultures. Previously we published a method to isolate human liver cells and investigated the suitability of these cells for their use in cell cultures in Experimental Biology and Medicine(1). Based on the broad interest in this technique the aim of this article was to provide a more detailed protocol for the liver cell isolation process including a video, which will allow an easy reproduction of this technique. Human liver cells were isolated from human liver tissue samples of surgical interventions by a two-step EGTA/collagenase P perfusion technique. PHH were separated from the NPC by an initial centrifugation at 50 x g. Density gradient centrifugation steps were used for removal of dead cells. Individual liver cell populations were isolated from the enriched NPC fraction using specific cell properties and cell sorting procedures. Beside the PHH isolation we were able to separate KC, LEC and HSC for further cultivation. Taken together, the presented protocol allows the isolation of PHH and NPC in high quality and quantity from one donor tissue sample. The access to purified liver cell populations could allow the creation of in vivo like human liver models.

  12. Prediction of fraction metabolized via CYP3A in humans utilizing cryopreserved human hepatocytes from a set of 12 single donors.

    Science.gov (United States)

    Desbans, C; Hilgendorf, C; Lutz, M; Bachellier, P; Zacharias, T; Weber, J C; Dolgos, H; Richert, L; Ungell, A-L

    2014-01-01

    1.  It has previously been demonstrated that metabolism of drugs via a single enzymatic pathway, particularly CYP3A4, is associated with increased risk for drug-drug interactions (DDI). Quantitative experimental systems as well as integrated prediction models to assess such risk during the preclinical phase are highly warranted. 2.  The present study was designed to systematically investigate the performance of human cryopreserved hepatocytes in suspension to predict fraction metabolized via CYP3A (fmCYP3A) by assessing the ketoconazole sensitive intrinsic clearance (CLint) for five prototypical CYP3A substrates with varying degree of CYP3A dependent CLint in twelve individual hepatocyte batches. 3.  We demonstrate that in contrast to well predicted mean hepatic metabolic clearance (CLH) and mean fmCYP3A data, the variability in CYP3A contribution for compounds having multiple metabolic pathways cannot be predicted from inhibition experiments using ketoconazole as inhibitor. Instead, data in the present paper indicate that the variability is larger after inhibition of CYP3A for compounds having multiple metabolic pathways. 4.  It is therefore recommended to estimate the average CLint and fmCYP3A for a given test compound in a series (n = 10) of individual human hepatocyte batches.

  13. Metabolism of Carfentanil, an Ultra-Potent Opioid, in Human Liver Microsomes and Human Hepatocytes by High-Resolution Mass Spectrometry.

    Science.gov (United States)

    Feasel, Michael G; Wohlfarth, Ariane; Nilles, John M; Pang, Shaokun; Kristovich, Robert L; Huestis, Marilyn A

    2016-11-01

    Carfentanil is an ultra-potent synthetic opioid. No human carfentanil metabolism data are available. Reportedly, Russian police forces used carfentanil and remifentanil to resolve a hostage situation in Moscow in 2002. This alleged use prompted interest in the pharmacology and toxicology of carfentanil in humans. Our study was conducted to identify human carfentanil metabolites and to assess carfentanil's metabolic clearance, which could contribute to its acute toxicity in humans. We used Simulations Plus's ADMET Predictor™ and Molecular Discovery's MetaSite™ to predict possible metabolite formation. Both programs gave similar results that were generally good but did not capture all metabolites seen in vitro. We incubated carfentanil with human hepatocytes for up to 1 h and analyzed samples on a Sciex 3200 QTRAP mass spectrometer to measure parent compound depletion and extrapolated that to represent intrinsic clearance. Pooled primary human hepatocytes were then incubated with carfentanil up to 6 h and analyzed for metabolite identification on a Sciex 5600+ TripleTOF (QTOF) high-resolution mass spectrometer. MS and MS/MS analyses elucidated the structures of the most abundant metabolites. Twelve metabolites were identified in total. N-Dealkylation and monohydroxylation of the piperidine ring were the dominant metabolic pathways. Two N-oxide metabolites and one glucuronide metabolite were observed. Surprisingly, ester hydrolysis was not a major metabolic pathway for carfentanil. While the human liver microsomal system demonstrated rapid clearance by CYP enzymes, the hepatocyte incubations showed much slower clearance, possibly providing some insight into the long duration of carfentanil's effects.

  14. Human Bone Marrow Mesenchymal Stem Cell-Derived Hepatocytes Improve the Mouse Liver after Acute Acetaminophen Intoxication by Preventing Progress of Injury

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    Peggy Stock

    2014-04-01

    Full Text Available Mesenchymal stem cells from human bone marrow (hMSC have the potential to differentiate into hepatocyte-like cells in vitro and continue to maintain important hepatocyte functions in vivo after transplantation into host mouse livers. Here, hMSC were differentiated into hepatocyte-like cells in vitro (hMSC-HC and transplanted into livers of immunodeficient Pfp/Rag2−/− mice treated with a sublethal dose of acetaminophen (APAP to induce acute liver injury. APAP induced a time- and dose-dependent damage of perivenous areas of the liver lobule. Serum levels of aspartate aminotransferase (AST increased to similar levels irrespective of hMSC-HC transplantation. Yet, hMSC-HC resided in the damaged perivenous areas of the liver lobules short-term preventing apoptosis and thus progress of organ destruction. Disturbance of metabolic protein expression was lower in the livers receiving hMSC-HC. Seven weeks after APAP treatment, hepatic injury had completely recovered in groups both with and without hMSC-HC. Clusters of transplanted cells appeared predominantly in the periportal portion of the liver lobule and secreted human albumin featuring a prominent quality of differentiated hepatocytes. Thus, hMSC-HC attenuated the inflammatory response and supported liver regeneration after acute injury induced by acetaminophen. They hence may serve as a novel source of hepatocyte-like cells suitable for cell therapy of acute liver diseases.

  15. Activation of the Nrf2 Pathway by Inorganic Arsenic in Human Hepatocytes and the Role of Transcriptional Repressor Bach1

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    Dan Liu

    2013-01-01

    Full Text Available Previous studies have proved that the environmental toxicant, inorganic arsenic, activates nuclear factor erythroid 2-related factor 2 (Nrf2 pathway in many different cell types. This study tried to explore the hepatic Nrf2 pathway upon arsenic treatment comprehensively, since liver is one of the major target organs of arsenical toxicity. Our results showed that inorganic arsenic significantly induced Nrf2 protein and mRNA expression in Chang human hepatocytes. We also observed a dose-dependent increase of antioxidant response element- (ARE- luciferase activity. Both the mRNA and protein levels of NAD(PH:quinone oxidoreductase 1 (NQO1 and heme oxygenase-1 (HO-1 were all upregulated dramatically. On the other hand, entry and accumulation of Nrf2 protein in the nucleus, while exportting the transcriptional repressor BTB and CNC homology 1 (Bach1 from nucleus to cytoplasm, were also confirmed by western blot and immunofluorescence assay. Our results therefore confirmed the arsenic-induced Nrf2 pathway activation in hepatocytes and also suggested that the translocation of Bach1 was associated with the regulation of Nrf2 pathway by arsenic. Hepatic Nrf2 pathway plays indispensable roles for cellular defenses against arsenic hepatotoxicity, and the interplay of Bach1 and Nrf2 may be helpful to understand the self-defensive responses and the diverse biological effects of arsenicals.

  16. Purinergic receptor functionality is necessary for infection of human hepatocytes by hepatitis delta virus and hepatitis B virus.

    Directory of Open Access Journals (Sweden)

    John M Taylor

    Full Text Available Hepatitis B virus (HBV and hepatitis delta virus (HDV are major sources of acute and chronic hepatitis. HDV requires the envelope proteins of HBV for the processes of assembly and infection of new cells. Both viruses are able to infect hepatocytes though previous studies have failed to determine the mechanism of entry into such cells. This study began with evidence that suramin, a symmetrical hexasulfated napthylurea, could block HDV entry into primary human hepatocytes (PHH and was then extrapolated to incorporate findings of others that suramin is one of many compounds that can block activation of purinergic receptors. Thus other inhibitors, pyridoxal-phosphate-6-azophenyl-2',4'-disulfonate (PPADS and brilliant blue G (BBG, both structurally unrelated to suramin, were tested and found to inhibit HDV and HBV infections of PHH. BBG, unlike suramin and PPADS, is known to be more specific for just one purinergic receptor, P2X7. These studies provide the first evidence that purinergic receptor functionality is necessary for virus entry. Furthermore, since P2X7 activation is known to be a major component of inflammatory responses, it is proposed that HDV and HBV attachment to susceptible cells, might also contribute to inflammation in the liver, that is, hepatitis.

  17. Foxa1 reduces lipid accumulation in human hepatocytes and is down-regulated in nonalcoholic fatty liver.

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    Marta Moya

    Full Text Available Triglyceride accumulation in nonalcoholic fatty liver (NAFL results from unbalanced lipid metabolism which, in the liver, is controlled by several transcription factors. The Foxa subfamily of winged helix/forkhead box (Fox transcription factors comprises three members which play important roles in controlling both metabolism and homeostasis through the regulation of multiple target genes in the liver, pancreas and adipose tissue. In the mouse liver, Foxa2 is repressed by insulin and mediates fasting responses. Unlike Foxa2 however, the role of Foxa1 in the liver has not yet been investigated in detail. In this study, we evaluate the role of Foxa1 in two human liver cell models, primary cultured hepatocytes and HepG2 cells, by adenoviral infection. Moreover, human and rat livers were analyzed to determine Foxa1 regulation in NAFL. Results demonstrate that Foxa1 is a potent inhibitor of hepatic triglyceride synthesis, accumulation and secretion by repressing the expression of multiple target genes of these pathways (e.g., GPAM, DGAT2, MTP, APOB. Moreover, Foxa1 represses the fatty acid transporter protein FATP2 and lowers fatty acid uptake. Foxa1 also increases the breakdown of fatty acids by inducing peroxisomal fatty acid β-oxidation and ketone body synthesis. Finally, Foxa1 is able to largely up-regulate UCP1, thereby dissipating energy and consistently decreasing the mitochondria membrane potential. We also report that human and rat NAFL have a reduced Foxa1 expression, possibly through a protein kinase C-dependent pathway. We conclude that Foxa1 is an antisteatotic factor that coordinately tunes several lipid metabolic pathways to block triglyceride accumulation in hepatocytes. However, Foxa1 is down-regulated in human and rat NAFL and, therefore, increasing Foxa1 levels could protect from steatosis. Altogether, we suggest that Foxa1 could be a novel therapeutic target for NAFL disease and insulin resistance.

  18. Human Hepatocyte Metabolism of Novel Synthetic Cannabinoids MN-18 and Its 5-Fluoro Analog 5F-MN-18.

    Science.gov (United States)

    Diao, Xingxing; Carlier, Jeremy; Zhu, Mingshe; Huestis, Marilyn A

    2017-08-18

    In 2014, 2 novel synthetic cannabinoids, MN-18 and its 5-fluoro analog, 5F-MN-18, were first identified in an ongoing survey of novel psychoactive substances in Japan. In vitro pharmacological assays revealed that MN-18 and 5F-MN-18 displayed high binding affinities to human CB1 and CB2 receptors, with Ki being 1.65-3.86 nmol/L. MN-18 and 5F-MN-18 were scheduled in Japan and some other countries in 2014. Despite increasing prevalence, no human metabolism data are currently available, making it challenging for forensic laboratories to confirm intake of MN-18 or 5F-MN-18. We incubated 10 μ mol/L of MN-18 and 5F-MN-18 in human hepatocytes for 3 h and analyzed the samples on a TripleTOF 5600(+) high-resolution mass spectrometer to identify appropriate marker metabolites. Data were acquired via full scan and information-dependent acquisition-triggered product ion scans with mass defect filter. In total, 13 MN-18 metabolites were detected, with the top 3 abundant metabolites being 1-pentyl-1H-indazole-3-carboxylic acid, pentyl-carbonylated MN-18, and naphthalene-hydroxylated MN-18. For 5F-MN-18, 20 metabolites were observed, with the top 3 abundant metabolites being 5`-OH-MN-18, MN-18 pentanoic acid, and 1-(5-fluoropentyl)-1H-indazole-3-carboxylic acid. We have characterized MN-18 and 5F-MN-18 metabolism with human hepatocytes and high-resolution mass spectrometry, and we recommend characteristic major metabolites for clinical and forensic laboratories to identify MN-18 and 5F-MN-18 intake and link observed adverse events to these novel synthetic cannabinoids. © 2017 American Association for Clinical Chemistry.

  19. Foxa1 Reduces Lipid Accumulation in Human Hepatocytes and Is Down-Regulated in Nonalcoholic Fatty Liver

    Science.gov (United States)

    Moya, Marta; Benet, Marta; Guzmán, Carla; Tolosa, Laia; García-Monzón, Carmelo; Pareja, Eugenia; Castell, José Vicente; Jover, Ramiro

    2012-01-01

    Triglyceride accumulation in nonalcoholic fatty liver (NAFL) results from unbalanced lipid metabolism which, in the liver, is controlled by several transcription factors. The Foxa subfamily of winged helix/forkhead box (Fox) transcription factors comprises three members which play important roles in controlling both metabolism and homeostasis through the regulation of multiple target genes in the liver, pancreas and adipose tissue. In the mouse liver, Foxa2 is repressed by insulin and mediates fasting responses. Unlike Foxa2 however, the role of Foxa1 in the liver has not yet been investigated in detail. In this study, we evaluate the role of Foxa1 in two human liver cell models, primary cultured hepatocytes and HepG2 cells, by adenoviral infection. Moreover, human and rat livers were analyzed to determine Foxa1 regulation in NAFL. Results demonstrate that Foxa1 is a potent inhibitor of hepatic triglyceride synthesis, accumulation and secretion by repressing the expression of multiple target genes of these pathways (e.g., GPAM, DGAT2, MTP, APOB). Moreover, Foxa1 represses the fatty acid transporter protein FATP2 and lowers fatty acid uptake. Foxa1 also increases the breakdown of fatty acids by inducing peroxisomal fatty acid β-oxidation and ketone body synthesis. Finally, Foxa1 is able to largely up-regulate UCP1, thereby dissipating energy and consistently decreasing the mitochondria membrane potential. We also report that human and rat NAFL have a reduced Foxa1 expression, possibly through a protein kinase C-dependent pathway. We conclude that Foxa1 is an antisteatotic factor that coordinately tunes several lipid metabolic pathways to block triglyceride accumulation in hepatocytes. However, Foxa1 is down-regulated in human and rat NAFL and, therefore, increasing Foxa1 levels could protect from steatosis. Altogether, we suggest that Foxa1 could be a novel therapeutic target for NAFL disease and insulin resistance. PMID:22238690

  20. A new splice variant of the major subunit of human asialoglycoprotein receptor encodes a secreted form in hepatocytes.

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    Jia Liu

    Full Text Available BACKGROUND: The human asialoglycoprotein receptor (ASGPR is composed of two polypeptides, designated H1 and H2. While variants of H2 have been known for decades, the existence of H1 variants has never been reported. PRINCIPAL FINDINGS: We identified two splice variants of ASGPR H1 transcripts, designated H1a and H1b, in human liver tissues and hepatoma cells. Molecular cloning of ASGPR H1 variants revealed that they differ by a 117 nucleotide segment corresponding to exon 2 in the ASGPR genomic sequence. Thus, ASGPR variant H1b transcript encodes a protein lacking the transmembrane domain. Using an H1b-specific antibody, H1b protein and a functional soluble ASGPR (sASGPR composed of H1b and H2 in human sera and in hepatoma cell culture supernatant were identified. The expression of ASGPR H1a and H1b in Hela cells demonstrated the different cellular loctions of H1a and H1b proteins at cellular membranes and in intracellular compartments, respectively. In vitro binding assays using fluorescence-labeled sASGPR or the substract ASOR revealed that the presence of sASGPR reduced the binding of ASOR to cells. However, ASOR itself was able to enhance the binding of sASGPR to cells expressing membrane-bound ASGPR. Further, H1b expression is reduced in liver tissues from patients with viral hepatitis. CONCLUSIONS: We conclude that two naturally occurring ASGPR H1 splice variants are produced in human hepatocytes. A hetero-oligomeric complex sASGPR consists of the secreted form of H1 and H2 and may bind to free substrates in circulation and carry them to liver tissue for uptake by ASGPR-expressing hepatocytes.

  1. Human umbilical cord mesenchymal stem cells and derived hepatocyte-like cells exhibit similar therapeutic effects on an acute liver failure mouse model.

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    Ruiping Zhou

    Full Text Available Mesenchymal stem cells (MSCs have exhibited therapeutic effects in multiple animal models so that are promising liver substitute for transplantation treatment of end-stage liver diseases. However, it has been shown that over-manipulation of these cells increased their tumorigenic potential, and that reducing the in vitro culture time could minimize the risk. In this study, we used a D-galactosamine plus lipopolysaccharide (Gal/LPS-induced acute liver failure mouse model, which caused death of about 50% of the mice with necrosis of more than 50% hepatocytes, to compare the therapeutic effects of human umbilical cord MSCs (hUCMSCs before and after induction of differentiation into hepatocyte (i-Heps. Induction of hUCMSCs to become i-Heps was achieved by treatment of the cells with a group of growth factors within 4 weeks. The resulted i-Heps exhibited a panel of human hepatocyte biomarkers including cytokeratin (hCK-18, α-fetoprotein (hAFP, albumin (hALB, and hepatocyte-specific functions glycogen storage and urea metabolism. We demonstrated that transplantation of both cell types through tail vein injection rescued almost all of the Gal/LPS-intoxicated mice. Although both cell types exhibited similar ability in homing at the mouse livers, the populations of the hUCMSCs-derived cells, as judged by expressing hAFP, hCK-18 and human hepatocyte growth factor (hHGF, were small. These observations let us to conclude that the hUCMSCs was as effective as the i-Heps in treatment of the mouse acute liver failure, and that the therapeutic effects of hUCMSCs were mediated largely via stimulation of host hepatocyte regeneration, and that delivery of the cells through intravenous injection was effective.

  2. Controlled and reversible induction of differentiation and activation of adult human hepatocytes by a biphasic culture technique

    Institute of Scientific and Technical Information of China (English)

    Marcus K.H. Auth; Wolf-Otto Bechstein; Roman A. Blaheta; Kim A. Boost; Kerstin Leckel; Wolf-Dietrich Beecken; Tobias Engl; Dietger Jonas; Elsie Oppermann; Philip Hilgard; Bernd H. Markus

    2005-01-01

    AIM: Clinical application of human hepatocytes (HC) is hampered by the progressive loss of growth and differentiation in vitro. The object of the study was to evaluate the effect of a biphasic culture technique on expression and activation of growth factor receptors and differentiation of human adult HC.METHODS: Isolated HC were sequentially cultured in a hormone enriched differentiation medium (DM) containing nicotinamide, insulin, transferrin, selenium, and dexamethasone or activation medium (AM) containing hepatocyte growth factor (HGF), epidermal growth factor (EGF), andgranulocyte-macrophage colony-stimulating factor (GMCSF). Expression, distribution and activation of the HC receptors (MET and EGFR) and the pattern of characteristic cytokeratin (CK) filaments were measured by fluorometry, confocal microscopy and Western blotting.RESULTS: In the biphasic culture system, HC underwent repeated cycles of activation (characterized by expression and activation of growth factor receptors) and re-differentiation (illustrated by distribution of typical filaments CK-18 but low or absent expression of CK-19). In AM increased expression of MET and EGFR was associated with receptor translocation into the cytoplasm and induction of atypical CK-19. In DM low expression of MET and EGFR was localized on the cell membrane and CK-19 was reduced. Receptor phosphorylation required embedding of HC in collagen type Ⅰ gel.CONCLUSION: Control and reversible modulation of growth factor receptor activation of mature human HC can be accomplishedin vitro, when defined signals from the extracellular matrix and sequential growth stimuli are provided. The biphasic technique helps overcome dedifferentiation, which occurs during continuous stimulation by means of growth factors.

  3. Direct infection and replication of naturally occurring hepatitis C virus genotypes 1, 2, 3 and 4 in normal human hepatocyte cultures.

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    Martina Buck

    Full Text Available BACKGROUND: Hepatitis C virus (HCV infection afflicts about 170 million individuals worldwide. However, the HCV life cycle is only partially understood because it has not been possible to infect normal human hepatocytes in culture. The current Huh-7 systems use cloned, synthetic HCV RNA expressed in hepatocellular carcinoma cells to produce virions, but these cells cannot be infected with naturally occurring HCV obtained from infected patients. METHODOLOGY/PRINCIPAL FINDINGS: Here, we describe a human hepatocyte culture permissible to the direct infection with naturally occurring HCV genotypes 1, 2, 3 and 4 in the blood of HCV-infected patients. The culture system mimics the biology and kinetics of HCV infection in humans, and produces infectious virions that can infect naïve human hepatocytes. CONCLUSIONS/SIGNIFICANCE: This culture system should complement the existing systems, and may facilitate the understanding of the HCV life cycle, its effects in the natural host cell, the hepatocyte, as well as the development of novel therapeutics and vaccines.

  4. Expression of human factor IX in rabbit hepatocytes by retrovirus-mediated gene transfer: Potential for gene therapy of hemophilia B

    Energy Technology Data Exchange (ETDEWEB)

    Thompson, A.R. (Univ. of Washington, Seattle (USA) Puget Sound Blood Center, Seattle, WA (USA)); Darlington, G. (Baylor College of Medicine, Houston, TX (USA)); Armentano, D.; Woo, S.L.C.

    1990-08-01

    Hemophilia B (Christmas disease) is a chromosome X-linked blood clotting disorder which results when factor IX is deficient or functionally defective. The enzyme is synthesized in the liver, and the existence of animal models for this genetic disease will permit the development of somatic gene therapy protocols aimed at transfer of the functional gene into the liver. The authors report the construction of an N2-based recombinant retroviral vector, NCMVFIX, for efficient transfer and expression of human factor IX cDNA in primary rabbit hepatocytes. In this construct the human cytomegalovirus immediate early promoter directs the expression of factor IX. Hepatocytes were isolated from 3-week-old New Zealand White rabbits, infected with the recombinant virus, and analyzed for secretion of active factor IX. The infected rabbit hepatocytes produced human factor IX that is indistinguishable from enzyme derived from normal human plasma. The recombinant protein is sufficiently {gamma}-carboxylated and is functionally active in clotting assays. These results establish the feasibility of using infected hepatocytes for the expression of this protein and are a step toward the goal of correcting hemophilia B by hepatic gene transfer.

  5. Rifampin Regulation of Drug Transporters Gene Expression and the Association of MicroRNAs in Human Hepatocytes

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    Eric A Benson

    2016-04-01

    Full Text Available Membrane drug transporters contribute to the disposition of many drugs. In human liver, drug transport is controlled by two main superfamilies of transporters, the solute carrier transporters (SLC and the ATP Binding Cassette transporters (ABC. Altered expression of these transporters due to drug-drug interactions can contribute to differences in drug exposure and possibly effect. In this study, we determined the effect of rifampin on gene expression of hundreds of membrane transporters along with all clinically relevant drug transporters. Methods: In this study, primary human hepatocytes (n=7 donors were cultured and treated for 24 hours with rifampin and vehicle control. RNA was isolated from the hepatocytes, mRNA expression was measured by RNA-seq, and miRNA expression was analyzed by Taqman OpenArray. The effect of rifampin on the expression of selected transporters was also tested in kidney cell lines. The impact of rifampin on the expression of 410 transporter genes from 19 different transporter gene families was compared with vehicle control. Results: Expression patterns of 12 clinically relevant drug transporter genes were changed by rifampin (FDR<0.05. For example, the expressions of ABCC2, ABCB1, and ABCC3 were increased 1.9-, 1.7-, and 1.2- fold, respectively. The effects of rifampin on four uptake drug transporters (SLCO1B3, SLC47A1, SLC29A1, SLC22A9 were negatively correlated with the rifampin effects on specific microRNA expression (SLCO1B3/miR-92a, SLC47A1/miR-95, SLC29A1/miR-30d#, and SLC22A9/miR-20; r<-0.79; p<0.05. Seven hepatic drug transporter genes (SLC22A1, SLC22A5, SLC15A1, SLC29A1, SLCO4C1, ABCC2, and ABCC4, whose expression was altered by rifampin in hepatocytes, were also present in a renal proximal tubular cell line, but in renal cells rifampin did not alter their gene expression. PXR expression was very low in the kidney cells; this may explain why rifampin induces gene expression in a tissue-specific manner

  6. HepatoNet1: a comprehensive metabolic reconstruction of the human hepatocyte for the analysis of liver physiology.

    Science.gov (United States)

    Gille, Christoph; Bölling, Christian; Hoppe, Andreas; Bulik, Sascha; Hoffmann, Sabrina; Hübner, Katrin; Karlstädt, Anja; Ganeshan, Ramanan; König, Matthias; Rother, Kristian; Weidlich, Michael; Behre, Jörn; Holzhütter, Herrmann-Georg

    2010-09-07

    We present HepatoNet1, the first reconstruction of a comprehensive metabolic network of the human hepatocyte that is shown to accomplish a large canon of known metabolic liver functions. The network comprises 777 metabolites in six intracellular and two extracellular compartments and 2539 reactions, including 1466 transport reactions. It is based on the manual evaluation of >1500 original scientific research publications to warrant a high-quality evidence-based model. The final network is the result of an iterative process of data compilation and rigorous computational testing of network functionality by means of constraint-based modeling techniques. Taking the hepatic detoxification of ammonia as an example, we show how the availability of nutrients and oxygen may modulate the interplay of various metabolic pathways to allow an efficient response of the liver to perturbations of the homeostasis of blood compounds.

  7. Galactosylated collagen matrix enhanced in vitro maturation of human embryonic stem cell-derived hepatocyte-like cells.

    Science.gov (United States)

    Ghodsizadeh, Arefeh; Hosseinkhani, Hossein; Piryaei, Abbas; Pournasr, Behshad; Najarasl, Mostafa; Hiraoka, Yosuke; Baharvand, Hossein

    2014-05-01

    Due to their important biomedical applications, functional human embryonic stem cell-derived hepatocyte-like cells (hESC-HLCs) are an attractive topic in the field of stem cell differentiation. Here, we have initially differentiated hESCs into functional hepatic endoderm (HE) and continued the differentiation by replating them onto galactosylated collagen (GC) and collagen matrices. The differentiation of hESC-HE cells into HLCs on GC substrate showed significant up-regulation of hepatic-specific genes such as ALB, HNF4α, CYP3A4, G6P, and ASGR1. There was more albumin secretion and urea synthesis, as well as more cytochrome p450 activity, in differentiated HLCs on GC compared to the collagen-coated substrate. These results suggested that GC substrate has the potential to be used for in vitro maturation of hESC-HLCs.

  8. Dose-dependent Cytotoxicity and Oxidative Stress Induced by "Naked" Fe3O4 Nanoparticles in Human Hepatocyte

    Institute of Scientific and Technical Information of China (English)

    LIN Xin-li; ZHAO Shu-hua; ZHANG Long; HU Gui-qin; SUN Zhi-wei; YANG Wen-sheng

    2012-01-01

    The dose-dependent cytotoxicity effect on human hepatocyte(HL-7702 cells) induced by "naked" Fe3O4 nanoparticles was assessed through cell viabilities and lactate dehydrogenase(LDH) activities.Three important oxidative indexes of the cells by glutathione peroxidase(GSH-Px),superoxide dismutase(SOD) and malondialdehyde(MDA) were determined.The good correlation of the cell viabilities with their GSH-Px,SOD and MDA levels indicated that the cytotoxicity is related to activation of oxidative stress induced by Fe3O4 nanoparticles.The oxidative stress also leads to corresponding DNA damage in a similar dose-dependent manner,followed by the changes of cell cycle and cell apoptosis.Such work provides important experimental data for the safety evaluation of superparamagnetic Fe3O4 nanoparticles.

  9. In vitro glucuronidation of 2,2-bis(bromomethyl)-1,3-propanediol by microsomes and hepatocytes from rats and humans.

    Science.gov (United States)

    Rad, Golriz; Hoehle, Simone I; Kuester, Robert K; Sipes, I Glenn

    2010-06-01

    2,2-Bis(bromomethyl)-1,3-propanediol (BMP) is a brominated flame retardant used in unsaturated polyester resins. In a 2-year bioassay BMP was shown to be a multisite carcinogen in rats and mice. Because glucuronidation is the key metabolic transformation of BMP by rats, in this study the in vitro hepatic glucuronidation of BMP was compared across several species. In addition, the glucuronidation activities of human intestinal microsomes and specific human hepatic UDP-glucuronosyltransferase (UGT) enzymes for BMP were determined. To explore other possible routes of metabolism for BMP, studies were conducted with rat and human hepatocytes. Incubation of hepatic microsomes with BMP in the presence of UDP-glucuronic acid resulted in the formation of a BMP monoglucuronide. The order of hepatic microsomal glucuronidation activity of BMP was rats, mice > hamsters > monkeys > humans. The rate of glucuronidation by rat hepatic microsomes was 90-fold greater than that of human hepatic microsomes. Human intestinal microsomes converted BMP to BMP glucuronide at a rate even lower than that of human hepatic microsomes. Among the human UGT enzymes tested, only UGT2B7 had detectable glucuronidation activity for BMP. BMP monoglucuronide was the only metabolite formed when BMP was incubated with suspensions of freshly isolated hepatocytes from male F-344 rats or with cryopreserved human hepatocytes. Glucuronidation of BMP in human hepatocytes was extremely low. Overall, the results support in vivo studies in rats in which BMP glucuronide was the only metabolite found. The poor glucuronidation capacity of humans for BMP suggests that the pharmacokinetic profile of BMP in humans will be dramatically different from that of rodents.

  10. Differential Response of Human Hepatocyte Chromatin to HDAC Inhibitors as a Function of Microenvironmental Glucose Level.

    Science.gov (United States)

    Felisbino, Marina Barreto; Alves da Costa, Thiago; Gatti, Maria Silvia Viccari; Mello, Maria Luiza Silveira

    2016-10-01

    Diabetes is a complex multifactorial disorder characterized by chronic hyperglycemia due to impaired insulin secretion. Recent observations suggest that the complexity of the disease cannot be entirely accounted for genetic predisposition and a compelling argument for an epigenetic component is rapidly emerging. The use of histone deacetylase inhibitor (HDACi) in clinical setting is an emerging area of investigation. In this study, we have aimed to understand and compare the response of hepatocyte chromatin to valproic acid (VPA) and trichostatin A (TSA) treatments under normoglycemic or hyperglycemic conditions to expand our knowledge about the consequences of HDACi treatment in a diabetes cell model. Under normoglycemic conditions, these treatments promoted chromatin remodeling, as assessed by image analysis and H3K9ac and H3K9me2 abundance. Simultaneously, H3K9ac marks shifted to the nuclear periphery accompanied by HP1 dissociation from the heterochromatin and a G1 cell cycle arrest. More striking changes in the cell cycle progression and mitotic ratios required drastic treatment. Under hyperglycemic conditions, high glucose per se promoted chromatin changes similar to those promoted by VPA and TSA. Nonetheless, these results were not intensified in cells treated with HDACis under hyperglycemic conditions. Despite the absence of morphological changes being promoted, HDACi treatment seems to confer a physiological meaning, ameliorating the cellular hyperglycemic state through reduction of glucose production. These observations allow us to conclude that the glucose level to which the hepatocytes are subjected affects how chromatin responds to HDACi and their action under high-glucose environment might not reflect on chromatin remodeling. J. Cell. Physiol. 231: 2257-2265, 2016. © 2016 Wiley Periodicals, Inc.

  11. Construction of IL-2 gene-modified human hepatocyte and its cultivation with microcarrier

    Institute of Scientific and Technical Information of China (English)

    Nan-Hong Tang; Yian-Ling Chen; Xiao-Qian Wang; Xiu-Jin Li; Feng-Zhi Yin; Xiao-Zhong Wang

    2003-01-01

    AIM: To construct interleukin-2 gene-modified humanhepatocyte line (L-02/IL-2) and investigate the changes ofthe function of liver cells and IL-2 secretion in culture withmicrocarrier, laying the foundation for further experimentationon hepatocyte transplantation.METHODS: hIL-2 gene was transduced into L-02hepatocytes by recombinant retroviral vector pLNCIL-2, andthe changes of morphology and clonogeneicity rate of thetransduced cells were observed, the secretion levels of hIL-2 in cultural supernatant were detected by ELISA and NeoRgene was amplified by PCR. The growth of L-02/IL-2, thespecial biochemistry items and the levels of IL-2 weredetected after cultivation with microcarrier.RESULTS: The clonogeneicity rate of the L-02/IL-2 cellswas lower than that of L-02/Neo cells and L-02 cells. Thelevels of hIL-2 could reach 32 000 pg/106 cells per day andkept secreting for more than ten weeks. NeoR gene segmentwas respectively obtained by PCR from both L-02/IL-2 andL-02/Neo cell's genomic DNA. At the 6th day in culture withmicrocarrier, the matrix-induced liver cell aggregates wereformed, the number of alive L-02/IL-2 cell were 16.8±0.53x106/flask and the levels of ALB and UREA were 52.54±1.28mg/L and 5.29±0.17 mmol/L, respectively. These data hadnot significantly changed as compared with those of L-02cells (P>0.05); However, the levels of IL-2 in IL-2/L-02 cellsremarkably exceeded that in L-02 cells in the whole cultureprocess (P<0.001).CONCLUSION: The IL-2 gene-modified hepatooyte line hasbeen successfully constructed. The L-02/IL-2 cellularaggregates cultured with microcarrier have a high capacityof IL-2 production as well as protein synthesis and aminoacid metabolism.

  12. Application of a higher throughput approach to derive apparent Michaelis-Menten constants of isoform-selective p450-mediated biotransformation reactions in human hepatocytes.

    Science.gov (United States)

    Li, Albert P; Schlicht, Kari E

    2014-01-01

    A higher throughput platform was developed for the determination of K(M) values for isoformselective P450 substrates in human hepatocytes via incubation of the hepatocytes with substrates in 384- well plates and metabolite quantification by RapidFire™ mass spectrometry. Isoform-selective P450 substrates were incubated at 8 concentrations in triplicate with cryopreserved human hepatocytes from 16 donors. The metabolic pathways examined were the CYP1A2-catalyzed tacrine 1-hydroxylation, CYP2B6-catalyzed bupropion hydroxylation, CYP2C8-catalyzed amodiaquine N-deethylation, CYP2C9- catalyzed diclofenac 4'-hydroxylation, CYP2D6-catalyzed dextromethorphan O-demethylation, and CYP3A4-catalyzed midazolam 1'-hydroxylation. Typical saturation enzyme kinetics was observed for all the pathways evaluated. Individual differences in the apparent V(max) and K(M) values were observed among the human hepatocytes from each of the 16 individual donors, with no statistically significant gender- or age-associated differences. A "composite" K(M) value was calculated for each of the pathways via normalizing the individual activities to their respective V(max) values to develop "relative activities" followed by Michaelis-Menten analysis of the mean relative activities of the 16 donors at each of the 8 substrate concentrations. The resulting "composite" K(M) values for the P450 substrates may be used to guide in vitro P450 inhibition and induction studies and kinetic modeling of in vivo drug-drug interaction.

  13. Epigallocatechin-3-gallate (EGCG): chemical and biomedical perspectives.

    Science.gov (United States)

    Nagle, Dale G; Ferreira, Daneel; Zhou, Yu-Dong

    2006-09-01

    The compound (-)-epigallocatechin-3-gallate (EGCG) is the major catechin found in green tea [Camellia sinensis L. Ktze. (Theaceae)]. This polyphenolic compound and several related catechins are believed to be responsible for the health benefits associated with the consumption of green tea. The potential health benefits ascribed to green tea and EGCG include antioxidant effects, cancer chemoprevention, improving cardiovascular health, enhancing weight loss, protecting the skin from the damage caused by ionizing radiation, and others. The compound EGCG has been shown to regulate dozens of disease-specific molecular targets. Many of these molecular targets are only affected by concentrations of EGCG that are far above the levels achieved by either drinking green tea or consuming moderate doses of green tea extract-based dietary supplements. In spite of this, well-designed double-blinded controlled clinical studies have recently demonstrated the efficacy of green tea extracts and purified EGCG products in patients. Therefore, this review highlights results from what the authors believe to be some of the most clinically significant recent studies and describes current developments in the stereoselective total synthesis of EGCG.

  14. Hepatic maturation of human iPS cell-derived hepatocyte-like cells by ATF5, c/EBPα, and PROX1 transduction.

    Science.gov (United States)

    Nakamori, Daiki; Takayama, Kazuo; Nagamoto, Yasuhito; Mitani, Seiji; Sakurai, Fuminori; Tachibana, Masashi; Mizuguchi, Hiroyuki

    2016-01-15

    Hepatocyte-like cells differentiated from human iPS cells (human iPS-HLCs) are expected to be utilized in drug development and research. However, recent hepatic characterization of human iPS-HLCs showed that these cells resemble fetal hepatocytes rather than adult hepatocytes. Therefore, in this study, we aimed to develop a method to enhance the hepatic function of human iPS-HLCs. Because the gene expression levels of the hepatic transcription factors (activating transcription factor 5 (ATF5), CCAAT/enhancer-binding protein alpha (c/EBPα), and prospero homeobox protein 1 (PROX1)) in adult liver were significantly higher than those in human iPS-HLCs and fetal liver, we expected that the hepatic functions of human iPS-HLCs could be enhanced by adenovirus (Ad) vector-mediated ATF5, c/EBPα, and PROX1 transduction. The gene expression levels of cytochrome P450 (CYP) 2C9, 2E1, alpha-1 antitrypsin, transthyretin, Na+/taurocholate cotransporting polypeptide, and uridine diphosphate glucuronosyl transferase 1A1 and protein expression levels of CYP2C9 and CYP2E1 were upregulated by ATF5, c/EBPα, and PROX1 transduction. These results suggest that the hepatic functions of the human iPS-HLCs could be enhanced by ATF5, c/EBPα, and PROX1 transduction. Our findings would be useful for the hepatic maturation of human iPS-HLCs.

  15. Quantitative analysis of individual hepatocyte growth factor receptor clusters in influenza A virus infected human epithelial cells using localization microscopy.

    Science.gov (United States)

    Wang, Qiaoyun; Dierkes, Rüdiger; Kaufmann, Rainer; Cremer, Christoph

    2014-04-01

    In this report, we applied a special localization microscopy technique (Spectral Precision Distance/Spatial Position Determination Microscopy/SPDM) to quantitatively analyze the effect of influenza A virus (IAV) infection on the spatial distribution of individual HGFR (Hepatocyte Growth Factor Receptor) proteins on the membrane of human epithelial cells at the single molecule resolution level. We applied this SPDM method to Alexa 488 labeled HGFR proteins with two different ligands. The ligands were either HGF (Hepatocyte Growth Factor), or IAV. In addition, the HGFR distribution in a control group of mock-incubated cells without any ligands was investigated. The spatial distribution of 1×10(6) individual HGFR proteins localized in large regions of interest on membranes of 240 cells was quantitatively analyzed and found to be highly non-random. Between 21% and 24% of the HGFR molecules were located in 44,304 small clusters with an average diameter of 54nm. The mean density of HGFR molecule signals per individual cluster was very similar in control cells, in cells with ligand only, and in IAV infected cells, independent of the incubation time. From the density of HGFR molecule signals in the clusters and the diameter of the clusters, the number of HGFR molecule signals per cluster was estimated to be in the range between 4 and 11 (means 5-6). This suggests that the membrane bound HGFR clusters form small molecular complexes with a maximum diameter of few tens of nm, composed of a relatively low number of HGFR molecules. This article is part of a Special Issue entitled: Viral Membrane Proteins - Channels for Cellular Networking. Copyright © 2013 Elsevier B.V. All rights reserved.

  16. Potency of individual bile acids to regulate bile acid synthesis and transport genes in primary human hepatocyte cultures.

    Science.gov (United States)

    Liu, Jie; Lu, Hong; Lu, Yuan-Fu; Lei, Xiaohong; Cui, Julia Yue; Ellis, Ewa; Strom, Stephen C; Klaassen, Curtis D

    2014-10-01

    Bile acids (BAs) are known to regulate their own homeostasis, but the potency of individual bile acids is not known. This study examined the effects of cholic acid (CA), chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), lithocholic acid (LCA) and ursodeoxycholic acid (UDCA) on expression of BA synthesis and transport genes in human primary hepatocyte cultures. Hepatocytes were treated with the individual BAs at 10, 30, and 100μM for 48 h, and RNA was extracted for real-time PCR analysis. For the classic pathway of BA synthesis, BAs except for UDCA markedly suppressed CYP7A1 (70-95%), the rate-limiting enzyme of bile acid synthesis, but only moderately (35%) down-regulated CYP8B1 at a high concentration of 100μM. BAs had minimal effects on mRNA of two enzymes of the alternative pathway of BA synthesis, namely CYP27A1 and CYP7B1. BAs increased the two major target genes of the farnesoid X receptor (FXR), namely the small heterodimer partner (SHP) by fourfold, and markedly induced fibroblast growth factor 19 (FGF19) over 100-fold. The BA uptake transporter Na(+)-taurocholate co-transporting polypeptide was unaffected, whereas the efflux transporter bile salt export pump was increased 15-fold and OSTα/β were increased 10-100-fold by BAs. The expression of the organic anion transporting polypeptide 1B3 (OATP1B3; sixfold), ATP-binding cassette (ABC) transporter G5 (ABCG5; sixfold), multidrug associated protein-2 (MRP2; twofold), and MRP3 (threefold) were also increased, albeit to lesser degrees. In general, CDCA was the most potent and effective BA in regulating these genes important for BA homeostasis, whereas DCA and CA were intermediate, LCA the least, and UDCA ineffective.

  17. Human induced hepatic lineage-oriented stem cells: autonomous specification of human iPS cells toward hepatocyte-like cells without any exogenous differentiation factors.

    Directory of Open Access Journals (Sweden)

    Tetsuya Ishikawa

    Full Text Available Preparing targeted cells for medical applications from human induced pluripotent stem cells (hiPSCs using growth factors, compounds, or gene transfer has been challenging. Here, we report that human induced hepatic lineage-oriented stem cells (hiHSCs were generated and expanded as a new type of hiPSC under non-typical coculture with feeder cells in a chemically defined hiPSC medium at a very high density. Self-renewing hiHSCs expressed markers of both human embryonic stem cells (hESCs and hepatocytes. Those cells were highly expandable, markedly enhancing gene expression of serum hepatic proteins and cytochrome P450 enzymes with the omission of FGF-2 from an undefined hiPSC medium. The hepatic specification of hiHSCs was not attributable to the genetic and epigenetic backgrounds of the starting cells, as they were established from distinct donors and different types of cells. Approximately 90% of hiHSCs autonomously differentiated to hepatocyte-like cells, even in a defined minimum medium without any of the exogenous growth factors necessary for hepatic specification. After 12 days of this culture, the differentiated cells significantly enhanced gene expression of serum hepatic proteins (ALB, SERPINA1, TTR, TF, FABP1, FGG, AGT, RBP4, and AHSG, conjugating enzymes (UGT2B4, UGT2B7, UGT2B10, GSTA2, and GSTA5, transporters (SULT2A1, SLC13A5, and SLCO2B1, and urea cycle-related enzymes (ARG1 and CPS1. In addition, the hepatocyte-like cells performed key functions of urea synthesis, albumin secretion, glycogen storage, indocyanine green uptake, and low-density lipoprotein uptake. The autonomous hepatic specification of hiHSCs was due to their culture conditions (coculture with feeder cells in a defined hiPSC medium at a very high density in self-renewal rather than in differentiation. These results suggest the feasibility of preparing large quantities of hepatocytes as a convenient and inexpensive hiPSC differentiation. Our study also suggests the

  18. Mitochondrial protein adducts formation and mitochondrial dysfunction during N-acetyl-m-aminophenol (AMAP)-induced hepatotoxicity in primary human hepatocytes

    Science.gov (United States)

    Xie, Yuchao; McGill, Mitchell R.; Du, Kuo; Dorko, Kenneth; Kumer, Sean C.; Schmitt, Timothy M.; Ding, Wen-Xing; Jaeschke, Hartmut

    2015-01-01

    3′-Hydroxyacetanilide or N-acetyl-meta-aminophenol (AMAP) is generally regarded as a non-hepatotoxic analog of acetaminophen (APAP). Previous studies demonstrated absence of toxicity after AMAP in mice, hamsters, primary mouse hepatocytes and several cell lines. In contrast, experiments with liver slices suggested that it may be toxic to human hepatocytes; however, the mechanism of toxicity is unclear. To explore this, we treated primary human hepatocytes (PHH) with AMAP or APAP for up to 48 h and measured several parameters to assess metabolism and injury. Although less toxic than APAP, AMAP dose-dependently triggered cell death in PHH as indicated by alanine aminotransferase (ALT) release and propidium iodide (PI) staining. Similar to APAP, AMAP also significantly depleted glutathione (GSH) in PHH and caused mitochondrial damage as indicated by glutamate dehydrogenase (GDH) release and the JC-1 assay. However, unlike APAP, AMAP treatment did not cause relevant c-jun-N-terminal kinase (JNK) activation in the cytosol or phospho-JNK translocation to mitochondria. To compare, AMAP toxicity was assessed in primary mouse hepatocytes (PMH). No cytotoxicity was observed as indicated by the lack of lactate dehydrogenase release and no PI staining. Furthermore, there was no GSH depletion or mitochondrial dysfunction after AMAP treatment in PMH. Immunoblotting for arylated proteins suggested that AMAP treatment caused extensive mitochondrial protein adducts formation in PHH but not in PMH. In conclusion, AMAP is hepatotoxic in PHH and the mechanism involves formation of mitochondrial protein adducts and mitochondrial dysfunction. PMID:26431796

  19. Therapeutic angiogenesis induced by human hepatocyte growth factor gene in hindlimb ischemia of dogs

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    A preclinical study of treating peripheral artery occlusive disease (PAD) was performed by using a hepatocyte growth factor (HGF) gene-expressing vector, plasmid pUDKH, in a dog model with complete ischemia of one hindlimb. After ligation of femoral artery of one hindlimb, pUDKH was transferred directly into the ischemic limb muscles. The angiogenic activity of the plasmid pUDKH was evaluated. On D 30 after injecting once of pUDKH at different doses into local muscles immediately after operation, the degree of augmentation of collateral vessel formation was significantly greater than that treated by blank vector. In addition, the blood flow rate of femoral artery in dogs treated with pUDKH was recovered on D 90, while the flow rate was only 1/5 to 1/3 in control dogs. The pulse amplitude of pUDKH-treated dogs was recovered on D 90, but it was hardly detectable in most of the control dogs. The side effects of intramuscular transfection of pUDKH were also investigated, and no significant positive change was found. It is suggested that angiogenesis induced by HGF gene has the potential for clinical use in the treatment of peripheral arterial diseases.

  20. Effects of artificial sweeteners on the AhR- and GR-dependent CYP1A1 expression in primary human hepatocytes and human cancer cells.

    Science.gov (United States)

    Kamenickova, Alzbeta; Pecova, Michaela; Bachleda, Petr; Dvorak, Zdenek

    2013-12-01

    Food constituents may cause a phenomenon of food-drug interactions. In the current study, we examined the effects of artificial sweeteners (aspartame, acesulfame, cyclamate, saccharin) on the aryl hydrocarbon receptor (AhR) and glucocorticoid receptor (GR)-dependent expression of CYP1A1 in human hepatocytes, hepatic HepG2 and intestinal LS174T cancer cell lines. Sweeteners were tested in concentrations up to those occurring in non-alcoholic beverages. Basal and ligand-inducible AhR- and GR-dependent reporter gene activation in stably transfected HepG2 and HeLa cells, respectively, were not affected by either of the sweeteners tested after 24h of incubation. The expression of CYP1A1 mRNA and protein in primary cultures of human hepatocytes and in LS174T and HepG2 cells was not induced by any of the tested sweeteners. Overall, aspartame, acesulfame, saccharin and cyclamate had no effects on CYP1A1 expression and transcriptional activities of AhR and GR. These data imply the safety of artificial sweeteners in terms of interference with AhR, GR and CYP1A1.

  1. Activation of the constitutive androstane receptor inhibits gluconeogenesis without affecting lipogenesis or fatty acid synthesis in human hepatocytes

    Energy Technology Data Exchange (ETDEWEB)

    Lynch, Caitlin; Pan, Yongmei; Li, Linhao [Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, Baltimore, MD 21201 (United States); Heyward, Scott; Moeller, Timothy [Bioreclamation In Vitro Technologies, Baltimore, MD 21227 (United States); Swaan, Peter W. [Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, Baltimore, MD 21201 (United States); Wang, Hongbing, E-mail: hwang@rx.umaryland.edu [Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, Baltimore, MD 21201 (United States)

    2014-08-15

    Objective: Accumulating evidence suggests that activation of mouse constitutive androstane receptor (mCAR) alleviates type 2 diabetes and obesity by inhibiting hepatic gluconeogenesis, lipogenesis, and fatty acid synthesis. However, the role of human (h) CAR in energy metabolism is largely unknown. The present study aims to investigate the effects of selective hCAR activators on hepatic energy metabolism in human primary hepatocytes (HPH). Methods: Ligand-based structure–activity models were used for virtual screening of the Specs database ( (www.specs.net)) followed by biological validation in cell-based luciferase assays. The effects of two novel hCAR activators (UM104 and UM145) on hepatic energy metabolism were evaluated in HPH. Results: Real-time PCR and Western blotting analyses reveal that activation of hCAR by UM104 and UM145 significantly repressed the expression of glucose-6-phosphatase and phosphoenolpyruvate carboxykinase, two pivotal gluconeogenic enzymes, while exerting negligible effects on the expression of genes associated with lipogenesis and fatty acid synthesis. Functional experiments show that UM104 and UM145 markedly inhibit hepatic synthesis of glucose but not triglycerides in HPH. In contrast, activation of mCAR by 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene, a selective mCAR activator, repressed the expression of genes associated with gluconeogenesis, lipogenesis, and fatty acid synthesis in mouse primary hepatocytes, which were consistent with previous observations in mouse model in vivo. Conclusion: Our findings uncover an important species difference between hCAR and mCAR in hepatic energy metabolism, where hCAR selectively inhibits gluconeogenesis without suppressing fatty acid synthesis. Implications: Such species selectivity should be considered when exploring CAR as a potential therapeutic target for metabolic disorders. - Highlights: • Novel hCAR activators were identified by computational and biological approaches. • The role

  2. Identification of early target genes of aflatoxin B1 in human hepatocytes, inter-individual variability and comparison with other genotoxic compounds

    Energy Technology Data Exchange (ETDEWEB)

    Josse, Rozenn; Dumont, Julie; Fautrel, Alain; Robin, Marie-Anne; Guillouzo, André, E-mail: andre.guillouzo@univ-rennes1.fr

    2012-01-15

    Gene expression profiling has recently emerged as a promising approach to identify early target genes and discriminate genotoxic carcinogens from non-genotoxic carcinogens and non-carcinogens. However, early gene changes induced by genotoxic compounds in human liver remain largely unknown. Primary human hepatocytes and differentiated HepaRG cells were exposed to aflatoxin B1 (AFB1) that induces DNA damage following enzyme-mediated bioactivation. Gene expression profile changes induced by a 24 h exposure of these hepatocyte models to 0.05 and 0.25 μM AFB1 were analyzed by using oligonucleotide pangenomic microarrays. The main altered signaling pathway was the p53 pathway and related functions such as cell cycle, apoptosis and DNA repair. Direct involvement of the p53 protein in response to AFB1 was verified by using siRNA directed against p53. Among the 83 well-annotated genes commonly modulated in two pools of three human hepatocyte populations and HepaRG cells, several genes were identified as altered by AFB1 for the first time. In addition, a subset of 10 AFB1-altered genes, selected upon basis of their function or tumor suppressor role, was tested in four human hepatocyte populations and in response to other chemicals. Although they exhibited large variable inter-donor fold-changes, several of these genes, particularly FHIT, BCAS3 and SMYD3, were found to be altered by various direct and other indirect genotoxic compounds and unaffected by non-genotoxic compounds. Overall, this comprehensive analysis of early gene expression changes induced by AFB1 in human hepatocytes identified a gene subset that included several genes representing potential biomarkers of genotoxic compounds. -- Highlights: ► Gene expression profile changes induced by aflatoxin B1 in human hepatocytes. ► AFB1 modulates various genes including tumor suppressor genes and proto-oncogenes. ► Important inter-individual variations in the response to AFB1. ► Some genes also altered by other

  3. Human hepatocyte growth factor promotes human bone marrow stem cells to differentiate into hepatocyte-like cells%上调hHGF促进人骨髓间充质干细胞向肝样细胞分化

    Institute of Scientific and Technical Information of China (English)

    陈丽; 崔洁; 侯军霞; 贺莉; 郑淑梅; 曾维政; 蒋明德

    2011-01-01

    This study aimed to investigate the effects of human hepatocyte growth factor (hHGF) gene on the human bone marrow stem cells (hBMSCs) developing into hepatocyte-like cells. hHGF gene was inserted into pcDNA3.1+, and then transfected into hBMSCs using Lipofectamine 2000. The expression of hHGF was confirmed to be up-regulated in the pcDNA3.1+-HGF transfected hBMSCs by using confocal laser, RT-PCR and Western blot respectively. Then the pcDNA3.1+-HGF transfected hBMSCs were cultured with the serum of alcoholic liver cirrhosis patients for 7 and 14 days. Western blot was performed to detect the alteration of α-fetoprotein (AFP) and albumin (ALB) expressions. At last, the recombinant plasmid pcDNA3.1+-HGF was constructed successfully,and could effectively increase both the hHGF mRNA and protein expressions in hBMSCs. The expression of AFP and ALB obviously increased in the pcDNA3.1+-HGF transfected hBMSCs after co-cultured with the serum of alcoholic liver cirrhosis patients for 7 and 14 days. We conclude that pcDNA3.1+-hHGF transfected hBMSCs could apparently up-regulate the expression of hHGF. Furthermore exogenous hHGF gene could promote hBMSCs to differentiate into hepatocyte-like cells, which may be a promising gene therapeutic method for terminal stage hepatopaths.%目的 探讨人促肝细胞生长因子(hHGF)对人骨髓间充质干细胞(hBMSCs)向肝样细胞分化的影响.方法 克隆hHGF基因,构建携带hHGF基因的真核表达质粒(pcDNA3.1(+)-hHGF),用脂质体法将pcDNA3.1(+)-hHGF转染hBMSCs,分别通过激光共聚焦、反转录聚合酶链反应(RT-PCR)、免疫蛋白印迹(Western blot)检测hBMSCs中表达hHGF的水平;用酒精性肝硬化患者血清诱导hHGF上调后的hBMSCs,采用Western blot分别检测诱导细胞内甲胎蛋白(AFP)及白蛋白(ALB)的表达变化.结果 成功构建pcDNA3.1(+)-hHGF表达质粒;激光共聚焦、RT-PCR、Western blot结果均表明转染后hBMSCs中hHGF分子呈高表达.Western blot

  4. Human hepatocytes loaded in 3D bioprinting generate mini-liver

    Institute of Scientific and Technical Information of China (English)

    Cheng Zhong; Hai-Yang Xie; Lin Zhou; Xiao Xu; Shu-Sen Zheng

    2016-01-01

    BACKGROUND: Because of an increasing discrepancy be-tween the number of potential liver graft recipients and the number of organs available, scientists are trying to create artiifcial liver to mimic normal liver function and therefore, to support the patient’s liver when in dysfunction. 3D printing technique meets this purpose. The present study was to test the feasibility of 3D hydrogel scaffolds for liver engineering. METHODS: We fabricated 3D hydrogel scaffolds with a bioprinter. The biocompatibility of 3D hydrogel scaffolds was tested. Sixty nude mice were randomly divided into four groups, with 15 mice in each group: control, hydrogel, hydro-gel with L02 (cell line HL-7702), and hydrogel with hepatocyte growth factor (HGF). Cells were cultured and deposited in scaffolds which were subsequently engrafted into livers after partial hepatectomy and radiation-induced liver damage (RILD). The engrafted tissues were examined after two weeks. The levels of alanine aminotransferase (ALT), aspartate amino-transferase (AST), albumin, total bilirubin, CYP1A2, CYP2C9, glutathione S-transferase (a-GST), and UDP-glucuronosyl transferase (UGT-2) were compared among the groups. He-matoxylin-eosin (HE) staining and immunohistochemistry of cKit and cytokeratin 18 (CK18) of engrafted tissues were evalu-ated. The survival time of the mice was also compared among the four groups. RESULTS: 3D hydrogel scaffolds did not impact the viability of cells. The levels of ALT, AST, albumin, total bilirubin, CY-P1A2, CYP2C9, a-GST and UGT-2 were signiifcantly improved in mice engrafted with 3D scaffold loaded with L02 compared with those in control and scaffold only (P CONCLUSION: 3D scaffold has the potential of recreating liver tissue and partial liver functions and can be used in the reconstruction of liver tissues.

  5. Identiifcation of Human Hepatocyte Proliferation Related Gene C2orf69

    Institute of Scientific and Technical Information of China (English)

    Ren-wen Zhang; Yong Qiao; Xiao-hua Hao; Hong-min Li; Hui Ren; Xiao-jing Zhang; Hong-shan Wei; Xiao-yuan Xu

    2014-01-01

    Objective To construct the prokaryotic expression vector pET-32a(+)-C2orf69 and induce the expression of recombinant proteins in vitro. Then the possible effects of recombinant protein on cell proliferation was observed and rabbit-anti-C2orf69 protein polyclonal antibodies was obtained. Methods Gene fragment of C2orf69 was ampliifed by PCR and then prokaryotic expression plasmid pET-32a(+)-C2orf69 was constructed. Recombinant protein C2orf69 expression was identiifed by SDS-PAGE and Western blot. The white-ear rabbits were immunized with purified recombinant protein C2orf69, and the potency and speciifcity of polyclonal antibody were evaluated by enzyme-linked immunosorbent assay (ELISA) and Western blot. Also, different liver cells were incubated with recombinant protein C2orf69 in vitro. Results C2orf69 gene fragment was successfully ampliifed, results of gene sequencing were consistent with the sequence in GenBank. Recombinant protein of C2orf69 was successfully induced and expressed. The polyclonal antibody titer was up to 1︰1 280 000 through enzyme-linked immunosorbent assay. Results of cell proliferation showed that the recombinant protein could inhibit the proliferation of different liver cells. Conclusions The recombinant protein C2orf69 could inhibit the proliferation of different liver cells, and we speculated that it may be a widely roled inhibitor of hepatocyte proliferation. Our experiment showed that the proliferation inhibition of cells may be realized by G1 phase extending and S phase shortening.

  6. Human CB1 Receptor Isoforms, present in Hepatocytes and β-cells, are Involved in Regulating Metabolism

    Science.gov (United States)

    González-Mariscal, Isabel; Krzysik-Walker, Susan M.; Doyle, Máire E.; Liu, Qing-Rong; Cimbro, Raffaello; Santa-Cruz Calvo, Sara; Ghosh, Soumita; Cieśla, Łukasz; Moaddel, Ruin; Carlson, Olga D.; Witek, Rafal P.; O’Connell, Jennifer F.; Egan, Josephine M.

    2016-01-01

    Therapeutics aimed at blocking the cannabinoid 1 (CB1) receptor for treatment of obesity resulted in significant improvements in liver function, glucose uptake and pancreatic β-cell function independent of weight loss or CB1 receptor blockade in the brain, suggesting that peripherally-acting only CB1 receptor blockers may be useful therapeutic agents. Neuropsychiatric side effects and lack of tissue specificity precluded clinical use of first-generation, centrally acting CB1 receptor blockers. In this study we specifically analyzed the potential relevance to diabetes of human CB1 receptor isoforms in extraneural tissues involved in glucose metabolism. We identified an isoform of the human CB1 receptor (CB1b) that is highly expressed in β-cells and hepatocytes but not in the brain. Importantly, CB1b shows stronger affinity for the inverse agonist JD-5037 than for rimonabant compared to CB1 full length. Most relevant to the field, CB1b is a potent regulator of adenylyl cyclase activity in peripheral metabolic tissues. CB1b blockade by JD-5037 results in stronger adenylyl cyclase activation compared to rimonabant and it is a better enhancer of insulin secretion in β-cells. We propose this isoform as a principal pharmacological target for the treatment of metabolic disorders involving glucose metabolism. PMID:27641999

  7. Cytotoxicity, oxidative stress, and genotoxicity in human hepatocyte and embryonic kidney cells exposed to ZnO nanoparticles

    Science.gov (United States)

    Guan, Rongfa; Kang, Tianshu; Lu, Fei; Zhang, Zhiguo; Shen, Haitao; Liu, Mingqi

    2012-10-01

    Traces of zinc oxide nanoparticles (ZnO NPs) used may be found in the liver and kidney. The aim of this study is to determine the optimal viability assay for using with ZnO NPs and to assess their toxicity to human hepatocyte (L02) and human embryonic kidney (HEK293) cells. Cellular morphology, mitochondrial function (MTT assay), and oxidative stress markers (malondialdehyde, glutathione (GSH) and superoxide dismutase (SOD)) were assessed under control and exposed to ZnO NPs conditions for 24 h. The results demonstrated that ZnO NPs lead to cellular morphological modifications, mitochondrial dysfunction, and cause reduction of SOD, depletion of GSH, and oxidative DNA damage. The exact mechanism behind ZnO NPs toxicity suggested that oxidative stress and lipid peroxidation played an important role in ZnO NPs-elicited cell membrane disruption, DNA damage, and subsequent cell death. Our preliminary data suggested that oxidative stress might contribute to ZnO NPs cytotoxicity.

  8. Human CB1 Receptor Isoforms, present in Hepatocytes and β-cells, are Involved in Regulating Metabolism.

    Science.gov (United States)

    González-Mariscal, Isabel; Krzysik-Walker, Susan M; Doyle, Máire E; Liu, Qing-Rong; Cimbro, Raffaello; Santa-Cruz Calvo, Sara; Ghosh, Soumita; Cieśla, Łukasz; Moaddel, Ruin; Carlson, Olga D; Witek, Rafal P; O'Connell, Jennifer F; Egan, Josephine M

    2016-09-19

    Therapeutics aimed at blocking the cannabinoid 1 (CB1) receptor for treatment of obesity resulted in significant improvements in liver function, glucose uptake and pancreatic β-cell function independent of weight loss or CB1 receptor blockade in the brain, suggesting that peripherally-acting only CB1 receptor blockers may be useful therapeutic agents. Neuropsychiatric side effects and lack of tissue specificity precluded clinical use of first-generation, centrally acting CB1 receptor blockers. In this study we specifically analyzed the potential relevance to diabetes of human CB1 receptor isoforms in extraneural tissues involved in glucose metabolism. We identified an isoform of the human CB1 receptor (CB1b) that is highly expressed in β-cells and hepatocytes but not in the brain. Importantly, CB1b shows stronger affinity for the inverse agonist JD-5037 than for rimonabant compared to CB1 full length. Most relevant to the field, CB1b is a potent regulator of adenylyl cyclase activity in peripheral metabolic tissues. CB1b blockade by JD-5037 results in stronger adenylyl cyclase activation compared to rimonabant and it is a better enhancer of insulin secretion in β-cells. We propose this isoform as a principal pharmacological target for the treatment of metabolic disorders involving glucose metabolism.

  9. Mathematical model of uptake and metabolism of arsenic(III in human hepatocytes - Incorporation of cellular antioxidant response and threshold-dependent behavior

    Directory of Open Access Journals (Sweden)

    Isukapalli Sastry S

    2011-01-01

    Full Text Available Abstract Background Arsenic is an environmental pollutant, potent human toxicant, and oxidative stress agent with a multiplicity of health effects associated with both acute and chronic exposures. A semi-mechanistic cellular-level toxicokinetic (TK model was developed in order to describe the uptake, biotransformation and clearance of arsenical species in human hepatocytes. Notable features of this model are the incorporation of arsenic-glutathione complex formation and a "switch-like" formulation to describe the antioxidant response of hepatocytes to arsenic exposure. Results The cellular-level TK model applies mass action kinetics in order to predict the concentrations of trivalent and pentavalent arsenicals in hepatocytes. The model simulates uptake of arsenite (iAsIII via aquaporin isozymes 9 (AQP9s, glutathione (GSH conjugation, methylation by arsenic methyltransferase (AS3MT, efflux through multidrug resistant proteins (MRPs and the induced antioxidant response via thioredoxin reductase (TR activity. The model was parameterized by optimization of model estimates for arsenite (iAsIII, monomethylated (MMA and dimethylated (DMA arsenicals concentrations with time-course experimental data in human hepatocytes for a time span of 48 hours, and dose-response data at 24 hours for a range of arsenite concentrations from 0.1 to 10 μM. Global sensitivity analysis of the model showed that at low doses the transport parameters had a dominant role, whereas at higher doses the biotransformation parameters were the most significant. A parametric comparison of the TK model with an analogous model developed for rat hepatocytes from the literature demonstrated that the biotransformation of arsenite (e.g. GSH conjugation has a large role in explaining the variation in methylation between rats and humans. Conclusions The cellular-level TK model captures the temporal modes of arsenical accumulation in human hepatocytes. It highlighted the key biological

  10. Omeprazole and lansoprazole enantiomers induce CYP3A4 in human hepatocytes and cell lines via glucocorticoid receptor and pregnane X receptor axis.

    Science.gov (United States)

    Novotna, Aneta; Dvorak, Zdenek

    2014-01-01

    Benzimidazole drugs lansoprazole and omeprazole are used for treatment of various gastrointestinal pathologies. Both compounds cause drug-drug interactions because they activate aryl hydrocarbon receptor and induce CYP1A genes. In the current paper, we examined the effects of lansoprazole and omeprazole enantiomers on the expression of key drug-metabolizing enzyme CYP3A4 in human hepatocytes and human cancer cell lines. Lansoprazole enantiomers, but not omeprazole, were equipotent inducers of CYP3A4 mRNA in HepG2 cells. All forms (S-, R-, rac-) of lansoprazole and omeprazole induced CYP3A4 mRNA and protein in human hepatocytes. The quantitative profiles of CYP3A4 induction by individual forms of lansoprazole and omeprazole exerted enantiospecific patterns. Lansoprazole dose-dependently activated pregnane X receptor PXR in gene reporter assays, and slightly modulated rifampicin-inducible PXR activity, with similar potency for each enantiomer. Omeprazole dose-dependently activated PXR and inhibited rifampicin-inducible PXR activity. The effects of S-omeprazole were much stronger as compared to those of R-omeprazole. All forms of lansoprazole, but not omeprazole, slightly activated glucocorticoid receptor and augmented dexamethasone-induced GR transcriptional activity. Omeprazole and lansoprazole influenced basal and ligand inducible expression of tyrosine aminotransferase, a GR-target gene, in HepG2 cells and human hepatocytes. Overall, we demonstrate here that omeprazole and lansoprazole enantiomers induce CYP3A4 in HepG2 cells and human hepatocytes. The induction comprises differential interactions of omeprazole and lansoprazole with transcriptional regulators PXR and GR, and some of the effects were enantiospecific. The data presented here might be of toxicological and clinical importance, since the effects occurred in therapeutically relevant concentrations.

  11. Long-term culture and cryopreservation does not affect the stability and functionality of human embryonic stem cell-derived hepatocyte-like cells.

    Science.gov (United States)

    Mandal, Arundhati; Raju, Sheena; Viswanathan, Chandra

    2016-02-01

    Human embryonic stem cells (hESCs) are predicted to be an unlimited source of hepatocytes which can pave the way for applications such as cell replacement therapies or as a model of human development or even to predict the hepatotoxicity of drug compounds. We have optimized a 23-d differentiation protocol to generate hepatocyte-like cells (HLCs) from hESCs, obtaining a relatively pure population which expresses the major hepatic markers and is functional and mature. The stability of the HLCs in terms of hepato-specific marker expression and functionality was found to be intact even after an extended period of in vitro culture and cryopreservation. The hESC-derived HLCs have shown the capability to display sensitivity and an alteration in the level of CYP enzyme upon drug induction. This illustrates the potential of such assays in predicting the hepatotoxicity of a drug compound leading to advancement of pharmacology.

  12. Adenovirus-mediated transfer of hepatocyte growth factor gene to human dental pulp stem cells under good manufacturing practice improves their potential for periodontal regeneration in swine

    OpenAIRE

    2015-01-01

    Introduction Periodontitis is one of the most widespread infectious diseases in humans. We previously promoted significant periodontal tissue regeneration in swine models with the transplantation of autologous periodontal ligament stem cells (PDLSCs) and PDLSC sheet. We also promoted periodontal tissue regeneration in a rat model with a local injection of allogeneic bone marrow mesenchymal stem cells. The purpose of the present study is to investigate the roles of the hepatocyte growth factor...

  13. Human hepatocytes support the hypertrophic but not the hyperplastic response to the murine nongenotoxic hepatocarcinogen sodium phenobarbital in an in vivo study using a chimeric mouse with humanized liver.

    Science.gov (United States)

    Yamada, Tomoya; Okuda, Yu; Kushida, Masahiko; Sumida, Kayo; Takeuchi, Hayato; Nagahori, Hirohisa; Fukuda, Takako; Lake, Brian G; Cohen, Samuel M; Kawamura, Satoshi

    2014-11-01

    High doses of sodium phenobarbital (NaPB), a constitutive androstane receptor (CAR) activator, have been shown to produce hepatocellular tumors in rodents by a mitogenic mode of action (MOA) involving CAR activation. The effect of 1-week dietary treatment with NaPB on liver weight and histopathology, hepatic CYP2B enzyme activity and CYP2B/3A mRNA expression, replicative DNA synthesis and selected genes related to cell proliferation, and functional transcriptomic and metabolomic analyses was studied in male CD-1 mice, Wistar Hannover (WH) rats, and chimeric mice with human hepatocytes. The treatment of chimeric mice with 1000-1500-ppm NaPB resulted in plasma levels around 3-5-fold higher than those observed in human subjects given therapeutic doses of NaPB. NaPB produced dose-dependent increases in hepatic CYP2B activity and CYP2B/3A mRNA levels in all animal models. Integrated functional metabolomic and transcriptomic analyses demonstrated that the responses to NaPB in the human liver were clearly different from those in rodents. Although NaPB produced a dose-dependent increase in hepatocyte replicative DNA synthesis in CD-1 mice and WH rats, no increase in replicative DNA synthesis was observed in human hepatocyte-originated areas of chimeric mice. In addition, treatment with NaPB had no effect on Ki-67, PCNA, GADD45β, and MDM2 mRNA expression in chimeric mice, whereas significant increases were observed in CD-1 mice and/or WH rats. However, increases in hepatocyte replicative DNA synthesis were observed in chimeric mice both in vivo and in vitro after treatment epidermal growth factor. Thus, although NaPB could activate CAR in both rodent and human hepatocytes, NaPB did not increase replicative DNA synthesis in human hepatocytes of chimeric mice, whereas it was mitogenic to rat and mouse hepatocytes. As human hepatocytes are refractory to the mitogenic effects of NaPB, the MOA for NaPB-induced rodent liver tumor formation is thus not relevant for humans. © The

  14. Differential effects of calcineurin inhibitors, tacrolimus and cyclosporin a, on interferon-induced antiviral protein in human hepatocyte cells.

    Science.gov (United States)

    Hirano, Kumi; Ichikawa, Tatsuki; Nakao, Kazuhiko; Matsumoto, Azusa; Miyaaki, Hisamitsu; Shibata, Hidetaka; Eguchi, Susumu; Takatsuki, Mitsuhisa; Ikeda, Masanori; Yamasaki, Hironori; Kato, Nobuyuki; Kanematsu, Takashi; Ishii, Nobuko; Eguchi, Katsumi

    2008-03-01

    The premise of our study is that selective inhibition of interferon (IFN) by calcineurin inhibitors contribute to the increased severity of hepatitis C virus (HCV) posttransplantation. Therefore, we examined the influence of calcineurin inhibitors in the human hepatocyte cell line on IFN-alpha-induced phosphorylation of Janus kinase (Jak) and signal transducers and activators of transcription (STAT), nuclear translocation of IFN-stimulated gene factor 3 (ISGF-3), IFN-stimulated regulatory element (ISRE)-contained promoter activity, and the expressions of antiviral proteins. Tacrolimus (Tac), but not cyclosporin A (CyA), had an inhibitory effect on IFN-alpha-induced double-stranded ribonucleic acid (RNA)-dependent protein kinase (PKR) in a dose-dependent manner. STAT-1 also acted in a similar fashion to PKR. IFN-alpha combined with Tac attenuated the ISRE-containing promoter gene activity as compared with IFN-alpha alone. In contrast, its expression in pretreated CyA was slightly attenuated. In pretreated Tac, but not CyA, the levels of IFN-alpha-induced tyrosine phosphorylated STAT-1 and -2 were clearly lower than those induced by IFN-alpha alone. Tac and CyA did not decrease the IFN-alpha-induced JAK-1 phosphorylation. The nuclear translocation rate of tyrosine phosphorylated STAT-1 was inhibited by pretreatment of both Tac and CyA by western blotting and immunohistochemistry. In an HCV replicon system, pretreated Tac diminished the replication inhibitory effect of IFN-alpha. In this study, we show that calcineurin inhibitors, especially Tac, are the negative regulators of IFN signaling in the hepatocyte; the greatest cause of such inhibition is the phosphorylation disturbance of STAT-1, next to inhibition of the nuclear translocation of STAT-1. In conclusion, disturbance of tyrosine phosphorylation of STAT-1 resulted in diminished ISRE-containing promoter activity and a decline in antiviral protein expression. Moreover, the replication of HCV was activated. This

  15. Investigation of the CYP2C9 induction profile in human hepatocytes by combining experimental and modelling approaches.

    Science.gov (United States)

    Belic, Ales; Temesvári, Manna; Kohalmy, Krisztina; Vrzal, Radim; Dvorak, Zdenek; Rozman, Damjana; Monostory, Katalin

    2009-12-01

    The goal of the present review is to characterise the induction profile of CYP2C9, a member of the cytochrome P450 superfamily. Since the mechanism of CYP2C9 induction is fairly complex, with parallel processes triggered by various inducers, an evaluation of the experimental results is often a great challenge. At least three nuclear receptors, the glucocrticoid receptor (GR), the pregnane X receptor (PXR) and the constitutive androstane receptor (CAR), are known to mediate the CYP2C9 gene induction in man. However, mathematical modelling and simulation can provide an appropriate tool for the interpretation of CYP2C9 regulatory mechanisms. As an example, we present modelling and simulation approaches of the CYP2C9 gene expression in human hepatocytes treated with well-known CYP2C9 inducers: the steroid hormone precursor dehydroepiandrosterone (DHEA) and the synthetic glucocorticoid dexamethasone (DXM). The results of the analysis suggest that in addition to the potent function of GR and the further involvement of PXR and CAR activated by DXM or DHEA, an additional factor might play a role in CYP2C9 regulation by DHEA. The novel potential candidate for DHEA action in CYP2C9 induction is likely to be the estrogen receptor. Additionally, the balance of DHEA sulphation-desulphation processes should also be considered in any description of DHEA-induced CYP2C9 profiles.

  16. Disturbance of gene expression in primary human hepatocytes by hepatotoxic pyrrolizidine alkaloids: A whole genome transcriptome analysis.

    Science.gov (United States)

    Luckert, Claudia; Hessel, Stefanie; Lenze, Dido; Lampen, Alfonso

    2015-10-01

    1,2-unsaturated pyrrolizidine alkaloids (PA) are plant metabolites predominantly occurring in the plant families Asteraceae and Boraginaceae. Acute and chronic PA poisoning causes severe hepatotoxicity. So far, the molecular mechanisms of PA toxicity are not well understood. To analyze its mode of action, primary human hepatocytes were exposed to a non-cytotoxic dose of 100 μM of four structurally different PA: echimidine, heliotrine, senecionine, senkirkine. Changes in mRNA expression were analyzed by a whole genome microarray. Employing cut-off values with a |fold change| of 2 and a q-value of 0.01, data analysis revealed numerous changes in gene expression. In total, 4556, 1806, 3406 and 8623 genes were regulated by echimidine, heliotrine, senecione and senkirkine, respectively. 1304 genes were identified as commonly regulated. PA affected pathways related to cell cycle regulation, cell death and cancer development. The transcription factors TP53, MYC, NFκB and NUPR1 were predicted to be activated upon PA treatment. Furthermore, gene expression data showed a considerable interference with lipid metabolism and bile acid flow. The associated transcription factors FXR, LXR, SREBF1/2, and PPARα/γ/δ were predicted to be inhibited. In conclusion, though structurally different, all four PA significantly regulated a great number of genes in common. This proposes similar molecular mechanisms, although the extent seems to differ between the analyzed PA as reflected by the potential hepatotoxicity and individual PA structure.

  17. Long-term exposure to abnormal glucose levels alters drug metabolism pathways and insulin sensitivity in primary human hepatocytes

    Science.gov (United States)

    Davidson, Matthew D.; Ballinger, Kimberly R.; Khetani, Salman R.

    2016-06-01

    Hyperglycemia in type 2 diabetes mellitus has been linked to non-alcoholic fatty liver disease, which can progress to inflammation, fibrosis/cirrhosis, and hepatocellular carcinoma. Understanding how chronic hyperglycemia affects primary human hepatocytes (PHHs) can facilitate the development of therapeutics for these diseases. Conversely, elucidating the effects of hypoglycemia on PHHs may provide insights into how the liver adapts to fasting, adverse diabetes drug reactions, and cancer. In contrast to declining PHH monocultures, micropatterned co-cultures (MPCCs) of PHHs and 3T3-J2 murine embryonic fibroblasts maintain insulin-sensitive glucose metabolism for several weeks. Here, we exposed MPCCs to hypo-, normo- and hyperglycemic culture media for ~3 weeks. While albumin and urea secretion were not affected by glucose level, hypoglycemic MPCCs upregulated CYP3A4 enzyme activity as compared to other glycemic states. In contrast, hyperglycemic MPCCs displayed significant hepatic lipid accumulation in the presence of insulin, while also showing decreased sensitivity to insulin-mediated inhibition of glucose output relative to a normoglycemic control. In conclusion, we show for the first time that PHHs exposed to hypo- and hyperglycemia can remain highly functional, but display increased CYP3A4 activity and selective insulin resistance, respectively. In the future, MPCCs under glycemic states can aid in novel drug discovery and mechanistic investigations.

  18. A Schisandra-Derived Compound Schizandronic Acid Inhibits Entry of Pan-HCV Genotypes into Human Hepatocytes

    Science.gov (United States)

    Qian, Xi-Jing; Zhang, Xiao-Lian; Zhao, Ping; Jin, Yong-Sheng; Chen, Hai-Sheng; Xu, Qing-Qiang; Ren, Hao; Zhu, Shi-Ying; Tang, Hai-Lin; Zhu, Yong-Zhe; Qi, Zhong-Tian

    2016-01-01

    Despite recent progress in the development of hepatitis C virus (HCV) inhibitors, cost-effective antiviral drugs, especially among the patients receiving liver transplantations, are still awaited. Schisandra is a traditional medicinal herb used to treat a range of liver disorders including hepatitis for thousands of years in China. To isolate the bioactive compounds of schisandra for the treatment of HCV infection, we screened a schisandra-extracts library and identified a tetracyclic triterpenoid, schizandronic acid (SZA), as a novel HCV entry inhibitor. Our findings suggested that SZA potently inhibited pan-HCV genotype entry into hepatoma cells and primary human hepatocytes without interfering virus binding on cell surface or internalization. However, virion-cell fusion process was impaired in the presence of SZA, along with the increased host membrane fluidity. We also found that SZA inhibited the spread of HCV to the neighboring cells, and combinations of SZA with interferon or telaprevir resulted in additive synergistic effect against HCV. Additionally, SZA diminished the establishment of HCV infection in vivo. The SZA target is different from conventional direct-acting antiviral agents, therefore, SZA is a potential therapeutic compound for the development of effective HCV entry inhibitors, especially for patients who need to prevent HCV reinfection during the course of liver transplantations. PMID:27252043

  19. In vitro evaluation of major in vivo drug metabolic pathways using primary human hepatocytes and HepaRG cells in suspension and a dynamic three-dimensional bioreactor system.

    Science.gov (United States)

    Darnell, Malin; Ulvestad, Maria; Ellis, Ewa; Weidolf, Lars; Andersson, Tommy B

    2012-10-01

    Major human specific metabolites, not detected during in vivo and in vitro preclinical studies, may cause unexpected drug interactions and toxicity in human and delays in clinical programs. Thus, reliable preclinical tools for the detection of major human metabolites are of high importance. The aim of this study was to compare major drug metabolic pathways in HepaRG cells, a human hepatoma cell line, to fresh human hepatocytes, cryopreserved human hepatocytes, and human in vivo data. Furthermore, the maintenance of cytochrome P450 (P450) and UDP-glucuronosyltransferase (UGT) activities in a dynamic three-dimensional (3D) bioreactor were evaluated over time by using HepaRG cells and human hepatocytes. (14)C-diclofenac and a candidate from AstraZeneca's drug development program, (14)C-AZD6610, which are metabolized by P450 and UGT in vivo, were used as model substrates. The proportion of relevant biotransformation pathways of the investigated drug was clearly different in the various cell systems. The hydroxylation route was favored in primary human hepatocytes, whereas the glucuronidation route was favored in HepaRG cells. The human in vivo metabolite profile of AZD6610 was best represented by human hepatocytes, whereas all major diclofenac metabolites were detected in HepaRG cells. Moreover, the metabolite profiles in cryopreserved and fresh human hepatocytes were essentially the same. The liver bioreactor using both fresh human hepatocytes and HepaRG cells retained biotransformation capacity over 1 week. Thus, the incubation time can be increased from a few hours in suspension to several days in 3D cultures, which opens up for detection of metabolites from slowly metabolized drugs.

  20. Secretory expression and characterization of a recombinant deleted variant of human hepatocyte growth factor in Pichia pastoris

    Institute of Scientific and Technical Information of China (English)

    Zhi-Min Liu; Hong-Liang Zhao; Chong Xue; Bing-Bing Deng; Wei Zhang; Xiang-Hua Xiong; Bing-Fen Yang; Xue-Qin Yao

    2005-01-01

    AIM: To study the secretory expression of human hepatocyte growth factor (hdHGF) gene in Pichia pastoris.METHODS: The full-length gene of human cDNA encoding the deleted variant of hdHGF was cloned by RT-PCR and overlapping-fragment PCR technique using mRNA of human placenta as a template. The cloned hdHGF cDNA was inserted into the Escherichia coliyeast shuttle vector of pPIC9. The constructed plasmid,pPIC9-hdHGF, was transformed into the GS115 cells of the methylotrophic yeast, P pastoris, using a chemical method. The Mut+ transformants were screened to obtain high-expression strains by the test and analysis of expressed products of shake-flask culture. A secretory form of rhdHGF was made with the aid of the leader peptide sequence of Saccharomyces cerevisiae α-factor.RESULTS: The expressed products, which showed a band of molecular mass of about 80 ku, were observed on 15% SDS-PAGE and identified by Western blotting and N-terminal amino acid sequencing. In the high cell density culture of 5 L fermentor by fed-batch culture protocol, the cell biomass was reached at approximately 135 g (DCW)/L. The productivity of secreted total supernant protein concentration attained a high-level expression of more than 8.0 g/L and the ratio of rhdHGF band area was about 12.3% of the total band area scanned by SDS-PAGE analysis, which estimated that the product of rhdHGF was 500-900 mg/L.CONCLUSION: The P pastoris system represents an attractive tool of generating large quantities of hdHGF for both research and industrial purposes.

  1. Mixed-ligand copper(II) complexes activate aryl hydrocarbon receptor AhR and induce CYP1A genes expression in human hepatocytes and human cell lines.

    Science.gov (United States)

    Kubešová, Kateřina; Dořičáková, Aneta; Trávníček, Zdeněk; Dvořák, Zdeněk

    2016-07-25

    The effects of four copper(II) mixed-ligand complexes [Cu(qui1)(L)]NO3·H2O (1-3) and [Cu(qui2)(phen)]NO3 (4), where qui1=2-phenyl-3-hydroxy-4(1H)-quinolinone, Hqui2=2-(4-amino-3,5-dichlorophenyl)-N-propyl-3-hydroxy-4(1H)-quinolinone-7-carboxamide, L=1,10-phenanthroline (phen) (1), 5-methyl-1,10-phenanthroline (mphen) (2), bathophenanthroline (bphen) (3), on transcriptional activities of steroid receptors, nuclear receptors and xenoreceptors have been studied. The complexes (1-4) did not influence basal or ligand-inducible activities of glucocorticoid receptor, androgen receptor, thyroid receptor, pregnane X receptor and vitamin D receptor, as revealed by gene reporter assays. The complexes 1 and 2 dose-dependently induced luciferase activity in stable gene reporter AZ-AhR cell line, and this induction was reverted by resveratrol, indicating involvement of aryl hydrocarbon receptor (AhR) in the process. The complexes 1, 2 and 3 induced CYP1A1 mRNA in LS180 cells and CYP1A1/CYP1A2 in human hepatocytes through AhR. Electrophoretic mobility shift assay EMSA showed that the complexes 1 and 2 transformed AhR in its DNA-binding form. Collectively, we demonstrate that the complexes 1 and 2 activate AhR and induce AhR-dependent genes in human hepatocytes and cancer cell lines. In conclusion, the data presented here might be of toxicological importance, regarding the multiple roles of AhR in human physiology and pathophysiology.

  2. EGCG protects endothelial cells against PCB 126-induced inflammation through inhibition of AhR and induction of Nrf2-regulated genes

    Energy Technology Data Exchange (ETDEWEB)

    Han, Sung Gu [Superfund Research Program, University of Kentucky, Lexington, KY 40536 (United States); Department of Animal and Food Sciences, College of Agriculture, University of Kentucky, Lexington, KY 40536 (United States); Han, Seong-Su [Department of Pathology, College of Medicine, University of Iowa, Iowa City, IA 52242 (United States); Toborek, Michal [Department of Neurosurgery, University of Kentucky, Lexington, KY 40536 (United States); Hennig, Bernhard, E-mail: bhennig@uky.edu [Superfund Research Program, University of Kentucky, Lexington, KY 40536 (United States); Department of Animal and Food Sciences, College of Agriculture, University of Kentucky, Lexington, KY 40536 (United States)

    2012-06-01

    Tea flavonoids such as epigallocatechin gallate (EGCG) protect against vascular diseases such as atherosclerosis via their antioxidant and anti-inflammatory functions. Persistent and widespread environmental pollutants, including polychlorinated biphenyls (PCB), can induce oxidative stress and inflammation in vascular endothelial cells. Even though PCBs are no longer produced, they are still detected in human blood and tissues and thus considered a risk for vascular dysfunction. We hypothesized that EGCG can protect endothelial cells against PCB-induced cell damage via its antioxidant and anti-inflammatory properties. To test this hypothesis, primary vascular endothelial cells were pretreated with EGCG, followed by exposure to the coplanar PCB 126. Exposure to PCB 126 significantly increased cytochrome P450 1A1 (Cyp1A1) mRNA and protein expression and superoxide production, events which were significantly attenuated following pretreatment with EGCG. Similarly, EGCG also reduced DNA binding of NF-κB and downstream expression of inflammatory markers such as monocyte chemotactic protein-1 (MCP-1) and vascular cell adhesion protein-1 (VCAM-1) after PCB exposure. Furthermore, EGCG decreased endogenous or base-line levels of Cyp1A1, MCP-1 and VCAM-1 in endothelial cells. Most of all, treatment of EGCG upregulated expression of NF-E2-related factor 2 (Nrf2)-controlled antioxidant genes, including glutathione S transferase (GST) and NAD(P)H:quinone oxidoreductase 1 (NQO1), in a dose-dependent manner. In contrast, silencing of Nrf2 increased Cyp1A1, MCP-1 and VCAM-1 and decreased GST and NQO1 expression, respectively. These data suggest that EGCG can inhibit AhR regulated genes and induce Nrf2-regulated antioxidant enzymes, thus providing protection against PCB-induced inflammatory responses in endothelial cells. -- Highlights: ► PCBs cause endothelial inflammation and subsequent atherosclerosis. ► Nutrition can modulate toxicity by environmental pollutants. ► We

  3. Integrated comparison of drug-related and drug-induced ultra performance liquid chromatography/mass spectrometry metabonomic profiles using human hepatocyte cultures.

    Science.gov (United States)

    Croixmarie, Vincent; Umbdenstock, Thierry; Cloarec, Olivier; Moreau, Amélie; Pascussi, Jean-Marc; Boursier-Neyret, Claire; Walther, Bernard

    2009-08-01

    The biochemical variations induced in human primary hepatocyte cultures by reference activators of xenoreceptor CAR (NR1I3) and PXR (NR1I2), i.e., rifampicin, phenobarbital, and 6-(4-chlorophenyl)imidazo[2,1-b] [1,3]thiazole-5-carbaldehyde O-3,4-dichlorobenzyl) oxime (CITCO), were investigated using a global metabonomics approach. Cultured human hepatocytes were treated with the three drugs before analysis of intracellular and extracellular media by ultra performance liquid chromatography/time-of-flight-mass spectrometry (UPLC/TOF-MS) technique, in order to list endogenous compounds potentially related to a PXR or CAR induction mechanism and to identify drug metabolites related to each treatment. The emphasis was put on the quality of the analytical data (dilution/filtration strategy before data processing) and on the appropriate pattern recognition techniques. In cellular media, the most significant variations seen in the data are not related to the treatments but to the source of hepatocytes, illustrating the importance of the genetic and/or environmental background in human liver experiments. However when applying classical multivariate statistical approaches (principal component analysis (PCA) and orthogonal partial least squares (O-PLS)), the statistical weight due to drug metabolites, present only in the treated groups, hinders the interpretation because of their predominance compared to most of the changes seen in endogenous metabolites. A new statistical approach, called shared and unique structure (SUS) plot, enabling the comparison of different treatments having the same control has been applied, allowing separation of clearly exogenous variables (drug metabolites) from endogenous biomarkers. Endogenous variables (either up- or down-regulated) have been attributed specifically to the impact of rifampicin (PXR ligand), CITCO (CAR ligand), and phenobarbital (CAR and PXR activator) on the biological regulation pathways of the hepatocytes. This global

  4. Epigallocatechin-3-Gallate (EGCG), a Green Tea Polyphenol, Stimulates Hepatic Autophagy and Lipid Clearance

    OpenAIRE

    Jin Zhou; Benjamin Livingston Farah; Rohit Anthony Sinha; Yajun Wu; Brijesh Kumar Singh; Boon-Huat Bay; Yang, Chung S.; Paul Michael Yen

    2014-01-01

    Epigallocatechin gallate (EGCG) is a major polyphenol in green tea that has been shown to have anti-inflammatory, anti-cancer, anti-steatotic effects on the liver. Autophagy also mediates similar effects; however, it is not currently known whether EGCG can regulate hepatic autophagy. Here, we show that EGCG increases hepatic autophagy by promoting the formation of autophagosomes, increasing lysosomal acidification, and stimulating autophagic flux in hepatic cells and in vivo. EGCG also increa...

  5. Hepatocyte differentiation of mesenchymalstemcells

    Institute of Scientific and Technical Information of China (English)

    Xu-Bo Wu; Ran Tao

    2012-01-01

    BACKGROUND: Liver cell transplantation and bioartiifcial liver may provide metabolic support of liver function temporary and are prospective treatments for patients with liver failure. Mesenchymal stem cells (MSCs) are expected to be an ideal cell source for transplantation or liver tissue engineering, however the hepatic differentiation of MSCs is still insufifcient for clinical application. DATA  SOURCES: A PubMed search on "mesenchymal stem cells","liver cell"and"hepatocyte differentiation"was performed on the topic, and the relevant articles published in the past ten years were reviewed. RESULTS:Hepatocyte-like cells differentiated from MSCs are a promising cell source for liver regeneration or tissue engineering. Although it is still a matter of debate as to whether MSC-derived hepatocytes may efifciently repopulate a host liver to provide adequate functional substitution, the majority of animal studies support that MSCs can become key players in liver-directed regenerative medicine. However the clinical application of human stem cells in the treatment of liver diseases is still in its infancy. CONCLUSIONS: Future studies are required to improve the efifcacy and consistency of hepatic differentiation from MSCs. It is necessary to better understand the mechanism to achieve transdifferentiation with high efifciency. More clinical trials are warranted to prove their efifcacy in the management of patients with liver failure.

  6. Differential expression of glutathione S-transferases P1-1 and A1-1 at protein and mRNA levels in hepatocytes derived from human bone marrow mesenchymal stem cells.

    Science.gov (United States)

    Allameh, Abdolamir; Esmaeli, Shahnaz; Kazemnejad, Somaieh; Soleimani, Masoud

    2009-06-01

    The aim of this study was to find out the profile of cellular glutathione (GSH) and GSH S-transferase (GST) in hepatocytes differentiated from adult mesenchymal stem cells (MSC). For this purpose, we have derived functionally active hepatocyte-like cells from normal human multipotent adult MSC. Then the differentiated cells were characterized by specific hepatic markers. The cellular GSH and GST catalytic activity toward 1-chloro-2,4-dinitrobenzene (CDNB) were determined in hepatocyte-like cells differentiated from MSC compared with undifferentiated MSC. Reverse transcription polymerase chain reaction (RT-PCR) and immunoblotting techniques were used to study GST-P1-1 and GST-A1-1 expression in differentiated and undifferentiated cells. The results showed that there is more than threefold increase in GST catalytic activity in hepatocytes recovered by day 14 of differentiation. GST-P1-1 mRNA expression was detected in both differentiated hepatocyte-like cells and their undifferentiated progenitors. Under similar conditions, only differentiated hepatocyte-like cells expressed GST-A1-1 mRNA. These results were further confirmed by showing that the undifferentiated cells expressed both GST-A and GST-P proteins. Unlike GST, the level of cellular GSH was declined (approximately 20%) in hepatocytes derived from MSC as compared to that of undifferentiated cells. These data may suggest that hepatogenic differentiation of human bone marrow MSC is accompanied with the regulation of factors participating in GSH conjugation pathway.

  7. Differential effects of omeprazole and lansoprazole enantiomers on aryl hydrocarbon receptor in human hepatocytes and cell lines.

    Science.gov (United States)

    Novotna, Aneta; Srovnalova, Alzbeta; Svecarova, Michaela; Korhonova, Martina; Bartonkova, Iveta; Dvorak, Zdenek

    2014-01-01

    Proton pump inhibitors omeprazole and lansoprazole contain chiral sulfur atom and they are administered as a racemate, i.e. equimolar mixture of S- and R-enantiomers. The enantiopure drugs esomeprazole and dexlansoprazole have been developed and introduced to clinical practice due to their improved clinical and therapeutic properties. Since omeprazole and lansoprazole are activators of aryl hydrocarbon receptor (AhR) and inducers of CYP1A genes, we examined their enantiospecific effects on AhR-CYP1A pathway in human cancer cells and primary human hepatocytes. We performed gene reporter assays for transcriptional activity of AhR, RT-PCR analyses for CYP1A1/2 mRNAs, western blots for CYP1A1/2 proteins and EROD assay for CYP1A1/2 catalytic activity. Lansoprazole and omeprazole enantiomers displayed differential effects on AhR-CYP1A1/2 pathway. In general, S-enantiomers were stronger activators of AhR and inducers of CYP1A genes as compared to R-enantiomers in lower concentrations, i.e. 1-10 µM for lansoprazole and 10-100 µM for omeprazole. In contrast, R-enantiomers were stronger AhR activators and CYP1A inducers than S-enantiomers in higher concentrations, i.e. 100 µM for lansoprazole and 250 µM for omeprazole. In conclusion, we provide the first evidence of enantiospecific effects of omeprazole and lansoprazole on AhR signaling pathway.

  8. Hepatocyte Growth Factor Suppresses Transforming Growth Factor-Beta-1 and Type III Collagen in Human Primary Renal Fibroblasts

    Directory of Open Access Journals (Sweden)

    Shan Mou

    2009-11-01

    Full Text Available Tubulointerstitial changes in the diabetic kidney correlate closely with renal fibrosis, and transforming growth factor-beta-1 (TGF-β1 is thought to play a key role in this process. In contrast, hepatocyte growth factor (HGF has shown therapeutic effects on injured renal tubules in animal models. This study was undertaken to test the hypothesis that the preventive effects of HGF may result from interventions in TGF-β1-mediated signaling and collagen III secretion. We examined the expression of HGF/HGF receptor (c-Met and TGF-β1 in renal fibroblasts at multiple time points. The effects of recombinant human HGF on TGF-β1 expression were studied by RT-PCR and Western blotting, and the levels of collagen III were measured by ELISA. In the high-glucose condition, the expression of HGF and c-Met in renal fibroblasts was detected as early as 6 hours following cell culture while the level of TGF-β1 peaked at 96 hours. The addition of recombinant human HGF to the culture media dose-dependently inhibited TGF-β1 mRNA expression and reduced collagen III secretion by 34%. These results indicate that, during hyperglycemia, HGF inhibits TGF-β1 signaling and type III collagen activation in interstitial fibroblasts. Furthermore, we should recognize that changes in the balance between HGF and TGF-β1 might be decisive in the pathogenesis of chronic renal fibrosis. Therefore, administration of HGF to restore this balance may offer a novel therapeutic intervention in managing renal fibrogenesis in diabetic nephropathy.

  9. Induction of hepatic CYP3A enzymes by pregnancy-related hormones: studies in human hepatocytes and hepatic cell lines.

    Science.gov (United States)

    Papageorgiou, Ioannis; Grepper, Susan; Unadkat, Jashvant D

    2013-02-01

    CYP3A activity is induced by approximately 2-fold during the third trimester of human pregnancy. Placental growth hormone (PGH), estrogens (primarily 17β-estradiol), cortisol, and progesterone have the potential to modulate CYP3A activity. Therefore, we determined whether the elevated plasma concentrations of these hormones during pregnancy induce hepatic CYP3A expression. We incubated sandwich-cultured human hepatocytes (SCHH) from premenopausal female donors (n = 2) with the physiologic (unbound, 1× total) and the 10× total third trimester hormone plasma concentrations (individually and in combination) and determined their effect on CYP3A activity and the transcripts of CYP3A4, CYP3A5, and the respective hormone receptors (growth hormone receptor, glucocorticoid receptor, and estrogen receptor alpha). Of all the hormones, cortisol was the most potent inducer of CYP3A activity and CYP3A4, CYP3A5 mRNA expression. The combination of PGH/growth hormone and cortisol induced CYP3A activity and expression significantly more than did cortisol alone. When incubated with the unbound or total plasma concentration of all the hormones, CYP3A activity in SCHH was induced to an extent comparable to that observed in vivo during the third trimester. These hormones had only a modest effect on the mRNA expression of the hormone receptors. The pattern of induction observed in SCHH was reproduced in HepaRG cells but not in HuH7/HepG2 cells. SCHH or HepaRG cells could be used to determine the mechanistic basis of CYP3A induction during pregnancy and to predict the magnitude of induction likely to be observed during the first and second trimesters, when phenotyping studies to measure in vivo CYP3A activity are logistically difficult to perform.

  10. Genomewide comparison of the inducible transcriptomes of nuclear receptors CAR, PXR and PPARα in primary human hepatocytes.

    Science.gov (United States)

    Kandel, Benjamin A; Thomas, Maria; Winter, Stefan; Damm, Georg; Seehofer, Daniel; Burk, Oliver; Schwab, Matthias; Zanger, Ulrich M

    2016-09-01

    The ligand-activated nuclear receptor pregnane X receptor (PXR, NR1I2) and the constitutive androstane receptor (CAR, NR1I3) are two master transcriptional regulators of many important drug metabolizing enzymes and transporter genes (DMET) in response to xenobiotics including many drugs. The peroxisome proliferator-activated receptor alpha (PPARα, NR1C1), the target of lipid lowering fibrate drugs, primarily regulates fatty acid catabolism and energy-homeostasis. Recent research has shown that there are substantial overlaps in the regulated genes of these receptors. For example, both CAR and PXR also modulate the transcription of key enzymes involved in lipid and glucose metabolism and PPARα also functions as a direct transcriptional regulator of important DMET genes including cytochrome P450s CYP3A4 and CYP2C8. Despite their important and widespread influence on liver metabolism, comparative data are scarce, particularly at a global level and in humans. The major objective of this study was to directly compare the genome-wide transcriptional changes elucidated by the activation of these three nuclear receptors in primary human hepatocytes. Cultures from six individual donors were treated with the prototypical ligands for CAR (CITCO), PXR (rifampicin) and PPARα (WY14,643) or DMSO as vehicle control. Genomewide mRNA profiles determined with Affymetrix microarrays were analyzed for differentially expressed genes and metabolic functions. The results confirmed known prototype target genes and revealed strongly overlapping sets of coregulated but also distinctly regulated and novel responsive genes and pathways. The results further specify the role of PPARα as a regulator of drug metabolism and the role of the xenosensors PXR and CAR in lipid metabolism and energy homeostasis. This article is part of a Special Issue entitled: Xenobiotic nuclear receptors: New Tricks for An Old Dog, edited by Dr. Wen Xie.

  11. HCMV Activates the IL-6-JAK-STAT3 Axis in HepG2 Cells and Primary Human Hepatocytes

    Science.gov (United States)

    Kumar, Amit; Tripathy, Manoj K.; Herbein, Georges

    2013-01-01

    Objectives There has been increased interest in the possible role of human cytomegalovirus (HCMV) in carcinogenesis during the last decade. HCMV seroprevalence was enhanced in patients with hepatocellular carcinoma (HCC) but a possible relationship between HCC and HCMV infection remained to be assessed. The aim of this work was to investigate the pro-tumor influence of HCMV on primary human hepatocytes (PHH) and HepG2 cells. Methods Following infection of PHH and HepG2 cells by two different strains of HCMV, we measured the production of IL-6 in culture supernatants by ELISA and the protein levels of STAT3, pSTAT3, JAK, cyclin D1, survivin, p53, p21, and Mdm2 by western Blotting in infected and uninfected cells. Cell proliferation and transformation were investigated using Ki67Ag expression measurement and soft-agar colony formation assay respectively. Results Infection of HepG2 cells and PHH by HCMV resulted in the production of IL-6 and the subsequent activation of the IL-6R-JAK-STAT3 pathway. HCMV increased the expression of cyclin D1 and survivin. Cell proliferation was enhanced in HepG2 and PHH infected with HCMV, despite a paradoxical overexpression of p53 and p21. More importantly, we observed the formation of colonies in soft agar seeded with PHH infected with HCMV and when we challenged the HepG2 cultures to form tumorspheres, we found that the HCMV-infected cultures formed 2.5-fold more tumorspheres than uninfected cultures. Conclusion HCMV activated the IL-6-JAK-STAT3 pathway in PHH and HepG2 cells, favored cellular proliferation, induced PHH transformation and enhanced HepG2 tumorsphere formation. Our observations raise the possibility that HCMV infection might be involved in the genesis of hepatocellular carcinoma. PMID:23555719

  12. An extended ΔCT-method facilitating normalisation with multiple reference genes suited for quantitative RT-PCR analyses of human hepatocyte-like cells.

    Directory of Open Access Journals (Sweden)

    Gesa Riedel

    Full Text Available Reference genes (RG as sample internal controls for gene transcript level analyses by quantitative RT-PCR (RT-qPCR must be stably expressed within the experimental range. A variety of in vitro cell culture settings with primary human hepatocytes, and Huh-7 and HepG2 cell lines, were used to determine candidate RG expression stability in RT-qPCR analyses. Employing GeNorm, BestKeeper and Normfinder algorithms, this study identifies PSMB6, MDH1 and some more RG as sufficiently unregulated, thus expressed at stable levels, in hepatocyte-like cells in vitro. Inclusion of multiple RG, quenching occasional regulations of single RG, greatly stabilises gene expression level calculations from RT-qPCR data. To further enhance validity and reproducibility of relative RT-qPCR quantifications, the ΔCT calculation can be extended (e-ΔCT by replacing the CT of a single RG in ΔCT with an averaged CT-value from multiple RG. The use of two or three RG--here identified suited for human hepatocyte-like cells--for normalisation with the straightforward e-ΔCT calculation, should improve reproducibility and robustness of comparative RT-qPCR-based gene expression analyses.

  13. 10-(6'-Plastoquinonyl)decyltriphenylphosphonium (SkQ1) Does Not Increase the Level of Cytochromes P450 in Rat Liver and Human Hepatocyte Cell Culture.

    Science.gov (United States)

    Myasoedova, K N; Silachev, D N; Petrov, A D

    2016-12-01

    Mitochondria-targeted antioxidant SkQ1 did not increase the content of cytochromes P450 in livers of rats that were given SkQ1 in drinking water for 5 days in a dose (2.5 µmol per kg body weight) that exceeded 10 times the SkQ1 therapeutic dose. SkQ1 did not affect the levels of cytochrome P450 forms CYP1A2, CYP2B6, and CYP3A4 in monolayer cultures of freshly isolated human hepatocytes, while specific inducers of these forms (omeprazole, phenobarbital, and rifampicin, respectively) significantly increased expression of the cytochromes P450 under the same conditions. We conclude that therapeutic doses of SkQ1 do not induce cytochromes P450 in liver, and the absence of the inducing effect cannot be explained by poor availability of hepatocytes to SkQ1 in vivo.

  14. Effect of human liver source on the functionality of isolated hepatocytes and liver slices

    NARCIS (Netherlands)

    Olinga, Peter; Hof, I.H; de Jong, Kurt; Slooff, M.JH; Meijer, D.K F; Groothuis, Geny; Merema, M.T.

    1998-01-01

    In vitro experiments using human liver tissue to study drug metabolism and transport are usually performed and interpreted without real consideration of the differences in procurement of the tissue, if it is obtained from different sources. Therefore, in this study the functionality of isolated hepa

  15. Can EGCG reduce abdominal fat in obese subjects?

    Science.gov (United States)

    Hill, Alison M; Coates, Alison M; Buckley, Jonathan D; Ross, Robert; Thielecke, Frank; Howe, Peter R C

    2007-08-01

    To evaluate metabolic effects of epigallocatechin gallate (EGCG) supplementation when combined with a program of regular aerobic exercise in overweight/obese post-menopausal women. Thirty-eight overweight or obese postmenopausal women exercised at moderate intensity, viz. walking three times per week for 45 min at 75% of age-predicted maximum heart rate (HR), and took a 150 mg capsule of EGCG (Teavigo) or placebo (lactose) twice daily for 12 weeks. Blood parameters (lipids, glucose and insulin), blood pressure, heart rate, arterial function and anthropometry were assessed at 0, 6 and 12 wk. At wk 0 and 12, body composition was assessed by dual energy X-ray absorptiometry (DXA) and abdominal fat was assessed by DXA and computed tomography (CT). Waist circumference (p fat (p fat (by DXA) (p EGCG can improve the health status of overweight individuals undergoing regular exercise by reducing HR and plasma glucose concentrations. Loss of body fat, however, may require a higher intake of EGCG, other catechins or addition of metabolic stimulants.

  16. The pore-forming toxin listeriolysin O mediates a novel entry pathway of L. monocytogenes into human hepatocytes.

    Directory of Open Access Journals (Sweden)

    Stephen Vadia

    2011-11-01

    Full Text Available Intracellular pathogens have evolved diverse strategies to invade and survive within host cells. Among the most studied facultative intracellular pathogens, Listeria monocytogenes is known to express two invasins-InlA and InlB-that induce bacterial internalization into nonphagocytic cells. The pore-forming toxin listeriolysin O (LLO facilitates bacterial escape from the internalization vesicle into the cytoplasm, where bacteria divide and undergo cell-to-cell spreading via actin-based motility. In the present study we demonstrate that in addition to InlA and InlB, LLO is required for efficient internalization of L. monocytogenes into human hepatocytes (HepG2. Surprisingly, LLO is an invasion factor sufficient to induce the internalization of noninvasive Listeria innocua or polystyrene beads into host cells in a dose-dependent fashion and at the concentrations produced by L. monocytogenes. To elucidate the mechanisms underlying LLO-induced bacterial entry, we constructed novel LLO derivatives locked at different stages of the toxin assembly on host membranes. We found that LLO-induced bacterial or bead entry only occurs upon LLO pore formation. Scanning electron and fluorescence microscopy studies show that LLO-coated beads stimulate the formation of membrane extensions that ingest the beads into an early endosomal compartment. This LLO-induced internalization pathway is dynamin-and F-actin-dependent, and clathrin-independent. Interestingly, further linking pore formation to bacteria/bead uptake, LLO induces F-actin polymerization in a tyrosine kinase-and pore-dependent fashion. In conclusion, we demonstrate for the first time that a bacterial pathogen perforates the host cell plasma membrane as a strategy to activate the endocytic machinery and gain entry into the host cell.

  17. The pore-forming toxin listeriolysin O mediates a novel entry pathway of L. monocytogenes into human hepatocytes.

    Science.gov (United States)

    Vadia, Stephen; Arnett, Eusondia; Haghighat, Anne-Cécile; Wilson-Kubalek, Elisabeth M; Tweten, Rodney K; Seveau, Stephanie

    2011-11-01

    Intracellular pathogens have evolved diverse strategies to invade and survive within host cells. Among the most studied facultative intracellular pathogens, Listeria monocytogenes is known to express two invasins-InlA and InlB-that induce bacterial internalization into nonphagocytic cells. The pore-forming toxin listeriolysin O (LLO) facilitates bacterial escape from the internalization vesicle into the cytoplasm, where bacteria divide and undergo cell-to-cell spreading via actin-based motility. In the present study we demonstrate that in addition to InlA and InlB, LLO is required for efficient internalization of L. monocytogenes into human hepatocytes (HepG2). Surprisingly, LLO is an invasion factor sufficient to induce the internalization of noninvasive Listeria innocua or polystyrene beads into host cells in a dose-dependent fashion and at the concentrations produced by L. monocytogenes. To elucidate the mechanisms underlying LLO-induced bacterial entry, we constructed novel LLO derivatives locked at different stages of the toxin assembly on host membranes. We found that LLO-induced bacterial or bead entry only occurs upon LLO pore formation. Scanning electron and fluorescence microscopy studies show that LLO-coated beads stimulate the formation of membrane extensions that ingest the beads into an early endosomal compartment. This LLO-induced internalization pathway is dynamin-and F-actin-dependent, and clathrin-independent. Interestingly, further linking pore formation to bacteria/bead uptake, LLO induces F-actin polymerization in a tyrosine kinase-and pore-dependent fashion. In conclusion, we demonstrate for the first time that a bacterial pathogen perforates the host cell plasma membrane as a strategy to activate the endocytic machinery and gain entry into the host cell.

  18. Studies on the mechanism of insulin resistance in the liver from humans with noninsulin-dependent diabetes. Insulin action and binding in isolated hepatocytes, insulin receptor structure, and kinase activity.

    OpenAIRE

    Caro, J F; Ittoop, O; Pories, W J; Meelheim, D; Flickinger, E G; Thomas, F; Jenquin, M; Silverman, J F; Khazanie, P G; Sinha, M. K.

    1986-01-01

    We have developed a method to isolate insulin-responsive human hepatocytes from an intraoperative liver biopsy to study insulin action and resistance in man. Hepatocytes from obese patients with noninsulin-dependent diabetes were resistant to maximal insulin concentration, and those from obese controls to submaximal insulin concentration in comparison to nonobese controls. Insulin binding per cell number was similar in all groups. However, insulin binding per surface area was decreased in the...

  19. Strategies to distinguish new synthetic cannabinoid FUBIMINA (BIM-2201) intake from its isomer THJ-2201: metabolism of FUBIMINA in human hepatocytes.

    Science.gov (United States)

    Diao, Xingxing; Scheidweiler, Karl B; Wohlfarth, Ariane; Zhu, Mingshe; Pang, Shaokun; Huestis, Marilyn A

    Since 2013, a new drugs-of-abuse trend attempts to bypass drug legislation by marketing isomers of scheduled synthetic cannabinoids (SCs), e.g., FUBIMINA (BIM-2201) and THJ-2201. It is much more challenging to confirm a specific isomer's intake and distinguish it from its structural analog because the isomers and their major metabolites usually have identical molecular weights and display the same product ions. Here, we investigated isomers FUBIMINA and THJ-2201 and propose strategies to distinguish their consumption. THJ-2201 was scheduled in the US, Japan, and Europe; however, FUBIMINA is easily available on the Internet. We previously investigated THJ-2201 metabolism in human hepatocytes, but human FUBIMINA metabolism is unknown. We aim to characterize FUBIMINA metabolism in human hepatocytes, recommend optimal metabolites to confirm its consumption, and propose strategies to distinguish between intakes of FUBIMINA and THJ-2201. FUBIMINA (10 μM) was incubated in human hepatocytes for 3 h, and metabolites were characterized with high-resolution mass spectrometry (HR-MS). We identified 35 metabolites generated by oxidative defluorination, further carboxylation, hydroxylation, dihydrodiol formation, glucuronidation, and their combinations. We recommend 5'-OH-BIM-018 (M34), BIM-018 pentanoic acid (M33), and BIM-018 pentanoic acid dihydrodiol (M7) as FUBIMINA specific metabolites. THJ-2201 produced specific metabolite markers 5'-OH-THJ-018 (F26), THJ-018 pentanoic acid (F25), and hydroxylated THJ-2201 (F13). Optimized chromatographic conditions to achieve different retention times and careful selection of specific product ion spectra enabled differentiation of isomeric metabolites, in this case FUBIMINA from THJ-2201. Our HR-MS approach should be applicable for differentiating future isomeric SCs, which is especially important when different isomers have different legal status.

  20. Serum-free culture of primary human hepatocytes in a miniaturized hollow-fibre membrane bioreactor for pharmacological in vitro studies.

    Science.gov (United States)

    Lübberstedt, Marc; Müller-Vieira, Ursula; Biemel, Klaus M; Darnell, Malin; Hoffmann, Stefan A; Knöspel, Fanny; Wönne, Eva C; Knobeloch, Daniel; Nüssler, Andreas K; Gerlach, Jörg C; Andersson, Tommy B; Zeilinger, Katrin

    2015-09-01

    Primary human hepatocytes represent an important cell source for in vitro investigation of hepatic drug metabolism and disposition. In this study, a multi-compartment capillary membrane-based bioreactor technology for three-dimensional (3D) perfusion culture was further developed and miniaturized to a volume of less than 0.5 ml to reduce demand for cells. The miniaturized bioreactor was composed of two capillary layers, each made of alternately arranged oxygen and medium capillaries serving as a 3D culture for the cells. Metabolic activity and stability of primary human hepatocytes was studied in this bioreactor in the presence of 2.5% fetal calf serum (FCS) under serum-free conditions over a culture period of 10 days. The miniaturized bioreactor showed functions comparable to previously reported data for larger variants. Glucose and lactate metabolism, urea production, albumin synthesis and release of intracellular enzymes (AST, ALT, GLDH) showed no significant differences between serum-free and serum-supplemented bioreactors. Activities of human-relevant cytochrome P450 (CYP) isoenzymes (CYP1A2, CYP3A4/5, CYP2C9, CYP2D6, CYP2B6) analyzed by determination of product formation rates from selective probe substrates were also comparable in both groups. Gene expression analysis showed moderately higher expression in the majority of CYP enzymes, transport proteins and enzymes of Phase II metabolism in the serum-free bioreactors compared to those maintained with FCS. In conclusion, the miniaturized bioreactor maintained stable function over the investigated period and thus provides a suitable system for pharmacological studies on primary human hepatocytes under defined serum-free conditions.

  1. Streptococcal Serum Opacity Factor Increases Hepatocyte Uptake of Human Plasma High Density Lipoprotein-Cholesterol1

    Science.gov (United States)

    Gillard, Baiba K.; Rosales, Corina; Pillai, Biju K.; Lin, Hu Yu; Courtney, Harry S.; Pownall, Henry J.

    2010-01-01

    Serum opacity factor (SOF), a virulence determinant of Streptococcus pyogenes, converts plasma high density lipoproteins (HDL) to three distinct species: lipid-free apolipoprotein (apo) A-I, neo HDL, a small discoidal HDL-like particle, and a large cholesteryl ester-rich microemulsion (CERM), that contains the cholesterol esters (CE) of up to ~400,000 HDL particles and apo E as its major protein. Similar SOF reaction products are obtained with HDL, total plasma lipoproteins and whole plasma. We hypothesized that hepatic uptake of CERM-CE via multiple apo E dependent receptors would be faster than that of HDL-CE. We tested our hypothesis using human hepatoma cells and lipoprotein receptor-specific Chinese hamster ovary (CHO) cells. [3H]CE uptake by HepG2 and Huh7 cells from HDL after SOF treatment, which transfers >90% of HDL-CE to CERM, was respectively 2.4 and 4.5 times faster than from control HDL. CERM-[3H]CE uptake was inhibited by LDL and HDL, suggestive of uptake by both the LDL receptor (LDL-R) and scavenger receptor class B type I (SR-BI). Studies in CHO cells specifically expressing LDL-R and SR-BI confirmed CERM-[3H]CE uptake by both receptors. RAP and heparin inhibit CERM-[3H]CE but not HDL-[3H]CE uptake thereby implicating LRP-1 and cell surface proteoglycans in this process. These data demonstrate that SOF treatment of HDL increases CE uptake via multiple hepatic apo E receptors. In so doing, SOF might increase hepatic disposal of plasma cholesterol in a way that is therapeutically useful. PMID:20879789

  2. Synergy between sulforaphane and selenium in the up-regulation of thioredoxin reductase and protection against hydrogen peroxide-induced cell death in human hepatocytes.

    Science.gov (United States)

    Li, Dan; Wang, Wei; Shan, Yujuan; Barrera, Lawrence N; Howie, Alexander F; Beckett, Geoffrey J; Wu, Kun; Bao, Yongping

    2012-07-15

    Dietary isothiocyanates and selenium are chemopreventive agents and potent inducers of antioxidant enzymes. It has been previously shown that sulforaphane and selenium have a synergistic effect on the upregulation of thioredoxin reductase-1 (TrxR-1) in human hepatoma HepG2 cells. In this paper, further evidence is presented to show that sulforaphane and selenium synergistically induce TrxR-1 expression in immortalised human hepatocytes. Sulforaphane was found to be more toxic toward hepatocytes than HepG2 cells with IC50=25.1 and 56.4 μM, respectively. Sulforaphane can protect against hydrogen peroxide-induced cell death and this protection was enhanced by co-treatment with selenium. Using siRNA to knock down TrxR-1 or Nrf2, sulforaphane (5 μM)-protected cell viability was reduced from 73% to 46% and 34%, respectively, suggesting that TrxR-1 is an important enzyme in protection against hydrogen peroxide-induced cell death. Sulforaphane-induced TrxR-1 expression was positively associated with significant levels of Nrf2 translocation into the nucleus, but co-treatment with selenium showed no significant increase in Nrf2 translocation. Moreover, MAPK (ERK, JNK and p38) and PI3K/Akt signalling pathways were found to play no significant role in sulforaphane-induced Nrf2 translocation into the nucleus. However, blocking ERK and JNK signalling pathways decreased sulforaphane-induced TrxR-1 mRNA by about 20%; whereas blocking p38 and PI3K/AKT increased TrxR-1 transcription. In summary, a combination of sulforaphane and selenium resulted in a synergistic upregulation of TrxR-1 that contributed to the enhanced protection against free radical-mediated oxidative damage in human hepatocytes. Copyright © 2012 Elsevier Ltd. All rights reserved.

  3. Monoacylglycerol O-acyltransferase 1 is regulated by peroxisome proliferator-activated receptor γ in human hepatocytes and increases lipid accumulation

    Energy Technology Data Exchange (ETDEWEB)

    Yu, Jung Hwan [Department of Biochemistry and Molecular Biology, Integrated Genomic Research Center for Metabolic Regulation, Institute of Genetic Science, Yonsei University College of Medicine, Seoul 120-752 (Korea, Republic of); Brain Korea 21 PLUS Project for Medical Science, Yonsei University, Seoul 120-752 (Korea, Republic of); Lee, Yoo Jeong [Division of Metabolic Disease, Center for Biomedical Sciences, National Institutes of Health, Cheongwon-gun, Chungbuk 363-951 (Korea, Republic of); Kim, Hyo Jung; Choi, Hyeonjin [Department of Biochemistry and Molecular Biology, Integrated Genomic Research Center for Metabolic Regulation, Institute of Genetic Science, Yonsei University College of Medicine, Seoul 120-752 (Korea, Republic of); Choi, Yoonjeong; Seok, Jo Woon [Department of Biochemistry and Molecular Biology, Integrated Genomic Research Center for Metabolic Regulation, Institute of Genetic Science, Yonsei University College of Medicine, Seoul 120-752 (Korea, Republic of); Brain Korea 21 PLUS Project for Medical Science, Yonsei University, Seoul 120-752 (Korea, Republic of); Kim, Jae-woo, E-mail: japol13@yuhs.ac [Department of Biochemistry and Molecular Biology, Integrated Genomic Research Center for Metabolic Regulation, Institute of Genetic Science, Yonsei University College of Medicine, Seoul 120-752 (Korea, Republic of); Brain Korea 21 PLUS Project for Medical Science, Yonsei University, Seoul 120-752 (Korea, Republic of); Severance Biomedical Science Institute, Yonsei University College of Medicine, Seoul 120-752 (Korea, Republic of)

    2015-05-08

    Monoacylglycerol O-acyltransferase (MGAT) is an enzyme that is involved in triglyceride synthesis by catalyzing the formation of diacylglycerol from monoacylglycerol and fatty acyl CoAs. Recently, we reported that MGAT1 has a critical role in hepatic TG accumulation and that its suppression ameliorates hepatic steatosis in a mouse model. However, the function of MGAT enzymes in hepatic lipid accumulation has not been investigated in humans. Unlike in rodents, MGAT3 as well as MGAT1 and MGAT2 are present in humans. In this study, we evaluated the differences between MGAT subtypes and their association with peroxisome proliferator-activated receptor γ (PPARγ), a regulator of mouse MGAT1 expression. In human primary hepatocytes, basal expression of MGAT1 was lower than that of MGAT2 or MGAT3, but was strongly induced by PPARγ overexpression. A luciferase assay as well as an electromobility shift assay revealed that human MGAT1 promoter activity is driven by PPARγ by direct binding to at least two regions of the promoter in 293T and HepG2 cells. Moreover, siRNA-mediated suppression of MGAT1 expression significantly attenuated lipid accumulation by PPARγ overexpression in HepG2 cells, as evidenced by oil-red-O staining. These results suggest that human MGAT1 has an important role in fatty liver formation as a target gene of PPARγ, and blocking MGAT1 activity could be an efficient therapeutic way to reduce nonalcoholic fatty liver diseases in humans. - Highlights: • PPARγ promotes MGAT1 expression in human primary hepatocytes. • PPARγ directly regulates MGAT1 promoter activity. • Human MGAT1 promoter has at least two PPARγ-binding elements. • Inhibition of MGAT1 expression attenuates hepatic lipid accumulation in humans.

  4. The right choice of antihypertensives protects primary human hepatocytes from ethanol- and recombinant human TGF-β1-induced cellular damage

    Directory of Open Access Journals (Sweden)

    Ehnert S

    2013-03-01

    Full Text Available Sabrina Ehnert,1 Teresa Lukoschek,2 Anastasia Bachmann,2 Juan J Martínez Sánchez,1 Georg Damm,3 Natascha C Nussler,4 Stefan Pscherer,5 Ulrich Stöckle,1 Steven Dooley,2 Sebastian Mueller,6 Andreas K Nussler11Eberhard Karls Universität Tübingen, BG Trauma Center, Tübingen, Germany; 2Mol Hepatology - Alcohol Associated Diseases, Department of Medicine II, Medical Faculty, Mannheim, Germany; 3Department of General, Visceral, and Transplantation Surgery, Charité University Medicine, Berlin, Germany; 4Clinic for General, Visceral, Endocrine Surgery and Coloproctology, Clinic Neuperlach, Städtisches Klinikum München GmbH, Munich, Germany; 5Department of Diabetology, Klinikum Traunstein, Kliniken Südostbayern AG, Traunstein, Germany; 6Department of Medicine, Salem Medical Center, Ruprecht-Karls-Universität, Heidelberg, GermanyBackground: Patients with alcoholic liver disease (ALD often suffer from high blood pressure and rely on antihypertensive treatment. Certain antihypertensives may influence progression of chronic liver disease. Therefore, the aim of this study is to investigate the impact of the commonly used antihypertensives amlodipine, captopril, furosemide, metoprolol, propranolol, and spironolactone on alcohol-induced damage toward human hepatocytes (hHeps.Methods: hHeps were isolated by collagenase perfusion. Reactive oxygen species (ROS were measured by fluorescence-based assays. Cellular damage was determined by lactate-dehydrogenase (LDH-leakage. Expression analysis was performed by reverse-transcription polymerase chain reaction and Western blot. Transforming growth factor (TGF-β signaling was investigated by a Smad3/4-responsive luciferase-reporter assay.Results: Ethanol and TGF-β1 rapidly increased ROS in hHeps, causing a release of 40%–60% of total LDH after 72 hours. All antihypertensives dose dependently reduced ethanol-mediated oxidative stress and cellular damage. Similar results were observed for TGF-β1-dependent

  5. Permissivity of primary human hepatocytes and different hepatoma cell lines to cell culture adapted hepatitis C virus.

    Directory of Open Access Journals (Sweden)

    Francois Helle

    Full Text Available Significant progress has been made in Hepatitis C virus (HCV culture since the JFH1 strain cloning. However, developing efficient and physiologically relevant culture systems for all viral genotypes remains an important goal. In this work, we aimed at producing a high titer JFH1 derived virus to test different hepatic cells' permissivity. To this end, we performed successive infections and obtained a JFH1 derived virus reaching high titers. Six potential adaptive mutations were identified (I599V in E2, R1373Q and M1611T in NS3, S2364P and C2441S in NS5A and R2523K in NS5B and the effect of these mutations on HCV replication and infectious particle production was investigated. This cell culture adapted virus enabled us to efficiently infect primary human hepatocytes, as demonstrated using the RFP-NLS-IPS reporter protein and intracellular HCV RNA quantification. However, the induction of a strong type III interferon response in these cells was responsible for HCV inhibition. The disruption of this innate immune response led to a strong infection enhancement and permitted the detection of viral protein expression by western blotting as well as progeny virus production. This cell culture adapted virus also enabled us to easily compare the permissivity of seven hepatoma cell lines. In particular, we demonstrated that HuH-7, HepG2-CD81, PLC/PRF/5 and Hep3B cells were permissive to HCV entry, replication and secretion even if the efficiency was very low in PLC/PRF/5 and Hep3B cells. In contrast, we did not observe any infection of SNU-182, SNU-398 and SNU-449 hepatoma cells. Using iodixanol density gradients, we also demonstrated that the density profiles of HCV particles produced by PLC/PRF/5 and Hep3B cells were different from that of HuH-7 and HepG2-CD81 derived virions. These results will help the development of a physiologically relevant culture system for HCV patient isolates.

  6. DNA damage induced by three major metabolites of 1,3-butadiene in human hepatocyte L02 cells.

    Science.gov (United States)

    Zhang, Pan-Pan; Wen, Ying; An, Jing; Yu, Ying-Xin; Wu, Ming-Hong; Zhang, Xin-Yu

    2012-09-18

    1,3-Butadiene (BD) is a carcinogenic air pollutant. Its bioactivation produces four major metabolites, i.e., 3,4-epoxy-1-butene (EB), 3,4-epoxy-1,2-butanediol (EBD), 1,2,3,4-diepoxybutane (DEB), and 3-butene-1,2-diol (BDD). Studies have been mostly focused on DEB due to its strong mutagenicity/carcinogenicity. In contrast, studies of genotoxicity of EB, EBD, and BDD have been limited. In particular, genotoxicity of EBD and BDD using strand breaks as the endpoint has not been investigated. To obtain a more complete understanding of BD toxicity, in the present study, we used comet assay to investigate DNA damage induced by EB, EBD, and BDD in human hepatocyte L02 cells, with the aim to determine their relative potencies, the types of DNA damage, and the possible pathway to form strand breaks. Using alkaline comet assay (pH>13), it was observed that EB and EBD caused similar concentration-dependent increases in DNA migration from 50 to 1000μM. However, BDD induced a statistically significant increase only at 1000μM, and the increase itself was very small. EBD was as potent as EB at lower concentrations (≤200μM), and was slightly less potent than EB at higher concentrations. The results indicated that these metabolites could generate strand breaks in cells with the rank order of the potencies being EB>≈EBD≫BDD. All three compounds failed to cause statistically significant increases in DNA migration in pre-lysed cells, suggesting that they did not produce strand breaks through chemical pathways under our experimental conditions. By using comet assays at pH 11.9 and pH 9, it was demonstrated that EB and EBD generated both single-strand breaks (SSB) and alkali-labile sites, but BDD produced only SSB. To our knowledge, this is the first report to investigate EBD- and BDD-induced strand breaks in cells. The results implied that EBD could play an important role in toxicity of BD.

  7. Obeticholic acid, a selective farnesoid X receptor agonist, regulates bile acid homeostasis in sandwich-cultured human hepatocytes.

    Science.gov (United States)

    Zhang, Yuanyuan; Jackson, Jonathan P; St Claire, Robert L; Freeman, Kimberly; Brouwer, Kenneth R; Edwards, Jeffrey E

    2017-08-01

    Farnesoid X receptor (FXR) is a master regulator of bile acid homeostasis through transcriptional regulation of genes involved in bile acid synthesis and cellular membrane transport. Impairment of bile acid efflux due to cholangiopathies results in chronic cholestasis leading to abnormal elevation of intrahepatic and systemic bile acid levels. Obeticholic acid (OCA) is a potent and selective FXR agonist that is 100-fold more potent than the endogenous ligand chenodeoxycholic acid (CDCA). The effects of OCA on genes involved in bile acid homeostasis were investigated using sandwich-cultured human hepatocytes. Gene expression was determined by measuring mRNA levels. OCA dose-dependently increased fibroblast growth factor-19 (FGF-19) and small heterodimer partner (SHP) which, in turn, suppress mRNA levels of cholesterol 7-alpha-hydroxylase (CYP7A1), the rate-limiting enzyme for de novo synthesis of bile acids. Consistent with CYP7A1 suppression, total bile acid content was decreased by OCA (1 μmol/L) to 42.7 ± 20.5% relative to control. In addition to suppressing de novo bile acids synthesis, OCA significantly increased the mRNA levels of transporters involved in bile acid homeostasis. The bile salt excretory pump (BSEP), a canalicular efflux transporter, increased by 6.4 ± 0.8-fold, and the basolateral efflux heterodimer transporters, organic solute transporter α (OSTα ) and OSTβ increased by 6.4 ± 0.2-fold and 42.9 ± 7.9-fold, respectively. The upregulation of BSEP and OSTα and OSTβ, by OCA reduced the intracellular concentrations of d8 -TCA, a model bile acid, to 39.6 ± 8.9% relative to control. These data demonstrate that OCA does suppress bile acid synthesis and reduce hepatocellular bile acid levels, supporting the use of OCA to treat bile acid-induced toxicity observed in cholestatic diseases. © 2017 Intercept Pharmaceuticals. Pharmacology Research & Perspectives published by John Wiley & Sons Ltd, British Pharmacological Society and

  8. Effects of L-carnitine against oxidative stress in human hepatocytes: involvement of peroxisome proliferator-activated receptor alpha

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    Li Jin-Lian

    2012-03-01

    Full Text Available Abstract Background Excessive oxidative stress and lipid peroxidation have been demonstrated to play important roles in the production of liver damage. L-carnitine is a natural substance and acts as a carrier for fatty acids across the inner mitochondrial membrane for subsequent beta-oxidation. It is also an antioxidant that reduces metabolic stress in the cells. Recent years L-carnitine has been proposed for treatment of various kinds of disease, including liver injury. This study was conducted to evaluate the protective effect of L-carnitine against hydrogen peroxide (H2O2-induced cytotoxicity in a normal human hepatocyte cell line, HL7702. Methods We analyzed cytotoxicity using MTT assay and lactate dehydrogenase (LDH release. Antioxidant activity and lipid peroxidation were estimated by reactive oxygen species (ROS levels, activities and protein expressions of superoxide dismutase (SOD and catalase (CAT, and malondialdehyde (MDA formation. Expressions of peroxisome proliferator-activated receptor (PPAR-alpha and its target genes were evaluated by RT-PCR or western blotting. The role of PPAR-alpha in L-carnitine-enhanced expression of SOD and CAT was also explored. Statistical analysis was performed by a one-way analysis of variance, and its significance was assessed by Dennett's post-hoc test. Results The results showed that L-carnitine protected HL7702 cells against cytotoxity induced by H2O2. This protection was related to the scavenging of ROS, the promotion of SOD and CAT activity and expression, and the prevention of lipid peroxidation in cultured HL7702 cells. The decreased expressions of PPAR-alpha, carnitine palmitoyl transferase 1 (CPT1 and acyl-CoA oxidase (ACOX induced by H2O2 can be attenuated by L-carnitine. Besides, we also found that the promotion of SOD and CAT protein expression induced by L-carnitine was blocked by PPAR-alpha inhibitor MK886. Conclusions Taken together, our findings suggest that L-carnitine could protect HL

  9. Computational and Biochemical Discovery of RSK2 as a Novel Target for Epigallocatechin Gallate (EGCG.

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    Hanyong Chen

    Full Text Available The most active anticancer component in green tea is epigallocatechin-3-gallate (EGCG. Protein interaction with EGCG is a critical step for mediating the effects of EGCG on the regulation of various key molecules involved in signal transduction. By using computational docking screening methods for protein identification, we identified a serine/threonine kinase, 90-kDa ribosomal S6 kinase (RSK2, as a novel molecular target of EGCG. RSK2 includes two kinase catalytic domains in the N-terminal (NTD and the C-terminal (CTD and RSK2 full activation requires phosphorylation of both terminals. The computer prediction was confirmed by an in vitro kinase assay in which EGCG inhibited RSK2 activity in a dose-dependent manner. Pull-down assay results showed that EGCG could bind with RSK2 at both kinase catalytic domains in vitro and ex vivo. Furthermore, results of an ATP competition assay and a computer-docking model showed that EGCG binds with RSK2 in an ATP-dependent manner. In RSK2+/+ and RSK2-/- murine embryonic fibroblasts, EGCG decreased viability only in the presence of RSK2. EGCG also suppressed epidermal growth factor-induced neoplastic cell transformation by inhibiting phosphorylation of histone H3 at Ser10. Overall, these results indicate that RSK2 is a novel molecular target of EGCG.

  10. Production of monodisperse epigallocatechin gallate (EGCG) microparticles by spray drying for high antioxidant activity retention.

    Science.gov (United States)

    Fu, Nan; Zhou, Zihao; Jones, Tyson Byrne; Tan, Timothy T Y; Wu, Winston Duo; Lin, Sean Xuqi; Chen, Xiao Dong; Chan, Peggy P Y

    2011-07-15

    Epigallocatechin gallate (EGCG) originated from green tea is well-known for its pharmaceutical potential and antiproliferating effect on carcinoma cells. For drug delivery, EGCG in a micro-/nanoparticle form is desirable for their optimized chemopreventive effect. In this study, first time reports that EGCG microparticles produced by low temperature spray drying can maintain high antioxidant activity. A monodisperse droplet generation system was used to realize the production of EGCG microparticles. EGCG microparticles were obtained with narrow size distribution and diameter of 30.24 ± 1.88 μM and 43.39 ± 0.69 μM for pure EGCG and lactose-added EGCG, respectively. The EC50 value (the amount of EGCG necessary to scavenge 50% of free radical in the medium) of spray dried pure EGCG particles obtained from different temperature is in the range of 3.029-3.075 μM compared to untreated EGCG with EC50 value of 3.028 μM. Varying the drying temperatures from 70°C and 130°C showed little detrimental effect on EGCG antioxidant activity. NMR spectrum demonstrated the EGCG did not undergo chemical structural change after spray drying. The major protective mechanism was considered to be: (1) the use of low temperature and (2) the heat loss from water evaporation that kept the particle temperature at low level. With further drier optimization, this monodisperse spray drying technique can be used as an efficient and economic approach to produce EGCG micro-/nanoparticles.

  11. On Preparation of High EGCG Catechinsby Means of Polyamide Adsorption%聚酰胺吸附法制备高 EGCG 儿茶素

    Institute of Scientific and Technical Information of China (English)

    罗赛; 龚正礼

    2016-01-01

    通过聚酰胺吸附法制备高 EGCG 儿茶素.以绿茶茶末为原料,经热水浸提,离心除渣,聚酰胺分离,浓缩干燥,得到 EGCG 儿茶素.结果表明,500 mL 聚酰胺吸附13.75 g EGCG 达到饱和;70%乙醇洗脱物中 EGCG 质量分数为63.200%;产品质量分数高于有机溶剂萃取产物;中试产品质量指标儿茶素超过75%,EGCG 超过55%,得率超过10%.得出该实验方法效果显著,工艺条件可行.%High EGCG catechins has been prepared by means of the polyamide adsorption .Firstly ,with the dust tea as raw materials ,extraction has been conducted by hot water and centrifugal cleaning .Secondly , the preparation has been done by means of polyamide adsorption .Lastly ,dryness has been concentrated and EGCG catechins got .Results show that 500 ml polyamide adsorption of 13 .75 g EGCG hasreached satu-ration .The EGCG content in the 70% ethanol elution is 63 .200% .The product is higher than that in organic sol-vent extracts .Pilot product quality in dicators catechin exceed 75% ,EGCG exceed 55% ,yield exceed 10% .It is concluded that the experimental method has led to significant results ,and that the process conditions is feasible .

  12. Neurorescue activity, APP regulation and amyloid-beta peptide reduction by novel multi-functional brain permeable iron- chelating- antioxidants, M-30 and green tea polyphenol, EGCG.

    Science.gov (United States)

    Avramovich-Tirosh, Yael; Reznichenko, Lydia; Mit, Tamar; Zheng, Hailin; Fridkin, Mati; Weinreb, Orly; Mandel, Silvia; Youdim, Moussa B H

    2007-09-01

    Accumulation of iron at sites where neurons degenerate in Parkinson's disease (PD) and Alzheimer's disease (AD) is thought to have a major role in oxidative stress induced process of neurodegeneration. The novel non-toxic lipophilic brain- permeable iron chelators, VK-28 (5- [4- (2- hydroxyethyl) piperazine-1-ylmethyl]- quinoline- 8- ol) and its multi-functional derivative, M-30 (5-[N-methyl-N-propargylaminomethyl]-8-hydroxyquinoline), as well as the main polyphenol constituent of green tea (-)-epigallocatechin-3-gallate (EGCG), which possesses iron metal chelating, radical scavenging and neuroprotective properties, offer potential therapeutic benefits for these diseases. M-30 and EGCG decreased apoptosis of human SH-SY5Y neuroblastoma cells in a neurorescue, serum deprivation model, via multiple protection mechanisms including: reduction of the pro-apoptotic proteins, Bad and Bax, reduction of apoptosis-associated Ser139 phosphorylated H2A.X and inhibition of the cleavage and activation of caspase-3. M-30 and EGCG also promoted morphological changes, resulting in axonal growth-associated protein-43 (GAP-43) implicating neuronal differentiation. Both compounds significantly reduced the levels of cellular holo-amyloid precursor protein (APP) in SH-SY5Y cells. The ability of theses novel iron chelators and EGCG to regulate APP are in line with the presence of an iron-responsive element (IRE) in the 5'-untranslated region (5'UTR) of APP. Also, EGCG reduced the levels of toxic amyloid-beta peptides in CHO cells over-expressing the APP "Swedish" mutation. The diverse molecular mechanisms and cell signaling pathways participating in the neuroprotective/neurorescue and APP regulation/processing actions of M-30 and EGCG, make these multifunctional compounds potential neuroprotective drugs for the treatment of neurodegenerative diseases, such as PD, AD, Huntington's disease and amyotrophic lateral sclerosis.

  13. Skin delivery of epigallocatechin-3-gallate (EGCG) and hyaluronic acid loaded nano-transfersomes for antioxidant and anti-aging effects in UV radiation induced skin damage.

    Science.gov (United States)

    Avadhani, Kiran S; Manikkath, Jyothsna; Tiwari, Mradul; Chandrasekhar, Misra; Godavarthi, Ashok; Vidya, Shimoga M; Hariharapura, Raghu C; Kalthur, Guruprasad; Udupa, Nayanabhirama; Mutalik, Srinivas

    2017-11-01

    The present work attempts to develop and statistically optimize transfersomes containing EGCG and hyaluronic acid to synergize the UV radiation-protective ability of both compounds, along with imparting antioxidant and anti-aging effects. Transfersomes were prepared by thin film hydration technique, using soy phosphatidylcholine and sodium cholate, combined with high-pressure homogenization. They were characterized with respect to size, polydispersity index, zeta potential, morphology, entrapment efficiency, Fourier Transform Infrared Spectroscopy (FTIR), Differential Scanning Calorimetry (DSC), X-ray Diffraction (XRD), in vitro antioxidant activity and ex vivo skin permeation studies. Cell viability, lipid peroxidation, intracellular ROS levels and expression of MMPs (2 and 9) were determined in human keratinocyte cell lines (HaCaT). The composition of the transfersomes was statistically optimized by Design of Experiments using Box-Behnken design with four factors at three levels. The optimized transfersome formulation showed vesicle size, polydispersity index and zeta potential of 101.2 ± 6.0 nm, 0.245 ± 0.069 and -44.8 ± 5.24 mV, respectively. FTIR and DSC showed no interaction between EGCG and the selected excipients. XRD results revealed no form conversion of EGCG in its transfersomal form. The optimized transfersomes were found to increase the cell viability and reduce the lipid peroxidation, intracellular ROS and expression of MMPs in HaCaT cells. The optimized transfersomal formulation of EGCG and HA exhibited considerably higher skin permeation and deposition of EGCG than that observed with plain EGCG. The results underline the potential application of the developed transfersomes in sunscreen cream/lotions for improvement of UV radiation-protection along with deriving antioxidant and anti-aging effects.

  14. In vitro profiling of the metabolism and drug-drug interaction of tofogliflozin, a potent and highly specific sodium-glucose co-transporter 2 inhibitor, using human liver microsomes, human hepatocytes, and recombinant human CYP.

    Science.gov (United States)

    Yamane, Mizuki; Kawashima, Kosuke; Yamaguchi, Koji; Nagao, Shunsuke; Sato, Mika; Suzuki, Masayuki; Honda, Kiyofumi; Hagita, Hitoshi; Kuhlmann, Olaf; Poirier, Agnes; Fowler, Stephen; Funk, Christoph; Simon, Sandrine; Aso, Yoshinori; Ikeda, Sachiya; Ishigai, Masaki

    2015-03-01

    Abstract 1. The metabolism and drug-drug interaction (DDI) risk of tofogliflozin, a potent and highly specific sodium-glucose co-transporter 2 inhibitor, were evaluated by in vitro studies using human liver microsomes, human hepatocytes, and recombinant human CYPs. 2. The main metabolite of tofogliflozin was the carboxylated derivative (M1) in human hepatocytes, which was the same as in vivo. The metabolic pathway of tofogliflozin to M1 was considered to be as follows: first, tofogliflozin was catalyzed to the primary hydroxylated derivative (M4) by CYP2C18, CYP4A11 and CYP4F3B, then M4 was oxidized to M1. 3. Tofogliflozin had no induction potential on CYP1A2 and CYP3A4. Neither tofogliflozin nor M1 had inhibition potential on CYPs, with the exception of a weak CYP2C19 inhibition by M1. 4. Not only are multiple metabolic enzymes involved in the tofogliflozin metabolism, but the drug is also excreted into urine after oral administration, indicating that tofogliflozin is eliminated through multiple pathways. Thus, the exposure of tofogliflozin would not be significantly altered by DDI caused by any co-administered drugs. Also, tofogliflozin seems not to cause significant DDI of co-administered drugs because tofogliflozin has no CYP induction or inhibition potency, and the main metabolite M1 has no clinically relevant CYP inhibition potency.

  15. Retinoic acid represses CYP7A1 expression in human hepatocytes and HepG2 cells by FXR/RXR-dependent and independent mechanisms.

    Science.gov (United States)

    Cai, Shi-Ying; He, Hongwei; Nguyen, Trong; Mennone, Albert; Boyer, James L

    2010-08-01

    Cholesterol 7alpha-hydroxylase (CYP7A1) plays a key role in maintaining lipid and bile salt homeostasis as it is the rate-limiting enzyme converting cholesterol to bile acids. Deficiency of CYP7A1 leads to hyperlipidemia in man and mouse. Hyperlipidemia is often seen in patients when treated with high-dose retinoic acid (RA), but the molecular mechanisms remain elusive. Our present study revealed that CYP7A1 mRNA expression is greatly repressed by RA in both human hepatocytes and HepG2 cells where increased fibroblast growth factor 19 (FGF19) and small heterodimer partner (SHP) expressions were also observed, suggesting farnesoid X receptor (FXR) and retinoid X receptor (RXR) were activated. Promoter reporter assays demonstrate that all-trans RA (atRA) specifically activated FXR/RXR. However, detailed molecular analyses indicate that this activation is through RXR, whose ligand is 9-cis RA. Knocking down of FXR or RXRalpha by small interference RNA (siRNA) in human hepatocytes increased CYP7A1 basal expression, but the repressive effect of atRA persisted, suggesting there are also FXR/RXR-independent mechanisms mediating atRA repression of CYP7A1 expression. Chromatin immunoprecipitation (ChIP) assay and cell transfection results indicate that PGC-1alpha plays a role in the FXR/RXR-independent mechanism. Our findings may provide a potential explanation for hyperlipidemic side effects observed in some patients treated with high-dose RA.

  16. Hyaluronic Acid/PLGA Core/Shell Fiber Matrices Loaded with EGCG Beneficial to Diabetic Wound Healing.

    Science.gov (United States)

    Shin, Yong Cheol; Shin, Dong-Myeong; Lee, Eun Ji; Lee, Jong Ho; Kim, Ji Eun; Song, Sung Hwa; Hwang, Dae-Youn; Lee, Jun Jae; Kim, Bongju; Lim, Dohyung; Hyon, Suong-Hyu; Lim, Young-Jun; Han, Dong-Wook

    2016-12-01

    During the last few decades, considerable research on diabetic wound healing strategies has been performed, but complete diabetic wound healing remains an unsolved problem, which constitutes an enormous biomedical burden. Herein, hyaluronic acid (HA)/poly(lactic-co-glycolic acid, PLGA) core/shell fiber matrices loaded with epigallocatechin-3-O-gallate (EGCG) (HA/PLGA-E) are fabricated by coaxial electrospinning. HA/PLGA-E core/shell fiber matrices are composed of randomly-oriented sub-micrometer fibers and have a 3D porous network structure. EGCG is uniformly dispersed in the shell and sustainedly released from the matrices in a stepwise manner by controlled diffusion and PLGA degradation over four weeks. EGCG does not adversely affect the thermomechanical properties of HA/PLGA-E matrices. The number of human dermal fibroblasts attached on HA/PLGA-E matrices is appreciably higher than that on HA/PLGA counterparts, while their proliferation is steadily retained on HA/PLGA-E matrices. The wound healing activity of HA/PLGA-E matrices is evaluated in streptozotocin-induced diabetic rats. After two weeks of surgical treatment, the wound areas are significantly reduced by the coverage with HA/PLGA-E matrices resulting from enhanced re-epithelialization/neovascularization and increased collagen deposition, compared with no treatment or HA/PLGA. In conclusion, the HA/PLGA-E matrices can be potentially exploited to craft strategies for the acceleration of diabetic wound healing and skin regeneration.

  17. Low dose EGCG treatment beginning in adolescence does not improve cognitive impairment in a Down syndrome mouse model.

    Science.gov (United States)

    Stringer, Megan; Abeysekera, Irushi; Dria, Karl J; Roper, Randall J; Goodlett, Charles R

    2015-11-01

    Down syndrome (DS) or Trisomy 21 causes intellectual disabilities in humans and the Ts65Dn DS mouse model is deficient in learning and memory tasks. DYRK1A is triplicated in DS and Ts65Dn mice. Ts65Dn mice were given up to ~20mg/kg/day epigallocatechin-3-gallate (EGCG), a Dyrk1a inhibitor, or water beginning on postnatal day 24 and continuing for three or seven weeks, and were tested on a series of behavioral and learning tasks, including a novel balance beam test. Ts65Dn as compared to control mice exhibited higher locomotor activity, impaired novel object recognition, impaired balance beam and decreased spatial learning and memory. Neither EGCG treatment improved performance of the Ts65Dn mice on these tasks. Ts65Dn mice had a non-significant increase in Dyrk1a activity in the hippocampus and cerebellum. Given the translational value of the Ts65Dn mouse model, further studies will be needed to identify the EGCG doses (and mechanisms) that may improve cognitive function.

  18. Dual roles of nuclear receptor liver X receptor α (LXRα) in the CYP3A4 expression in human hepatocytes as a positive and negative regulator.

    Science.gov (United States)

    Watanabe, Keisuke; Sakurai, Kaori; Tsuchiya, Yuri; Yamazoe, Yasushi; Yoshinari, Kouichi

    2013-08-01

    CYP3A4 is a major drug-metabolizing enzyme in humans, whose expression levels show large inter-individual variations and are associated with several factors such as genetic polymorphism, physiological and disease status, diet and xenobiotic exposure. Nuclear receptor pregnane X receptor (PXR) is a key transcription factor for the xenobiotic-mediated transcription of CYP3A4. In this study, we have investigated a possible involvement of liver X receptor α (LXRα), a critical regulator of cholesterol homeostasis, in the hepatic CYP3A4 expression since several recent reports suggest the involvement of CYP3A enzymes in the cholesterol metabolism in humans and mice. Reporter assays using wild-type and mutated CYP3A4 luciferase reporter plasmids and electrophoretic mobility shift assays revealed that LXRα up-regulated CYP3A4 through the known DNA elements critical for the PXR-dependent CYP3A4 transcription, suggesting LXRα as a positive regulator for the CYP3A4 expression and a crosstalk between PXR and LXRα in the expression. In fact, reporter assays showed that LXRα activation attenuated the PXR-dependent CYP3A4 transcription. Moreover, a PXR agonist treatment-dependent increase in CYP3A4 mRNA levels was suppressed by co-treatment with an LXRα agonist in human primary hepatocytes and HepaRG cells. The suppression was not observed when LXRα expression was knocked-down in HepaRG cells. In conclusion, the present results suggest that sterol-sensitive LXRα positively regulates the basal expression of CYP3A4 but suppresses the xenobiotic/PXR-dependent CYP3A4 expression in human hepatocytes. Therefore, nutritional, physiological and disease conditions affecting LXRα might be one of the determinants for the basal and xenobiotic-responsive expression of CYP3A4 in human livers.

  19. Met-Independent Hepatocyte Growth Factor-mediated regulation of cell adhesion in human prostate cancer cells

    Directory of Open Access Journals (Sweden)

    Davis Rodney

    2006-07-01

    Full Text Available Abstract Background Prostate cancer cells communicate reciprocally with the stromal cells surrounding them, inside the prostate, and after metastasis, within the bone. Each tissue secretes factors for interpretation by the other. One stromally-derived factor, Hepatocyte Growth Factor (HGF, was found twenty years ago to regulate invasion and growth of carcinoma cells. Working with the LNCaP prostate cancer progression model, we found that these cells could respond to HGF stimulation, even in the absence of Met, the only known HGF receptor. The new HGF binding partner we find on the cell surface may help to clarify conflicts in the past literature about Met expression and HGF response in cancer cells. Methods We searched for Met or any HGF binding partner on the cells of the PC3 and LNCaP prostate cancer cell models, using HGF immobilized on agarose beads. By using mass spectrometry analyses and sequencing we have identified nucleolin protein as a novel HGF binding partner. Antibodies against nucleolin (or HGF were able to ameliorate the stimulatory effects of HGF on met-negative prostate cancer cells. Western blots, RT-PCR, and immunohistochemistry were used to assess nucleolin levels during prostate cancer progression in both LNCaP and PC3 models. Results We have identified HGF as a major signaling component of prostate stromal-conditioned media (SCM and have implicated the protein nucleolin in HGF signal reception by the LNCaP model prostate cancer cells. Antibodies that silence either HGF (in SCM or nucleolin (on the cell surfaces eliminate the adhesion-stimulatory effects of the SCM. Likewise, addition of purified HGF to control media mimics the action of SCM. C4-2, an LNCaP lineage-derived, androgen-independent human prostate cancer cell line, responds to HGF in a concentration-dependent manner by increasing its adhesion and reducing its migration on laminin substratum. These HGF effects are not due to shifts in the expression levels of

  20. Improved human endometrial stem cells differentiation into functional hepatocyte-like cells on a glycosaminoglycan/collagen-grafted polyethersulfone nanofibrous scaffold.

    Science.gov (United States)

    Khademi, Farzaneh; Ai, Jafar; Soleimani, Masoud; Verdi, Javad; Mohammad Tavangar, Seyed; Sadroddiny, Esmaeil; Massumi, Mohammad; Mahmoud Hashemi, Seyed

    2017-11-01

    Liver tissue engineering (TE) is rapidly emerging as an effective technique which combines engineering and biological processes to compensate for the shortage of damaged or destroyed liver tissues. We examined the viability, differentiation, and integration of hepatocyte-like cells on an electrospun polyethersulfone (PES) scaffold, derived from human endometrial stem cells (hEnSCs). Natural polymers were separately grafted on plasma-treated PES nanofibers, that is, collagen, heparan sulfate (HS) and collagen-HS. Galactosilated PES (PES-Gal) nanofibrous were created. The engineering and cell growth parameters were considered and compared with each sample. The cellular studies revealed increased cell survival, attachment, and normal morphology on the bioactive natural polymer-grafted scaffolds after 30 days of hepatic differentiation. The chemical and molecular assays displayed hepatocyte differentiation. These cells were also functional, showing glycogen storage, α-fetoprotein, and albumin secretion. The HS nanoparticle-grafted PES nanofibers demonstrated a high rate of cell proliferation, differentiation, and integration. Based on the observations mentioned above, engineered tissue is a good option in the future, for the commercial production of three-dimensional liver tissues for clinical purposes. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 2516-2529, 2017. © 2016 Wiley Periodicals, Inc.

  1. EXPERIMENTAL HEPATOCYTE XENOTRANSPLANTATION – A COMPREHENSIVE REVIEW OF THE LITERATURE

    Science.gov (United States)

    Zhou, Huidong; Liu, Hong; Ezzelarab, Mohamed; Schmelzer, Eva; Wang, Yi; Gerlach, Jörg; Gridelli, Bruno; Cooper, David K. C.

    2015-01-01

    Background Hepatocyte transplantation is a potential therapy for certain diseases of the liver, including hepatic failure. However, there is a limited supply of human livers as a source of cells and, after isolation, human hepatocytes can be difficult to expand in culture, limiting the number available for transplantation. Hepatocytes from other species, e.g., the pig, have therefore emerged as a potential alternative source. We searched the literature through the end of 2014 to assess the current status of experimental research into hepatocyte xenotransplantation. Literature search and results The literature search identified 51 reports of in vivo cross-species transplantation of hepatocytes in a variety of experimental models. Most studies investigated the transplantation of human (n=23) or pig (n=19) hepatocytes. No studies explored hepatocytes from genetically-engineered pigs. The spleen was the most common site of transplantation (n=23), followed by the liver (through the portal vein [n=6]) and peritoneal cavity (n=19). In 47 studies (92%), there was evidence of hepatocyte engraftment and function across a species barrier. Conclusions The data provided by this literature search strengthen the hypothesis that xenotransplantation of hepatocytes is feasible and potentially successful as a clinical therapy for certain liver diseases, including hepatic failure. By excluding vascular structures, hepatocytes isolated from genetically-engineered pig livers may address some of the immunological problems of xenotransplantation. PMID:25950141

  2. Experimental hepatocyte xenotransplantation--a comprehensive review of the literature.

    Science.gov (United States)

    Zhou, Huidong; Liu, Hong; Ezzelarab, Mohamed; Schmelzer, Eva; Wang, Yi; Gerlach, Jörg; Gridelli, Bruno; Cooper, David K C

    2015-01-01

    Hepatocyte transplantation (Tx) is a potential therapy for certain diseases of the liver, including hepatic failure. However, there is a limited supply of human livers as a source of cells and, after isolation, human hepatocytes can be difficult to expand in culture, limiting the number available for Tx. Hepatocytes from other species, for example, the pig, have therefore emerged as a potential alternative source. We searched the literature through the end of 2014 to assess the current status of experimental research into hepatocyte xenoTx. The literature search identified 51 reports of in vivo cross-species Tx of hepatocytes in a variety of experimental models. Most studies investigated the Tx of human (n = 23) or pig (n = 19) hepatocytes. No studies explored hepatocytes from genetically engineered pigs. The spleen was the most common site of Tx (n = 23), followed by the liver (through the portal vein [n = 6]) and peritoneal cavity (n = 19). In 47 studies (92%), there was evidence of hepatocyte engraftment and function across a species barrier. The data provided by this literature search strengthen the hypothesis that xenoTx of hepatocytes is feasible and potentially successful as a clinical therapy for certain liver diseases, including hepatic failure. By excluding vascular structures, hepatocytes isolated from genetically engineered pig livers may address some of the immunological problems of xenoTx.

  3. Bioprinting of human pluripotent stem cells and their directed differentiation into hepatocyte-like cells for the generation of mini-livers in 3D.

    Science.gov (United States)

    Faulkner-Jones, Alan; Fyfe, Catherine; Cornelissen, Dirk-Jan; Gardner, John; King, Jason; Courtney, Aidan; Shu, Wenmiao

    2015-10-21

    We report the first investigation into the bioprinting of human induced pluripotent stem cells (hiPSCs), their response to a valve-based printing process as well as their post-printing differentiation into hepatocyte-like cells (HLCs). HLCs differentiated from both hiPSCs and human embryonic stem cells (hESCs) sources were bioprinted and examined for the presence of hepatic markers to further validate the compatibility of the valve-based bioprinting process with fragile cell transfer. Examined cells were positive for nuclear factor 4 alpha and were demonstrated to secrete albumin and have morphology that was also found to be similar to that of hepatocytes. Both hESC and hiPSC lines were tested for post-printing viability and pluripotency and were found to have negligible difference in terms of viability and pluripotency between the printed and non-printed cells. hESC-derived HLCs were 3D printed using alginate hydrogel matrix and tested for viability and albumin secretion during the remaining differentiation and were found to be hepatic in nature. 3D printed with 40-layer of HLC-containing alginate structures reached peak albumin secretion at day 21 of the differentiation protocol. This work demonstrates that the valve-based printing process is gentle enough to print human pluripotent stem cells (hPSCs) (both hESCs and hiPSCs) while either maintaining their pluripotency or directing their differentiation into specific lineages. The ability to bioprint hPSCs will pave the way for producing organs or tissues on demand from patient specific cells which could be used for animal-free drug development and personalized medicine.

  4. Hepatobiliary Clearance Prediction: Species Scaling From Monkey, Dog, and Rat, and In Vitro-In Vivo Extrapolation of Sandwich-Cultured Human Hepatocytes Using 17 Drugs.

    Science.gov (United States)

    Kimoto, Emi; Bi, Yi-An; Kosa, Rachel E; Tremaine, Larry M; Varma, Manthena V S

    2017-09-01

    Hepatobiliary elimination can be a major clearance pathway dictating the pharmacokinetics of drugs. Here, we first compared the dose eliminated in bile in preclinical species (monkey, dog, and rat) with that in human and further evaluated single-species scaling (SSS) to predict human hepatobiliary clearance. Six compounds dosed in bile duct-cannulated (BDC) monkeys showed biliary excretion comparable to human; and the SSS of hepatobiliary clearance with plasma fraction unbound correction yielded reasonable predictions (within 3-fold). Although dog SSS also showed reasonable predictions, rat overpredicted hepatobiliary clearance for 13 of 24 compounds. Second, we evaluated the translatability of in vitro sandwich-cultured human hepatocytes (SCHHs) to predict human hepatobiliary clearance for 17 drugs. For drugs with no significant active uptake in SCHH studies (i.e., with or without rifamycin SV), measured intrinsic biliary clearance was directly scalable with good predictability (absolute average fold error [AAFE] = 1.6). Drugs showing significant active uptake in SCHH, however, showed improved predictability when scaled based on extended clearance term (AAFE = 2.0), which incorporated sinusoidal uptake along with a global scaling factor for active uptake and the canalicular efflux clearance. In conclusion, SCHH is a useful tool to predict human hepatobiliary clearance, whereas BDC monkey model may provide further confidence in the prospective predictions. Copyright © 2017 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

  5. Monoacylglycerol O-acyltransferase 1 is regulated by peroxisome proliferator-activated receptor γ in human hepatocytes and increases lipid accumulation.

    Science.gov (United States)

    Yu, Jung Hwan; Lee, Yoo Jeong; Kim, Hyo Jung; Choi, Hyeonjin; Choi, Yoonjeong; Seok, Jo Woon; Kim, Jae-woo

    2015-05-08

    Monoacylglycerol O-acyltransferase (MGAT) is an enzyme that is involved in triglyceride synthesis by catalyzing the formation of diacylglycerol from monoacylglycerol and fatty acyl CoAs. Recently, we reported that MGAT1 has a critical role in hepatic TG accumulation and that its suppression ameliorates hepatic steatosis in a mouse model. However, the function of MGAT enzymes in hepatic lipid accumulation has not been investigated in humans. Unlike in rodents, MGAT3 as well as MGAT1 and MGAT2 are present in humans. In this study, we evaluated the differences between MGAT subtypes and their association with peroxisome proliferator-activated receptor γ (PPARγ), a regulator of mouse MGAT1 expression. In human primary hepatocytes, basal expression of MGAT1 was lower than that of MGAT2 or MGAT3, but was strongly induced by PPARγ overexpression. A luciferase assay as well as an electromobility shift assay revealed that human MGAT1 promoter activity is driven by PPARγ by direct binding to at least two regions of the promoter in 293T and HepG2 cells. Moreover, siRNA-mediated suppression of MGAT1 expression significantly attenuated lipid accumulation by PPARγ overexpression in HepG2 cells, as evidenced by oil-red-O staining. These results suggest that human MGAT1 has an important role in fatty liver formation as a target gene of PPARγ, and blocking MGAT1 activity could be an efficient therapeutic way to reduce nonalcoholic fatty liver diseases in humans.

  6. Modulation of nuclear T3 binding by T3 in a human hepatocyte cell-line (Chang-liver) - T3 stimulation of cell growth but not of malic enzyme, glucose-6-phosphatdehydrogenase or 6-phosphogluconate-dehydrogenase

    DEFF Research Database (Denmark)

    Matzen, L E; Kristensen, S R; Kvetny, J

    1991-01-01

    The T3 modulation of nuclear T3 binding (NBT3), the T3 effect on cell growth, and the T3 and insulin effects on malic enzyme (ME), glucose-6-phosphat-dehydrogenase (G6PD) and 6-phosphogluconat-dehydrogenase (G6PD) were studied in a human hepatocyte cell-line (Chang-liver). T3 was bound to a high...

  7. Effect of (-)-epigallocatechin-3-gallate on glucose-induced human serum albumin glycation.

    Science.gov (United States)

    Li, M; Hagerman, A E

    2015-01-01

    (-)-Epigallocatechin-3-gallate (EGCg) is a naturally occurring polyphenol found in plant-based foods and beverages such as green tea. Although EGCg can eliminate carbonyl species produced by glucose autoxidation and thus can inhibit protein glycation, it is also reported to be a pro-oxidant that stimulates protein glycation in vitro. To better understand the balance between antioxidant and pro-oxidant features of EGCg, we evaluated EGCg-mediated bioactivities in a human serum albumin (HSA)/glucose model by varying three different parameters (glucose level, EGCg concentration, and time of exposure to EGCg). Measurements of glycation-induced fluorescence, protein carbonyls, and electrophoretic mobility showed that the level of HSA glycation was positively related to the glucose level over the range 10-100 mM during a 21-day incubation at 37°C and pH: 7.4. Under mild glycemic pressure (10 mM), long exposure to EGCg enhanced HSA glycation, while brief exposure to low concentrations of EGCg did not. Under high glycemic pressure (100 mM glucose), long exposure to EGCg inhibited glycation. For the first time we showed that brief exposure to EGCg reversed glycation-induced fluorescence, indicating a restorative effect. In conclusion, our research identified glucose level, EGCg concentration, and time of exposure as critical factors dictating EGCg bioactivities in HSA glycation. EGCg did not affect HSA glycation under normal physiological conditions but had a potential therapeutic effect on HSA severely damaged by glycation.

  8. Mangifera indica L. extract and mangiferin modulate cytochrome P450 and UDP-glucuronosyltransferase enzymes in primary cultures of human hepatocytes.

    Science.gov (United States)

    Rodeiro, Idania; José Gómez-Lechón, M; Perez, Gabriela; Hernandez, Ivones; Herrera, José Alfredo; Delgado, Rene; Castell, José V; Teresa Donato, M

    2013-05-01

    The aqueous stem bark extract of Mangifera indica L. (MSBE) has been reported to have antioxidant, anti-inflammatory and analgesic properties. In previous studies, we showed that MSBE and mangiferin, its main component, lower the activity of some cytochrome P-450 (P450) enzymes in rat hepatocytes and human liver microsomes. In the present study, the effects of MSBE and mangiferin on several P450 enzymes and UDP-glucuronosyltransferases (UGTs) in human-cultured hepatocytes have been examined. After hepatocytes underwent a 48-h treatment with sub-cytotoxic concentrations of the products (50-250 µg/mL), a concentration-dependent decrease of the activity of the five P450 enzymes measured (CYP1A2, 2A6, 2C9, 2D6 and 3A4) was observed. For all the activities, a reduction of at least 50% at the highest concentration (250 µg/mL) was observed. In addition, UGT activities diminished. MSBE considerably reduced UGT1A9 activity (about 60% at 250 µg/mL) and lesser effects on the other UGTs. In contrast, 250 µg/mL mangiferin had greater effects on UGT1A1 and 2B7 than on UGT1A9 (about 55% vs. 35% reduction, respectively). Quantification of specific mRNAs revealed reduced CYP3A4 and 3A5 mRNAs content, and an increase in CYP1A1, CYP1A2, UGT1A1 and UGT1A9 mRNAs. No remarkable effects on the CYP2A6, 2B6, 2C9, 2C19, 2D6 and 2E1 levels were observed. Our results suggest that the activity and/or expression of major P450 and UGT enzymes is modulated by MSBE and that potential herb-drugs interactions could arise after a combined intake of this extract with conventional medicines. Therefore, the potential safety risks of this natural product derived by altering the ADMET properties of co-administered drugs should be examined.

  9. EGCG Protects against 6-OHDA-Induced Neurotoxicity in a Cell Culture Model

    Directory of Open Access Journals (Sweden)

    Dan Chen

    2015-01-01

    Full Text Available Background. Parkinson’s disease (PD is a progressive neurodegenerative disease that causes severe brain dopamine depletion. Disruption of iron metabolism may be involved in the PD progression. Objective. To test the protective effect of (−-epigallocatechin-3-gallate (EGCG against 6-hydroxydopamine- (6-OHDA- induced neurotoxicity by regulating iron metabolism in N27 cells. Methods. Protection by EGCG in N27 cells was assessed by SYTOX green assay, MTT, and caspase-3 activity. Iron regulatory gene and protein expression were measured by RT-PCR and Western blotting. Intracellular iron uptake was measured using 55Fe. The EGCG protection was further tested in primary mesencephalic dopaminergic neurons by immunocytochemistry. Results. EGCG protected against 6-OHDA-induced cell toxicity. 6-OHDA treatment significantly (p<0.05 increased divalent metal transporter-1 (DMT1 and hepcidin and decreased ferroportin 1 (Fpn1 level, whereas pretreatment with EGCG counteracted the effects. The increased 55Fe (by 96%, p<0.01 cell uptake confirmed the iron burden by 6-OHDA and was reduced by EGCG by 27% (p<0.05, supporting the DMT1 results. Pretreatment with EGCG and 6-OHDA significantly increased (p<0.0001 TH+ cell count (~3-fold and neurite length (~12-fold compared to 6-OHDA alone in primary mesencephalic neurons. Conclusions. Pretreatment with EGCG protected against 6-OHDA-induced neurotoxicity by regulating genes and proteins involved in brain iron homeostasis, especially modulating hepcidin levels.

  10. A 3-day EGCG-supplementation reduces interstitial lactate concentration in skeletal muscle of overweight subjects.

    Science.gov (United States)

    Most, Jasper; van Can, Judith G P; van Dijk, Jan-Willem; Goossens, Gijs H; Jocken, Johan; Hospers, Jeannette J; Bendik, Igor; Blaak, Ellen E

    2015-12-09

    Green tea, particularly epigallocatechin-3-gallate (EGCG), may affect body weight and composition, possibly by enhancing fat oxidation. The aim of this double-blind, randomized placebo-controlled cross-over study was to investigate whether 3-day supplementation with EGCG (282 mg/day) stimulates fat oxidation and lipolysis in 24 overweight subjects (age = 30 ± 2 yrs, BMI = 27.7 ± 0.3 kg/m(2)). Energy expenditure, substrate metabolism and circulating metabolites were determined during fasting and postprandial conditions. After 6 h, a fat biopsy was collected to examine gene expression. In 12 subjects, skeletal muscle glycerol, glucose and lactate concentrations were determined using microdialysis. EGCG-supplementation did not alter energy expenditure and substrate oxidation compared to placebo. Although EGCG reduced postprandial circulating glycerol concentrations (P = 0.015), no difference in skeletal muscle lipolysis was observed. Fasting (P = 0.001) and postprandial (P = 0.003) skeletal muscle lactate concentrations were reduced after EGCG-supplementation compared to placebo, despite similar tissue blood flow. Adipose tissue leptin (P = 0.05) and FAT/CD36 expression (P = 0.08) were increased after EGCG compared to placebo. In conclusion, 3-day EGCG-supplementation decreased postprandial plasma glycerol concentrations, but had no significant effects on skeletal muscle lipolysis and whole-body fat oxidation in overweight individuals. Furthermore, EGCG decreased skeletal muscle lactate concentrations, which suggest a shift towards a more oxidative muscle phenotype.

  11. Epigallocatechin-3-gallate (EGCG), a green tea polyphenol, stimulates hepatic autophagy and lipid clearance.

    Science.gov (United States)

    Zhou, Jin; Farah, Benjamin Livingston; Sinha, Rohit Anthony; Wu, Yajun; Singh, Brijesh Kumar; Bay, Boon-Huat; Yang, Chung S; Yen, Paul Michael

    2014-01-01

    Epigallocatechin gallate (EGCG) is a major polyphenol in green tea that has been shown to have anti-inflammatory, anti-cancer, anti-steatotic effects on the liver. Autophagy also mediates similar effects; however, it is not currently known whether EGCG can regulate hepatic autophagy. Here, we show that EGCG increases hepatic autophagy by promoting the formation of autophagosomes, increasing lysosomal acidification, and stimulating autophagic flux in hepatic cells and in vivo. EGCG also increases phosphorylation of AMPK, one of the major regulators of autophagy. Importantly, siRNA knockdown of AMPK abrogated autophagy induced by EGCG. Interestingly, we observed lipid droplet within autophagosomes and autolysosomes and increased lipid clearance by EGCG, suggesting it promotes lipid metabolism by increasing autophagy. In mice fed with high-fat/western style diet (HFW; 60% energy as fat, reduced levels of calcium, vitamin D3, choline, folate, and fiber), EGCG treatment reduces hepatosteatosis and concomitantly increases autophagy. In summary, we have used genetic and pharmacological approaches to demonstrate EGCG induction of hepatic autophagy, and this may contribute to its beneficial effects in reducing hepatosteatosis and potentially some other pathological liver conditions.

  12. Epigallocatechin-3-gallate (EGCG, a green tea polyphenol, stimulates hepatic autophagy and lipid clearance.

    Directory of Open Access Journals (Sweden)

    Jin Zhou

    Full Text Available Epigallocatechin gallate (EGCG is a major polyphenol in green tea that has been shown to have anti-inflammatory, anti-cancer, anti-steatotic effects on the liver. Autophagy also mediates similar effects; however, it is not currently known whether EGCG can regulate hepatic autophagy. Here, we show that EGCG increases hepatic autophagy by promoting the formation of autophagosomes, increasing lysosomal acidification, and stimulating autophagic flux in hepatic cells and in vivo. EGCG also increases phosphorylation of AMPK, one of the major regulators of autophagy. Importantly, siRNA knockdown of AMPK abrogated autophagy induced by EGCG. Interestingly, we observed lipid droplet within autophagosomes and autolysosomes and increased lipid clearance by EGCG, suggesting it promotes lipid metabolism by increasing autophagy. In mice fed with high-fat/western style diet (HFW; 60% energy as fat, reduced levels of calcium, vitamin D3, choline, folate, and fiber, EGCG treatment reduces hepatosteatosis and concomitantly increases autophagy. In summary, we have used genetic and pharmacological approaches to demonstrate EGCG induction of hepatic autophagy, and this may contribute to its beneficial effects in reducing hepatosteatosis and potentially some other pathological liver conditions.

  13. Comparative transcriptional network modeling of three PPAR-α/γ co-agonists reveals distinct metabolic gene signatures in primary human hepatocytes.

    Directory of Open Access Journals (Sweden)

    Renée Deehan

    Full Text Available AIMS: To compare the molecular and biologic signatures of a balanced dual peroxisome proliferator-activated receptor (PPAR-α/γ agonist, aleglitazar, with tesaglitazar (a dual PPAR-α/γ agonist or a combination of pioglitazone (Pio; PPAR-γ agonist and fenofibrate (Feno; PPAR-α agonist in human hepatocytes. METHODS AND RESULTS: Gene expression microarray profiles were obtained from primary human hepatocytes treated with EC(50-aligned low, medium and high concentrations of the three treatments. A systems biology approach, Causal Network Modeling, was used to model the data to infer upstream molecular mechanisms that may explain the observed changes in gene expression. Aleglitazar, tesaglitazar and Pio/Feno each induced unique transcriptional signatures, despite comparable core PPAR signaling. Although all treatments inferred qualitatively similar PPAR-α signaling, aleglitazar was inferred to have greater effects on high- and low-density lipoprotein cholesterol levels than tesaglitazar and Pio/Feno, due to a greater number of gene expression changes in pathways related to high-density and low-density lipoprotein metabolism. Distinct transcriptional and biologic signatures were also inferred for stress responses, which appeared to be less affected by aleglitazar than the comparators. In particular, Pio/Feno was inferred to increase NFE2L2 activity, a key component of the stress response pathway, while aleglitazar had no significant effect. All treatments were inferred to decrease proliferative signaling. CONCLUSIONS: Aleglitazar induces transcriptional signatures related to lipid parameters and stress responses that are unique from other dual PPAR-α/γ treatments. This may underlie observed favorable changes in lipid profiles in animal and clinical studies with aleglitazar and suggests a differentiated gene profile compared with other dual PPAR-α/γ agonist treatments.

  14. 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.

  15. Assessment of Protein Binding of 5-Hydroxythalidomide Bioactivated in Humanized Mice with Human P450 3A-Chromosome or Hepatocytes by Two-Dimensional Electrophoresis/Accelerator Mass Spectrometry.

    Science.gov (United States)

    Yamazaki, Hiroshi; Suemizu, Hiroshi; Kazuki, Yasuhiro; Oofusa, Ken; Kuribayashi, Shunji; Shimizu, Makiko; Ninomiya, Shinichi; Horie, Toru; Shibata, Norio; Guengerich, F Peter

    2016-08-15

    Bioactivation of 5-hydroxy-[carbonyl-(14)C]thalidomide, a known metabolite of thalidomide, by human artificial or native cytochrome P450 3A enzymes, and nonspecific binding in livers of mice was assessed using two-dimensional electrophoresis combined with accelerator mass spectrometry. The apparent major target proteins were liver microsomal cytochrome c oxidase subunit 6B1 and ATP synthase subunit α in mice containing humanized P450 3A genes or transplanted humanized liver. Liver cytosolic retinal dehydrogenase 1 and glutathione transferase A1 were targets in humanized mice with P450 3A and hepatocytes, respectively. 5-Hydroxythalidomide is bioactivated by human P450 3A enzymes and trapped with proteins nonspecifically in humanized mice.

  16. Itraconazole cis-diastereoisomers activate aryl hydrocarbon receptor AhR and pregnane X receptor PXR and induce CYP1A1 in human cell lines and human hepatocytes.

    Science.gov (United States)

    Stepankova, Martina; Pastorkova, Barbora; Bachleda, Petr; Dvorak, Zdenek

    2017-04-05

    Triazole antimycotic itraconazole contains in its structure three chiral centres; therefore, it forms eight stereoisomers. Commercial preparations of itraconazole are a mixture of four cis-diastereoisomers. There is much evidence that efficacy, adverse effects, and toxicity of chiral drugs may be stereospecific. Therefore, we have prepared 4 pure cis-diastereoisomers of itraconazole and investigated their effects on transcriptional activities of xenoreceptors aryl hydrocarbon receptor AhR and pregnane X receptor PXR. Gene reporter assays showed that itraconazole dose-dependently activated both AhR and PXR, and the activation of AhR but not of PXR was enantiospecific. Itraconazole diastereoisomers transformed AhR and PXR into their DNA-binding forms, as demonstrated by electromobility shift assays. Cytochrome P450 CYP1A1 mRNA and protein were induced by itraconazole diastereoisomers in human hepatoma cells HepG2, human skin cells HaCaT, and in primary human hepatocytes. The expression of CYP3A4 in human intestinal LS180 cells was not influenced by itraconazole, but we observed downregulation of CYP3A4 in human hepatocytes. Collectively, we show that itraconazole is a dual activator of AhR and PXR, with differential effects on the target genes for xenoreceptors. The enantiospecific pattern was observed only in gene reporter assays for AhR. The data presented here might be of toxicological and clinical importance.

  17. New IVIVE method for the prediction of total human clearance and relative elimination pathway contributions from in vitro hepatocyte and microsome data.

    Science.gov (United States)

    Riede, Julia; Poller, Birk; Umehara, Ken-ichi; Huwyler, Jörg; Camenisch, Gian

    2016-04-30

    Total human clearance is a key determinant for the pharmacokinetic behavior of drug candidates. Our group recently introduced the Extended Clearance Model (ECM) as an accurate in vitro-in vivo extrapolation (IVIVE) method for the prediction of hepatic clearance. Yet, knowledge about relative elimination pathway contributions is needed in order to predict the total human clearance of drug candidates. In the present work, a training set of 18 drug compounds was used to describe the affiliations between in vitro sinusoidal uptake clearance and the fractional contributions of hepatic (metabolic and biliary) or renal clearance to overall in vivo elimination. By means of these quantitative relationships and using a validation set of 10 diverse drug molecules covering different (sub)classes of the Extended Clearance Concept Classification System (ECCCS), the relative contributions of elimination pathways were calculated and demonstrated to well correlate with human reference data. Likewise, ECM- and pathway-based predictions of total clearances from both data sets demonstrated a strong correlation with the observed clinical values with 26 out of 28 compounds within a three-fold deviation. Hence, total human clearance and relative contributions of elimination pathways were successfully predicted by the presented method using solely hepatocyte and microsome in vitro data.

  18. CD81 is a central regulator of cellular events required for hepatitis C virus infection of human hepatocytes.

    Science.gov (United States)

    Brazzoli, Michela; Bianchi, Alessia; Filippini, Sara; Weiner, Amy; Zhu, Qing; Pizza, Mariagrazia; Crotta, Stefania

    2008-09-01

    Infection with hepatitis C virus (HCV) is still a major public health problem, and the events leading to hepatocyte infection are not yet fully understood. Combining confocal microscopy with biochemical analysis and studies of infection requirements using pharmacological inhibitors and small interfering RNAs, we show here that engagement of CD81 activates the Rho GTPase family members Rac, Rho, and Cdc42 and that the block of these signaling pathways drastically reduces HCV infectivity. Activation of Rho GTPases mediates actin-dependent relocalization of the HCV E2/CD81 complex to cell-cell contact areas where CD81 comes into contact with the tight-junction proteins occludin, ZO-1, and claudin-1, which was recently described as an HCV coreceptor. Finally, we show that CD81 engagement activates the Raf/MEK/ERK signaling cascade and that this pathway affects postentry events of the virus life cycle. In conclusion, we describe a range of cellular events that are manipulated by HCV to coordinate interactions with its multiple coreceptors and to establish productive infections and find that CD81 is a central regulator of these events.

  19. Effectiveness of xenotransplantation of human fetal hepatocytes in spleen of rats with acute liver failure induced by CCL4

    Directory of Open Access Journals (Sweden)

    Abdukhakim Khadjibaev

    2013-04-01

    Full Text Available Human’s fetal hepatocytes (HFH were intrasplenic transplanted white non-pedigree rats with acute liver failure (ALF challenged by single per oral administration of hepatotropic toxin diluted in oil ССl4 at a dose 10 ml/kg (volumetric correlation 1:1 (10 mL/kg body weight as a 1:1 mixture of CCl4 and mineral oil. Transplantation had positive effect on all biochemical blood parameters of the studying animals. Morphologic study showed that reparative-restorative processes were arising in hepatic parenchyma after administration of HFH into splenic pulp of rats with model of ALF on days 14-21. Substantial and main factor in restoration of parenchyma was restoration of micro topographic interrelations in acinus as well as polyploidy of hepatic cells expressed in increase of hepatocytes’ nuclei sizes and hypertrophy of cells themselves. It is an indirect confirmation of engraftment of HFH in liver of rats with model of ALF.

  20. Glucagon and cAMP inhibit cholesterol 7alpha-hydroxylase (CYP7A1) gene expression in human hepatocytes: discordant regulation of bile acid synthesis and gluconeogenesis.

    Science.gov (United States)

    Song, Kwang-Hoon; Chiang, John Y L

    2006-01-01

    The gene encoding cholesterol 7alpha-hydroxylase (CYP7A1) is tightly regulated to control bile acid synthesis and maintain lipid homeostasis. Recent studies in mice suggest that bile acid synthesis is regulated by the fasted-to-fed cycle, and fasting induces CYP7A1 gene expression in parallel to the induction of peroxisome proliferators-activated receptor gamma co-activator 1alpha (PGC-1alpha) and phosphoenolpyruvate carboxykinase (PEPCK). How glucagon regulates CYP7A1 gene expression in the human liver is not clear. Here we show that glucagon and cyclic adenosine monophosphate (cAMP) strongly repressed CYP7A1 mRNA expression in human primary hepatocytes. Reporter assays confirmed that cAMP and protein kinase A (PKA) inhibited human CYP7A1 gene transcription, in contrast to their stimulation of the PEPCK gene. Mutagenesis analysis identified a PKA-responsive region located within the previously identified HNF4alpha binding site in the human CYP7A1 promoter. Glucagon and cAMP increased HNF4alpha phosphorylation and reduced the amount of HNF4alpha present in CYP7A1 chromatin. Our findings suggest that glucagon inhibited CYP7A1 gene expression via PKA phosphorylation of HNF4alpha, which lost its ability to bind the CYP7A1 gene and resulted in inhibition of human CYP7A1 gene transcription. In conclusion, this study unveils a species difference in nutrient regulation of the human and mouse CYP7A1 gene and suggests a discordant regulation of bile acid synthesis and gluconeogenesis by glucagon in human livers during fasting.

  1. Prediction of the metabolic clearance of benzophenone-2, and its interaction with isoeugenol and coumarin using cryopreserved human hepatocytes in primary culture.

    Science.gov (United States)

    de Sousa, Georges; Teng, Sophie; Salle-Siri, Romain; Pery, Alexandre; Rahmani, Roger

    2016-04-01

    Benzophenone-2 (BP2) is widely used as a UV screen in both industrial products and cosmetic formulations, where it is frequently found associated with fragrance compounds, such as isoeugenol and coumarin. BP2 is now recognized as an endocrine disruptor, but to date, no information has been reported on its fate in humans. The intrinsic clearance (Clint) and metabolic interactions of BP2 were explored using cryopreserved human hepatocytes in primary cultures. In vitro kinetic experiments were performed to estimate the Michaelis-Menten parameters. The substrate depletion method demonstrated that isoeugenol was cleared more rapidly than BP2 or coumarin (Clint = 259, 94.7 and 0.40 μl/min/10(6) cells respectively). This vitro model was also used to study the metabolic interactions between BP2 and isoeugenol and coumarin. Coumarin exerted no effects on either isoeugenol or BP2 metabolism, because of its independent metabolic pathway (CYP2A6). Isoeugenol appeared to be a potent competitive substrate inhibitor of BP2 metabolism, equivalent to the specific UGT1A1 substrate: estradiol. Despite the fact that inhibition of UGT by xenobiotics is not usually considered to be a major concern, the involvement of UGT1A1 in BP2 metabolism may have pharmacokinetic and pharmacological consequences, due to the its polymorphisms in humans and its pure estrogenic effect.

  2. Effects of physiological levels of the green tea extract Epigallocatechin gallate (EGCG on breast cancer cells

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    Li eZeng

    2014-05-01

    Full Text Available Physiological concentrations of the green tea extract EGCG caused growth inhibition in oestrogen receptor α (ERα-positive MCF7 cells, that was associated with down-regulation of the ERα and reduced insulin-like growth factor (IGF binding protein-2 (IGFBP-2 abundance and increased protein abundance of the tumour suppressor genes p53/p21. In contrast to MCF7 cells that have wt p53, EGCG alone did not change cell proliferation or death significantly in another ERα-positive cell line T47D that possesses mutant p53. EGCG increased ERα protein levels and as a consequence, the cells responded significantly better to an ERα antagonist Tamoxifen (TAM in the presence of EGCG. EGCG significantly increased cell death in an ERα-negative cell line, MDA-MB-231 that also possesses mutant p53. EGCG significantly increased the ERα and IGF-I receptor (IGF-IR levels and thereby enhanced the sensitivities of the cells to Tamoxifen and a blocking antibody targeting the IGF-1R (αIR3. In contrast to MCF7, T47D and MDA-MB-231 breast cancer cells that exhibited significant changes in key molecules involved in breast growth and survival upon treatment with physiological levels of EGCG, the growth, survival and levels of these proteins in non-malignant breast epithelial cells, MCF10A cells, were not affected.

  3. Green tea polyphenol epigallocatechin-3-gallate (EGCG) induced intermolecular cross-linking of membrane proteins.

    Science.gov (United States)

    Chen, Rong; Wang, Jian-Bo; Zhang, Xian-Qing; Ren, Jing; Zeng, Cheng-Ming

    2011-03-15

    Increasing evidence has demonstrated that EGCG possesses prooxidant potential in biological systems, including modifying proteins, breaking DNA strands and inducing the generation of reactive oxygen species. In the present study, the prooxidant effect of EGCG on erythrocyte membranes was investigated. SDS-PAGE and NBT-staining assay were utilized to detect the catechol-protein adducts that generated upon treating the membranes with EGCG. The results indicated that EGCG was able to bind covalently to sulfhydryl groups of membrane proteins, leading to the formation of protein aggregates with intermolecular cross-linking. We suggested that the catechol-quinone originated from the oxidation of EGCG acted as a cross-linker on which peptide chains were combined through thiol-S-alkylation at the C2- and C6-sites of the gallyl ring. EGC showed similar effects as EGCG on the ghost membranes, whereas ECG and EC did not, suggesting that a structure with a gallyl moiety is a prerequisite for a catechin to induce the aggregation of membrane proteins and to deplete membrane sulfhydryls. EDTA and ascorbic acid inhibited the EGCG-induced aggregation of membrane proteins by blocking the formation of catechol-quinone. The information of the present study may provide a fresh insight into the prooxidant effect and cytotoxicity of tea catechins.

  4. Epigallocatechin gallate (EGCG) suppresses lipopolysaccharide-induced inflammatory bone resorption, and protects against alveolar bone loss in mice.

    Science.gov (United States)

    Tominari, Tsukasa; Matsumoto, Chiho; Watanabe, Kenta; Hirata, Michiko; Grundler, Florian M W; Miyaura, Chisato; Inada, Masaki

    2015-01-01

    Epigallocatechin gallate (EGCG), a major polyphenol in green tea, possesses antioxidant properties and regulates various cell functions. Here, we examined the function of EGCG in inflammatory bone resorption. In calvarial organ cultures, lipopolysaccharide (LPS)-induced bone resorption was clearly suppressed by EGCG. In osteoblasts, EGCG suppressed the LPS-induced expression of COX-2 and mPGES-1 mRNAs, as well as prostaglandin E2 production, and also suppressed RANKL expression, which is essential for osteoclast differentiation. LPS-induced bone resorption of mandibular alveolar bones was attenuated by EGCG in vitro, and the loss of mouse alveolar bone mass was inhibited by the catechin in vivo.

  5. In vitro evaluation of hepatotoxic drugs in human hepatocytes from multiple donors: Identification of P450 activity as a potential risk factor for drug-induced liver injuries.

    Science.gov (United States)

    Utkarsh, Doshi; Loretz, Carol; Li, Albert P

    2016-08-01

    A possible risk factor for drug-induced hepatotoxicity is drug metabolizing enzyme activity, which is known to vary among individuals due to genetic (genetic polymorphism) and environmental factors (environmental pollutants, foods, and medications that are inhibitors or inducers of drug metabolizing enzymes). We hypothesize that hepatic cytochrome P450-dependent monooxygenase (CYP) activity is one of the key risk factors for drug induced liver injuries (DILI) in the human population, especially for drugs that are metabolically activated to cytotoxic/reactive metabolites. Human hepatocytes from 19 donors were evaluated for the activities of 8 major P450 isoforms: CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4. Extensive individual variations were observed, consistent with what is known to be in the human population. As CYP3A4 is known to be one of the most important P450 isoforms for drug metabolism, studies were performed to evaluate the relationship between the in vitro cytotoxicity of hepatotoxic drugs and CYP3A4 activity. In a proof of concept study, hepatocytes from six donors (lots) representing the observed range of CYP3A4 activities were chosen for the evaluation of in vitro hepatotoxicity of four drugs known to be associated with acute liver failure: acetaminophen, cyclophosphamide, ketoconazole, and tamoxifen. The hepatocytes were cultured in collagen-coated plates and treated with the hepatotoxicants for approximately 24 h, followed by viability determination based on cellular adenosine triphosphate (ATP) contents. HH1023, the lot of hepatocytes with the highest CYP3A4 activity, was found to be the most sensitive to the cytotoxicity of all 4 hepatotoxic drugs, thereby suggesting that high CYP3A4 activity may be a risk factor. To further validate the relationship, a second study was performed with hepatocytes from 16 donors. In this study, the hepatocytes were quantified for CYP3A4 activity at the time of treatment. Results of the

  6. EGCG attenuates autoimmune arthritis by inhibition of STAT3 and HIF-1α with Th17/Treg control.

    Directory of Open Access Journals (Sweden)

    Eun-Ji Yang

    Full Text Available Epigallocatechin-3-gallate (EGCG is a green tea polyphenol exerting potent anti-oxidant and anti-inflammatory effects by inhibiting signaling and gene expression. The objective of the study was to evaluate the effect of EGCG on interleukin (IL-1 receptor antagonist knockout (IL-1RaKO autoimmune arthritis models. IL-1RaKO arthritis models were injected intraperitoneally with EGCG three times per week after the first immunization. EGCG decreased the arthritis index and showed protective effects against joint destruction in the IL-1RaKO arthritis models. The expression of pro-inflammatory cytokines, oxidative stress proteins, and p-STAT3 (Y705 and p-STAT3 (S727, mTOR and HIF-1α were significantly lower in mice treated with EGCG. EGCG reduced osteoclast markers in vivo and in vitro along with anti-osteoclastic activity was observed in EGCG-treated IL-1RaKO mice. The proportion of Foxp3(+ Treg cells increased in the spleens of mice treated with EGCG, whereas the proportion of Th17 cells reduced. In vitro, p-STAT3 (Y705 and p-STAT3 (S727, HIF1α and glycolytic pathway molecules were decreased by EGCG. EGCG suppressed the activation of mTOR and subsequently HIF-1α, which is considered as a metabolic check point of Th17/Treg differentiation supporting the therapeutic potential of EGCG in autoimmune arthritis.

  7. Evaluation of epigallocatechin-3-gallate (EGCG) cross-linked collagen membranes and concerns on osteoblasts

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    Chu, Chenyu; Deng, Jia [State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu 610041 (China); Xiang, Lin; Wu, Yingying [State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu 610041 (China); Department of Oral Implantology, West China Hospital of Stomatology, Sichuan University, Chengdu 610041 (China); Wei, Xiawei [State Key Laboratory of Biotherapy and Laboratory for Aging Research, West China Hospital, Sichuan University, and Collaborative Innovation Center for Biotherapy, Chengdu, Sichuan 610041 (China); Qu, Yili [State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu 610041 (China); Department of Oral Implantology, West China Hospital of Stomatology, Sichuan University, Chengdu 610041 (China); Man, Yi, E-mail: manyi780203@126.com [State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu 610041 (China); Department of Oral Implantology, West China Hospital of Stomatology, Sichuan University, Chengdu 610041 (China)

    2016-10-01

    Collagen membranes have ideal biological and mechanical properties for supporting infiltration and proliferation of osteoblasts and play a vital role in guided bone regeneration (GBR). However, pure collagen can lead to inflammation, resulting in progressive bone resorption. Therefore, a method for regulating the level of inflammatory cytokines at surgical sites is paramount for the healing process. Epigallocatechin-3-gallate (EGCG) is a component extracted from green tea with numerous biological activities including an anti-inflammatory effect. Herein, we present a novel cross-linked collagen membrane containing different concentrations of EGCG (0.0064%, 0.064%, and 0.64%) to regulate the level of inflammatory factors secreted by pre-osteoblast cells; improve cell proliferation; and increase the tensile strength, wettability, and thermal stability of collagen membranes. Scanning electron microscope images show that the surfaces of collagen membranes became smoother and the collagen fiber diameters became larger with EGCG treatment. Measurement of the water contact angle demonstrated that introducing EGCG improved membrane wettability. Fourier transform infrared spectroscopy analyses indicated that the backbone of collagen was intact, and the thermal stability was significant improved in differential scanning calorimetry. The mechanical properties of 0.064% and 0.64% EGCG-treated collagen membranes were 1.5-fold greater than those of the control. The extent of cross-linking was significantly increased, as determined by a 2,4,6-trinitrobenzenesulfonic acid solution assay. The Cell Counting Kit-8 (CCK-8) and live/dead assays revealed that collagen membrane cross-linked by 0.0064% EGCG induced greater cell proliferation than pure collagen membranes. Additionally, real-time polymerase chain reaction and enzyme-linked immunosorbent assay results showed that EGCG significantly affected the production of inflammatory factors secreted by MC3T3-E1 cells. Taken together, our

  8. Design of a Vitronectin-Based Recombinant Protein as a Defined Substrate for Differentiation of Human Pluripotent Stem Cells into Hepatocyte-Like Cells.

    Directory of Open Access Journals (Sweden)

    Masato Nagaoka

    Full Text Available Maintenance and differentiation of human pluripotent stem cells (hPSCs usually requires culture on a substrate for cell adhesion. A commonly used substratum is Matrigel purified from Engelbreth-Holm-Swarm sarcoma cells, and consists of a complex mixture of extracellular matrix proteins, proteoglycans, and growth factors. Several studies have successfully induced differentiation of hepatocyte-like cells from hPSCs. However, most of these studies have used Matrigel as a cell adhesion substrate, which is not a defined culture condition. In an attempt to generate a substratum that supports undifferentiated properties and differentiation into hepatic lineage cells, we designed novel substrates consisting of vitronectin fragments fused to the IgG Fc domain. hPSCs adhered to these substrates via interactions between integrins and the RGD (Arg-Gly-Asp motif, and the cells maintained their undifferentiated phenotypes. Using a previously established differentiation protocol, hPSCs were efficiently differentiated into mesendodermal and hepatic lineage cells on a vitronectin fragment-containing substrate. We found that full-length vitronectin did not support stable cell adhesion during the specification stage. Furthermore, the vitronectin fragment with the minimal RGD-containing domain was sufficient for differentiation of human induced pluripotent stem cells into hepatic lineage cells under completely defined conditions that facilitate the clinical application of cells differentiated from hPSCs.

  9. Curcumin inhibits prosurvival pathways in chronic lymphocytic leukemia B cells and may overcome their stromal protection in combination with EGCG.

    Science.gov (United States)

    Ghosh, Asish K; Kay, Neil E; Secreto, Charla R; Shanafelt, Tait D

    2009-02-15

    Chronic lymphocytic leukemia (CLL) is incurable with current chemotherapy treatments. Curcumin (diferuloylmethane), an active ingredient in the spice turmeric, inhibits tumor metastasis, invasion, and angiogenesis in tumor cell lines. We evaluated the effects of curcumin on the viability of primary CLL B cells and its ability to overcome stromal mediated protection. The in vitro effect of curcumin on primary CLL B cells was evaluated using fluorescence activated cell sorter analysis and Western blotting. For some experiments, CLL B cells were cocultured with human stromal cells to evaluate the effects of curcumin on leukemia cells cultured in their microenvironment. Finally, the effect of curcumin in combination with the green tea extract epigallocatechin-3 gallate (EGCG) was evaluated. Curcumin induced apoptosis in CLL B cells in a dose-dependent (5-20 micromol/L) manner and inhibited constitutively active prosurvival pathways, including signal transducers and activators of transcription 3 (STAT3), AKT, and nuclear factor kappaB. Moreover, curcumin suppressed expression of the anti-apoptotic proteins Mcl-1 and X-linked inhibitor of apoptosis protein (XIAP), and up-regulated the pro-apoptotic protein BIM. Coculture of CLL B cells with stromal cells resulted in elevated levels of STAT3, increased expression of Mcl-1 and XIAP, and decreased sensitivity to curcumin. When curcumin was administered simultaneously with EGCG, antagonism was observed for most patient samples. In contrast, sequential administration of these agents led to substantial increases in CLL B-cell death and could overcome stromal protection. Curcumin treatment was able to overcome stromal protection of CLL B cells on in vitro testing and to synergize with EGCG when administered in a sequential fashion. Additional evaluation of curcumin as a potential therapeutic agent for treatment of CLL seems warranted.

  10. Human Muse cells, non-tumorigenic pluripotent-like stem cells, have the capacity for liver regeneration by specific homing and replenishment of new hepatocytes in liver fibrosis mouse model.

    Science.gov (United States)

    Iseki, Masahiro; Kushida, Yoshihiro; Wakao, Shohei; Akimoto, Takahiro; Mizuma, Masamichi; Motoi, Fuyuhiko; Asada, Ryuta; Shimizu, Shinobu; Unno, Michiaki; Chazenbalk, Gregorio; Dezawa, Mari

    2016-11-02

    Muse cells, a novel type of non-tumorigenic pluripotent-like stem cells reside in the bone marrow, skin and adipose tissue, are collectable as cells positive for pluripotent surface marker SSEA-3. They are able to differentiate into cells representative of all three germ layers. The capacity of intravenously injected human bone marrow-Muse cells to repair the liver fibrosis model of immunodeficient mice was evaluated in this study. They exhibited the ability for differentiation spontaneously into hepatoblast/hepatocyte-lineage cells and high migration toward the serum and liver tissue of carbon tetrachloride-treated mice in vitro. In vivo, they specifically accumulated into the liver, but not into other organs except the lower rate in the lung at 2 weeks after intravenous injection into the liver fibrosis model. After homing, Muse cells spontaneously differentiated in vivo into HepPar-1 (71.1±15.2%), human albumin (54.3±8.2%) and anti-trypsin (47.9±4.6%)-positive cells without fusing with host hepatocytes, and expressed mature functional markers such as human-CYP1A2, and human-Glc-6-Pase, at 8 weeks. Recovery in serum total bilirubin and albumin, and significant attenuation of fibrosis were recognized with statistical differences between the Muse group and control groups which received the vehicle or the same number of non-Muse cells, namely cells other than Muse cells in bone marrow mesenchymal stem cells. Thus, unlike ES and iPS cells, Muse cells are unique in their efficient migration and integration into damaged liver only by intravenous injection, nontumorigenicity, and spontaneous differentiation into hepatocytes, rendering induction into hepatocytes prior to transplantation unnecessary. They are suggested to repair liver fibrosis in two simple steps; expansion after collection from the bone marrow and intravenous injection. Such feasible strategy might provide impressive regenerative performance to liver disease patients.

  11. Hepatocyte Growth Factor Effects on Mesenchymal Stem Cells Derived from Human Arteries: A Novel Strategy to Accelerate Vascular Ulcer Wound Healing

    Directory of Open Access Journals (Sweden)

    Sabrina Valente

    2016-01-01

    Full Text Available Vascular ulcers are a serious complication of peripheral vascular disease, especially in diabetics. Several approaches to treat the wounds are proposed but they show poor outcomes and require long healing times. Hepatocyte Growth Factor/Scatter Factor (HGF/SF is a pleiotropic cytokine exerting many biological activities through the c-Met receptor. This study was aimed at verifying whether HGF/SF influences proliferation, migration, and angiogenesis on mesenchymal stem cells isolated from human arteries (hVW-MSCs. hVW-MSCs were exposed to NIBSC HGF/SF (2.5, 5, 10, and 70 ng/mL from 6 hrs to 7 days. HGF and c-MET mRNA and protein expression, cell proliferation (Alamar Blue and Ki–67 assay, migration (scratch and transwell assays, and angiogenesis (Matrigel were investigated. hVW-MSCs displayed stemness features and expressed HGF and c-MET. HGF/SF did not increase hVW-MSC proliferation, whereas it enhanced the cell migration, the formation of capillary-like structures, and the expression of angiogenic markers (vWF, CD31, and KDR. The HGF/SF effects on hVW-MSC migration and angiogenic potential are of great interest to accelerate wound healing process. Local delivery of HGF/SF could therefore improve the healing of unresponsive vascular ulcers.

  12. Hepatocyte Growth Factor Effects on Mesenchymal Stem Cells Derived from Human Arteries: A Novel Strategy to Accelerate Vascular Ulcer Wound Healing.

    Science.gov (United States)

    Valente, Sabrina; Ciavarella, Carmen; Pasanisi, Emanuela; Ricci, Francesca; Stella, Andrea; Pasquinelli, Gianandrea

    2016-01-01

    Vascular ulcers are a serious complication of peripheral vascular disease, especially in diabetics. Several approaches to treat the wounds are proposed but they show poor outcomes and require long healing times. Hepatocyte Growth Factor/Scatter Factor (HGF/SF) is a pleiotropic cytokine exerting many biological activities through the c-Met receptor. This study was aimed at verifying whether HGF/SF influences proliferation, migration, and angiogenesis on mesenchymal stem cells isolated from human arteries (hVW-MSCs). hVW-MSCs were exposed to NIBSC HGF/SF (2.5, 5, 10, and 70 ng/mL) from 6 hrs to 7 days. HGF and c-MET mRNA and protein expression, cell proliferation (Alamar Blue and Ki-67 assay), migration (scratch and transwell assays), and angiogenesis (Matrigel) were investigated. hVW-MSCs displayed stemness features and expressed HGF and c-MET. HGF/SF did not increase hVW-MSC proliferation, whereas it enhanced the cell migration, the formation of capillary-like structures, and the expression of angiogenic markers (vWF, CD31, and KDR). The HGF/SF effects on hVW-MSC migration and angiogenic potential are of great interest to accelerate wound healing process. Local delivery of HGF/SF could therefore improve the healing of unresponsive vascular ulcers.

  13. Notopterygium forbesii boiss extract and its active constituents increase reactive species and heme oxygenase-1 in human fetal hepatocytes: mechanisms of action.

    Science.gov (United States)

    Tang, Soon Yew; Wang, Huansong; Zhang, Wenxia; Halliwell, Barry

    2008-12-01

    Notopterygium forbesii Boiss (NF) has been used as a traditional Chinese medicine for the treatment of common cold and rheumatism. However, there has been limited research on the biological properties of NF, and the mechanisms of action remain unknown. Here, we aimed to study the mechanism of NF-induced heme oxygenase-1 (HO-1) in human fetal hepatocytes (HFHs) and to identify the constituents responsible. Exposure of HFHs to NF causes oxidative stress with the accumulation of reactive species, which in turn leads to the phosphorylation of p38 MAPK and nuclear accumulation of Nrf2 transcription factor, and eventually increased levels of HO-1 mRNA and protein. The increases in reactive species and HO-1 protein are inhibited by agonists of glucocorticoid receptors (GR), such as RU28362, prednisolone, and dexamethasone, as well as by N-acetyl-L-cysteine and SB203580 (a p38 inhibitor), suggesting a role of GR in NF-induced increases in reactive species and HO-1. Assay-guided fractionation of NF led to three active compounds, phenethyl ferulate, bergaptol, and isoimperatorin, that were found to increase oxidative stress and HO-1 protein levels in HFHs. The induction of HO-1 protein in response to moderate oxidative stress may explain some of the beneficial pharmacological effects of NF.

  14. Effects on Proliferation and Migration of the Human Colon Carcinoma Cell Line SW620 by Silencing of Hepatocyte Growth Factor Expression

    Institute of Scientific and Technical Information of China (English)

    Yi-tao JIA; Lei ZHANG; Yan LI; Ya-di WANG; Wei GUO; Lei CAO; Zhong-xin LI

    2010-01-01

    OBJECTIVE Hepatocyte growth factor (HGF) expression is closely related to the progression and poor prognosis of colorectal cancer patients. In this study, we investigated the effects on proliferation and migration of the human colon carcinoma cell line SW620 by silencing HGF expression. METHODS HGF was silenced using specifi c HGF α/β siRNA.The proliferation, migration, cell cycle and ultrastructure of SW620 cells were examined. RESULTS The transfection efficiency was 70%–80%. The expression rate of HGF in the experimental group was signifi cantly lower than that in the negative and blank control groups (P <0.05). The proliferation inhibition rate in the experimental group at 24, 48, 72 and 96 h after transfection was 14.2%, 50.2%, 39.5% and 23.2%, respectively. The migratory ability of cells in the experimental group was significantly inhibited compared with that in the negative control or blank control groups (58.2% vs. 2.1% or 0%, P < 0.05).CONCLUSION The application of RNA interference to silence the expression of HGF in the colon carcinoma cell line SW620 effectively inhibits the proliferation and migration of tumor cells.

  15. The human hepatocyte cell lines IHH and HepaRG : models to study glucose, lipid and lipoprotein metabolism

    NARCIS (Netherlands)

    Samanez, Carolina Huaman; Caron, Sandrine; Briand, Olivier; Dehondt, Helene; Duplan, Isabelle; Kuipers, Folkert; Hennuyer, Nathalie; Clavey, Veronique; Staels, Bart

    2012-01-01

    Metabolic diseases reach epidemic proportions. A better knowledge of the associated alterations in the metabolic pathways in the liver is necessary. These studies need in vitro human cell models. Several human hepatoma models are used, but the response of many metabolic pathways to physiological sti

  16. The human hepatocyte cell lines IHH and HepaRG : models to study glucose, lipid and lipoprotein metabolism

    NARCIS (Netherlands)

    Samanez, Carolina Huaman; Caron, Sandrine; Briand, Olivier; Dehondt, Helene; Duplan, Isabelle; Kuipers, Folkert; Hennuyer, Nathalie; Clavey, Veronique; Staels, Bart

    Metabolic diseases reach epidemic proportions. A better knowledge of the associated alterations in the metabolic pathways in the liver is necessary. These studies need in vitro human cell models. Several human hepatoma models are used, but the response of many metabolic pathways to physiological

  17. DNA adducts of the tobacco carcinogens 2-amino-9H-pyrido[2,3-b]indole and 4-aminobiphenyl are formed at environmental exposure levels and persist in human hepatocytes.

    Science.gov (United States)

    Nauwelaërs, Gwendoline; Bellamri, Medjda; Fessard, Valérie; Turesky, Robert J; Langouët, Sophie

    2013-09-16

    Aromatic amines and structurally related heterocyclic aromatic amines (HAAs) are produced during the combustion of tobacco or during the high-temperature cooking of meat. Exposure to some of these chemicals may contribute to the etiology of several common types of human cancers. 2-Amino-9H-pyrido[2,3-b]indole (AαC) is the most abundant HAA formed in mainstream tobacco smoke: it arises in amounts that are 25-100 times greater than the levels of the arylamine, 4-aminobiphenyl (4-ABP), a human carcinogen. 2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) is a prevalent HAA formed in cooked meats. AαC and MeIQx are rodent carcinogens; however, their carcinogenic potency in humans is unknown. A preliminary assessment of the carcinogenic potential of these HAAs in humans was conducted by examining the capacity of primary human hepatocytes to form DNA adducts of AαC and MeIQx, in comparison to 4-ABP, followed by the kinetics of DNA adduct removal by cellular enzyme repair systems. The principal DNA adducts formed were N-(deoxyguanosin-8-yl) (dG-C8) adducts. Comparable levels of DNA adducts were formed with AαC and 4-ABP, whereas adduct formation was ∼5-fold lower for MeIQx. dG-C8-AαC and dG-C8-4-ABP were formed at comparable levels in a concentration-dependent manner in human hepatocytes treated with procarcinogens over a 10,000-fold concentration range (1 nM-10 μM). Pretreatment of hepatocytes with furafylline, a selective inhibitor of cytochrome P450 1A2, resulted in a strong diminution of DNA adducts signifying that P450 1A2 is a major P450 isoform involved in bioactivation of these procarcinogens. The kinetics of adduct removal varied for each hepatocyte donor. Approximately half of the DNA adducts were removed within 24 h of treatment; however, the remaining lesions persisted over 5 days. The high levels of AαC present in tobacco smoke and its propensity to form persistent DNA adducts in human hepatocytes suggest that AαC can contribute to DNA damage

  18. Pluripotent stem cell-derived hepatocyte-like cells.

    Science.gov (United States)

    Schwartz, R E; Fleming, H E; Khetani, S R; Bhatia, S N

    2014-01-01

    Liver disease is an important clinical problem, impacting over 30 million Americans and over 600 million people worldwide. It is the 12th leading cause of death in the United States and the 16th worldwide. Due to a paucity of donor organs, several thousand Americans die yearly while waiting for liver transplantation. Unfortunately, alternative tissue sources such as fetal hepatocytes and hepatic cell lines are unreliable, difficult to reproduce, and do not fully recapitulate hepatocyte phenotype and functions. As a consequence, alternative cell sources that do not have these limitations have been sought. Human embryonic stem (hES) cell- and induced pluripotent stem (iPS) cell-derived hepatocyte-like cells may enable cell based therapeutics, the study of the mechanisms of human disease and human development, and provide a platform for screening the efficacy and toxicity of pharmaceuticals. iPS cells can be differentiated in a step-wise fashion with high efficiency and reproducibility into hepatocyte-like cells that exhibit morphologic and phenotypic characteristics of hepatocytes. In addition, iPS-derived hepatocyte-like cells (iHLCs) possess some functional hepatic activity as they secrete urea, alpha-1-antitrypsin, and albumin. However, the combined phenotypic and functional traits exhibited by iHLCs resemble a relatively immature hepatic phenotype that more closely resembles that of fetal hepatocytes rather than adult hepatocytes. Specifically, iHLCs express fetal markers such as alpha-fetoprotein and lack key mature hepatocyte functions, as reflected by drastically reduced activity (~0.1%) of important detoxification enzymes (i.e. CYP2A6, CYP3A4). These key differences between iHLCs and primary adult human hepatocytes have limited the use of stem cells as a renewable source of functional adult hepatocytes for in vitro and in vivo applications. Unfortunately, the developmental pathways that control hepatocyte maturation from a fetal into an adult hepatocyte are

  19. First metabolic profile of PV8, a novel synthetic cathinone, in human hepatocytes and urine by high-resolution mass spectrometry.

    Science.gov (United States)

    Swortwood, Madeleine J; Ellefsen, Kayla N; Wohlfarth, Ariane; Diao, Xingxing; Concheiro-Guisan, Marta; Kronstrand, Robert; Huestis, Marilyn A

    2016-07-01

    Novel psychoactive substances (NPS) are ever changing on the drug market, making it difficult for toxicology laboratory methods to stay current with so many new drugs. Recently, PV8, a synthetic pyrrolidinophenone, was detected in seized products in Japan (2013), The Netherlands (2014), and Germany (2014). There are no controlled PV8 administration studies, and no pharmacodynamic and pharmacokinetic data. The objective was to determine PV8's metabolic stability in human liver microsome (HLM) incubation and its metabolism following human hepatocyte incubation and high-resolution mass spectrometry (HRMS) with a Thermo Scientific Q-Exactive. Data were acquired with a full-scan data-dependent mass spectrometry method. Scans were thoroughly data mined with different data processing algorithms and analyzed in WebMetaBase. PV8 exhibited a relatively short 28.8 min half-life, with an intrinsic 24.2 μL/min/mg microsomal clearance. This compound is predicted to be an intermediate clearance drug with an estimated human 22.7 mL/min/kg hepatic clearance. Metabolic pathways identified in vitro included: hydroxylation, ketone reduction, carboxylation, N-dealkylation, iminium formation, dehydrogenation, N-oxidation, and carbonylation. The top three in vitro metabolic pathways were di-hydroxylation > ketone reduction > γ-lactam formation. Authentic urine specimen analyses revealed the top three metabolic pathways were aliphatic hydroxylation > ketone reduction + aliphatic hydroxylation > aliphatic carboxylation, although the most prominent peak was parent PV8. These data provide useful urinary metabolite targets (aliphatic hydroxylation, aliphatic hydroxylation + ketone reduction, aliphatic carboxylation, and di-hydroxylation) for forensic and clinical testing, and focus reference standard companies' synthetic efforts to provide commercially available standards needed for PV8 biological specimen testing. Graphical Abstract Top four PV8 metabolites identified in vitro

  20. The spectral properties of (--epigallocatechin 3-O-gallate (EGCG fluorescence in different solvents: dependence on solvent polarity.

    Directory of Open Access Journals (Sweden)

    Vladislav Snitsarev

    Full Text Available (--Epigallocatechin 3-O-gallate (EGCG a molecule found in green tea and known for a plethora of bioactive properties is an inhibitor of heat shock protein 90 (HSP90, a protein of interest as a target for cancer and neuroprotection. Determination of the spectral properties of EGCG fluorescence in environments similar to those of binding sites found in proteins provides an important tool to directly study protein-EGCG interactions. The goal of this study is to examine the spectral properties of EGCG fluorescence in an aqueous buffer (AB at pH=7.0, acetonitrile (AN (a polar aprotic solvent, dimethylsulfoxide (DMSO (a polar aprotic solvent, and ethanol (EtOH (a polar protic solvent. We demonstrate that EGCG is a highly fluorescent molecule when excited at approximately 275 nm with emission maxima between 350 and 400 nm depending on solvent. Another smaller excitation peak was found when EGCG is excited at approximately 235 nm with maximum emission between 340 and 400 nm. We found that the fluorescence intensity (FI of EGCG in AB at pH=7.0 is significantly quenched, and that it is about 85 times higher in an aprotic solvent DMSO. The Stokes shifts of EGCG fluorescence were determined by solvent polarity. In addition, while the emission maxima of EGCG fluorescence in AB, DMSO, and EtOH follow the Lippert-Mataga equation, its fluorescence in AN points to non-specific solvent effects on EGCG fluorescence. We conclude that significant solvent-dependent changes in both fluorescence intensity and fluorescence emission shifts can be effectively used to distinguish EGCG in aqueous solutions from EGCG in environments of different polarity, and, thus, can be used to study specific EGCG binding to protein binding sites where the environment is often different from aqueous in terms of polarity.

  1. Effects of mesenchymal stem cells transfected with human hepatocyte growth factor gene on healing of burn wounds

    Institute of Scientific and Technical Information of China (English)

    HA Xiao-qin; L(U) Tong-de; HUI Ling; Dong Fang

    2010-01-01

    Objective: To explore the effects of bone marrow-derived mesenchymal stem cells (BMSCs)transfected with adenoviral vector carrying hepatocyte growth factor (HGF, Ad-HGF) on burn wound healing.Methods: BMSCs from male Wistar rats were separated and purified with Percoll separating medium by density gradient centrifugation and cultured with DMEM containing 20% fetal bovine serum (FBS). Then BMSCs were transfected with Ad-HGF at the optimal gene transduction efficiency of 100 multiplicity of infection (MOI). The efficiency of transfection and the expression of HGF in the suspension were detected by flow cytometry and enzyme linked immunosorbent assay (ELISA) respectively. Thirtytwo female rats were subjected to 90℃ water for 12 seconds to induce a partial thickness skin burn. The animals were randomly divided into mesenchymal stem cells (MSCs) treatment group (Group A), Ad-HGF treatment group (Group B),Ad-HGF-modified MSCs treatment group (Group C) and saline control group (Group D). On days 3, 5, 7, 14 and 21 postburn, HE and Sirius red stain were performed to observe the burn wound healing and collagen content. The content of hydroxyproline in wounds was also detected.Transplanted cells and the expression of(sex-determining region Y) SRY gene were detected by in situ hybridization and polymerase chain reaction (PCR), while the expression of HGF in wound tissues was detected by ELISA.Results: The result of flow cytometry showed that the transfection efficiency was 86.41% at 100 MOI. Compared with the control group, the content of HGF in the supernatant after transfection increased time-dependently and peaked at 48 h, showing significant differences at 24 h, 48 h,72 h and 96 h (P<0.01 ). Results of HE stain revealed that the range of re-epidermidalization in Group C was significantly larger than that in other groups in the first week. Three weeks postburn, the epidermis was significantly thicker in Group C than in other groups and the nails of dermis inserted into

  2. Effect of human umbilical cord mesenchymal stem cell-secretion on proliferation and apoptosis in hepatocytes%人脐带间充质干细胞分泌物对肝细胞增殖和凋亡的影响

    Institute of Scientific and Technical Information of China (English)

    黎娇; 朱争艳; 杜智; 骆莹; 王鹏; 高英堂

    2010-01-01

    目的 探讨人脐带间充质干细胞(human umbilical cord mesenchymal stem cells,HUC MSC)旁分泌物质在体外对肝细胞再生和凋亡的影响.方法 利用Ⅳ型胶原酶和胰酶消化法从脐带中分离间充质干细胞,制备含有HUCMSC旁分泌物质的条件培养基(mesenchymal stem cells-conditioned medium,MSC-CM),采用低浓度胶原酶原位循环灌流法分离肝细胞.试验分为对照组、2%MSC-CM组和8%MSC-CM组三组.采用MTT比色法观察不同浓度MSC-CM对正常肝细胞增殖的影响.测定上清中尿素、白蛋白的含量,观察不同浓度MSC-CM对肝细胞功能的影响.利用放线菌素D和肿瘤坏死因子α诱导肝细胞凋亡,采用细胞活性分析试剂盒检测不同浓度MSC-CM对肝细胞凋亡的影响.结果 与对照组比较,2%MSC-CM组吸光度(A)540nm值(P<0.01)以及上清尿素和白蛋白含量显著升高(P<0.01),肝细胞存活率增加(P<0.05);8%MSC-CM组与对照组无显著差异.结论 低浓度的MSC-CM在体外可以刺激正常肝细胞再生,抑制受损肝细胞凋亡,改善肝细胞功能.%Objective To investigate the effect of human umbilical cord mesenchymal stem cell paracrine substance on proliferation and apoptosis of liver cells in vitro. Methods Mesenchymal stem cells (MSC)were separated from human umbilical cord with type Ⅳ collagenase and trypsogen digestion method and cultured in vitro. The human umbilical cord mesenchymal stem cells-conditioned medium(MSC-CM) which contain paracrine substance of human umbilical cord mesenchymal stem cells (HUCMSC) was prepared. Hepatocytes were isolated from SD rats by low concentration collagenase perfusion procedure. There were three groups in the experiment, control group, 2% MSC-CM group and 8% MSC-CM group. The proliferation of normal hepatocytes were assayed with MTT method. We detected the urea and albumin level in culture supernatant to assay the hepatocyte function under different concentration MSC-CM. Hepatocytes were

  3. Cholangiocarcinomas can originate from hepatocytes in mice.

    Science.gov (United States)

    Fan, Biao; Malato, Yann; Calvisi, Diego F; Naqvi, Syed; Razumilava, Nataliya; Ribback, Silvia; Gores, Gregory J; Dombrowski, Frank; Evert, Matthias; Chen, Xin; Willenbring, Holger

    2012-08-01

    Intrahepatic cholangiocarcinomas (ICCs) are primary liver tumors with a poor prognosis. The development of effective therapies has been hampered by a limited understanding of the biology of ICCs. Although ICCs exhibit heterogeneity in location, histology, and marker expression, they are currently thought to derive invariably from the cells lining the bile ducts, biliary epithelial cells (BECs), or liver progenitor cells (LPCs). Despite lack of experimental evidence establishing BECs or LPCs as the origin of ICCs, other liver cell types have not been considered. Here we show that ICCs can originate from fully differentiated hepatocytes. Using a mouse model of hepatocyte fate tracing, we found that activated NOTCH and AKT signaling cooperate to convert normal hepatocytes into biliary cells that act as precursors of rapidly progressing, lethal ICCs. Our findings suggest a previously overlooked mechanism of human ICC formation that may be targetable for anti-ICC therapy.

  4. Food Inhibits the Oral Bioavailability of the Major Green Tea Antioxidant Epigallocatechin Gallate in Humans

    Directory of Open Access Journals (Sweden)

    Nenad Naumovski

    2015-05-01

    Full Text Available The bioavailability of the most abundant and most active green tea antioxidant, epigallocatechin gallate (EGCG remains uncertain. Therefore, the systemic absorption of EGCG was tested in healthy fasted humans. It was administered as capsules with water or with a light breakfast, or when incorporated within a strawberry sorbet. The results for plasma EGCG clearly revealed that taking EGCG capsules without food was better; the AUC was 2.7 and 3.9 times higher than when EGCG capsules were taken with a light breakfast (p = 0.044 or with EGCG imbedded in the strawberry sorbet (p = 0.019, respectively. This pattern was also observed for Cmax and Cav. Therefore, ingesting food at the same time as EGCG, whether it was imbedded or not in food, substantially inhibited the absorption of the catechin. As with some types of medications that are affected by food, it appears that EGCG should be taken without food in order to maximise its systemic absorption. Therefore, based on these findings, ingesting EGCG with water on an empty stomach is the most appropriate method for the oral delivery of EGCG in clinical trials where EGCG is to be investigated as a potential bioactive nutraceutical in humans.

  5. Skatole (3-Methylindole Is a Partial Aryl Hydrocarbon Receptor Agonist and Induces CYP1A1/2 and CYP1B1 Expression in Primary Human Hepatocytes.

    Directory of Open Access Journals (Sweden)

    Martin Krøyer Rasmussen

    Full Text Available Skatole (3-methylindole is a product of bacterial fermentation of tryptophan in the intestine. A significant amount of skatole can also be inhaled during cigarette smoking. Skatole is a pulmonary toxin that induces the expression of aryl hydrocarbon receptor (AhR regulated genes, such as cytochrome P450 1A1 (CYP1A1, in human bronchial cells. The liver has a high metabolic capacity for skatole and is the first organ encountered by the absorbed skatole; however, the effect of skatole in the liver is unknown. Therefore, we investigated the impact of skatole on hepatic AhR activity and AhR-regulated gene expression. Using reporter gene assays, we showed that skatole activates AhR and that this is accompanied by an increase of CYP1A1, CYP1A2 and CYP1B1 expression in HepG2-C3 and primary human hepatocytes. Specific AhR antagonists and siRNA-mediated AhR silencing demonstrated that skatole-induced CYP1A1 expression is dependent on AhR activation. The effect of skatole was reduced by blocking intrinsic cytochrome P450 activity and indole-3-carbinole, a known skatole metabolite, was a more potent inducer than skatole. Finally, skatole could reduce TCDD-induced CYP1A1 expression, suggesting that skatole is a partial AhR agonist. In conclusion, our findings suggest that skatole and its metabolites affect liver homeostasis by modulating the AhR pathway.

  6. Human adult chondrocytes express hepatocyte growth factor (HGF) isoforms but not HgF: potential implication of osteoblasts on the presence of HGF in cartilage.

    Science.gov (United States)

    Guévremont, Melanie; Martel-Pelletier, Johanne; Massicotte, Frédéric; Tardif, Ginette; Pelletier, Jean-Pierre; Ranger, Pierre; Lajeunesse, Daniel; Reboul, Pascal

    2003-06-01

    HGF is increased in human OA cartilage, possibly from Ob's. RT-PCR shows HGF isoforms are differently regulated between chondrocytes and Ob. A paracrine cross-talk between subchondral bone and cartilage may occur during OA. Recently, hepatocyte growth factor (HGF) has been identified by immunohistochemistry in cartilage and more particularly in the deep zone of human osteoarthritic (OA) cartilage. By investigating HGF expression in cartilage, we found that chondrocytes did not express HGF; however, they expressed the two truncated isoforms, namely HGF/NK1 and HGF/NK2. Because the only other cells localized near the deep zone are osteoblasts from the subchondral bone plate, we hypothesized that they were expressing HGF. Indeed, we found that HGF was synthesized by osteoblasts from the subchondral bone plate. Moreover, OA osteoblasts produced five times more HGF than normal osteoblasts and almost no HGF/NK1, unlike normal osteoblasts. Because prostaglandin E2 (PGE2) and pro-inflammatory cytokines such as interleukin (IL)-1 and IL-6 are involved in OA progression, we investigated whether these factors impact HGF produced by normal osteoblasts. PGE2 was the only factor tested that was able to stimulate HGF synthesis. However, the addition of NS398, a selective inhibitor of cyclo-oxygenase-2 (COX-2) had no effect on HGF produced by OA osteoblasts. HGF/NK2 had a moderate stimulating effect on HGF production by normal osteoblasts, whereas osteocalcin was not modulated by either HGF or HGF/NK2. When investigating signaling routes that might be implicated in OA osteoblast-produced HGF, we found that protein kinase A was at least partially involved. In summary, this study raises the hypothesis that the HGF found in articular cartilage is produced by osteoblasts, diffuses into the cartilage, and may be implicated in the OA process.

  7. A food contaminant ochratoxin A suppresses pregnane X receptor (PXR)-mediated CYP3A4 induction in primary cultures of human hepatocytes.

    Science.gov (United States)

    Doricakova, Aneta; Vrzal, Radim

    2015-11-04

    Ochratoxin A (OCHA) is a mycotoxin, which can be found in food such as coffee, wine, cereals, meat, nuts. Since it is absorbed via gastrointestinal tract, it is reasonable to anticipate that the liver will be the first organ to which OCHA comes into the contact before systemic circulation. Many xenobiotics are metabolically modified after the passage of the liver to biologically more active substances, sometimes with more harmful activity. Promoting own metabolism is often achieved via transcriptional regulation of biotransformation enzymes through ligand-activated transcription factors. Pregnane X receptor (PXR) belongs to such a group of regulators and it was demonstrated to be activated by many compounds of synthetic as well as natural origin. Our intention was to investigate if OCHA is capable of activating the PXR with consequent induction of PXR-regulated CYP3A4 gene. We found that OCHA does not activate PXR but displays antagonist-like behavior when combined with rifampicin (RIF) in gene reporter assay in human embryonal kidney cells (Hek293T). It was very weak inducer of CYP3A4 mRNA in primary cultures of human hepatocytes and it antagonized RIF-mediated CYP3A4 induction of mRNA as well as protein. In addition, it caused the decline of PXR protein as well as mRNA which was faster than that with actinomycin D, a transcription inhibitor. Since we found that OCHA induced the expression of miR-148a, which was described to regulate PXR expression, we conclude that antagonist-like behavior of OCHA is not due to the antagonism itself but due to the downregulation of PXR gene expression. Herein we provide important findings which bring a piece of puzzle into the understanding of mechanism of toxic action of ochratoxin A.

  8. Distinguishing Intake of New Synthetic Cannabinoids ADB-PINACA and 5F-ADB-PINACA with Human Hepatocyte Metabolites and High-Resolution Mass Spectrometry.

    Science.gov (United States)

    Carlier, Jeremy; Diao, Xingxing; Scheidweiler, Karl B; Huestis, Marilyn A

    2017-05-01

    ADB-PINACA and its 5-fluoropentyl analog 5F-ADB-PINACA are among the most potent synthetic cannabinoids tested to date, with several severe intoxication cases. ADB-PINACA and 5F-ADB-PINACA have a different legal status, depending on the country. Synthetic cannabinoid metabolites predominate in urine, making detection of specific metabolites the most reliable way for proving intake in clinical and forensic specimens. However, there are currently no data on ADB-PINACA and 5F-PINACA metabolism. The substitution of a single fluorine atom distinguishes the 2 molecules, which may share common major metabolites. For some legal applications, distinguishing between ADB-PINACA and 5F-PINACA intake is critical. For this reason, we determined the human metabolic fate of the 2 analogs. ADB-PINACA and 5F-PINACA were incubated for 3 h with pooled cryopreserved human hepatocytes, followed by liquid chromatography-high-resolution mass spectrometry analysis. Data were processed with Compound Discoverer. We identified 19 and 12 major ADB-PINACA and 5F-ADB-PINACA metabolites, respectively. Major metabolic reactions included pentyl hydroxylation, hydroxylation followed by oxidation (ketone formation), and glucuronidation of ADB-PINACA, and oxidative defluorination followed by carboxylation of 5F-ADB-PINACA. We recommend ADB-PINACA ketopentyl and hydroxypentyl, and ADB-PINACA 5-hydroxypentyl and pentanoic acid, as optimal markers for ADB-PINACA and 5F-ADB-PINACA intake, respectively. Since the 2 compounds present positional isomers as the primary metabolites, monitoring unique product ions and optimized chromatographic conditions are required for a clear distinction between ADB-PINACA and 5F-ADB-PINACA intake. © 2016 American Association for Clinical Chemistry.

  9. Application of exogenous mixture of glutathione and stable isotope labeled glutathione for trapping reactive metabolites in cryopreserved human hepatocytes. Detection of the glutathione conjugates using high resolution accurate mass spectrometry.

    Science.gov (United States)

    Mezine, Igor; Bode, Chris; Raughley, Bethany; Bhoopathy, Sid; Roberts, Kenneth J; Owen, Albert J; Hidalgo, Ismael J

    2013-08-25

    Metabolites (including reactive metabolites) of troglitazone were generated by incubation with cryopreserved human hepatocytes and trapped in the presence of an exogenous mixture of unlabeled and stable isotope labeled (SIL: [1,2-(13)C, (15)N]-glycine) glutathione (GSH/SIL-GSH). The incubation samples were analyzed using liquid chromatography-high resolution accurate mass spectrometry (LC-HRAMS) implemented on a LTQ Orbitrap mass spectrometer. The GSH conjugates of the reactive metabolites were detected via a characteristic mono-isotopic pattern (peaks separated by 3.0037u). Analysis of the incubation samples led to detection of a number of previously described GSH conjugates, as well as two novel methylated GSH conjugates, which were partially characterized based on accurate mass measurements and MS/MS data. The addition of exogenous GSH led to an increase in the apparent level of detected GSH conjugates. Kinetic isotopic measurements showed that the rates of incorporation of exogenous GSH are conjugate-specific. In conclusion, this approach, based on the use of a mixture of GSH/SIL-GSH, allows facile capture and detection of reactive metabolites in human hepatocytes. Moreover, the data suggest that routine addition of glutathione to the assay medium may be advisable for experiments with cryopreserved hepatocytes. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  10. A New Splice Variant of the Major Subunit of Human Asialoglycoprotein Receptor Encodes a Secreted Form in Hepatocytes

    OpenAIRE

    Jia Liu; Bin Hu; Yan Yang; Zhiyong Ma; Yuan Yu; Shenpei Liu; Baoju Wang; Xiping Zhao; Mengji Lu; Dongliang Yang

    2010-01-01

    BACKGROUND: The human asialoglycoprotein receptor (ASGPR) is composed of two polypeptides, designated H1 and H2. While variants of H2 have been known for decades, the existence of H1 variants has never been reported. PRINCIPAL FINDINGS: We identified two splice variants of ASGPR H1 transcripts, designated H1a and H1b, in human liver tissues and hepatoma cells. Molecular cloning of ASGPR H1 variants revealed that they differ by a 117 nucleotide segment corresponding to exon 2 in the ASGPR geno...

  11. Assessment of competitive and mechanism-based inhibition by clarithromycin: use of domperidone as a CYP3A probe-drug substrate and various enzymatic sources including a new cell-based assay with freshly isolated human hepatocytes.

    Science.gov (United States)

    Michaud, Veronique; Turgeon, Jacques

    2010-04-01

    Clarithromycin is involved in a large number of clinically relevant drug-drug interactions. Discrepancies are observed between the magnitude of drug interactions predicted from in vitro competitive inhibition studies and changes observed clinically in the plasma levels of affected CYP3A substrates. The formation of metabolic-intermediate complexes has been proposed to explain these differences. The objectives of our study were: 1) to determine the competitive inhibition potency of clarithromycin on the metabolism of domperidone as a CYP3A probe drug using human recombinant CYP3A4 and CYP3A5 isoenzymes, human liver microsomes and cultured human hepatocytes; 2) to establish the modulatory role of cytochrome b5 on the competitive inhibition potency of clarithromycin; 3) to demonstrate the clarithromycin-induced formation of CYP450 metabolic-intermediate complexes in human liver microsomes; and 4) to determine the extent of CYP3A inhibition due to metabolic-intermediate complex formation using human liver microsomes and cultured human hepatocytes. At high concentrations (100 µM), clarithromycin had weak competitive inhibition potency towards CYP3A4 and CYP3A5. Inhibition potency was further decreased by the addition of cytochrome b5 (9-19%). Clarithromycin-induced metabolic-intermediate complexes were revealed by spectrophotometry analysis using human liver microsomes while time- and concentration-dependent mechanism-based inhibitions were quantified using isolated hepatocytes. These results indicate that mechanism-based but not competitive inhibition of CYP3As is the major underlying mechanism of drug-drug interactions observed clinically with clarithromycin. Drug interactions between clarithromycin and several CYP3A substrates are predicted to be insidious; the risk of severe adverse events should increase over time and persist for a few days after cessation of the drug.

  12. COUPLING OF THE ANTIVIRAL DRUG ARA-AMP TO LACTOSAMINATED ALBUMIN LEADS TO SPECIFIC UPTAKE IN RAT AND HUMAN HEPATOCYTES

    NARCIS (Netherlands)

    JANSEN, RW; KRUIJT, JK; VANBERKEL, TJC; MEIJER, DKF

    1993-01-01

    We covalently coupled 9-beta-D-arabinofuranosyladenine 5'-monophosphate (ara-AMP) to the carrier molecule lactosaminated human serum albumin using a water-soluble carbodiimide with a two-step conjugation method (pH 4.5 and pH 7.5) instead of the commonly used single-step conjugation at pH 7.5. This

  13. A long-term hepatitis B viremia model generated by transplanting nontumorigenic immortalized human hepatocytes in Rag-2-deficient mice

    NARCIS (Netherlands)

    Parashar, B; Moshage, H; Tanaka, KE; Engelhardt, D; Rabbani, E; Roy-Chowdhury, N; Roy-Chowdhury, J

    2000-01-01

    Development of new therapies for human hepatitis B virus infection (HBV) would be greatly facilitated by the availability of a suitable small-animal model for HBV virus production in vivo. To develop a murine model for HBV production, we established an immortalized, cloned liver cell line by transfe

  14. Effective analysis on treatment of rat acute liver failure by human fetal hepatocyte transplantation%人胚胎肝细胞移植治疗大鼠急性肝衰竭的效果分析

    Institute of Scientific and Technical Information of China (English)

    刘凯歌; 尚红利; 赵慧; 牛春燕; 汪雯

    2011-01-01

    目的 探讨人胚胎肝细胞移植治疗急性肝衰竭(ALF)的疗效及能否成为移植细胞源.方法 采用四甲基偶氮唑盐法检测人胚胎肝细胞增殖情况;免疫细胞化学检测其ALB、细胞角蛋白-18(CK-18)的表达; D-氨基半乳糖(D-gal)药物诱导ALF的实验动物模型;人胚胎肝细胞移植治疗ALF的动物模型,包括移植组与对照组在肝功能指标[ALT、AST、碱性磷酸酶(ALP)、总胆红素(TBIL)]的差异性比较.免疫组织化学法检测植入脾脏中的人胚胎肝细胞ALB及CK-18的表达.结果 培养第5天左右细胞数量达到最高峰,每天完成4~5次分裂,胚胎肝细胞的生长曲线呈抛物线型.免疫细胞化学检测提示其具有表达ALB、CK-18的功能; D-gal 1.6 g/kg腹腔注射72 h后大鼠ALF模型制备成功;移植第3~5天后,移植组与对照组比较,肝功能(ALT、AST、ALP、TBIL)指标差异有统计学意义(P<0.01).植入脾内的肝细胞具有分泌并表达CK-18、ALB的功能.结论 人胚胎肝细胞脾内移植能有效治疗ALF,并可能成为肝细胞移植的靶细胞源.%Objective To investigate the effect of treating rat acute liver failure and alternative sources of cells for transplantation by human fetal hepatocyte transplantation Methods The proliferation,expression of albumin and cytokeratin-18 of human fetal hepatocyte were detected by MTT and immunocytochemistry.The animal models of rat acute liver failure were made by D-galactosamine.Hepatic functions.such as alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase and total bilirubin were measured between the treated and the control groups.The expression of albumin and cytokeratin-18 of human fetal hepatocyte transplanted in spleen were detected by immunohistochemistry.Results The cell population reached a peak, when human fetal hepatocyte were cultivated for 5 d.The cell division had 4 - 5 times per day,its growth curve present parabola.Immunocytochemistry revealed the

  15. EGCG inhibits cardiomyocyte apoptosis in pressure overload-induced cardiac hypertrophy and protects cardiomyocytes from oxidative stress in rats

    Institute of Scientific and Technical Information of China (English)

    Rui SHENG; Zhen-lun GU; Mei-lin XIE; Wen-xuan ZHOU; Ci-yi GUO

    2007-01-01

    Aim: To investigate the effects of epigallocatechin gallate (EGCG) on pressure overload and hydrogen peroxide (H2O2) induced cardiac myocyte apoptosis. Methods: Cardiac hypertrophy was established in rats by abdominal aortic constriction. EGCG 25, 50 and 100 mg/kg were administered intragastrically (ig). Cultured newborn rat cardiomyocytes were preincubated with EGCG, and oxidative stress injury was induced by H2O2. Results: In cardiac hypertrophy induced by AC in rats, relative to the model group, EGCG 25, 50 and 100 mg/kg ig for 6weeks dose-dependently reduced systolic blood pressure (SBP) and heart weight indices, decreased malondialdehyde (MDA) content, and increased superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX) activity, both in serum and in the myocardium. Also, treatment with EGCG 50 and 100 mg/kg markedly improved cardiac structure and inhibited fibrosis in HE and van Gieson (VG) stain, and reduced apoptotic myocytes in the hypertrophic myocardium detected by terminal transferase-mediated dUTP-biotin nick end-labeling (TUNEL) assay. Inthe Western blot analysis, EGCG significantly inhibited pressure overload-inducedp53 increase and bcl-2 decrease. In H2O2-induced cardiomyocyte injury, when preincubated with myocytes for 6-48 h, EGCG 12.5-200 mg/L increased cell viability determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) assay. EGCG also attenuated H2O2-induced lactate dehydrogenase (LDH) release and MDA formation. Meanwhile, EGCG 50 and 100 mg/L significantly inhibited the cardiomyocyte apoptotic rate in flow cytometry. Conclusion: EGCG inhibits cardiac myocyte apoptosis and oxidative stress in pressure overload in-duced cardiac hypertrophy. Also, EGCG prevented cardiomyocyte apoptosis from oxidative stress in vitro. The mechanism might be related to the inhibitory effects of EGCG on p53 induction and bcl-2 decrease.

  16. Alginate microencapsulated hepatocytes optimised for transplantation in acute liver failure.

    Directory of Open Access Journals (Sweden)

    Suttiruk Jitraruch

    Full Text Available BACKGROUND AND AIM: Intraperitoneal transplantation of alginate-microencapsulated human hepatocytes is an attractive option for the management of acute liver failure (ALF providing short-term support to allow native liver regeneration. The main aim of this study was to establish an optimised protocol for production of alginate-encapsulated human hepatocytes and evaluate their suitability for clinical use. METHODS: Human hepatocyte microbeads (HMBs were prepared using sterile GMP grade materials. We determined physical stability, cell viability, and hepatocyte metabolic function of HMBs using different polymerisation times and cell densities. The immune activation of peripheral blood mononuclear cells (PBMCs after co-culture with HMBs was studied. Rats with ALF induced by galactosamine were transplanted intraperitoneally with rat hepatocyte microbeads (RMBs produced using a similar optimised protocol. Survival rate and biochemical profiles were determined. Retrieved microbeads were evaluated for morphology and functionality. RESULTS: The optimised HMBs were of uniform size (583.5±3.3 µm and mechanically stable using 15 min polymerisation time compared to 10 min and 20 min (p<0.001. 3D confocal microscopy images demonstrated that hepatocytes with similar cell viability were evenly distributed within HMBs. Cell density of 3.5×10(6 cells/ml provided the highest viability. HMBs incubated in human ascitic fluid showed better cell viability and function than controls. There was no significant activation of PBMCs co-cultured with empty or hepatocyte microbeads, compared to PBMCs alone. Intraperitoneal transplantation of RMBs was safe and significantly improved the severity of liver damage compared to control groups (empty microbeads and medium alone; p<0.01. Retrieved RMBs were intact and free of immune cell adherence and contained viable hepatocytes with preserved function. CONCLUSION: An optimised protocol to produce GMP grade alginate

  17. 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.

  18. First Metabolic Profile of XLR-11, a Novel Synthetic Cannabinoid, Obtained by Using Human Hepatocytes and High-Resolution Mass Spectrometry

    Science.gov (United States)

    Wohlfarth, Ariane; Pang, Shaokun; Zhu, Mingshe; Gandhi, Adarsh S.; Scheidweiler, Karl B.; Liu, Hua-fen; Huestis, Marilyn A.

    2015-01-01

    BACKGROUND Since the mid-2000s synthetic cannabinoids have been abused as recreational drugs, prompting scheduling of these substances in many countries. To circumvent legislation, manufacturers constantly market new compounds; [1-(5-fluoropentyl)indol-3-yl]-(2,2,3,3-tetramethylcyclopropyl)methanone (XLR-11), the fluorinated UR-144 analog, is one of the most recent and widely abused drugs, and its use is now linked with acute kidney injury. Our goal was to investigate XLR-11 metabolism for identification of major urinary targets in analytical methods and to clarify the origin of metabolites when one or more parent synthetic cannabinoids can be the source. METHODS We incubated 10 μmol/L XLR-11 with pooled human hepatocytes and sampled after 1 and 3 h. Samples were analyzed by high-resolution mass spectrometry with a TOF scan followed by information-dependent acquisition triggered product ion scans with dynamic background subtraction and mass defect filters. Scans were thoroughly data mined with different data processing algorithms (Metabolite Pilot 1.5). RESULTS XLR-11 underwent phase I and II metabolism, producing more than 25 metabolites resulting from hydroxylation, carboxylation, hemiketal and hemiacetal formation, internal dehydration, and further glucuronidation of some oxidative metabolites. No sulfate or glutathione conjugation was observed. XLR-11 also was defluorinated, forming UR-144 metabolites. On the basis of mass spectrometry peak areas, we determined that the major metabolites were 2′-carboxy-XLR-11, UR-144 pentanoic acid, 5-hydroxy-UR-144, hydroxy-XLR-11 glucuronides, and 2′-carboxy-UR-144 pentanoic acid. Minor metabolites were combinations of the biotransformations mentioned above, often glucuronidated. CONCLUSIONS These are the first data defining major urinary targets of XLR-11 metabolism that could document XLR-11 intake in forensic and clinical investigations. PMID:24014837

  19. In Vitro Model for Hepatotoxicity Studies Based on Primary Human Hepatocyte Cultivation in a Perfused 3D Bioreactor System

    Directory of Open Access Journals (Sweden)

    Fanny Knöspel

    2016-04-01

    Full Text Available Accurate prediction of the potential hepatotoxic nature of new pharmaceuticals remains highly challenging. Therefore, novel in vitro models with improved external validity are needed to investigate hepatic metabolism and timely identify any toxicity of drugs in humans. In this study, we examined the effects of diclofenac, as a model substance with a known risk of hepatotoxicity in vivo, in a dynamic multi-compartment bioreactor using primary human liver cells. Biotransformation pathways of the drug and possible effects on metabolic activities, morphology and cell transcriptome were evaluated. Formation rates of diclofenac metabolites were relatively stable over the application period of seven days in bioreactors exposed to 300 µM diclofenac (300 µM bioreactors (300 µM BR, while in bioreactors exposed to 1000 µM diclofenac (1000 µM BR metabolite concentrations declined drastically. The biochemical data showed a significant decrease in lactate production and for the higher dose a significant increase in ammonia secretion, indicating a dose-dependent effect of diclofenac application. The microarray analyses performed revealed a stable hepatic phenotype of the cells over time and the observed transcriptional changes were in line with functional readouts of the system. In conclusion, the data highlight the suitability of the bioreactor technology for studying the hepatotoxicity of drugs in vitro.

  20. In Vitro Model for Hepatotoxicity Studies Based on Primary Human Hepatocyte Cultivation in a Perfused 3D Bioreactor System.

    Science.gov (United States)

    Knöspel, Fanny; Jacobs, Frank; Freyer, Nora; Damm, Georg; De Bondt, An; van den Wyngaert, Ilse; Snoeys, Jan; Monshouwer, Mario; Richter, Marco; Strahl, Nadja; Seehofer, Daniel; Zeilinger, Katrin

    2016-04-16

    Accurate prediction of the potential hepatotoxic nature of new pharmaceuticals remains highly challenging. Therefore, novel in vitro models with improved external validity are needed to investigate hepatic metabolism and timely identify any toxicity of drugs in humans. In this study, we examined the effects of diclofenac, as a model substance with a known risk of hepatotoxicity in vivo, in a dynamic multi-compartment bioreactor using primary human liver cells. Biotransformation pathways of the drug and possible effects on metabolic activities, morphology and cell transcriptome were evaluated. Formation rates of diclofenac metabolites were relatively stable over the application period of seven days in bioreactors exposed to 300 µM diclofenac (300 µM bioreactors (300 µM BR)), while in bioreactors exposed to 1000 µM diclofenac (1000 µM BR) metabolite concentrations declined drastically. The biochemical data showed a significant decrease in lactate production and for the higher dose a significant increase in ammonia secretion, indicating a dose-dependent effect of diclofenac application. The microarray analyses performed revealed a stable hepatic phenotype of the cells over time and the observed transcriptional changes were in line with functional readouts of the system. In conclusion, the data highlight the suitability of the bioreactor technology for studying the hepatotoxicity of drugs in vitro.

  1. Effect of EGCG on lipid absorption and plasma lipid levels in rats.

    Science.gov (United States)

    Raederstorff, Daniel G; Schlachter, Manfred F; Elste, Volker; Weber, Peter

    2003-06-01

    Catechins, compounds derived from green tea, have been shown to reduce plasma cholesterol levels and the rate of cholesterol absorption. We investigated the dose response and the mechanism of action of epigallocatechin gallate (EGCG) on these parameters in rats. Wistar rats were fed a diet high in cholesterol and fat containing either none, 0.25% (0.2 g/day/kg BW), 0.5% (0.4 g/day/kg/BW) or 1.0% (0.7 g/day/kg BW) of EGCG. After 4 weeks of treatment, total cholesterol and low density lipoprotein plasma levels were significantly reduced in the group fed 1% EGCG when compared to the no treatment group. Plasma triglycerides and high-density lipoprotein levels did not change significantly. Following a single oral application of a liquid test-meal, intestinal cholesterol absorption in Wistar rats was 79.3% in the control group. In the group treated with 0.1 g/kg BW EGCG intestinal cholesterol absorption decreased to 73.7% and in the group treated with 0.5 g/kg BW of EGCG intestinal cholesterol absorption fell significantly to 62.7% (P = 0.005). Total fat absorption was very efficient in the control group (99.5% of the applied dose) and decreased significantly but moderately in the group treated with the highest doses of EGCG (0.75, 1 g/kg BW). In an in-vitro biliary micelle model, the addition of 55 microM to 1300 microM EGCG not only decreased cholesterol solubility dose-dependently in these micelles but also altered the size of the mixed lecithin/taurocholate/cholesterol micelles as demonstrated by light scattering. This study provides evidence suggesting that the cholesterol-lowering effect of green tea is mainly elicited by EGCG, one of the most abundant catechins contained in green tea. It is suggested that one of the underlying mechanisms by which EGCG affects lipid metabolism is by interfering with the micellar solubilization of cholesterol in the digestive tract, which then in turn decreased cholesterol absorption.

  2. Resveratrol, but not EGCG, in the diet suppresses DMBA-induced mammary cancer in rats

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    Whitsett Timothy

    2006-05-01

    Full Text Available Abstract Despite the advent of new and aggressive therapeutics, breast cancer remains a leading killer among women; hence there is a need for the prevention of this disease. Several naturally occurring polyphenols have received much attention for their health benefits, including anti-carcinogenic properties. Two of these are resveratrol, a component of red grapes, and epigallocatechin-3-gallate (EGCG, the major catechin found in green tea. In this study, we tested the hypothesis that these two polyphenols protect against chemically-induced mammary cancer by modulating mammary gland architecture, cell proliferation, and apoptosis. Female Sprague-Dawley CD rats were exposed to either resveratrol (1 g/kg AIN-76A diet, EGCG (0.065% in the drinking water, or control diet (AIN-76A for the entirety of their life starting at birth. At 50 days postpartum, rats were treated with 60 mg dimethylbenz[a]anthracene (DMBA/kg body weight to induce mammary cancer. Resveratrol, but not EGCG, suppressed mammary carcinogenesis (fewer tumors per rat and longer tumor latency. Analysis of mammary whole mounts from 50-day-old rats revealed that resveratrol, but not EGCG, treatment resulted in more differentiated lobular structures. Bromodeoxyuridine (BrdU incorporation studies showed that resveratrol treatment caused a significant reduction in proliferative cells in mammary terminal ductal structures at 50 days postpartum, making them less susceptible to carcinogen insult. The epithelial cells of terminal end buds in the mammary glands of resveratrol-treated rats also showed an increase in apoptotic cells compared to the control or EGCG-treated rats as measured by a DNA fragmentation assay. At the given doses, resveratrol treatment resulted in a serum resveratrol concentration of 2.00 μM, while treatment with EGCG resulted in a serum EGCG concentration of 31.06 nM. 17β-Estradiol, progesterone, and prolactin concentrations in the serum were not significantly affected

  3. EGCG mediated downregulation of NF-AT and macrophage infiltration in experimental hepatic steatosis.

    Science.gov (United States)

    Krishnan, Thulasi Raman; Velusamy, Prema; Srinivasan, Ashokkumar; Ganesan, Thellamudhu; Mangaiah, Suresh; Narasimhan, Kishorekumar; Chakrapani, Lakshmi Narasimhan; J, Thanka; Walter, Charles Emmanuel Jebaraj; Durairajan, Sankari; Nathakattur Saravanabavan, Sanddhya; Periandavan, Kalaiselvi

    2014-09-01

    Increased fat consumption in industrialized countries has resulted in hepatic steatosis that upregulates atherogenic aspirant genes, leading to atherosclerosis and mortality. Although extensive studies have been carried out to elucidate the atheroprotective efficacy of epigallocatechin-3-gallate (EGCG), the effect of EGCG on hepatic steatosis has not been studied comprehensively. Hence, the current study was designed to find out the effect of EGCG on hepatic events that prelude atherosclerosis with special reference to macrophage infiltration. Male albino rats of Wistar strain were used in this study. Basic biochemical assays along with the protein expression of CAMs, NF-κB, TNF-α and NF-AT were assayed in the current study. EGCG supplementation significantly reverted the alterations in both biochemical and histological parameters and is shown to reduce the TNF-α mediated NF-AT expression and thereby its downstream targets like ICAM-1 and E-selectin expression to a greater extent than NF-κB mediated downstream targets like VCAM-1 and P-selectin in hypercholesterolemic rat liver. Our results suggest that EGCG influences the early events of atherosclerosis that occur; thereby modulating the NF-AT pathway and thereby mitigating the hypercholesterolemic stress. Copyright © 2014 Elsevier Inc. All rights reserved.

  4. In vitro proliferation of primary rat hepatocytes expressing ureogenesis activity by coculture with STO cells.

    Science.gov (United States)

    Takagi, Mutsumi; Kondo, Hiroki; Yoshida, Toshiomi

    2002-01-01

    A coculture system for in vitro proliferation of rat primary hepatocytes with feeder cells was investigated with the view of constructing a hybrid artificial liver module using human hepatocytes. A mouse cell line, STO, was apparently effective compared with other kinds of feeder cells such as FLS5 and MRC5 in increasing the total cell number in the coculture of rat primary hepatocytes. The parenchymal hepatocyte, which are polygonal cells, spread well as the cultivation progressed. Monitoring of parenchymal hepatocyte colonies under a microscope showed that the area of each colony increased as the single culture and cocultures progressed. The adhesion area per hepatocyte increased little during the coculture with STO cells, while the adhesion area increased markedly in the single culture of hepatocytes. The density of hepatocytes increased more than two times during the coculture for 5 d, while it decreased during the single culture. The production rate of urea by hepatocytes markedly increased during the coculture, while the rate in the single culture was lower than the coculture and increased only slightly. The specific rate of urea production by hepatocytes in the coculture did not decrease even as the hepatocytes grew. Consequently, rat primary hepatocytes could proliferate in vitro by coculture with STO cells without a decrease in urea production activity.

  5. Y-27632 Increases Sensitivity of PANC-1 Cells to Epigallocatechin Gallate (EGCG) in Regulating Cell Proliferation and Migration

    Science.gov (United States)

    Liu, Xing; Bi, Yongyi

    2016-01-01

    Background The study aimed to investigate the inhibitory effect of (1R,4r)-4-((R)-1-aminoethyl)-N-(pyridin-4-yl) cyclohexanecarboxamide (Y-27632) and (−)-epigallocatechin-3-gallate (EGCG) on the proliferation and migration of PANC-1 cells. EGCG, found in green tea, has been previously shown to be one of the most abundant and powerful catechins in cancer prevention and treatment. Y-27632, a selective inhibitor of rho-associated protein kinase 1, is widely used in treating cardiovascular disease, inflammation, and cancer. Material/Methods PANC-1 cells, maintained in Dulbecco’s Modified Eagle’s Medium, were treated with dimethyl sulfoxide (control) as well as different concentrations (20, 40, 60, and 80 μg/mL) of EGCG for 48 h. In addition, PANC-1 cells were treated separately with 60 μg/mL EGCG, 20 μM Y-27632, and EGCG combined with Y-27632 (60 μg/mL EGCG + 20 μM Y-27632) for 48 h. The effect of EGCG and Y-27632 on the proliferation and migration of PANC-1 cells was evaluated using Cell Counting Kit-8 and transwell migration assays. The expression of peroxisome proliferator–activated receptor alpha (PPARα) and Caspase-3 mRNA was determined by Quantitative real-time polymerase chain reaction (RT-qPCR). Results EGCG (20–80 μg/mL) inhibited cell viability in a dose-dependent manner. Y-27632 enhanced the sensitivity of PANC-1 cells to EGCG (by increasing the expression of PPARα and Caspase-3 mRNA) and suppressed cell proliferation. PANC-1 cell migration was inhibited by treatment with a combination of EGCG and Y-27632. Conclusions Y-27632 increases the sensitivity of PANC-1 cells to EGCG in regulating cell proliferation and migration, which is likely to be related to the expression of PPARα mRNA and Caspase-3 mRNA. PMID:27694793

  6. Viabilidade do fígado bioartificial utilizando hepatócitos humanos imunoprotegidos por macroencapsulação Assessment of bioartificial liver using human hepatocytes immunoprotected by macroencapsulation

    Directory of Open Access Journals (Sweden)

    João Eduardo Leal Nicoluzzi

    2004-10-01

    Full Text Available OBJETIVO: O transplante de hepatócitos xenogênicos encapsulados pode ser utilizado no futuro em situações como a insuficiência hepática fulminante. Porém, observa-se perda precoce da expressão de genes hepatocitários específicos em hepatócitos humanos. O objetivo deste estudo é avaliar a influência da resposta imunológica na perda da expressão genética hepatocitária de hepatócitos humanos encapsulados e transplantados em ratos. MÉTODO: Hepatócitos humanos foram isolados de fragmentos hepáticos, encapsulados em fibras e transplantados em ratos. Nos dias 3, 7 e 14 após o transplante as fibras foram coletadas e avaliadas a morfologia por microscopia óptica e eletrônica, e a expressão dos genes por biologia molecular. O ARNm da albumina humana foi quantificado por RT-PCR e Northern blot. A resposta imunológica contra os hepatócitos foi avaliada através do ADN hepatocitário na busca de apoptose do núcleo celular e pelo aumento da expressão do CMH de classe I. RESULTADOS: Os aspectos morfológicos dos hepatócitos mantiveram-se normais até o sétimo dia após o transplante. Não se observaram células envolvidas com resposta imunológica do receptor nas fibras. Os transcritos da albumina foram detectados até D-14. Entre os dias 3 e 7 estavam em 30% em relação ao dia 0. A análise do ADN mostrou bandas preservadas sem a presença de fenômenos de apoptose nos diferentes dias. Não ocorreu aumento da expressão do CMH de classe I. CONCLUSÕES: Hepatócitos humanos encapsulados e transplantados em ratos permanecem viáveis apesar da diminuição da expressão de determinados genes. Este fenômeno, não se deve à resposta imunológica do receptor, mas ao próprio processo de isolamento celular.BACKGROUND: Xenogeneic hepatocytes encapsulated in semipermeable membranes could be used in the future; however, encapsulated human hepatocytes presented an early decrease of hepatocyte gene expression. The objective of this study

  7. Legalon-SIL downregulates HCV core and NS5A in human hepatocytes expressing full-length HCV

    Institute of Scientific and Technical Information of China (English)

    Marjan Mehrab-Mohseni; Hossein Sendi; Nury Steuerwald; Sriparna Ghosh; Laura W Schrum; Herbert L Bonkovsky

    2011-01-01

    AIM: To determine the effect of Legalon-SIL (LS) on hepatitis C virus (HCV) core and NS5A expression and on heme oxygenase-1 (HMOX-1) and its transcriptionalregulators in human hepatoma cells expressing full length HCV genotype 1b.METHODS: CON1 cells were treated with 50 μmol/or 200 μmol/L LS. Cells were harvested after 2, 6 and 24 h. HCV RNA and protein levels were determined byquantitative real-time polymerase chain reaction and Western blotting, respectively.RESULTS: HCV RNA (core and NS5A regions) was decreased after 6 h with LS 200 μmol/L (P < 0.05).Both 50 and 200 μmol/L LS decreased HCV RNA levels[core region (by 55% and 88%, respectively) and NS5A region (by 62% and 87%, respectively) after 24 h compared with vehicle (dimethyl sulphoxide) control (P< 0.01). Similarly HCV core and NS5A protein were decreased(by 85%, P < 0.01 and by 65%, P < 0.05, respectively)by LS 200 μmol/L. Bach1 and HMOX-1 RNAwere also downregulated by LS treatment (P < 0.01),while Nrf2 protein was increased (P < 0.05).CONCLUSION: Our results demonstrate that treatment with LS downregulates HCV core and NS5A expression in CON1 cells which express full length HCVgenotype 1b, and suggests that LS may prove to be a valuable alternative or adjunctive therapy for the treatment of HCV infection.

  8. Streptococcal serum opacity factor increases the rate of hepatocyte uptake of human plasma high-density lipoprotein cholesterol.

    Science.gov (United States)

    Gillard, Baiba K; Rosales, Corina; Pillai, Biju K; Lin, Hu Yu; Courtney, Harry S; Pownall, Henry J

    2010-11-16

    Serum opacity factor (SOF), a virulence determinant of Streptococcus pyogenes, converts plasma high-density lipoproteins (HDL) to three distinct species: lipid-free apolipoprotein (apo) A-I, neo HDL, a small discoidal HDL-like particle, and a large cholesteryl ester-rich microemulsion (CERM) that contains the cholesterol esters (CE) of up to ∼400000 HDL particles and apo E as its major protein. Similar SOF reaction products are obtained with HDL, total plasma lipoproteins, and whole plasma. We hypothesized that hepatic uptake of CERM-CE via multiple apo E-dependent receptors would be faster than that of HDL-CE. We tested our hypothesis using human hepatoma cells and lipoprotein receptor-specific Chinese hamster ovary (CHO) cells. The uptake of [(3)H]CE by HepG2 and Huh7 cells from HDL after SOF treatment, which transfers >90% of HDL-CE to CERM, was 2.4 and 4.5 times faster, respectively, than from control HDL. CERM-[(3)H]CE uptake was inhibited by LDL and HDL, suggestive of uptake by both the LDL receptor (LDL-R) and scavenger receptor class B type I (SR-BI). Studies in CHO cells specifically expressing LDL-R and SR-BI confirmed CERM-[(3)H]CE uptake by both receptors. RAP and heparin inhibit CERM-[(3)H]CE but not HDL-[(3)H]CE uptake, thereby implicating LRP-1 and cell surface proteoglycans in this process. These data demonstrate that SOF treatment of HDL increases the rate of CE uptake via multiple hepatic apo E receptors. In so doing, SOF might increase the level of hepatic disposal of plasma cholesterol in a way that is therapeutically useful.

  9. Study on Expression Conditions of EGCG-O-Methyltransferase in Recombinant Escherichia coli Bacteria%EGCG-O-甲基转移酶(EOMT)在重组大肠杆菌中的表达条件研究

    Institute of Scientific and Technical Information of China (English)

    费冬梅; 林智; 吕海鹏; 张悦; 谭俊峰; 郭丽

    2011-01-01

    EGCG3"Me could be produced from EGCG catalyzed by EGCG-O-Methyltransferase. Taken the yield of EGCG3"Me as main index, the present study focused on the producing conditions of EGCG-O-Methyltransferase induced by IPTG in recombinant E. Coli bacteria. Results showed that the optimum producing conditions were as follows: the concentration of IPTG was 0.05 mmol/L, the induction time was 20 h, the initial pH of medium was 7.0 and the induction temperature was 20℃.%本研究以EGCG-O-甲基转移酶(EOMT)催化EGCG生成EGCG3”Me的产量为主要指标,探讨了重组大肠杆菌内EGCG-O-甲基转移酶的诱导表达条件.结果表明,当诱导剂IPTG终浓度为0.05 mmol/L,诱导时间为20h,培养基初始pH为7.0,以及诱导温度为20℃时,EOMT的表达效果最佳.

  10. Potential Role of (-)-epigallocatechin-3-gallate (EGCG) in the Secondary Prevention of Alzheimer Disease.

    Science.gov (United States)

    Xicota, Laura; Rodríguez-Morató, Jose; Dierssen, Mara; de la Torre, Rafael

    2015-08-25

    Medical advances in the last decades have increased the average life expectancy, but also the incidence and prevalence of age-associated neurodegenerative diseases. Alzheimer disease (AD) is one of the most common neurodegenerative diseases and the most prevalent type of dementia. A plethora of different mechanisms contribute to AD, among which oxidative stress plays a key role in its development and progression. So far, there are no pharmacological treatments available and the current medications are mainly symptomatic. In the last years, dietary polyphenols have gained research attention due to their interesting biological activities, and more specifically their antioxidant properties. (-)-epigallocatechin-3-gallate (EGCG) is a natural flavanol that has been extensively studied regarding its potential effects in AD. In this review we present the current in vitro and in vivo experimentation regarding the use of EGCG in AD. We also review the complex mechanisms of action of EGCG, not only limited to its antioxidant activity, which may explain its beneficial health effects.

  11. EGCG ameliorates diet-induced metabolic syndrome associating with the circadian clock.

    Science.gov (United States)

    Mi, Yashi; Qi, Guoyuan; Fan, Rong; Ji, Xiaohua; Liu, Zhigang; Liu, Xuebo

    2017-06-01

    In response to the daily light-dark (LD) cycle, organisms on Earth have evolved with the approximately 24-h endogenous oscillations to coordinate behavioral and physiological processes, including feeding, sleep, and metabolism homeostasis. Circadian desynchrony triggered by an energy-dense diet rich in fats and fructose is intimately connected with a series of metabolic disorders. Previous studies revealed that (-)-Epigallocatechin-3-gallate (EGCG) could mitigate metabolic misalignment; however, only a few reports have focused on its potential effect on directly manipulating circadian rhythms to ameliorate metabolic syndrome. Our goal was to investigate the regulating effect of EGCG treatment on metabolic misalignment triggered by a high-fat and high-fructose diet (HFFD) associating with the circadian clock. Our results indicated that HFFD treatment partially exhibited poor circadian oscillations of the core clock gene and the clock-controlled gene in the liver and fat relative to the control group. EGCG administration may ameliorate the diet-dependent decline in circadian function by controlling the Sirt1-PGC1αloop, implying the existence of an EGCG-entrainable oscillator. Subsequently, reducing fatty acid synthesis and elevating β-oxidation in the liver coupled with the increasing brown adipose tissue (BAT) energy expenditure observed in the EGCG group of mice prevented the adipocyte hypertrophy and fat accumulations common to BAT and white adipose tissue (WAT) derived from the HFFD mice. This study is the first to provide compelling evidences that EGCG may ameliorate diet-induced metabolic misalignment by regulating the rhythmic expression of the circadian clock genes in the liver and fat. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. A 3-day EGCG-supplementation reduces interstitial lactate concentration in skeletal muscle of overweight subjects

    OpenAIRE

    Jasper Most; van Can, Judith G P; Jan-Willem van Dijk; Goossens, Gijs H.; Johan Jocken; Hospers, Jeannette J.; Igor Bendik; Ellen E. Blaak

    2015-01-01

    Green tea, particularly epigallocatechin-3-gallate (EGCG), may affect body weight and composition, possibly by enhancing fat oxidation. The aim of this double-blind, randomized placebo-controlled cross-over study was to investigate whether 3-day supplementation with EGCG (282mg/day) stimulates fat oxidation and lipolysis in 24 overweight subjects (age = 30 ± 2yrs, BMI = 27.7 ± 0.3 kg/m2). Energy expenditure, substrate metabolism and circulating metabolites were determined during fasting and p...

  13. Separation and purification of epigallocatechin-3-gallate (EGCG) from green tea using combined macroporous resin and polyamide column chromatography.

    Science.gov (United States)

    Jin, Xin; Liu, Mingyan; Chen, Zaixing; Mao, Ruikun; Xiao, Qinghuan; Gao, Hua; Wei, Minjie

    2015-10-01

    Epigallocatechin-3-gallate (EGCG) is a major bioactive ingredient of green tea that produces beneficial neuroprotective effects. In this paper, to optimize the EGCG enrichment, thirteen macroporous resins with different chemical and physical properties were systemically evaluated. Among the thirteen tested resins, the H-bond resin HPD826 exhibited best adsorption/desorption capabilities and desorption ratio, as well as weakest affinity for caffeine. The absorption of EGCG on the HPD826 resin followed the pseudo-second-order kinetics and Langmuir isotherm model. The separation parameters of EGCG were optimized by dynamic adsorption/desorption experiments with the HPD826 resin column. Under the optimal condition, the content of EGCG in the 30% ethanol eluent increased by 5.8-fold from 7.7% to 44.6%, with the recovery yield of 72.1%. After further purification on a polyamide column, EGCG with 74.8% purity was obtained in the 40-50% ethanol fraction with a recovery rate of 88.4%. In addition, EGCG with 95.1% purity could be easily obtained after one-step crystallization in distilled water. Our study suggests that the combined macroporous resin and polyamide column chromatography is a simple method for large-scale separation and purification of EGCG from natural plants for food and pharmaceutical applications.

  14. A Fast Assembly of (-)-Epigallocatechin-3-gallate [(-)-EGCG] via Intra-and Inter-molecular Mitsunobu Reaction

    Institute of Scientific and Technical Information of China (English)

    DING Tong-Jian; WANG Xiao-Long; CAO Xiao-Ping

    2006-01-01

    This paper described a concise construction of (-)-EGCG (1f) (with an overall yield of 20% for seven steps based on the starting cinnamyl alcohol derivative 3), featuring asymmetric dihydroxylation (ADH), intra- and intermolecular Mitsunobu reaction as key steps. Our strategy disclosed herein constitutes a new effective general synthetic approach toward the analogues of (-)-EGCG (1f).

  15. Curcumin and EGCG Suppress Apurinic/Apyrimidinic Endonuclease 1 and Induce Complete Remission in B-cell Non-Hodgkin's lymphoma Patients

    Directory of Open Access Journals (Sweden)

    Hashem M. Neenaa

    2011-12-01

    Full Text Available ABSTRACT:Background: Follicular lymphoma (FL is the most common subtype of indolent lymphoma. FL is still considered to be an incurable disease and palliation of symptoms is an acceptable approach to the expected pattern of repeated relapses due to developing resistance to chemotherapy agents. Apurinic/apyrimidinic endonuclease/redox factor-1 (APE1/Ref-1 is a multifunctional protein involved in DNA base excision repair (BER of oxidative DNA damage and in redox regulation of a number of transcription factors. It was observed that cytoplasmic APE1 induced COX-2 expression through NF-êB activation. It has been shown that chemopreventive agents potentiate the efficacy of chemotherapy through the regulation of multiple signaling pathways, including NF-êB, c-Myc, cyclooxygenase-2, apoptosis, and others, suggesting a multitargeted nature of chemopreventive agents. We hypothesized that curcumin, a polyphenolic antioxidant derived from the spice turmeric, and epigallocatechin gallate (EGCG from green tea would potentiate the effect of chemotherapy in B-cell lymphoma.Objective: We examined the role of human apurinic/apyrimidinic endonuclease 1 (APE1 in resistance and prognosis in patients with FL. Our major objective was to update the safety and efficacy results of the antitumor effect of combination of curcumin and EGCG therapy in relapsed or resistant indolent or transformed non-Hodgkin follicular lymphoma patients and their peripheral blood mononuclear cells (PBMCs compared with healthy donors’ controls.Methods: Thirty patients with FL with over-expression of constitutive active NF-êB in their PBMCs received regular CHOP and consumed capsules compatible with curcumin doses between 0.9 and 5.4 g daily for up to 9 months and 9.0 g/day green tea whole extract "1000 mg tablets of green tea whole extract containing 200 mg EGCG. We designed a dose-escalation Functional Foods in Health and Disease 2011, 1(12:525-544 study to explore the efficacy of CHOP

  16. Green tea EGCG suppresses T cell proliferation by impairing IL-2/IL-2R signaling leading to inhibition of cell cycle

    Science.gov (United States)

    Epigallocatechin-3-gallate (EGCG), an active ingredient of green tea, has been indicated to have various health benefits. However, little is known about the effect of EGCG on immune function. In this study, we investigated the effect of in vitro EGCG supplementation at physiologically relevant level...

  17. Green tea EGCG suppresses T cell proliferation through impairment of IL-2/IL-2 receptor signaling

    Science.gov (United States)

    Studies have suggested a benefit of consuming green tea in promoting general health and reducing the risk of certain diseases. However, little is known about the effect of green tea on immune function. In this study we determined the effect of epigallocatechin-3-gallate (EGCG), the major active comp...

  18. [Study on inhibitory effect of EGCG on Calcium oxalate nephrolithiasis in rats and its related mechanism].

    Science.gov (United States)

    Zhou, Yong; Wang, Shuo; Tang, Chun-bo

    2015-04-01

    In the study, the inhibitory effect of epigallocatechin gallate (EGCG) on Calcium oxalate nephrolithiasis and its possible mechanism were investigated. The rat Calcium oxalate nephrolithiasis model was induced through the combined oral administration of ethylene glycol and ammonium chloride, which was intervened with EGCG. Rat blood samples were collected to detect blood creatinine (Cr), blood urea nitrogen (BUN) and blood calcium. Rat urine samples were collected to observe and compare 24-hour urine volume, oxalic acid (Ox) and calcium in urine. Renal samples were collected to prepare tissue slices and observe the pathological changes in Calcium oxalate nephrolithiasis. The expression of osteopontin (OPN) in renal tissues was evaluated by Real-time PCR and Western blot. According to the results, compared with normal rats, rats in the nephrolithiasis model showed significant increases in Cr, BUN, urine Calcium, urine Ox and renal OPN expression (P nephrolithiasis, those processed with EGCG revealed remarkable declines in Cr, BUN, urine Calcium and urine Ox (P nephrolithiasis rats showed significant pathological changes in Calcium oxalate calculus. After ECCG treatment, the renal pathological changes and OPN expression attenuated significantly in a concentration-dependent manner. The results showed that EGCG inhibits the formation of calcium oxalate nephrolithiasis in rats and shows a notable protective effect on renal functions.

  19. (-) Epigallocatechin Gallate (EGCG) Prevents Lipid Changes and Collagen Abnormalities in Chronic Ethanol-Fed Rats.

    Science.gov (United States)

    Kaviarasan, S; Viswanathan, P; Ravichandran, M K; Anuradha, C V

    2008-01-01

    ABSTRACT The objective of the study is to examine the influence of (-) epigallocatechin gallate (EGCG), a green tea component, on lipid and collagen abnormalities in chronic ethanol-fed rats. Solubility properties, aldehyde content, fluorescence, and peroxidation were analyzed in collagen samples isolated from liver. Chronic alcoholism (6 g/kg/day x 60 days) was associated with fatty liver and collagen accumulation. Significant alterations in the levels of lipids (cholesterol, phospholipids, free fatty acids, and triglycerides) and total collagen were observed in liver. Collagen obtained from ethanol-fed rats showed alterations in solubility properties, increased fluorescence, peroxidation, and aldehyde content. Coadministration of EGCG along with ethanol significantly reduced the levels of liver lipids and collagen, improved the solubility properties of collagen, and caused a reduction in cross-linking as evidenced by a decrease in fluorescence, peroxidation, and aldehyde content. Histology of liver sections of ethanol-fed rats showed accumulation of fat and collagen, which were largely prevented by EGCG administration. The possible mechanisms in the protective action of EGCG in alcoholic liver disease are suggested and discussed.

  20. A toxicogenomics-based parallelogram approach to evaluate the relevance of coumarin-induced responses in primary human hepatocytes in vitro for humans in vivo

    NARCIS (Netherlands)

    Kienhuis, A.S.; Poll, M.C.G. van de; Jong, C.H.C. de; Gottschalk, R.; Herwijnen, M. van; Boorsma, A.; Kleinjans, J.C.S.; Stierum, R.H.; Delft, J.H.M. van

    2009-01-01

    A compound for which marked species differences have been reported in laboratory animals and humans is coumarin. In rats, metabolites of coumarin are highly toxic, whereas in humans, the compound is mainly metabolized to non-toxic metabolites. In the present study, a toxicogenomics-based parallelogr

  1. Culture of cryopreserved rat hepatocyte

    Institute of Scientific and Technical Information of China (English)

    Haitao Yin; Gaojun Teng; Lifeng Wang; Baorui Liu; Xiaoping Qian

    2006-01-01

    Objective: To study the method of cryopreserving rat hepatocytes and double collagen gel culture measurement after its cryopreservation. Methods: Rat hepatocytes, isolated by two-step perfusion with collagenase using an extra corporeal perfusion apparatus, were cryopreserved in double collagen gel with culture medium added by epidermal growth factor(EGF).The expression of cell function and cellular morphology were examined during culture. Results: The hepatocytes cryopreserved in double collagen gel concluding EGF showed good morphology and biological characteristics. After thawing, the MTT metabolism and protein synthesis of hepatocytes in sandwich ± EGF groups were better than those in control group. And the morphology and function of hepatocytes in sandwich group was better than that in EGF group(P < 0.05). Conclusion: Double collagen gel culture can keep hepatocyte's activities. Thawed hepatocytes can be cultivated with collagenous matrix, which provides an environment that more closely resembles that in vivo and maintain the expression of certain liver-specific function of hepatocytes.

  2. Evaluation of multiple mechanism-based toxicity endpoints in primary cultured human hepatocytes for the identification of drugs with clinical hepatotoxicity: Results from 152 marketed drugs with known liver injury profiles.

    Science.gov (United States)

    Zhang, Jie; Doshi, Utkarsh; Suzuki, Ayako; Chang, Ching-Wei; Borlak, Jürgen; Li, Albert P; Tong, Weida

    2016-08-05

    We report here the results of a collaborative research program to develop a robust and reliable in vitro system to allow an accurate definition of the drug-induced liver injury (DILI) potential of new drug entities during drug development. The in vitro hepatotoxic potential of 152 drugs with known DILI profiles were evaluated in primary cultured human hepatocytes with four mechanistically-relevant endpoints: cellular ATP depletion, reactive oxygen species (ROS), glutathione (GSH) depletion, and caspase activation for apoptosis. The drugs, 80 in the testing set and 72 in the validation set, were classified based on serious clinical/regulatory outcomes as defined by reported acute liver failure, black-box warning, and/or withdrawal. The drugs were further sub-categorized for dominant types of liver injury. Logistic regression models were performed to calculate the area under the receiver operating characteristics curve (AUROC) and to evaluate the prediction potential of the selected endpoints for serious clinical/regulatory outcomes. The ROS/ATP ratio was found to yield an excellent AUROC in both the testing (0.8989, P drugs associated with severe from non-severe DILI cases (p drugs in primary human hepatocytes using the ROS/ATP ratio endpoint may aid the definition of their potential to cause severe DILI.

  3. Evaluation of plasma antioxidant activity in rats given excess EGCg with reference to endogenous antioxidants concentrations and assay methods.

    Science.gov (United States)

    Yokotani, Kaori; Umegaki, Keizo

    2017-02-01

    The contribution of (-)-epigallocatechin gallate (EGCg) intake to in vivo antioxidant activity is unclear, even with respect to plasma. In this study, we examined how administration of EGCg contributes to plasma antioxidant activity, relative to its concentration, endogenous antioxidants, and assay methods, namely oxygen radical absorbance capacity (ORAC) and ferric reducing/antioxidant power (FRAP). Administration of EGCg (500 mg/kg) to rats increased plasma EGCg (4μmol/L as free form) and ascorbic acid (1.7-fold), as well as ORAC (1.2-fold) and FRAP (3-fold) values. The increase in plasma ascorbic acid following EGCg administration was accompanied by its relocation from the adrenal glands and lymphocytes into plasma, and was related to the increase in FRAP. Plasma deproteinization and assays in plasma model solutions revealed that protein levels significantly contributed to ORAC values, where plasma ascorbic acid was not influenced by deproteinization, differences in FRAP values with and without deproteinization were estimated to determine the contribution of enhanced ascorbic acid attributable to EGCg administration. These results will help to understand the points that should be considered when evaluating EGCg antioxidant activity in plasma.

  4. EGCG debilitates the persistence of EBV latency by reducing the DNA binding potency of nuclear antigen 1

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Ya-Lin; Tsai, Hsing-Lyn [Department of Life Sciences, Tzu-Chi University, 701 Chung-Yang Rd. Sec 3, Hualien 97004, Taiwan (China); Peng, Chih-Wen, E-mail: pengcw@mail.tcu.edu.tw [Department of Life Sciences, Tzu-Chi University, 701 Chung-Yang Rd. Sec 3, Hualien 97004, Taiwan (China)

    2012-01-20

    Highlights: Black-Right-Pointing-Pointer Two cell-based reporter platforms were established for screening of EBNA1 inhibitors. Black-Right-Pointing-Pointer EGCG acts as an inhibitor to block EBNA1 binding with the cognate oriP sequence. Black-Right-Pointing-Pointer EGCG debilitates EBNA1-dependent transcription enhancement and episome maintenance. Black-Right-Pointing-Pointer EGCG impairs persistence of EBV latency. Black-Right-Pointing-Pointer EGCG is a potent anti-EBV agent for targeting the latent cascade of EBV. -- Abstract: Because the expression of EBNA1 is prevalent in all EBV-associated tumors, it has become one of the most attractive drug targets for the discovery of anti-EBV compounds. In a cell-based reporter system, EBNA1 consistently upregulated the transcription of an oriP-Luc mini-EBV episome by 6- to 8-fold. The treatment of cells with 50 {mu}M EGCG effectively blocked the binding of EBNA1 to oriP-DNA both in vivo and in vitro, which led to the abrogation of EBNA1-dependent episome maintenance and transcriptional enhancement. Importantly, the anti-EBNA1 effects caused by EGCG ultimately impaired the persistence of EBV latent infection. Our data suggest that the inhibition of EBNA1 activity by EGCG could be a promising starting point for the development of new protocols for anti-EBV therapy.

  5. Inhibition of Proliferation and Induction of Apoptosis In Human Gastric Cancer SGC-7901 Cell Line by Epigallo Catechingallate(EGCG)%表没食子儿茶素没食子酸酯抑制人胃癌细胞增殖和诱导凋亡作用的研究

    Institute of Scientific and Technical Information of China (English)

    黄磊

    2010-01-01

    目的:研究表没食子儿茶素没食子酸酯(EGCG)对体外培养人胃癌细胞系SGC-7901增殖抑制作用,并初步探讨其诱导SGC-7901细胞凋亡的分子机制.方法:采用平皿克隆形成实验法测定不同剂量EGCG对SGC-7901细胞锚定依赖性生长能力的影响;Wester Blot法检测不同剂量EGCG处理人胃癌细胞(SGC-7901)48 h前后NF-κB(p65)、Bcl-2、Bax和Caspase-3蛋白表达变化情况.结果:平皿克隆形成实验法显示:EGCG可抑制人胃癌细胞SGC-7901生长,且呈剂量依赖性,其对SGC-7901细胞IC50值为63.5 μg/mL;Wester Blot显示随着药物浓度升高,EGCG可以逐渐下调NF-κB和Bcl-2的蛋白表达,上调Bax、Caspase-3蛋白表达.结论:EGCG具有诱导人胃癌SGC-7901细胞凋亡的作用,其机制可能与抑制核转录因子NF-B活化,导致Bax/Bcl-2比值的上调,促进Caspase-3的活化有关.

  6. Modeling Nonalcoholic Fatty Liver Disease with Human Pluripotent Stem Cell-Derived Immature Hepatocyte-Like Cells Reveals Activation of PLIN2 and Confirms Regulatory Functions of Peroxisome Proliferator-Activated Receptor Alpha

    Science.gov (United States)

    Graffmann, Nina; Ring, Sarah; Kawala, Marie-Ann; Wruck, Wasco; Ncube, Audrey; Trompeter, Hans-Ingo

    2016-01-01

    Nonalcoholic fatty liver disease (NAFLD/steatosis) is a metabolic disease characterized by the incorporation of fat into hepatocytes. In this study, we developed an in vitro model for NAFLD based on hepatocyte-like cells (HLCs) differentiated from human pluripotent stem cells. We induced fat storage in these HLCs and detected major expression changes of metabolism-associated genes, as well as an overall reduction of liver-related microRNAs. We observed an upregulation of the lipid droplet coating protein Perilipin 2 (PLIN2), as well as of numerous genes of the peroxisome proliferator-activated receptor (PPAR) pathway, which constitutes a regulatory hub for metabolic processes. Interference with PLIN2 and PPARα resulted in major alterations in gene expression, especially affecting lipid, glucose, and purine metabolism. Our model recapitulates many metabolic changes that are characteristic for NAFLD. It permits the dissection of disease-promoting molecular pathways and allows us to investigate the influences of distinct genetic backgrounds on disease progression. PMID:27308945

  7. General review on in vitro hepatocyte models and their applications.

    Science.gov (United States)

    Guguen-Guillouzo, Christiane; Guillouzo, Andre

    2010-01-01

    In vitro hepatocyte models represent very useful systems in both fundamental research and various application areas. Primary hepatocytes appear as the closest model for the liver in vivo. However, they are phenotypically unstable, have a limited life span and in addition, exhibit large interdonor variability when of human origin. Hepatoma cell lines appear as an alternative but only the HepaRG cell line exhibits various functions, including major cytochrome P450 activities, at levels close to those found in primary hepatocytes. In vitro hepatocyte models have brought a substantial contribution to the understanding of the biochemistry, physiology, and cell biology of the normal and diseased liver and in various application domains such as xenobiotic metabolism and toxicity, virology, parasitology, and more generally cell therapies. In the future, new well-differentiated hepatocyte cell lines derived from tumors or from either embryonic or adult stem cells might be expected and although hepatocytes will continue to be used in various fields, these in vitro liver models should allow marked advances, especially in cell-based therapies and predictive and mechanistic hepatotoxicity of new drugs and other chemicals. All models will benefit from new developments in throughput screening based on cell chips coupled with high-content imaging and in toxicogenomics technologies.

  8. Hepatocyte xenotransplantation for treating liver disease.

    Science.gov (United States)

    Bonavita, André Gustavo; Quaresma, Kátia; Cotta-de-Almeida, Vinícius; Pinto, Marcelo Alves; Saraiva, Roberto Magalhães; Alves, Luiz Anastácio

    2010-01-01

    The treatment of acute and chronic liver failure is still a challenge despite modern therapeutic innovations. While liver transplantation can restore liver function and improve patient survival, donor shortages limit this treatment to a small number of patients. Cellular xenotransplantation has emerged as an alternative for treating liver failure. Xenohepatocytes could be readily available in sufficient quantities to treat patients in critical condition and thereby reduce the donor shortage. The use of isolated encapsulated or non-encapsulated cells can reduce the immunorejection response. Several studies using animal models of acute or chronic liver failure have demonstrated improved survival and recovery of liver function after xenotransplantation of adult hepatocytes. Porcine liver cells are a potential source of xenohepatocytes due to similarities with human physiology and the great number of hepatocytes that can be obtained. The recent development of less immunogenic transgenic pigs, new immunosuppressive drugs, and cellular encapsulation systems represents important advances in the field of cellular xenotransplantation. In this study, we review the work carried out in animal models that deals with the advantages and limitations of hepatocyte xenotransplantation, and we propose new studies needed in this field.

  9. Effect of EGCG on treatment and prevention of cancer%EGCG在肿瘤防治中的作用

    Institute of Scientific and Technical Information of China (English)

    李晶晶; 胡成平

    2013-01-01

    表没食子儿茶素没食子酸酯(EGCG)是绿茶的主要组成成分,国内外多年的研究表明其对多种肿瘤均有防治作用.本文将从EGCG在肿瘤的发生发展、肿瘤的转移浸润、肿瘤的血管生成及肿瘤的耐药等方面来阐述EGCG的肿瘤防治作用.%(-)-Epigallocatechin gallate (EGCG),is the major component of green tea.According to many investigations from home and abroad,EGCG has the effect on treatment and prevention of a variety of cancers.This article concerns the following aspects of anticancer effect of EGCG: tumor development,metastasis,invasion,angiogenesis,drug resistance,and others.

  10. Microvesicles derived from human Wharton's jelly mesenchymal stem cells promote human renal cancer cell growth and aggressiveness through induction of hepatocyte growth factor.

    Directory of Open Access Journals (Sweden)

    Tao Du

    Full Text Available In our previous study, microvesicles (MVs released from human Wharton's jelly mesenchymal stem cells (hWJ-MSCs retard the growth of bladder cancer cells. We would like to know if MVs have a similar effect on human renal cell carcinoma (RCC. By use of cell culture and the BALB/c nu/nu mice xeno-graft model, the influence of MVs upon the growth and aggressiveness of RCC (786-0 was assessed. Cell counting kit-8 (CCK-8 assay, incidence of tumor, tumor size, Ki-67 or TUNEL staining was used to evaluate tumor cell growth in vitro or in vivo. Flow cytometry assay (in vitro or examination of cyclin D1 expression (in vivo was carried out to determine the alteration of cell cycle. The aggressiveness was analyzed by Wound Healing Assay (in vitro or MMP-2 and MMP-9 expression (in vivo. AKT/p-AKT, ERK1/2/p-ERK1/2 or HGF/c-MET expression was detected by real-time PCR or western blot. Our data demonstrated that MVs promote the growth and aggressiveness of RCC both in vitro and in vivo. In addition, MVs facilitated the progression of cell cycle from G0/1 to S. HGF expression in RCC was greatly induced by MVs, associated with activation of AKT and ERK1/2 signaling pathways. RNase pre-treatment abrogated all effects of MVs. In summary, induction of HGF synthesis via RNA transferred by MVs activating AKT and ERK1/2 signaling is one of crucial contributors to the pro-tumor effect.

  11. The effects of EGCG on fat oxidation and endurance performance in male cyclists.

    Science.gov (United States)

    Dean, Sara; Braakhuis, Andrea; Paton, Carl

    2009-12-01

    Researchers have long been investigating strategies that can increase athletes' ability to oxidize fatty acids and spare carbohydrate, thus potentially improving endurance capacity. Green-tea extract (epigallocatechin-3-gallate; EGCG) has been shown to improve endurance capacity in mice. If a green-tea extract can stimulate fat oxidation and as a result spare glycogen stores, then athletes may benefit through improved endurance performance. Eight male cyclists completed a study incorporating a 3-way crossover, randomized, placebo-controlled, double-blinded, diet-controlled research design. All participants received 3 different treatments (placebo 270 mg, EGCG 270 mg, and placebo 270 mg + caffeine 3 mg/kg) over a 6-day period and 1 hr before exercise testing. Each participant completed 3 exercise trials consisting of 60 min of cycling at 60% maximum oxygen uptake (VO2(max)) immediately followed by a self-paced 40-km cycling time trial. The study found little benefit in consuming green-tea extract on fat oxidation or cycling performance, unlike caffeine, which did benefit cycling performance. The physiological responses observed during submaximal cycling after caffeine ingestion were similar to those reported previously, including an increase in heart rate (EGCG 147 +/- 17, caffeine 146 +/- 19, and placebo 144 +/- 15 beats/min), glucose at the 40-min exercise time point (placebo 5.0 +/- 0.8, EGCG 5.4 +/- 1.0, and caffeine 5.8 +/- 1.0 mmol/L), and resting plasma free fatty acids and no change in the amount of carbohydrate and fat being oxidized. Therefore, it was concluded that green-tea extract offers no additional benefit to cyclists over and above those achieved by using caffeine.

  12. Current status of hepatocyte xenotransplantation.

    Science.gov (United States)

    Meier, Raphael P H; Navarro-Alvarez, Nalu; Morel, Philippe; Schuurman, Henk-Jan; Strom, Stephen; Bühler, Leo H

    2015-11-01

    The treatment of acute liver failure, a condition with high mortality, comprises optimal clinical care, and in severe cases liver transplantation. However, there are limitations in availability of organ donors. Hepatocyte transplantation is a promising alternative that could fill the medical need, in particular as the bridge to liver transplantation. Encapsulated porcine hepatocytes represent an unlimited source that could function as a bioreactor requiring minimal immunosuppression. Besides patients with acute liver failure, patients with alcoholic hepatitis who are unresponsive to a short course of corticosteroids are a target for hepatocyte transplantation. In this review we present an overview of the innate immune barriers in hepatocyte xenotransplantation, including the role of complement and natural antibodies; the role of phagocytic cells and ligands like CD47 in the regulation of phagocytic cells; and the role of Natural Killer cells. We present also some illustrations of physiological species incompatibilities in hepatocyte xenotransplantation, such as incompatibilities in the coagulation system. An overview of the methodology for cell microencapsulation is presented, followed by proof-of-concept studies in rodent and nonhuman primate models of fulminant liver failure: these studies document the efficacy of microencapsulated porcine hepatocytes which warrants progress towards clinical application. Lastly, we present an outline of a provisional clinical trial, that upon completion of preclinical work could start within the upcoming 2-3 years.

  13. (-)-Epigallocatechin-3-gallate (EGCG) modulates neurological function when intravenously infused in acute and, chronically injured spinal cord of adult rats.

    Science.gov (United States)

    Renno, Waleed M; Al-Khaledi, Ghanim; Mousa, Alyaa; Karam, Shaima M; Abul, Habib; Asfar, Sami

    2014-02-01

    Spinal cord injury (SCI) causes severe and long lasting motor and sensory deficits, chronic pain, and autonomic dysreflexia. (-)-epigallocatechin-3-gallate (EGCG) has shown to produce neuroprotective effect in a broad range of neurodegenerative disease animal models. This study designed to test the efficacy of intravenous infusion of EGCG for 36 h, in acutely injured rats' spinal cord: within first 4 h post-injury and, in chronically SC injured rats: after one year of injury. Functional outcomes measured using standard BBB scale, The Louisville Swim Scale (LSS) and, pain behavior assessment tests. 72 Female adult rats subjected to moderate thoracic SCI using MASCIS Impactor, blindly randomized as the following: (I) Acute SCI + EGCG (II) Acute SCI + saline. (III) Chronic SCI + EGCG. (IV) Chronic SCI + saline and, sham SCI animals. EGCG i.v. treatment of acute and, chronic SCI animals resulted in significantly better recovery of motor and sensory functions, BBB and LSS (P spinal cord increased (P < 0.001). Percent areas of GAP-43 and GFAP immunohistochemistry showed significant (P < 0.05) increase. We conclude that the therapeutic window of opportunity for EGCG to depict neurological recovery in SCI animals, is viable up to one year post SCI when intravenously infused for 36 h.

  14. Green tea polyphenol EGCG reverse cisplatin resistance of A549/DDP cell line through candidate genes demethylation.

    Science.gov (United States)

    Zhang, Youwei; Wang, Xiang; Han, Liang; Zhou, Yizhou; Sun, Sanyuan

    2015-02-01

    Epigallocatechin-3-gallate (EGCG), the major polyphenol in green tea, has been extensively studied as a potential demethylating agent. Our hypothesis is that EGCG could resensitize non-small-cell lung cancer (NSCLC) cells to cisplatin (DDP) through candidate genes demethylation. The A549/DDP cell line was established by continuous exposure of A549 cells to increasing concentrations of DDP. MTT, colony formation assay, flow cytometric analysis, Hoechst staining, real time-PCR, quantitative methylation-specific PCR and in vivo experiments were performed in this study. EGCG+DDP treatment significantly caused proliferation inhibition, cell cycle arrest in G1 phase, increase of apoptosis in A549/DDP cells, along with inhibition of DNA methyltransferase (DNMT) activity and histone deacetylase (HDAC) activity, reversal of hypermethylated status and downregulated expression of GAS1, TIMP4, ICAM1 and WISP2 gene in A549/DDP cells. Furthermore, pre-treatment with EGCG followed by DDP caused significant tumor inhibition in vivo. Methylation levels of GAS1, TIMP4, ICAM1 and WISP2 were decreased and their expression levels were increased in EGCG-treatment groups, but only combinatorial treatment group caused growth inhibition. In conclusion, we identified EGCG pretreatment resensitized cells to DDP, along with the demethylation and restoration of expression of candidate genes.

  15. The green tea catechin epigallocatechin gallate (EGCG blocks cell motility, chemotaxis and development in Dictyostelium discoideum.

    Directory of Open Access Journals (Sweden)

    Kyle J McQuade

    Full Text Available Catechins, flavanols found at high levels in green tea, have received significant attention due to their potential health benefits related to cancer, autoimmunity and metabolic disease, but little is known about the mechanisms by which these compounds affect cellular behavior. Here, we assess whether the model organism Dictyostelium discoideum is a useful tool with which to characterize the effects of catechins. Epigallocatechin gallate (EGCG, the most abundant and potent catechin in green tea, has significant effects on the Dictyostelium life cycle. In the presence of EGCG aggregation is delayed, cells do not stream and development is typically stalled at the loose aggregate stage. The developmental effects very likely result from defects in motility, as EGCG reduces both random movement and chemotaxis of Dictyostelium amoebae. These results suggest that catechins and their derivatives may be useful tools with which to better understand cell motility and development in Dictyostelium and that this organism is a useful model to further characterize the activities of catechins.

  16. Poly (Ethylene Glycol-Block-Brush Poly (L-Lysine Copolymer as an Efficient Nanocarrier for Human Hepatocyte Growth Factor with Enhanced Bioavailability and Anti-Ischemia Reperfusion Injury Efficacy

    Directory of Open Access Journals (Sweden)

    Fei Tong

    2017-08-01

    Full Text Available Background/Aims: The aim of this study was to assess the effect of human hepatocyte growth factor (hHGF-loaded poly (ethylene glycol-b-brush poly (l-lysine (PEG-b-P(ELG-g-PLL copolymer on ischemia/reperfusion (I/R injury to different organs. Methods: The isoelectric point (pI of hHGF is 5.5, and hHGF combined with PEG-b-P(ELG-g-PLL copolymer via electrostatic interactions at pH 7.4. The synthesized PEG-b-P(ELG-g-PLL copolymer was analyzed using 1H nuclear magnetic resonance (1H NMR and gel permeation chromatography (GPC. The hHGF/PEG-b-P(ELG-g-PLL complex was evaluated using a nanoparticle size instrument and transmission electron microscopy (TEM. In addition, vivo performance of hHGF/PEG-b-P(ELG-g-PLL complex was evaluated using plasma hHGF concentration and different organs ischemia reperfusion injury in rats. Results: An in vitro investigation showed that PEG-b-P(ELG-g-PLL could serve as a potential hHGF nanocarrier with efficient encapsulation and sustained release. An additional in vivo investigation revealed that the hHGF/PEG-b-P(ELG-g-PLL complex could prolong increases in plasma hHGF concentration and protect different organs (the brain, heart and kidney against I/R injury. Conclusion: Poly (ethylene glycol-block-brush poly (l-lysine copolymer as an efficient nanocarrier for human hepatocyte growth factor with enhanced bioavailability and anti-ischemia reperfusion injury efficacy.

  17. 人肝细胞/微孔聚丙烯杂化界面的生物相容性%Biocompatibility of human hepatocyte/microporous polypropylene hybridization

    Institute of Scientific and Technical Information of China (English)

    张瑞; 彭承宏; 韩宝三; 王兆海; 吴旭波; 吴薇; 高长有

    2008-01-01

    背景:生物反应器膜材料不仅具备双向物质交换、良好的理化特性,还要具有良好的生物相容性.目的:评价人肝细胞/微孔聚丙烯杂化界面,即接技改性微孔聚丙烯超滤膜的生物相容性.设计、时间及地点:动物实验观察,于2005-09/2007-10在上海交通大学医学院附属瑞金医院器官移植中心(上海消化外科研究所)实验室和浙江大学高分子材料研究所共同完成.材料:选用孔径为0.2 μm,分子截流量为Mr 50000-100000的微孔聚丙烯超滤平面薄膜为实验模型,利用光化学接枝聚合改性技术,在聚丙烯超滤膜表面通过化学键的形式接枝亲水性丙烯酰胺基团,成功地构建一种人肿细胞/微孔聚丙烯杂化界面,即新型的生物人工肝生物反应器膜--接枝改性微孔聚丙烯超滤膜.方法:参照国际标准化组织ISO10993-1:1992医疗器械生物学评价标准和要求进行溶血试验、细胞毒性试验、全身急性毒性试验、热原试验、皮肤和皮内刺激试验,评价接枝改性微孔聚丙烯超滤膜的生物相容性.主要观察指标:接枝改性微孔聚丙烯超滤膜浸提液的溶血试验、细胞毒性试验、全身急性毒性试验、热原试验、皮肤和皮内刺激试验结果.结果:接枝改性微孔聚丙烯超滤膜的溶血率为1.90% 50 000-100 000 molecular blockage, were used. Photochemical graft polymerization modification technique was adopted to graft hydrophilic acrylamide group through chemical bonds on MPP surface and succeeded in constructing an interface of human hepatocyte/microporous polypropylene, i.e. bioreactor membrane of bioartificial liver, graft modified MPP.METHODS: The biocompatibility of modified MPP was evaluated by hemolysis test, cytotoxicity test, acute systemic toxicity test, pyrogen test, skin sensitization and percutaneous stimulation test according to the requirements and biological evaluation criteria of medical device of ISO10993-1:1992.MAIN

  18. Detection of differentially expressed genes reversing MDR of HCC induced by EGCG%EGCG逆转肝癌耐多药差异基因表达

    Institute of Scientific and Technical Information of China (English)

    韦艳; 张海英; 梁钢

    2011-01-01

    Objective To investigate the mechanism of reversing effect of epigallocatechin gallate (EGCG) on multidrug resistance (MDR) of human hepatocellular carcinoma ( HCC ) cell lines Bel-FU, and to provide the reference for screening natural drugs for reversing the MDR of HCC at transcriptional level. Methods Human HCC cell line BEL-7402 and multidrug-resistant HCC cell line Bel-Fu were cultured for the study. The MDR of Bel-Fu cells and its drug sensitivity to EGCG were determined by methyl thiazolyl tetrazolium (MTT) assay. Confined to the scope of the development of HCC,the 42 gene expression profiles of BEL-7402 group, BeL-FU group, and BeL-FU EGCG group were detected by real-time quantitative-PCR array. The expression profiles of gene Hras, MDR1 were comfirmed by real-time quantitative polymerase chain reaction. Results The resistance index (IR) of Bel-Fu to 5 -fluorouracil, cytarabine, vincristin, doxorubicin, daunorubicin was 89.6,3.9,37.2,16. 5 ,and 3.7,respectively. The 50% inhibitory concentration(IC50) and 10% inhibitory concentration(IC10) of EGCG was 587.4lμg/mL and 83. 1 μg/mL,respectively. Compared with those in BEL-7402 group,5 differentially expressed genes were up-regulated in Bel-Fu group. Compared with those in Bel-Fu group, 6 differentially expressed genes were down-regulated in Bel-Fu EGCG group. Compared with those in BEL-7402 group, the expression profiles of gene Hras and MDR1 in Bel-Fu group showed 2. 87 and 2. 74 times of increase. Compared with those in Bel-Fu group,the expression profiles of gene Hras and MDR1 in the Bel-Fu EGCG group decreased by 2. 37 and 2. 63 times.Conclusion EGCG could reverse the MDR of human hepatocellular carcinoma cell lines Bel-FU.%目的 探讨表没食子儿茶素没食子酸酯(EGCG)逆转人肝癌细胞耐多药的机制,为进一步在基因转录水平上高通量筛选逆转肝癌耐多药天然药物提供一定的参考依据.方法 采用人肝癌细胞Bel-7402及其耐5-氟尿嘧啶Bel-FU细胞,甲

  19. Green tea compound epigallo-catechin-3-gallate (EGCG) increases neuronal survival in adult hippocampal neurogenesis in vivo and in vitro.

    Science.gov (United States)

    Ortiz-López, L; Márquez-Valadez, B; Gómez-Sánchez, A; Silva-Lucero, M D C; Torres-Pérez, M; Téllez-Ballesteros, R I; Ichwan, M; Meraz-Ríos, M A; Kempermann, G; Ramírez-Rodríguez, G B

    2016-05-13

    Epigallo-catechin-3-gallate (EGCG), found in the leaves of Camellia sinensis (green tea), has antioxidant- and scavenger-functions and acts neuroprotectively. It has been publicized as anti-aging remedy but data on potential cellular mechanisms are scarce. Recent studies claimed that EGCG specifically promotes neural precursor cell proliferation in the dentate gyrus of C57Bl/6 mice, without changes at the level of immature and mature new neurons. We here analyzed the effects of EGCG on adult hippocampal neurogenesis in male Balb/C mice and saw a different pattern. Two weeks of treatment with EGCG (0, 0.625, 1.25, 2.5, 5 and 10mg/kg) showed a dose-response curve that peaked at 2.5mg/kg of EGCG with significantly increased cell survival without affecting cell proliferation but decreasing apoptotic cells. Also, EGCG increased the population of doublecortin-(DCX)-expressing cells that comprises the late intermediate progenitor cells (type-2b and -3) as well as immature neurons. After EGCG treatment, the young DCX-positive neurons showed more elaborated dendritic trees. EGCG also significantly increased net neurogenesis in the adult hippocampus and increased the hippocampal levels of phospho-Akt. Ex vivo, EGCG exerted a direct effect on survival and neuronal differentiation of adult hippocampal precursor cells, which was absent, when PI3K, a protein upstream of Akt, was blocked. Our results thus support a pro-survival and a pro-neurogenic role of EGCG. In the context of the conflicting published results, however, potential genetic modifiers must be assumed. These might help to explain the overall variability of study results with EGCG. Our data do indicate, however, that natural compounds such as EGCG can in principle modulate brain plasticity.

  20. Therapeutic induction of angiogenesis by direct myocardial administration of an adenovirus vector encoding human hepatocyte growth factor gene and its safety

    Institute of Scientific and Technical Information of China (English)

    WU Danli; ZHANG Yourong; LAO Miaofen; YUAN Lizhen; WANG Lan; HA Xiaoqin; WU Zuze(WU Cutse)

    2004-01-01

    After the study in vitro and in rats, we assessed further the effects and safety of local angiogen therapy using intramyocardial delivery of an adenovirus carrying hepatocyte growth factor gene (Ad-HGF) in a canine ischemia model. The angiogenic activity of Ad-HGF was evaluated from three aspects. First, the augmentation of collateral vessel development was assessed by angiography 30 d after surgery. The results showed that the density of collateral vessels in treated group was higher than that of control group. Secondly, infarct size was evaluated by TTC staining and image analysis. The results showed that the infarct size of treated group was smaller than that of control group. Thirdly, the myocardial regional blood flow was determined by the method of colored microspheres. The results showed that the blood flow recovered to the level before ligation in treated group, but that of the control group was lower than normal level. In addition, during the study of chronic toxicity, we tested the anti-adenovirus antibodies by neutralization method. The antibodies yielded after the fourth injection decreased slowly from peak level and disappeared 12 weeks after drug withdrawal. Overall, Ad-HGF can promote angiogenesis in ischemic myocardium and reduce infarct size.So this method may be considered as a therapeutic angiogenesis induction strategy for ischemic disease including myocardial infarction and peripheral artery disease. At the same time, Ad-HGF could induce the yield of anti-adenovirus antibodies to neutralize adenovirus, which may be the mechanism of adenovirus clearance.

  1. Human Mesenchymal Stem Cells Provide Protection against Radiation-Induced Liver Injury by Antioxidative Process, Vasculature Protection, Hepatocyte Differentiation, and Trophic Effects

    Directory of Open Access Journals (Sweden)

    Sabine Francois

    2013-01-01

    Full Text Available To evaluate the potential therapeutic effect of the infusion of hMSCs for the correction of liver injuries, we performed total body radiation exposure of NOD/SCID mice. After irradiation, mir-27b level decreases in liver, increasing the directional migration of hMSCs by upregulating SDF1α. A significant increase in plasmatic transaminases levels, apoptosis process in the liver vascular system, and in oxidative stress were observed. hMSC injection induced a decrease in transaminases levels and oxidative stress, a disappearance of apoptotic cells, and an increase in Nrf2, SOD gene expression, which might reduce ROS production in the injured liver. Engrafted hMSCs expressed cytokeratin CK18 and CK19 and AFP genes indicating possible hepatocyte differentiation. The presence of hMSCs expressing VEGF and Ang-1 in the perivascular region, associated with an increased expression of VEGFr1, r2 in the liver, can confer a role of secreting cells to hMSCs in order to maintain the endothelial function. To explain the benefits to the liver of hMSC engraftment, we find that hMSCs secreted NGF, HGF, and anti-inflammatory molecules IL-10, IL1-RA contributing to prevention of apoptosis, increasing cell proliferation in the liver which might correct liver dysfunction. MSCs are potent candidates to repair and protect healthy tissues against radiation damages.

  2. Metformin stimulates FGF21 expression in primary hepatocytes

    DEFF Research Database (Denmark)

    Nygaard, Eva B; Vienberg, Sara G; Ørskov, Cathrine

    2012-01-01

    incubated with Compound C (an AMPK inhibitor) in the presence of metformin. A strong dose-dependent increase in FGF21 expression was observed in both rat and human hepatocytes treated with metformin. This effect was blocked by addition of the AMPK-inhibitor Compound C. The study shows that metformin...

  3. Combination of erlotinib and EGCG induces apoptosis of head and neck cancers through posttranscriptional regulation of Bim and Bcl-2.

    Science.gov (United States)

    Haque, Abedul; Rahman, Mohammad Aminur; Chen, Zhuo Georgia; Saba, Nabil F; Khuri, Fadlo R; Shin, Dong M; Ruhul Amin, A R M

    2015-07-01

    Combinatorial approaches using two or more compounds are gaining increasing attention for cancer therapy. We have previously reported that the combination of the EGFR-TKI erlotinib and epigallocatechin-3-gallate (EGCG) exhibited synergistic chemopreventive effects in head and neck cancers by inducing the expression of Bim, p21, p27, and by inhibiting the phosphorylation of ERK and AKT and expression of Bcl-2. In the current study, we further investigated the mechanism of regulation of Bim, Bcl-2, p21 and p27, and their role in apoptosis. shRNA-mediated silencing of Bim significantly inhibited apoptosis induced by the combination of erlotinib and EGCG (p = 0.005). On the other hand, overexpression of Bcl-2 markedly protected cells from apoptosis (p = 0.003), whereas overexpression of constitutively active AKT only minimally protected cells from apoptosis induced by the combination of the two compounds. Analysis of mRNA expression by RT-PCR revealed that erlotinib, EGCG and their combination had no significant effects on the mRNA expression of Bim, p21, p27 or Bcl-2 suggesting the post-transcriptional regulation of these molecules. Furthermore, we found that erlotinib or the combination of EGCG and erlotinib inhibited the phosphorylation of Bim and stabilized Bim after inhibition of protein translation by cycloheximide. Taken together, our results strongly suggest that the combination of erlotinib and EGCG induces apoptosis of SCCHN cells by regulating Bim and Bcl-2 at the posttranscriptional level.

  4. Blockade of the formation of insoluble ubiquitinated protein aggregates by EGCG3"Me in the alloxan-induced diabetic kidney.

    Directory of Open Access Journals (Sweden)

    Shuxian Cai

    Full Text Available BACKGROUND: Renal accumulation of reactive carbonyl compounds (RCCs has been linked to the progression of diabetic nephropathy. We previously demonstrated that carbonyl stress induces the formation of amino-carbonyl cross-links and sharply increases the content of β-sheet-rich structures, which is the seed of insoluble aggregates formation, and tea catechin (--epigallocatechin 3-gallate (EGCG can reverse this process in vitro and in vivo. In this study, methylated derivative (--epigallocatechin-3-O-(3-O-methyl-gallate (EGCG3"Me was hypothesized to neutralize carbonyl stress mediating the formation of insoluble ubiquitinated protein (IUP aggregates, and reduce the early development of diabetic nephropathy. METHODS AND RESULTS: Diabetes was induced in mice by intraperitoneally injecting alloxan monohydrate (200 mg/kg/d twice and administering EGCG3"Me by gavage for 15 d. Reagent case and western blot results showed that, in diabetic kidneys, the carbonyl proteins in the serum increased; and in insoluble protein fraction, 4-hydroxynonenal-modified proteins, IUP aggregates and p62 accumulated; FT-IR study demonstrated that the lipid content, anti-parallel β-sheet structure and aggregates increased. EGCG3"Me treatment could effectively reverse this process, even better than the negative control treatment. CONCLUSIONS: EGCG3"Me exhibiting anti-β-sheet-rich IUP aggregate properties, maybe represents a new strategy to impede the progression of diabetic nephropathy and other diabetic complications.

  5. EGCG Enhances Cisplatin Sensitivity by Regulating Expression of the Copper and Cisplatin Influx Transporter CTR1 in Ovary Cancer.

    Directory of Open Access Journals (Sweden)

    Xuemin Wang

    Full Text Available Cisplatin is one of the first-line platinum-based chemotherapeutic agents for treatment of many types of cancer, including ovary cancer. CTR1 (copper transporter 1, a transmembrane solute carrier transporter, has previously been shown to increase the cellular uptake and sensitivity of cisplatin. It is hypothesized that increased CTR1 expression would enhance the sensitivity of cancer cells to cisplatin (cDDP. The present study demonstrates for the first time that (--epigallocatechin-3-gallate (EGCG, a major polyphenol from green tea, can enhance CTR1 mRNA and protein expression in ovarian cancer cells and xenograft mice. EGCG inhibits the rapid degradation of CTR1 induced by cDDP. The combination of EGCG and cDDP increases the accumulation of cDDP and DNA-Pt adducts, and subsequently enhances the sensitivity of ovarian cancer SKOV3 and OVCAR3 cells to the chemotherapeutic agent. In the OVCAR3 ovarian cancer xenograft nude mice model, the combination of the lower concentration of cDDP and EGCG strongly repressed the tumor growth and exhibited protective effect on the nephrotoxicity induced by cisplatin. Overall, these findings uncover a novel chemotherapy mechanism of EGCG as an adjuvant for the treatment of ovarian cancer.

  6. CYCLOSPORINE-A BLOCKS BILE-ACID SYNTHESIS IN CULTURED-HEPATOCYTES BY SPECIFIC-INHIBITION OF CHENODEOXYCHOLIC ACID SYNTHESIS

    NARCIS (Netherlands)

    PRINCEN, HMG; WOLTHERS, BG; VONK, RJ; KUIPERS, F

    1991-01-01

    Bile acid synthesis, determined by conversion of [4-C-14]cholesterol into bile acids in rat and human hepatocytes and by measurement of mass production of bile acids in rat hepatocytes, was dose-dependently decreased by cyclosporin A, with 52% (rat) and 45% (human) inhibition at 10-mu-M. The decreas

  7. Cytotoxic effects of 109 reference compounds on rat H4IIE and human HepG2 hepatocytes. III: Mechanistic assays on oxygen consumption with MitoXpress and NAD(P)H production with Alamar Blue™.

    Science.gov (United States)

    Schoonen, Willem G E J; Stevenson, Joe C R; Westerink, Walter M A; Horbach, G Jean

    2012-04-01

    In vitro toxicity screening can reduce the attrition rate of drug candidates in the pharmaceutical industry in the early development process. The focus in this study is to compare the sensitivity for cytotoxicity of a time-resolved fluoro metric oxygen probe with that of a fluoro metric Alamar Blue™ (AB) assay. Both assays measure mitochondrial activity by either oxygen consumption (LUX-A65N-1 (MitoXpress, Luxcel) probe) or NADH/FADH conversion (AB). Both assays were carried out with increasing concentrations of 109 reference compounds using rat H4IIE and human HepG2 hepatocytes at incubation periods of 24, 48 and 72 h. Prior to this study, the influence on medium with either glucose or galactose was studied to analyze the rate of glycolysis and oxygen consumption, which latter process may be impaired in hepatoma cells. Inhibitors of oxygen consumption in combination with a glucose up-take inhibitor showed the largest consumption rate differences in the presence of 5mM of glucose. The choice for the 109 reference compounds was based on the so-called Multicentre Evaluation for In vitro Cytotoxicity (MEIC) and on diverse drug categories. For 59 toxic reference compounds, an evaluation for both assays was carried up to 10(-3)M. Toxicity was demonstrated with MitoXpress for 23 (39%) and 36 (61%) compounds in H4IIE and HepG2 cells, respectively, and with AB for 44 (75%) and 40 (68%) compounds. For 50 more pharmaceutical drugs more physiological concentrations were used up to 3.16×10(-5)M, and only 19 (38%) of these compounds appeared to be toxic in both assays. In conclusion, overall 63 (58%) and 60 (55%) compounds showed toxic effects with the MitoXpress and AB assays on rat H4IIE and human HepG2 hepatocytes, respectively. AB assays were more sensitive with respect to H4IIE cells and MitoXpress assays with respect to HepG2 cells. At all tested time intervals, MitoXpress showed its sensitivity, while AB is more sensitive at 48 and 72 h. With AB more toxic compounds

  8. Reversible regulation of cell cycle-related genes by epigallocatechin gallate for hibernation of neonatal human tarsal fibroblasts.

    Science.gov (United States)

    Bae, Jung Yoon; Kanamune, Jun; Han, Dong-Wook; Matsumura, Kazuaki; Hyon, Suong-Hyu

    2009-01-01

    We investigated the hibernation effect of epigallocatechin-3-O-gallate (EGCG) on neonatal human tarsal fibroblasts (nHTFs) by analyzing the expression of cell cycle-related genes. EGCG application to culture media moderately inhibited the growth of nHTFs, and the removal of EGCG from culture media led to complete recovery of cell growth. EGCG resulted in a slight decrease in the cell population of the S and G(2)/M phases of cell cycle with concomitant increase in that of the G(0)/G(1) phase, but this cell cycle profile was restored to the initial level after EGCG removal. The expression of cyclin D1 (CCND1), CCNE2, CCN-dependent kinase 6 (CDK6), and CDK2 was restored, whereas that of CCNA, CCNB1, and CDK1 was irreversibly attenuated. The expression of a substantial number of genes analyzed by cDNA microarray was affected by EGCG application, and these affected expression levels were restored to the normal levels after EGCG removal. We also found the incorporation of FITC-EGCG into the cytosol of nHTFs and its further nuclear translocation, which might lead to the regulation of the exogenous signals directed to genes for cellular responses including proliferation and cell cycle progression. These results suggest that EGCG temporarily affects not only genes related to the cell cycle but also various other cellular functions.

  9. The variant hepatocyte nuclear factor 1 activates the P1 promoter of the human alpha-folate receptor gene in ovarian carcinoma.

    Science.gov (United States)

    Tomassetti, Antonella; Mangiarotti, Fabio; Mazzi, Mimma; Sforzini, Sabrina; Miotti, Silvia; Galmozzi, Enrico; Elwood, Patrick C; Canevari, Silvana

    2003-02-01

    The alpha folate receptor (alpha FR) is a membrane glycoprotein that binds folates, and mediates their uptake and that of antifolate drugs. alpha FR is absent on ovarian surface epithelium (OSE) but is detectable during early transforming events in this epithelium, with increasing expression levels in association with tumor progression. Analysis of transcriptional regulation of the alpha FR gene have revealed two promoter regions, P1 and P4, flanking exons 1 and 4, respectively, and a requirement for three SP1 sites and an INR element for optimal P4 activity. Here, we focused on the P1 transcription regulation in ovarian carcinoma cells. RNase protection assay indicated that the 5'-untranslated region is heterogeneous because of different start sites and alternative splicing of exon 3. A core region of the P1 promoter was sufficient for maximal promoter activity in ovarian carcinoma cell lines but not in OSE cells or in alpha FR-nonexpressing cell lines. Deletion and mutation analysis of this core promoter identified a cis-regulatory element at position +27 to +33 of the untranslated exon 1, which is responsible for maximum P1 activity. This element formed an abundant DNA-protein complex with nuclear proteins from ovarian cancer cells but not from other cell lines or OSE cells. Competition experiments and supershift assays demonstrated binding of the P1 cis-regulatory element by a transcription factor involved in embryonic development, the variant hepatocyte nuclear factor-1 (vHNF1). Analysis of RNA from various cell lines and surgical specimens confirmed that vHNF1 is expressed in ovarian carcinomas. Thus, vHNF1 regulates tissue-specific transcription in ovarian carcinoma.

  10. Antiallergic tea catechin, (-)-epigallocatechin-3-O-(3-O-methyl)-gallate, suppresses FcepsilonRI expression in human basophilic KU812 cells.

    Science.gov (United States)

    Fujimura, Yoshinori; Tachibana, Hirofumi; Maeda-Yamamoto, Mari; Miyase, Toshio; Sano, Mitsuaki; Yamada, Koji

    2002-09-25

    We previously found that the O-methylated derivative of (-)-epigallocatechin-3-O-gallate (EGCg), (-)-epigallocatechin-3-O-(3-O-methyl)-gallate (EGCG' '3Me), has potent antiallergic activity. The high-affinity IgE receptor, FcepsilonRI, is found at high levels on basophils and mast cells and plays a key role in a series of acute and chronic human allergic reactions. To understand the mechanism of action for the antiallergic EGCG' '3Me, the effect of EGCG' '3Me on the cell surface expression of FcepsilonRI in human basophilic KU812 cells was examined. Flow cytometric analysis showed that EGCG' '3Me was able to decrease the cell surface expression of FcepsilonRI. Moreover, immunoblot analysis revealed that total cellular expression of the FcepsilonRI alpha chain decreased upon treatment with EGCG' '3Me. FcepsilonRI is a tetrameric structure comprising one alpha chain, one beta chain, and two gamma chains. The level of mRNA production of each subunit in KU812 cells was investigated. EGCG' '3Me reduced FcepsilonRI alpha and gamma mRNA levels. The cross-linkage of FcepsilonRI causes the activation of basophils, which leads to the secretion of inflammatory mediators including histamine. EGCG' '3Me treatment inhibited the FcepsilonRI cross-linking-induced histamine release. These results suggested that EGCG' '3Me can negatively regulate basophil activation through the suppression of FcepsilonRI expression.

  11. Lineage tracing reveals conversion of liver sinusoidal endothelial cells into hepatocytes.

    Science.gov (United States)

    Tan, Zhaoli; Chen, Keyan; Shao, Yong; Gao, Lihua; Wang, Yan; Xu, Jianming; Jin, Yang; Hu, Xianwen; Wang, Youliang

    2016-09-01

    Although liver sinusoidal endothelial cells (LSECs) have long been known to contribute to liver regeneration following injury, the exact role of these cells in liver regeneration remains poorly understood. In this work, we performed lineage tracing of LSECs in mice carrying Tie2-Cre or VE-cadherin-Cre constructs to facilitate fate-mapping of LSECs in liver regeneration. Some YFP-positive LSECs were observed to convert into hepatocytes following a two-thirds partial hepatectomy (PH). Furthermore, human umbilical vein endothelial cells (HUVECs) could be triggered to convert into cells that closely resembled hepatocytes when cultured with serum from mice that underwent an extended PH. These findings suggest that mature non-hepatocyte LSECs play an essential role in mammalian liver regeneration by converting to hepatocytes. The conversion of LSECs to hepatocyte-like (iHep) cells may provide a new approach to tissue engineering.

  12. Co-operation of the transcription factor hepatocyte nuclear factor-4 with Sp1 or Sp3 leads to transcriptional activation of the human haem oxygenase-1 gene promoter in a hepatoma cell line.

    Science.gov (United States)

    Takahashi, Shigeru; Matsuura, Naomi; Kurokawa, Takako; Takahashi, Yuji; Miura, Takashi

    2002-11-01

    We reported previously that the 5'-flanking region (nucleotides -1976 to -1655) of the human haem oxygenase-1 ( hHO-1 ) gene enhances hHO-1 promoter activity in human hepatoma HepG2 cells, but not in HeLa cells [Takahashi, Takahashi, Ito, Nagano, Shibahara and Miura (1999) Biochim. Biophys. Acta 1447, 231-235]. To define more precisely the regulatory elements involved, in the present study we have functionally dissected this region and localized the enhancer to a 50 bp fragment (-1793 to -1744). Site-direct mutagenesis analysis revealed that two regions were responsible for this enhancer activity, i.e. a hepatocyte nuclear factor-4 (HNF-4) homologous region and a GC box motif homologous region. Mutation in either region alone moderately decreased enhancer activity. However, mutations in both regions reduced promoter activity to the basal level. Electrophoretic mobility-shift assays demonstrated that the P5-2 fragment (-1793 to -1744) interacted with at least two nuclear factors, i.e. HNF-4 and Sp1/Sp3. Co-transfection experiments using Drosophila SL2 cells revealed that HNF-4 and Sp1/Sp3 synergistically stimulated the enhancer activity of the P5-2 fragment. These results indicate that co-operation of HNF-4 with Sp1 or Sp3 leads to the activation of hHO-1 gene expression in hepatoma cells.

  13. Electroanalysis of the interaction between (-)-epigallocatechin-3-gallate (EGCG) and amyloid-β in the presence of copper.

    Science.gov (United States)

    Zhang, Biao; Cheng, Xin R; da Silva, Iranaldo S; Hung, Vinci W S; Veloso, Anthony J; Angnes, Lúcio; Kerman, Kagan

    2013-03-01

    The misfolding of amyloid-beta (Aβ) peptide is one of the pathological hallmarks of Alzheimer's disease (AD). Polyphenols are strong antioxidants and metal chelators, with characteristics that are of beneficial therapeutic values for their development as candidates targeting neurodegenerative and metal-induced diseases. We have demonstrated here the electrochemical properties of a green tea component, (-)-epigallocatechin-3-gallate (EGCG), and its potent activity on Aβ peptides. Characterization of early interactions (≤48 h) between EGCG and Aβ was conducted using square wave voltammetry (SWV). The interaction of Cu(ii) ions with the Tyr-10 residue of Aβ was shown to be affected by surrounding His residues. Morphological changes due to the binding of EGCG and Cu(II) were also elucidated using transmission electron microscopy (TEM). Electroanalytical techniques are promising for facilitating the investigation of metals and flavonoids in drug screening studies.

  14. Response of MiRNA-22-3p and MiRNA-149-5p to Folate Deficiency and the Differential Regulation of MTHFR Expression in Normal and Cancerous Human Hepatocytes

    Science.gov (United States)

    Li, Chao; Ni, Juan; Liu, Yao-Xian; Wang, Han; Liang, Zi-Qing; Wang, Xu

    2017-01-01

    Background/Aims Folic acid (FA) is a core micronutrient involved in DNA synthesis/methylation, and the metabolism of FA is responsible for genomic stability. MicroRNAs may affect gene expression during folate metabolism when cellular homeostasis is changed. This study aimed to reveal the relationship between FA deficiency and the expression of miR-22-p/miR-149-5p and the targeted regulation of miR-22-3p/miR-149-5p on the key folate metabolic gene Methylenetetrahydrofolate reductase (MTHFR). Methods Normal (HL-7702 cells) and cancerous (QGY-7703 cells) human hepatocytes were intervened in modified RPMI 1640 with FA deficiency for 21 days. The interaction between MTHFR and the tested miRNAs was verified by Dual-Luciferase Reporter Assays. The changes in the expression of miR-22-3p/miR-149-5p in response to FA deficiency were detected by Poly (A) Tailing RT-qPCR, and the expression of MTHFR at both the transcriptional and translational levels was determined by RT-qPCR and Western blotting, respectively. Result MiR-22-3p/miR-149-5p directly targeted the 3’UTR sequence of the MTHFR gene. FA deficiency led to an upregulation of miR-22-3p/miR-149-5p expression in QGY-7703/HL-7702 cells, while the transcription of MTHFR was decreased in QGY-7703 cells but elevated in HL-7702 cells. Western blotting showed that FA deficiency resulted in a decline of the MTHFR protein in QGY-7703 cells, whereas in HL-7702 cells, the MTHFR protein level remained constant. Conclusion The results suggested that miR-22-3p/miR-149-5p exert different post-transcriptional effects on MTHFR under conditions of FA deficiency in normal and cancerous human hepatocytes. The results also implied that miR-22-3p/miR-149-5p might exert anticancer effects in cases of long-term FA deficiency. PMID:28045918

  15. Autophagy inhibition contributes to the synergistic interaction between EGCG and doxorubicin to kill the hepatoma Hep3B cells.

    Directory of Open Access Journals (Sweden)

    Li Chen

    Full Text Available (--Epigallocatechin-3-O-gallate(EGCG, the highest catechins from green tea, has promisingly been found to sensitize the efficacy of several chemotherapy agents like doxorubicin (DOX in hepatocellular carcinoma (HCC treatment. However, the detailed mechanisms by which EGCG augments the chemotherapeutic efficacy remain unclear. Herein, this study was designed to determine the synergistic impacts of EGCG and DOX on hepatoma cells and particularly to reveal whether the autophagic flux is involved in this combination strategy for the HCC. Electron microscopy and fluorescent microscopy confirmed that DOX significantly increased autophagic vesicles in hepatoma Hep3B cells. Western blot and trypan blue assay showed that the increasing autophagy flux by DOX impaired about 45% of DOX-induced cell death in these cells. Conversely, both qRT-PCR and western blotting showed that EGCG played dose-dependently inhibitory role in autophagy signaling, and that markedly promoted cellular growth inhibition. Amazingly, the combined treatment caused a synergistic effect with 40 to 60% increment on cell death and about 45% augmentation on apoptosis versus monotherapy pattern. The DOX-induced autophagy was abolished by this combination therapy. Rapamycin, an autophagic agonist, substantially impaired the anticancer effect of either DOX or combination with EGCG treatment. On the other hand, using small interference RNA targeting chloroquine autophagy-related gene Atg5 and beclin1 to inhibit autophagy signal, hepatoma cell death was dramatically enhanced. Furthermore, in the established subcutaneous Hep3B cells xenograft tumor model, about 25% reduction in tumor growth as well as 50% increment of apoptotic cells were found in combination therapy compared with DOX alone. In addition, immunohistochemistry analysis indicated that the suppressed tendency of autophagic hallmark microtubule-associated protein light chain 3 (LC3 expressions was consistent with thus combined

  16. Effect of Brewing Duration on the Antioxidant and Hepatoprotective Abilities of Tea Phenolic and Alkaloid Compounds in a t-BHP Oxidative Stress-Induced Rat Hepatocyte Model

    Directory of Open Access Journals (Sweden)

    Laura Braud

    2015-08-01

    Full Text Available Tea is an interesting source of antioxidants capable of counteracting the oxidative stress implicated in liver diseases. We investigated the impact of antioxidant molecules provided by a mixture of teas’ leaves (green, oolong, pu-erh after different infusion durations in the prevention of oxidative stress in isolated rat hepatocytes, by comparison with pure epigallocatechin-3-gallate (EGCG, the main representative of tea catechins. Dried aqueous tea extracts (ATE obtained after 5, 15 and 30 min infusion time were characterized for total polyphenols (gallic acid equivalent, catechins, gallic acid and caffeine (HPLC-DAD/ESI-MS contents, and for scavenging ability against 2,2-diphenyl-1-picrylhydrazyl free radical. Hepatoprotection was evaluated through hepatocyte viability tests using tert-butyl hydroperoxide as a stress inducer, (3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide, neutral red uptake, real-time cellular impedance and mitochondrial function tests. We showed that a 5-min incubation time is sufficient for an optimal bioaccessibility of tea compounds with the highest antioxidative ability, which decreases for longer durations. A 4-h pretreatment of cells with ATE significantly prevented cell death by regulating reactive oxygen species production and maintaining mitochondrial integrity. Pure EGCG, at doses similar in ATE (5–12 µM, was inefficient, suggesting a plausible synergy of several water-soluble tea compounds to explain the ATE beneficial effects.

  17. 人骨髓干细胞异体内诱导分化为肝细胞的实验%Xenogenic differentiation of human bone marrow stem cells into hepatocytes

    Institute of Scientific and Technical Information of China (English)

    陈建锋; 高毅; 蒋泽生; 李浩

    2006-01-01

    BACKGROUND: How to obtain human-derived hepatocyte of high quality is the key problem for both bioartificial liver and hepatocyte transplantation. Bone marrow stem cells (BMSCs) can differentiate hepatocyte under proper condition, which provides a new think for obtaining hepatocyte.OBJECTIVE: To investigate the methods of the trans-differentiation of human BMSCs into hepatocyte in rats so as to provide a new think for clinical transplantation of liver and source of bioartificial liver.DESIGN: Randomized controlled study.SETTING: General Surgery of Zhujiang Hospital of Southern Medical University.MATERIALS: The experiment was conducted at Central Laboratory of Zhujia ng Hospital of Southern Medical University from May 2004 to February 2005. Totally 40 male SD rats of clean grade were divided randomly into five groups: model group, modeling + 14-day transplantation group of human BMSCs, modeling + 28-day transplantation group of human BMSCs, modeling + 14-day transplantation group of CL-1 cell (human hepatocyte family), and modeling + 28-day transplantation group of CL-1 cell (human hepatocyte family) with 8 in each group.acetaminofIuorene + carbon tetrachloride + cyclophosphamide were esand differentiated into hepatocyte with remedial liver regeneration. Human BMSCs were observed for 14 days in modeling + 14-day transplantation group of human BMSCs, for 28 days in modeling + 28-day transplantation group of human BMSCs, for 14 days in modeling + 14-day transplantation group of CL-1 cell and for 28 days in modeling + 28-day transplantation group of CL-1 cell. However, cells in model group function of rats was measured at normal state, before and after transplantation. The expressions of human albumin mRNA in liver were detected by immunohistochemistry staining, polymerase chain reaction (PCR) and real time reverse transcription polymerase chain reaction (RT-PCR) respectively.of human albumin mRNA in liver.transplantation of human BMSCs on hepatic function and content

  18. Green tea catechin, epigallocatechin-3-gallate, attenuates the cell viability of human non-small-cell lung cancer A549 cells via reducing Bcl-xL expression.

    Science.gov (United States)

    Sonoda, Jun-Ichiro; Ikeda, Ryuji; Baba, Yasutaka; Narumi, Keiko; Kawachi, Akio; Tomishige, Erisa; Nishihara, Kazuya; Takeda, Yasuo; Yamada, Katsushi; Sato, Keizo; Motoya, Toshiro

    2014-07-01

    Clinical and epidemiological studies have indicated that the consumption of green tea has a number of beneficial effects on health. Epigallocatechin-3-gallate (EGCg), the major polyphenolic compound present in green tea, has received much attention as an active ingredient. Among the numerous promising profiles of EGCg, the present study focused on the anticancer effects. Apoptosis induced by EGCg and subsequent cell growth suppression have been demonstrated in a number of cell culture studies. However, the underlying mechanism of apoptotic cell death remains unclear. Thus, the aim of the present study was to identify the major molecule that mediates proapoptotic cell death by EGCg. The effect of EGCg on cell proliferation and the induction of mRNA that modulates apoptotic cell death was evaluated in the A549 human non-small-cell lung cancer cell line. In addition, morphological changes were assessed by microscopy in A549 cells that had been treated with 100 μM EGCg for 24 h. The MTT assay revealed that cell proliferation was significantly reduced by EGCg in a dose-dependent manner (3-100 μM). The mRNA expression level of B-cell lymphoma-extra large (Bcl-xL) was decreased in A549 cells following 24 h incubation with 100 μM EGCg. Therefore, the results indicated that the inhibition of cell proliferation by EGCg may be achieved via suppressing the expression of the cell death-inhibiting gene, Bcl-xL.

  19. Preventive Effects of Epigallocatechin-3-O-Gallate against Replicative Senescence Associated with p53 Acetylation in Human Dermal Fibroblasts

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    Dong-Wook Han

    2012-01-01

    Full Text Available Considering the various pharmacological activities of epigallocatechin-3-O-gallate (EGCG including anticancer, and anti-inflammatory, antidiabetic, and so forth, relatively less attention has been paid to the antiaging effect of EGCG on primary cells. In this study, the preventive effects of EGCG against serial passage-induced senescence were investigated in primary cells including rat vascular smooth muscle cells (RVSMCs, human dermal fibroblasts (HDFs, and human articular chondrocytes (HACs. The involvement of Sirt1 and acetylated p53 was examined as an underlying mechanism for the senescence preventive activity of EGCG in HDFs. All cells were employed with the initial passage number (PN between 3 and 7. For inducing senescence, the cells were serially passaged at the predetermined times and intervals in the absence or presence of EGCG (50 or 100 μM. Serial passage-induced senescence in RVSMCs and HACs was able to be significantly prevented at 50 μM EGCG, while in HDFs, 100 μM EGCG could significantly prevent senescence and recover their cell cycle progression close to the normal level. Furthermore, EGCG was found to prevent serial passage- and H2O2-induced senescence in HDFs by suppressing p53 acetylation, but the Sirt1 activity was unaffected. In addition, proliferating HDFs showed similar cellular uptake of FITC-conjugated EGCG into the cytoplasm with their senescent counterparts but different nuclear translocation of it from them, which would partly account for the differential responses to EGCG in proliferating versus senescent cells. Taking these results into consideration, it is suggested that EGCG may be exploited to craft strategies for the development of an antiaging or age-delaying agent.

  20. 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

  1. Induction of digitoxigenin monodigitoxoside UDP-glucuronosyltransferase activity by glucocorticoids and other inducers of cytochrome P-450p in primary monolayer cultures of adult rat hepatocytes and in human liver.

    Science.gov (United States)

    Schuetz, E G; Hazelton, G A; Hall, J; Watkins, P B; Klaassen, C D; Guzelian, P S

    1986-06-25

    We have recently proposed that glucocorticoids induce cytochrome P-450p, a liver microsomal hemoprotein originally isolated from rats treated with the antiglucocorticoid pregnenolone 16 alpha-carbonitrile (PCN), through a mechanism that involves a stereospecific recognition system clearly distinguishable from the classic glucocorticoid receptor (Schuetz, E. G., Wrighton, S. A., Barwick, J. L., and Guzelian, P. S. (1984) J. Biol. Chem. 259, 1999-2012). We now report that digitoxigenin monodigitoxoside UDP-glucuronosyltransferase (DIG UDP-glucuronosyltransferase), a liver microsomal enzyme activity induced by PCN in rats, is also inducible, as is P-450p, in primary monolayer cultures of adult rat hepatocytes. DIG UDP-glucuronosyltransferase activity closely resembled reported characteristics of induction of P-450p in its time course of induction, concentration-response relationships, exclusivity of induction by steroids with glucocorticoid properties, unusual rank order of potency of glucocorticoid agonists, unusually high ED50 for induction by glucocorticoids, enhanced induction rather than inhibition by anti-glucocorticoids in the presence of glucocorticoids, and finally, induction by nonsteroidal inducers of P-450p. DIG UDP-glucuronosyltransferase activity was also readily detected in human liver microsomes and was elevated in two patients who had received inducers of P-450p. We conclude that the liver enzymes controlled by the postulated PCN recognition system include not only P-450p but also one or more UDP-glucuronosyltransferases.

  2. EGCG Inhibits Proliferation, Invasiveness and Tumor Growth by Up-Regulation of Adhesion Molecules, Suppression of Gelatinases Activity, and Induction of Apoptosis in Nasopharyngeal Carcinoma Cells

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    Chih-Yeu Fang

    2015-01-01

    Full Text Available (−-Epigallocatechin-3-gallate (EGCG, a major green tea polyphenol, has been shown to inhibit the proliferation of a variety of tumor cells. Epidemiological studies have shown that drinking green tea can reduce the incidence of nasopharyngeal carcinoma (NPC, yet the underlying mechanism is not well understood. In this study, the inhibitory effect of EGCG was tested on a set of Epstein Barr virus-negative and -positive NPC cell lines. Treatment with EGCG inhibited the proliferation of NPC cells but did not affect the growth of a non-malignant nasopharyngeal cell line, NP460hTert. Moreover, EGCG treated cells had reduced migration and invasive properties. The expression of the cell adhesion molecules E-cadherin and β-catenin was found to be up-regulated by EGCG treatment, while the down-regulation of matrix metalloproteinases (MMP-2 and MMP-9 were found to be mediated by suppression of extracellular signal-regulated kinase (ERK phosphorylation and AP-1 and Sp1 transactivation. Spheroid formation by NPC cells in suspension was significantly inhibited by EGCG. Oral administration of EGCG was capable of suppressing tumor growth in xenografted mice bearing NPC tumors. Treatment with EGCG was found to elevate the expression of p53 and p21, and eventually led to apoptosis of NPC cells via caspase 3 activation. The nuclear translocation of NF-κB and β-catenin was also suppressed by EGCG treatment. These results indicate that EGCG can inhibit the proliferation and invasiveness, and induce apoptosis, of NPC cells, making it a promising agent for chemoprevention or adjuvant therapy of NPC.

  3. Upregulation of CYP 450s expression of immortalized hepatocyte-like cells derived from mesenchymal stem cells by enzyme inducers

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    Limsrichamrern Somchai

    2011-09-01

    Full Text Available Abstract Background The strenuous procurement of cultured human hepatocytes and their short lives have constrained the cell culture model of cytochrome P450 (CYP450 induction, xenobiotic biotransformation, and hepatotoxicity. The development of continuous non-tumorous cell line steadily containing hepatocyte phenotypes would substitute the primary hepatocytes for these studies. Results The hepatocyte-like cells have been developed from hTERT plus Bmi-1-immortalized human mesenchymal stem cells to substitute the primary hepatocytes. The hepatocyte-like cells had polygonal morphology and steadily produced albumin, glycogen, urea and UGT1A1 beyond 6 months while maintaining proliferative capacity. Although these hepatocyte-like cells had low basal expression of CYP450 isotypes, their expressions could be extensively up regulated to 80 folds upon the exposure to enzyme inducers. Their inducibility outperformed the classical HepG2 cells. Conclusion The hepatocyte-like cells contained the markers of hepatocytes including CYP450 isotypes. The high inducibility of CYP450 transcripts could serve as a sensitive model for profiling xenobiotic-induced expression of CYP450.

  4. Hepatocytes: critical for glucose homeostasis.

    Science.gov (United States)

    Klover, Peter J; Mooney, Robert A

    2004-05-01

    Maintaining blood glucose levels within a narrow range is a critical physiological function requiring multiple metabolic pathways and involving several cell types, including a prominent role for hepatocytes. Under hormonal control, hepatocytes can respond to either feeding or fasting conditions by storing or producing glucose as necessary. In the fasting state, the effects of glucagon avoid hypoglycemia by stimulating glucogenesis and glycogenolysis and initiating hepatic glucose release. Postprandially, insulin prevents hyperglycemia, in part, by suppressing hepatic gluconeogenesis and glycogenolysis and facilitating hepatic glycogen synthesis. Both transcriptional regulation of rate limiting enzymes and modulation of enzyme activity through phosphorylation and allosteric regulation are involved. Type 2 diabetes mellitus is the most common serious metabolic condition in the world, and results from a subnormal response of tissues to insulin (insulin resistance) and a failure of the insulin-secreting beta cells to compensate. In type 2 diabetes, glucose is overproduced by the hepatocyte and is ineffectively metabolized by other organs. Impairments in the insulin signal transduction pathway appear to be critical lesions contributing to insulin resistance and type 2 diabetes.

  5. Impact of EGCG Supplementation on the Progression of Diabetic Nephropathy in Rats: An Insight into Fibrosis and Apoptosis.

    Science.gov (United States)

    Mohan, Thangarajeswari; Velusamy, Prema; Chakrapani, Lakshmi Narasimhan; Srinivasan, Ashok Kumar; Singh, Abhilasha; Johnson, Thanka; Periandavan, Kalaiselvi

    2017-09-13

    Apoptosis is an active response of cells to altered microenvironments, which is characterized by cell shrinkage, chromatin condensation, and DNA fragmentation, in a variety of cell types such as renal epithelial cells, endothelial cells, mesangial cells, and podocytes. Hyperglycemia is among the microenvironmental factors that may facilitate apoptosis, which plays a decisive role in the initiation of diabetic nephropathy. Transforming growth factor-β emerges as a powerful fibrogenic factor in the development of renal hypertrophy. Although, a number of potential treatment strategies exist for diabetic nephropathy, considering the ease of use and bioavailability, phytochemicals stands distinct as the preeminent option. EGCG, a green tea catechin is one such phytochemical which possesses hypoglycemic and antifibrotic activity. The present study aims to explore the potential of EGCG to prevent apoptosis in a high-fat diet and STZ induced diabetic nephropathy rats by assessing renal function, pro-fibrotic marker, and the expression of apoptotic and antiapoptotic proteins. Our results validate EGCG as a potential antiapoptotic agent evidently by improving renal function via down regulating TGF-β, consequently ameliorating diabetic nephropathy. In accordance with this, EGCG might be regarded as a prospective therapeutic candidate in modulating diabetic nephropathy, thus being a promising treatment.

  6. Better Management of Alcohol Liver Disease Using a ‘Microstructured Synbox’ System Comprising L. plantarum and EGCG

    Science.gov (United States)

    Rishi, Praveen; Arora, Sumeha; Kaur, Ujjwal Jit; Chopra, Kanwaljit; Kaur, Indu Pal

    2017-01-01

    Synergistic combination of probiotics with carbohydrate based prebiotics is widely employed for the treatment of various gut related disorders. However, such carbohydrate based prebiotics encourage the growth of pathogens and probiotics, equally. Aim of the study was (i) to explore the possibility of using epigallocatechin gallate (EGCG) a phenolic compound, as a prebiotic for L.plantarum; (ii) to develop and evaluate a microstructured synbox (microencapsulating both probiotic and EGCG together) in rat model of alcohol liver disease (ALD); and, (iii) to confirm whether the combination can address issues of EGCG bioavailability and probiotic survivability in adverse gut conditions. Growth enhancing effect of EGCG on L. plantarum (12.8±0.5 log 10 units) was significantly (p≤0.05) better than inulin (11.4±0.38 log 10 units), a natural storage carbohydrate. The formulated synbox significantly modulated the levels of alcohol, endotoxin, hepatic enzymes and restored the hepatoarchitecture in comparison to simultaneous administration of free agents. Additionally, using a battery of techniques, levels of various cellular and molecular markers viz. NF-kB/p50, TNF-α, IL12/p40, and signalling molecules TLR4, CD14, MD2, MyD88 and COX-2 were observed to be suppressed. Developed microbead synbox, as a single delivery system for both the agents showed synergism and hence, holds promise as a therapeutic option for ALD management. PMID:28060832

  7. Asialoglycoprotein receptor mediated hepatocyte targeting - strategies and applications.

    Science.gov (United States)

    D'Souza, Anisha A; Devarajan, Padma V

    2015-04-10

    Hepatocyte resident afflictions continue to affect the human population unabated. The asialoglycoprotein receptor (ASGPR) is primarily expressed on hepatocytes and minimally on extra-hepatic cells. This makes it specifically attractive for receptor-mediated drug delivery with minimum concerns of toxicity. ASGPR facilitates internalization by clathrin-mediated endocytosis and exhibits high affinity for carbohydrates specifically galactose, N-acetylgalactosamine and glucose. Isomeric forms of sugar, galactose density and branching, spatial geometry and galactose linkages are key factors influencing ligand-receptor binding. Popular ligands for ASGPR mediated targeting are carbohydrate polymers, arabinogalactan and pullulan. Other ligands include galactose-bearing glycoproteins, glycopeptides and galactose modified polymers and lipids. Drug-ligand conjugates provide a viable strategy; nevertheless ligand-anchored nanocarriers provide an attractive option for ASGPR targeted delivery and are widely explored. The present review details various ligands and nanocarriers exploited for ASGPR mediated delivery of drugs to hepatocytes. Nanocarrier properties affecting ASGPR mediated uptake are discussed at length. The review also highlights the clinical relevance of ASGPR mediated targeting and applications in diagnostics. ASGPR mediated hepatocyte targeting provides great promise for improved therapy of hepatic afflictions.

  8. 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.

  9. Strategies for immortalization of primary hepatocytes

    Science.gov (United States)

    Eva, Ramboer; Bram, De Craene; Joery, De Kock; Tamara, Vanhaecke; Geert, Berx; Vera, Rogiers; Mathieu, Vinken

    2014-01-01

    The liver has the unique capacity to regenerate in response to a damaging event. Liver regeneration is hereby largely driven by hepatocyte proliferation, which in turn relies on cell cycling. The hepatocyte cell cycle is a complex process that is tightly regulated by several well-established mechanisms. In vitro, isolated hepatocytes do not longer retain this proliferative capacity. However, in vitro cell growth can be boosted by immortalization of hepatocytes. Well-defined immortalization genes can be artificially overexpressed in hepatocytes or the cells can be conditionally immortalized leading to controlled cell proliferation. This paper discusses the current immortalization techniques and provides a state-of-the-art overview of the actually available immortalized hepatocyte-derived cell lines and their applications. PMID:24911463

  10. Epigallocatechin-3-gallate regulates cell growth, cell cycle and phosphorylated nuclear factor-KB in human dermal fibroblasts

    Institute of Scientific and Technical Information of China (English)

    Dong-Wook HAN; Mi Hee LEE; Hak Hee KIM; Suong-Hyu HYON; Jong-Chul PARK

    2011-01-01

    Aim: To investigate the effects of (-)epigallocatechin-3-gallate (EGCG), the main polyphenol in green tea, on cell growth, cell cycle and phosphorylated nuclear factor-kB (pNF-KB) expression in neonatal human dermal fibroblasts (nHDFs).Methods: The proliferation and cell-cycle of nHDFs were determined using WST-8 cell growth assay and flow cytometry, respectively. The apoptosis was examined using DNA ladder and Annexin V-FITC assays. The expression levels of pNF-kB and cell cycle-related genes and proteins in nHDFs were measured using cDNA microarray analyses and Western blot. The cellular uptake of EGCG was examined using fluorescence (FITC)-Iabeled EGCG (FITC-EGCG) in combination with confocal microscopy.Results: The effect of EGCG on the growth of nHDFs depended on the concentration tested. At a low concentration (200 μmol/L), EGCG resulted in a slight decrease in the proportion of ceils in the S and G/M phases of cell cycle with a concomitant increase in the proportion of cells in G/G phase. At the higher doses (400 and 800 pmol/L), apoptosis was induced. The regulation of EGCG on the expression of pNF-kB was also concentration-dependent, whereas it did not affect the unphosphorylated NF-kB expression, cDNA microarray analysis showed that cell cycle-related genes were down-regulated by EGCG (200 μmol/L). The expression of cyclins A/B and cyclin-dependent kinase 1 was reversibly regulated by EGCG (200 μmol/L). FITC-EGCG was found to be internalized into the cyto-plasm and translocated into the nucleus of nHDFs.Conclusion: EGCG, through uptake into cytoplasm, reversibly regulated the cell growth and expression of cell cycle-related proteins and genes in normal fibroblasts.

  11. Comparative metabolism of 1,2,3,3,3-pentafluoropropene in male and female mouse, rat, dog, and human liver microsomes and cytosol and male rat hepatocytes via oxidative dehalogenation and glutathione S-conjugation pathways.

    Science.gov (United States)

    Han, Xing; Szostek, Bogdan; Yang, Ching-Hui; Cheatham, Steve F; Mingoia, Robert T; Nabb, Diane L; Gannon, Shawn A; Himmelstein, Matthew W; Jepson, Gary W

    2011-07-01

    In vitro metabolism of 1,2,3,3,3-pentafluoropropene (PFP) was investigated in the present study. PFP was metabolized via cytochrome P450-catalyzed oxidative dehalogenation in liver microsomes and glutathione transferase (GST)-catalyzed conjugation in liver microsomes and cytosol. Two oxidation products, 2,3,3,3-tetrafluoropropionaldehyde (TPA) and 3,3,3-trifluoropyruvaldehyde (TFPA), and two GSH conjugates, S-(2,3,3,3-tetrafluoropropenyl)-GSH (TFPG) and S-(1,2,3,3,3-pentafluoropropyl)-GSH (PFPG) were identified. Enzyme kinetic parameters for the formation of TFPA, TFPG, and PFPG were obtained in male and female rat, mouse, dog, and human liver microsomes and cytosol and were confirmed using freshly isolated male rat hepatocytes. For the TFPA pathway, dog microsomes exhibited much larger K(m) values than rat, mouse, and human microsomes. Sex differences in the rates of metabolism within a given species were minor and generally were less than 2-fold. Across the species, liver microsomes were the primary subcellular fraction for GSH S-conjugation and the apparent reaction rates for the formation of TFPG were much greater than those for PFPG in liver microsomes. PFPG was unstable and had a half-life of approximately 3.9 h in a phosphate buffer (pH 7.4 and 37°C). The intrinsic clearance values for the formation of TFPA were much greater than those for the formation of GSH S-conjugates, suggesting that cytochrome P450-mediated oxidation is the primary pathway for the metabolism of PFP at relatively low PFP concentrations. Because saturation of the GST-mediated reactions was not reached at the highest possible PFP concentration, GSH S-conjugation may become a much more important pathway at higher PFP concentrations (relative to the K(m) for TFPA).

  12. RNAi screening in primary human hepatocytes of genes implicated in genome-wide association studies for roles in type 2 diabetes identifies roles for CAMK1D and CDKAL1, among others, in hepatic glucose regulation.

    Directory of Open Access Journals (Sweden)

    Steven Haney

    Full Text Available Genome-wide association (GWA studies have described a large number of new candidate genes that contribute to of Type 2 Diabetes (T2D. In some cases, small clusters of genes are implicated, rather than a single gene, and in all cases, the genetic contribution is not defined through the effects on a specific organ, such as the pancreas or liver. There is a significant need to develop and use human cell-based models to examine the effects these genes may have on glucose regulation. We describe the development of a primary human hepatocyte model that adjusts glucose disposition according to hormonal signals. This model was used to determine whether candidate genes identified in GWA studies regulate hepatic glucose disposition through siRNAs corresponding to the list of identified genes. We find that several genes affect the storage of glucose as glycogen (glycolytic response and/or affect the utilization of pyruvate, the critical step in gluconeogenesis. Of the genes that affect both of these processes, CAMK1D, TSPAN8 and KIF11 affect the localization of a mediator of both gluconeogenesis and glycolysis regulation, CRTC2, to the nucleus in response to glucagon. In addition, the gene CDKAL1 was observed to affect glycogen storage, and molecular experiments using mutant forms of CDK5, a putative target of CDKAL1, in HepG2 cells show that this is mediated by coordinate regulation of CDK5 and PKA on MEK, which ultimately regulates the phosphorylation of ribosomal protein S6, a critical step in the insulin signaling pathway.

  13. RNAi screening in primary human hepatocytes of genes implicated in genome-wide association studies for roles in type 2 diabetes identifies roles for CAMK1D and CDKAL1, among others, in hepatic glucose regulation.

    Science.gov (United States)

    Haney, Steven; Zhao, Juan; Tiwari, Shiwani; Eng, Kurt; Guey, Lin T; Tien, Eric

    2013-01-01

    Genome-wide association (GWA) studies have described a large number of new candidate genes that contribute to of Type 2 Diabetes (T2D). In some cases, small clusters of genes are implicated, rather than a single gene, and in all cases, the genetic contribution is not defined through the effects on a specific organ, such as the pancreas or liver. There is a significant need to develop and use human cell-based models to examine the effects these genes may have on glucose regulation. We describe the development of a primary human hepatocyte model that adjusts glucose disposition according to hormonal signals. This model was used to determine whether candidate genes identified in GWA studies regulate hepatic glucose disposition through siRNAs corresponding to the list of identified genes. We find that several genes affect the storage of glucose as glycogen (glycolytic response) and/or affect the utilization of pyruvate, the critical step in gluconeogenesis. Of the genes that affect both of these processes, CAMK1D, TSPAN8 and KIF11 affect the localization of a mediator of both gluconeogenesis and glycolysis regulation, CRTC2, to the nucleus in response to glucagon. In addition, the gene CDKAL1 was observed to affect glycogen storage, and molecular experiments using mutant forms of CDK5, a putative target of CDKAL1, in HepG2 cells show that this is mediated by coordinate regulation of CDK5 and PKA on MEK, which ultimately regulates the phosphorylation of ribosomal protein S6, a critical step in the insulin signaling pathway.

  14. Development of mouse hepatocyte lines permissive for hepatitis C virus (HCV.

    Directory of Open Access Journals (Sweden)

    Hussein Hassan Aly

    Full Text Available The lack of a suitable small animal model for the analysis of hepatitis C virus (HCV infection has hampered elucidation of the HCV life cycle and the development of both protective and therapeutic strategies against HCV infection. Human and mouse harbor a comparable system for antiviral type I interferon (IFN induction and amplification, which regulates viral infection and replication. Using hepatocytes from knockout (ko mice, we determined the critical step of the IFN-inducing/amplification pathways regulating HCV replication in mouse. The results infer that interferon-beta promoter stimulator (IPS-1 or interferon A receptor (IFNAR were a crucial barrier to HCV replication in mouse hepatocytes. Although both IFNARko and IPS-1ko hepatocytes showed a reduced induction of type I interferons in response to viral infection, only IPS-1-/- cells circumvented cell death from HCV cytopathic effect and significantly improved J6JFH1 replication, suggesting IPS-1 to be a key player regulating HCV replication in mouse hepatocytes. We then established mouse hepatocyte lines lacking IPS-1 or IFNAR through immortalization with SV40T antigen. Expression of human (hCD81 on these hepatocyte lines rendered both lines HCVcc-permissive. We also found that the chimeric J6JFH1 construct, having the structure region from J6 isolate enhanced HCV replication in mouse hepatocytes rather than the full length original JFH1 construct, a new finding that suggests the possible role of the HCV structural region in HCV replication. This is the first report on the entry and replication of HCV infectious particles in mouse hepatocytes. These mouse hepatocyte lines will facilitate establishing a mouse HCV infection model with multifarious applications.

  15. High expression of MDR1, MRP1, and MRP3 in the hepatic progenitor cell compartment and hepatocytes in severe human liver disease

    NARCIS (Netherlands)

    Ros, J.E.; Libbrecht, L; Geuken, M; Jansen, PLM; Roskams, TAD

    An increase in bile ductular structures is observed in diverse human liver diseases. These structures harbour the progenitor cell compartment of the liver. Since ATP-binding cassette (ABC) transporters may have a cytoprotective role in liver disease, an immunohistochemical study was performed on

  16. Functional involvement of proteins, interacting with sphingolipids, in sphingolipid transport to the canalicular membrane in the human hepatocytic cell line, HepG2?

    NARCIS (Netherlands)

    Zegers, MMP; Zaal, KJM; Hoekstra, D

    A photoreactive sphingolipid precursor was used to investigate the potential involvement of protein-lipid interactions that may convey specificity to sphingolipid transport in the human hepatoma cell line, HepG2, A I-125-labeled, photoreactive ceramide, I-125-N-3-Cer, was incubated with the cells

  17. Optimization on condition of epigallocatechin-3-gallate (EGCG) nanoliposomes by response surface methodology and cellular uptake studies in Caco-2 cells

    Science.gov (United States)

    Luo, Xiaobo; Guan, Rongfa; Chen, Xiaoqiang; Tao, Miao; Ma, Jieqing; Zhao, Jin

    2014-06-01

    The major component in green tea polyphenols, epigallocatechin-3-gallate (EGCG), has been demonstrated to prevent carcinogenesis. To improve the effectiveness of EGCG, liposomes were used as a carrier in this study. Reverse-phase evaporation method besides response surface methodology is a simple, rapid, and beneficial approach for liposome preparation and optimization. The optimal preparation conditions were as follows: phosphatidylcholine-to-cholesterol ratio of 4.00, EGCG concentration of 4.88 mg/mL, Tween 80 concentration of 1.08 mg/mL, and rotary evaporation temperature of 34.51°C. Under these conditions, the experimental encapsulation efficiency and size of EGCG nanoliposomes were 85.79% ± 1.65% and 180 nm ± 4 nm, which were close with the predicted value. The malondialdehyde value and the release test in vitro indicated that the prepared EGCG nanoliposomes were stable and suitable for more widespread application. Furthermore, compared with free EGCG, encapsulation of EGCG enhanced its inhibitory effect on tumor cell viability at higher concentrations.

  18. Detection of Dichlorvos Adducts in a Hepatocyte Cell Line

    Science.gov (United States)

    2014-06-30

    G6019) was obtained from Sigma (St. Louis, MS), human actin gamma recombinant protein (ACTG1, H00000071) and lactate dehydrogenase A recombi - nant...microRNA and mRNA expression profiling analysis of dichlorvos cytotoxicity in porcine kidney epithelial PK15 cells. DNA Cell Biol. 2011, 30 (12), 1073...Dissociation of DDVP-induced DNA strand breaks from oxidative damage in isolated rat hepatocytes. Toxicology 1996, 108 (1−2), 49−56. (40) Clapp, C.; Portt, L

  19. 提高裸鼠体内人胎肝细胞移植物存活率的方法探讨%Methodological exploration of improving the survival rate of human fetal hepatocytes transplanted into nude mice

    Institute of Scientific and Technical Information of China (English)

    唐智敏; 詹春姣; 金文银; 李航森; 石淑仙

    2000-01-01

    Objectives To explore whether human fetal hepatocytes(HFH)transplanted into nude mice survived and study the methods for raising successful rate of transplantation.Methods The suspension of human fetal hepatocytes was transplanted into the abdominal cavity.liver.speen and mesenterium respectively.Three months later,histological observation was performed.Results The HFH could be found on the transplanting sites of spleen and mesenterium with only 10% successful rate of transplantation. Intraperitoneal injection of dexamethasone after transplantation could increase the Successful rate of the transplantation to 6/10.Under this circumstances.if HFH in cellular Sphere culture was used as transplantation material,successful rate of transplantation could reach 90%.Based on methods mentioned above,a lobe of liver in nude mice was resected,successful rate of transplantation could reach 100%,and HFH transplanted into the spleens of nude mice still survived 6 months after transplantation.Conclusions HFH transplanted in nude mice could survive for at least 6 months.If transplantationmaterials were made up into cellular sphere cultures,combining with using dexamethasone,the survival rate of transplantation might significantly be increased.%目的 探讨人胎肝细胞移植入裸鼠体内能否存活及提高移植物存活率的方法.方法 将人胎肝细胞悬液分别移植至裸鼠的腹腔、肝脏、脾脏及肠系膜,3个月后进行移植部位的组织学观察.结果 仅移植入脾脏和肠系膜的组中各有1只(1/10)受者可观察到存活的移植物,而移植入肝脏组无法区分裸鼠肝细胞和人胎肝细胞,其它组均未观察到移植物;移植后腹腔注射地塞米松,可将移植成功率提高到6/10;若将人胎肝细胞采用球体细胞培养技术制成细胞球团行脾脏内移植,术后腹腔注射地塞米松,则移植成功率可达9/10,术后6个月仍可见存活的移植物.结论 人胎肝细胞移植到

  20. Establishment of a functional cell line expressing both subunits of H1a and H2c of human hepatocyte surface molecule ASGPR.

    Science.gov (United States)

    Hu, Bin; Yang, Yan; Liu, Jia; Ma, Zhiyong; Huang, Hongping; Liu, Shenpei; Yu, Yuan; Hao, Youhua; Wang, Baoju; Lu, Mengji; Yang, Dongliang

    2010-10-01

    To better understand the effect of a new split variant of human asialoglycoprotein receptor (ASGPR H1b) on ASGPR ligands' binding ability, we established a functional cell line which expresses ASGPR. The full lengths of ASGPRH1a and H2c fragments from human liver were amplified by reverse transcript PCR (RT-PCR) and inserted into eukaryotic expression vector pIRES2EGFP, pCDNA3.1 (Zeo+) respectively. The recombinants were co-transfected into HeLa cells. After selection by using Neocin and Zeocin, a stably transfected cell line was established, which was designated 4-1-6. The transcription and expression of ASGPRH1a and H2c in 4-1-6 were confirmed by RT-PCR, Western blotting and immunofluorescence. The endocytosis function of the artificial "ASGPR" on the surface of 4-1-6 was tested by FACS. It was found that the cell line 4-1-6 could bind ASGPR natural ligand molecular asialo-orosomucoid (ASOR). After the eukaryotic plasmid H1b/pCDNA3.1 (neo) was transfected into cell line 4-1-6, H1b did not down-regulate the ligand binding ability of ASGPR. The eukaryotic expression plasmid H1b/pcDNA3.1 (neo) and H2c/pcDNA3.1 (neo) were co-transfected transiently into Hela cell. Neither single H1b nor H1b and H2c could bind ASOR. In conclusion, a functional cell line of human asialoglycoprotein receptor (ASGPR) which expresses both H1a and H2c stably was established. The new split variant H1b has no effect on ASGPR binding to ASOR. ASGPRH1b alone can't bind to ASOR, it yet can't form functional complex with ASGPRH2c.

  1. Hepatocyte IKK2 protects Mdr2-/- mice from chronic liver failure.

    Directory of Open Access Journals (Sweden)

    Hanno Ehlken

    Full Text Available Mice lacking the Abc4 protein encoded by the multidrug resistance-2 gene (Mdr2(-/- develop chronic periductular inflammation and cholestatic liver disease resulting in the development of hepatocellular carcinoma (HCC. Inhibition of NF-κB by expression of an IκBα super-repressor (IκBαSR transgene in hepatocytes was shown to prevent HCC development in Mdr2(-/- mice, suggesting that NF-κB acts as a tumour promoter in this model of inflammation-associated carcinogenesis. On the other hand, inhibition of NF-κB by hepatocyte specific ablation of IKK2 resulted in increased liver tumour development induced by the chemical carcinogen DEN. To address the role of IKK2-mediated NF-κB activation in hepatocytes in the pathogenesis of liver disease and HCC in Mdr2(-/- mice, we generated Mdr2-deficient animals lacking IKK2 specifically in hepatocytes using the Cre-loxP system. Mdr2(-/- mice lacking IKK2 in hepatocytes developed spontaneously a severe liver disease characterized by cholestasis, major hyperbilirubinemia and severe to end-stage fibrosis, which caused muscle wasting, loss of body weight, lethargy and early spontaneous death. Cell culture experiments showed that primary hepatocytes lacking IKK2 were more sensitive to bile acid induced death, suggesting that hepatocyte-specific IKK2 deficiency sensitized hepatocytes to the toxicity of bile acids under conditions of cholestasis resulting in greatly exacerbated liver damage. Mdr2(-/-IKK2(Hep-KO mice remarkably recapitulate chronic liver failure in humans and might be of special importance for the study of the mechanisms contributing to the pathogenesis of end-stage chronic liver disease or its implications on other organs.IKK2-mediated signaling in hepatocytes protects the liver from damage under conditions of chronic inflammatory cholestasis and prevents the development of severe fibrosis and liver failure.

  2. Application of Green Tea Catechin for Inducing the Osteogenic Differentiation of Human Dedifferentiated Fat Cells in Vitro

    Directory of Open Access Journals (Sweden)

    Koji Kaida

    2015-11-01

    Full Text Available Despite advances in stem cell biology, there are few effective techniques to promote the osteogenic differentiation of human primary dedifferentiated fat (DFAT cells. We attempted to investigate whether epigallocatechin-3-gallate (EGCG, the main component of green tea catechin, facilitates early osteogenic differentiation and mineralization on DFAT cells in vitro. DFAT cells were treated with EGCG (1.25–10 μM in osteogenic medium (OM with or without 100 nM dexamethasone (Dex for 12 days (hereafter two osteogenic media were designated as OM(Dex and OM. Supplementation of 1.25 μM EGCG to both the media effectively increased the mRNA expression of collagen 1 (COL1A1 and runt-related transcription factor 2 (RUNX2 and also increased proliferation and mineralization. Compared to OM(Dex with EGCG, OM with EGCG induced earlier expression for COL1A1 and RUNX2 at day 1 and higher mineralization level at day 12. OM(Dex with 10 μM EGCG remarkably hampered the proliferation of the DFAT cells. These results suggest that OM(without Dex with EGCG might be a preferable medium to promote proliferation and to induce osteoblast differentiation of DFAT cells. Our findings provide an insight for the combinatory use of EGCG and DFAT cells for bone regeneration and stem cell-based therapy.

  3. Application of Green Tea Catechin for Inducing the Osteogenic Differentiation of Human Dedifferentiated Fat Cells in Vitro.

    Science.gov (United States)

    Kaida, Koji; Honda, Yoshitomo; Hashimoto, Yoshiya; Tanaka, Masahiro; Baba, Shunsuke

    2015-11-25

    Despite advances in stem cell biology, there are few effective techniques to promote the osteogenic differentiation of human primary dedifferentiated fat (DFAT) cells. We attempted to investigate whether epigallocatechin-3-gallate (EGCG), the main component of green tea catechin, facilitates early osteogenic differentiation and mineralization on DFAT cells in vitro. DFAT cells were treated with EGCG (1.25-10 μM) in osteogenic medium (OM) with or without 100 nM dexamethasone (Dex) for 12 days (hereafter two osteogenic media were designated as OM(Dex) and OM). Supplementation of 1.25 μM EGCG to both the media effectively increased the mRNA expression of collagen 1 (COL1A1) and runt-related transcription factor 2 (RUNX2) and also increased proliferation and mineralization. Compared to OM(Dex) with EGCG, OM with EGCG induced earlier expression for COL1A1 and RUNX2 at day 1 and higher mineralization level at day 12. OM(Dex) with 10 μM EGCG remarkably hampered the proliferation of the DFAT cells. These results suggest that OM(without Dex) with EGCG might be a preferable medium to promote proliferation and to induce osteoblast differentiation of DFAT cells. Our findings provide an insight for the combinatory use of EGCG and DFAT cells for bone regeneration and stem cell-based therapy.

  4. Combined low dose ionizing radiation and green tea-derived epigallocatechin-3-gallate treatment induces human brain endothelial cells death.

    Science.gov (United States)

    McLaughlin, Nancy; Annabi, Borhane; Lachambre, Marie-Paule; Kim, Kwang Sik; Bahary, Jean-Paul; Moumdjian, Robert; Béliveau, Richard

    2006-11-01

    The microvasculature of brain tumors has been proposed as the primary target for ionizing radiation (IR)-induced apoptosis. However, the contribution of low dose IR-induced non-apoptotic cell death pathways has not been investigated. This study aimed to characterize the effect of IR on human brain microvascular endothelial cells (HBMEC) and to assess the combined effect of epigallocatechin-3-gallate (EGCg), a green tea-derived anti-angiogenic molecule. HBMEC were treated with EGCg, irradiated with a sublethal (effective in EGCg pretreated-cells reaching 70% cell death. Analysis of cell cycle revealed that IR-induced cell accumulation in G2-phase. Expression of cyclin-dependent kinase inhibitors p21(CIP/Waf1) and p27(Kip) were increased by EGCg and IR. Although random DNA fragmentation increased by approximately 40% following combined EGCg/IR treatments, the synergistic reduction of cell survival was not related to increased pro-apoptotic caspase-3, caspase-9 and cytochrome C proteins. Cell necrosis increased 5-fold following combined EGCg/IR treatments while no changes in early or late apoptosis were observed. Our results suggest that the synergistic effects of combined EGCg/IR treatments may be related to necrosis, a non-apoptotic cell death pathway. Strategies sensitizing brain tumor-derived EC to IR may enhance the efficacy of radiotherapy and EGCg may represent such a potential agent.

  5. Mechanisms and prevention of hepatocyte cell death

    NARCIS (Netherlands)

    Vrenken, Titia Eveline

    2008-01-01

    In many liver diseases, hepatocyte damage occurs upon exposure to toxic bile acids, inflammatory cytokines and increased levels of reactive oxygen species (ROS). Detailed information of the signaling pathways involved in hepatocyte damage will facilitate the discovery of new targets for intervention

  6. Recombinant Laminins Drive the Differentiation and Self-Organization of hESC-Derived Hepatocytes

    Directory of Open Access Journals (Sweden)

    Kate Cameron

    2015-12-01

    Full Text Available Stem cell-derived somatic cells represent an unlimited resource for basic and translational science. Although promising, there are significant hurdles that must be overcome. Our focus is on the generation of the major cell type of the human liver, the hepatocyte. Current protocols produce variable populations of hepatocytes that are the product of using undefined components in the differentiation process. This serves as a significant barrier to scale-up and application. To tackle this issue, we designed a defined differentiation process using recombinant laminin substrates to provide instruction. We demonstrate efficient hepatocyte specification, cell organization, and significant improvements in cell function and phenotype. This is driven in part by the suppression of unfavorable gene regulatory networks that control cell proliferation and migration, pluripotent stem cell self-renewal, and fibroblast and colon specification. We believe that this represents a significant advance, moving stem cell-based hepatocytes closer toward biomedical application.

  7. Recombinant Laminins Drive the Differentiation and Self-Organization of hESC-Derived Hepatocytes.

    Science.gov (United States)

    Cameron, Kate; Tan, Rosanne; Schmidt-Heck, Wolfgang; Campos, Gisela; Lyall, Marcus J; Wang, Yu; Lucendo-Villarin, Baltasar; Szkolnicka, Dagmara; Bates, Nicola; Kimber, Susan J; Hengstler, Jan G; Godoy, Patricio; Forbes, Stuart J; Hay, David C

    2015-12-08

    Stem cell-derived somatic cells represent an unlimited resource for basic and translational science. Although promising, there are significant hurdles that must be overcome. Our focus is on the generation of the major cell type of the human liver, the hepatocyte. Current protocols produce variable populations of hepatocytes that are the product of using undefined components in the differentiation process. This serves as a significant barrier to scale-up and application. To tackle this issue, we designed a defined differentiation process using recombinant laminin substrates to provide instruction. We demonstrate efficient hepatocyte specification, cell organization, and significant improvements in cell function and phenotype. This is driven in part by the suppression of unfavorable gene regulatory networks that control cell proliferation and migration, pluripotent stem cell self-renewal, and fibroblast and colon specification. We believe that this represents a significant advance, moving stem cell-based hepatocytes closer toward biomedical application.

  8. Hepatocyte isolation from resected benign tissues: Results of a 5-year experience

    Science.gov (United States)

    Meng, Fan-Ying; Liu, Li; Liu, Jun; Li, Chun-You; Wang, Jian-Ping; Yang, Feng-Hui; Chen, Zhi-Shui; Zhou, Ping

    2016-01-01

    AIM To analyze retrospectively a 5-year experience of human hepatocyte isolation from resected liver tissues with benign disease. METHODS We established a method of modified four-step retrograde perfusion to isolate primary human hepatocytes. Samples were collected from the resected livers of patients with intrahepatic duct calculi (n = 7) and liver hemangioma (n = 17). Only the samples weighing ≥ 15 g were considered suitable for hepatocyte isolation. By using the standard trypan blue exclusion technique, hepatocyte viability and yield were immediately determined after isolation. RESULTS Twenty-four liver specimens, weighing 15-42 g, were immediately taken from the margin of the removed samples and transferred to the laboratory for hepatocyte isolation. Warm ischemia time was 5-35 min and cold ischemia time was 15-45 min. For the 7 samples of intrahepatic duct calculi, the method resulted in a hepatocyte yield of 3.49 ± 2.31 × 106 hepatocytes/g liver, with 76.4% ± 10.7% viability. The 17 samples of liver hemangioma had significantly higher yield of cells (5.4 ± 1.71 × 106 cells/g vs 3.49 ± 2.31 × 106 cells/g, P 0.05). We obtained a cell yield of 5.31 ± 1.87 × 106 hepatocytes/g liver when the samples weighed > 20 g. However, for the tissues weighing ≤ 20 g, a reduction in yield was found (3.08 ± 1.86 × 106 cells/g vs 5.31 ± 1.87 × 106 cells/g, P < 0.05). CONCLUSION Benign diseased livers are valuable sources for large-number hepatocyte isolation. Our study represents the largest number of primary human hepatocytes isolated from resected specimens from patients with benign liver disease. We evaluated the effect of donor liver characteristics on cell isolation, and we found that samples of liver hemangioma can provide better results than intrahepatic duct calculi, in terms of cell yield. Furthermore, the size of the tissues can affect the outcome of hepatocyte isolation. PMID:27688659

  9. Low-Density Lipoprotein Uptake Demonstrates a Hepatocyte Phenotype in the Dog, but Is Nonspecific.

    Science.gov (United States)

    Gow, Adam G; Muirhead, Rhona; Hay, David C; Argyle, David J

    2016-01-01

    Low-density lipoprotein (LDL) uptake is one of a number of tests used to demonstrate hepatocyte-like function after stem cell differentiation. Use of two compounds, LDL and acetylated LDL (AcLDL), has been described despite each having different mechanisms of uptake. Three primary hepatocyte cultures and three sets of mesenchymal stromal cell (MSC) cultures, derived from both adipose tissue and bone marrow, were harvested from dogs. Those cells were compared to commercially available human and mouse bone marrow-derived MSCs. LDL receptor expression was demonstrated by gene expression and immunofluorescence in all primary hepatocyte cultures, undifferentiated canine bone marrow MSCs and canine adipose MSCs. Undifferentiated human and mouse bone marrow MSCs also expressed the LDL receptor. In vitro, canine hepatocytes took up labeled LDL, but not AcLDL. All undifferentiated MSCs took up LDL, but not AcLDL. In conclusion, LDL and not AcLDL is a test of canine hepatocyte-like phenotype, but this is not tissue or species specific and, therefore, is not informative assay when testing proof of MSC to hepatocyte differentiation.

  10. The Research Progress of Epigallocatechin Gallate (EGCG) on Weight Loss Mechanism%表没食子儿茶素没食子酸酯(EGCG)减肥机制的研究进展

    Institute of Scientific and Technical Information of China (English)

    魏冰; 白厚增; 杨则宜

    2012-01-01

    In recent years, obesity has become a major social problem in developed and developing countries. As the main component of green tea catechins, more and more studies showed that as a safe diet supplement, epigallocate-chin gallate (EGCG) can reduce obesity induced by high fat diet through reducing the lipid absorption, increasing energy metabolism and fatty acid oxidation, inhibiting adipocyte differentiation and lipid formation. The effect of EGCG administration on fat reducing and it's mechanism was reviewed in this paper.%近年来,肥胖已经成为困扰发达国家和发展中国家的一个重大社会问题.作为绿茶儿茶素类的主要成分,越来越多的研究证明了表没食子儿茶素没食子酸酯(EGCG)可通过减少脂质吸收,增加能量代谢和脂肪酸氧化,抑制脂肪细胞分化和脂肪生成等机制改善高脂饮食诱导的肥胖,该方法安全可靠.本文就EGCG减肥及其可能机制作一综述.

  11. SHP-2 tyrosine phosphatase inhibits p73-dependent apoptosis and expression of a subset of p53 target genes induced by EGCG

    OpenAIRE

    2007-01-01

    Green tea polyphenol, epigallocatechin-3-gallate (EGCG) differentially regulates the cellular growth of cancer cells in a p53-dependent manner through apoptosis and/or cell cycle arrest. In an effort to further elucidate the mechanism of differential growth regulation by EGCG, we have investigated the role of the tyrosine phosphatase, SHP-2. Comparing the responses of mouse embryonic fibroblasts (MEFs), expressing either WT or functionally inactive/truncated SHP-2, we find that inactivation o...

  12. Novel epigallocatechin-3-gallate (EGCG derivative as a new therapeutic strategy for reducing neuropathic pain after chronic constriction nerve injury in mice.

    Directory of Open Access Journals (Sweden)

    Xavier Xifró

    Full Text Available Neuropathic pain is common in peripheral nerve injury and often fails to respond to ordinary medication. Here, we investigated whether the two novel epigallocatechin-3-gallate (EGCG polyphenolic derivatives, compound 23 and 30, reduce the neuropathic pain in mice chronic constriction nerve injury (CCI. First, we performed a dose-response study to evaluate nociceptive sensation after administration of EGCG and its derivatives 23 and 30, using the Hargreaves test at 7 and 21 days after injury (dpi. We daily administered EGCG, 23 and 30 (10 to 100 mg/Kg; i.p. during the first week post-CCI. None of the doses of compound 23 caused significant pain diminution, whereas 50mg/kg was optimal for both EGCG and 30 to delay the latency of paw withdrawal. With 50 mg/Kg, we showed that EGCC prevented the thermal hyperalgesia from 7 to 21 dpi and compound 30 from 14 to 56 dpi. To evaluate the molecular mechanisms underpinning why EGCG and compound 30 differentially prevented the thermal hyperalgesia, we studied several biochemical parameters in the dorsal horn of the spinal cord at 14 and 56 dpi. We showed that the effect observed with EGCG and compound 30 was related to the inhibition of fatty acid synthase (FASN, a known target of these polyphenolic compounds. Additionally, we observed that EGCG and compound 30 reduced the expression of CCI-mediated inflammatory proteins and the nuclear localization of nuclear factor-kappa B at 14 dpi, but not at 56 dpi. We also strongly detected a decrease of synaptic plasma membrane levels of N-methyl-D-asparte receptor 2B in CCI-mice treated with compound 30 at 56 dpi. Altogether, compound 30 reduced the chronic thermal hyperalgesia induced by CCI better than the natural compound EGCG. Thus, our findings provide a rationale for the preclinical development of compound 30 as an agent to treat neuropathic pain.

  13. Data in support of the negative influence of divalent cations on (-)-epigallocatechin-3-gallate (EGCG)-mediated inhibition of matrix metalloproteinase-2 (MMP-2).

    Science.gov (United States)

    Deb, Gauri; Batra, Sahil; Limaye, Anil M

    2016-03-01

    In this data article we have provided evidence for the negative influence of divalent cations on (-)-epigallocatechin-3-gallate (EGCG)-mediated inhibition of matrix metalloproteinase-2 (MMP-2) activity in cell-free experiments. Chelating agents, such as EDTA and sodium citrate alone, did not affect MMP-2 activity. While EDTA enhanced, excess of divalent cations interfered with EGCG-mediated inhibition of MMP-2.

  14. Synthesis of Gelatin-γ-Polyglutamic Acid-Based Hydrogel for the In Vitro Controlled Release of Epigallocatechin Gallate (EGCG from Camellia sinensis

    Directory of Open Access Journals (Sweden)

    John Philip Domondon Garcia

    2013-12-01

    Full Text Available The antioxidant property and other health benefits of the most abundant catechin, epigallocatechin gallate (EGCG, are limited because of poor stability and permeability across intestine. Protecting the EGCG from the harsh gastrointestinal tract (GIT environment can help to increase its bioavailability following oral administration. In this study, EGCG was loaded to hydrogel prepared from ionic interaction between an optimized concentration of gelatin and γ-polyglutamic acid (γ-PGA, with ethylcarbodiimide (EDC as the crosslinker. Physicochemical characterization of hydrogel was done using Fourier transform-infrared spectroscopy (FT-IR, differential scanning calorimetry (DSC and scanning electron microscopy (SEM. The dependence of the swelling degree (SD of the hydrogel to the amount of gelatin, γ-PGA, EDC, swelling time and pH was determined. A high SD of the crosslinked hydrogel was noted at pH 4.5, 6.8 and 9.0 compared to pH 7.4, which describes pH-responsiveness. Approximately 67% of the EGCG from the prepared solution was loaded to the hydrogel after 12 h post-loading, in which loading efficiency was related to the amount of EDC. The in vitro release profile of EGCG at pH 1.2, 6.8 and 7.4, simulating GIT conditions, resulted in different sustained release curves. Wherein, the released EGCG was not degraded instantly compared to free-EGCG at controlled temperature of 37 °C at different pH monitored against time. Therefore, this study proves the potential of pH-responsive gelatin-γ-PGA-based hydrogel as a biopolymer vehicle to deliver EGCG.

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

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    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

  16. Identification of epigallocatechin-3-gallate in green tea polyphenols as a potent inducer of p53-dependent apoptosis in the human lung cancer cell line A549.

    Science.gov (United States)

    Yamauchi, Rieko; Sasaki, Kaori; Yoshida, Kenichi

    2009-08-01

    The effects of green tea polyphenols on cultured cancer cells have been well characterized, especially the effects of epigallocatechin-3-gallate (EGCg), since EGCg suppresses oncogenic signaling pathways and induces cell cycle arrest or apoptosis by regulating cell cycle-associated proteins. In the present study, we attempted to identify signaling pathways or target molecules regulated by each of or a mixture of green tea polyphenols, including epicatechin (EC), epicatechin-3-gallate (ECg), epigallocatechin (EGC), and EGCg, in the human lung cancer cell line A549. ECg, EGC, and a catechin mixture, in addition to EGCg, significantly decreased cell viability. In contrast, caspase 3/7 activity, an apoptosis indicator, was specifically induced by EGCg. By conducting a series of luciferase-based reporter assays, we revealed that the catechin mixture only up-regulates the p53 reporter. EGCg was a more potent inducer of p53-dependent transcription, and this induction was further supported by the induced level of p53 protein. RNA interference (RNAi)-mediated p53 knockdown completely abolished EGCg-induced apoptosis. Finally, a proteome and western blot analysis using approximately 70 different antibodies failed to detect up-regulated proteins in catechin mixture-treated A549 cells. Taken together, these results indicate that EGCg, among several green tea polyphenols, is a potent apoptosis inducer that functions exclusively through a p53-dependent pathway in A549 cells.

  17. Hepatocyte-specific IKK-β activation enhances VLDL-triglyceride production in APOE*3-Leiden mice.

    Science.gov (United States)

    van Diepen, Janna A; Wong, Man C; Guigas, Bruno; Bos, Jasper; Stienstra, Rinke; Hodson, Leanne; Shoelson, Steven E; Berbée, Jimmy F P; Rensen, Patrick C N; Romijn, Johannes A; Havekes, Louis M; Voshol, Peter J

    2011-05-01

    Low-grade inflammation in different tissues, including activation of the nuclear factor κB pathway in liver, is involved in metabolic disorders such as type 2 diabetes and cardiovascular diseases (CVDs). In this study, we investigated the relation between chronic hepatocyte-specific overexpression of IkB kinase (IKK)-β and hypertriglyceridemia, an important risk factor for CVD, by evaluating whether activation of IKK-β only in the hepatocyte affects VLDL-triglyceride (TG) metabolism directly. Transgenic overexpression of constitutively active human IKK-β specifically in hepatocytes of hyperlipidemic APOE*3-Leiden mice clearly induced hypertriglyceridemia. Mechanistic in vivo studies revealed that the hypertriglyceridemia was caused by increased hepatic VLDL-TG production rather than a change in plasma VLDL-TG clearance. Studies in primary hepatocytes showed that IKK-β overexpression also enhances TG secretion in vitro, indicating a direct relation between IKK-β activation and TG production within the hepatocyte. Hepatic lipid analysis and hepatic gene expression analysis of pathways involved in lipid metabolism suggested that hepatocyte-specific IKK-β overexpression increases VLDL production not by increased steatosis or decreased FA oxidation, but most likely by carbohydrate-responsive element binding protein-mediated upregulation of Fas expression. These findings implicate that specific activation of inflammatory pathways exclusively within hepatocytes induces hypertriglyceridemia. Furthermore, we identify the hepatocytic IKK-β pathway as a possible target to treat hypertriglyceridemia.

  18. Hepatitis B virus polymerase inhibits RIG-I- and Toll-like receptor 3-mediated beta interferon induction in human hepatocytes through interference with interferon regulatory factor 3 activation and dampening of the interaction between TBK1/IKKepsilon and DDX3.

    Science.gov (United States)

    Yu, Shiyan; Chen, Jieliang; Wu, Min; Chen, Hui; Kato, Nobuyuki; Yuan, Zhenghong

    2010-08-01

    Hepatitis B virus (HBV) infection remains one of the most serious health problems worldwide. Whilst studies have shown that HBV impairs interferon (IFN) production from dendritic cells in chronic hepatitis B patients, it remains unknown whether HBV inhibits IFN production in human hepatocytes. Using transient transfection assays in a primary human hepatocyte cell line (PH5CH8), this study demonstrated that HBV polymerase inhibits IFN-beta promoter activity induced by Newcastle disease virus, Sendai virus or poly(I : C) in a dose-dependent manner, whilst ectopic expression of the HBV core and X proteins had no effect on IFN-beta promoter activity. In addition, HBV polymerase blocked cellular IFN-beta expression and consequent antiviral immunity revealed by an infection protection assay. Furthermore, overexpression of key molecules on the IFN-beta induction axis, together with HBV polymerase, resulted in a block of IFN-beta promoter activity triggered by RIG-I, IPS-1, TRIF, TBK1 and IKKepsilon, but not by an IFN regulatory factor 3 dominant-positive mutant (IRF3-5D), suggesting that HBV polymerase prevents IFN-beta expression at the TBK1/IKKepsilon level. Further studies showed that HBV polymerase inhibited phosphorylation, dimerization and nuclear translocation of IRF3, in response to Sendai virus infection. Finally, it was shown that HBV polymerase-mediated dampening of the interaction between TBK1/IKKepsilon and DDX3 may be involved in the inhibitory effect on IFN-beta induction. Taken together, these findings reveal a novel role of HBV polymerase in HBV counteraction of IFN-beta production in human hepatocytes.

  19. Clinical applications of hepatocyte transplantation

    Institute of Scientific and Technical Information of China (English)

    Giada Pietrosi; Giovanni Battista Vizzini; Salvatore Gruttadauria; Bruno Gridelli

    2009-01-01

    The shortage of organ donors is a problem worldwide, with approximately 15% of adult patients with lifethreatening liver diseases dying while on the waiting list. The use of cell transplantation for liver disease is an attempt to correct metabolic defects, or to support liver function as a bridge to liver transplantation and, as such, has raised a number of expectations. Most of the available studies briefly reported here focus on adult hepatocyte transplantation (HT), and the results are neither reproducible nor comparable, because the means of infusion, amount of injected cells and clinical variability differ among the studies. To better understand the specific role of HT in the management of end-stage liver disease, it is important that controlled studies, designed on the principles of evidence-based medicine, be done in order to guarantee the reproducibility of results.

  20. Primary 3-dimensional culture of mouse hepatocytes

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Complex 3-dimensional structures with good functions have been obtained under the primary mixcoculture of mouse hepatocytes with mouse liver fibroblasts without serum. Albumin secretion is kept above 10 μg/106 cells and urea synthesis reaches 25 μg/106 on the 7th day of culture. Avoiding serum affection, liver fibroblasts' effects on hepatocytes' viability, functions and 3-dimensional structure forming in primary serum-free culture have been studied. Important effects of the mesenchyma, especially the direct adherence of fibroblasts to hepatocytes, are shown.

  1. 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.

  2. Significant improvement of survival by intrasplenic hepatocyte transplantation in totally hepatectomized rats.

    Science.gov (United States)

    Vogels, B A; Maas, M A; Bosma, A; Chamuleau, R A

    1996-01-01

    The effect of intrasplenic hepatocyte transplantation (HTX) was studied in an experimental model of acute liver failure in rats with chronic liver atrophy. Rats underwent a portacaval shunt operation on Day -14 to induce liver atrophy, and underwent total hepatectomy on Day 0 as a start of acute liver failure. Intrasplenic hepatocyte or sham transplantation was performed on Day -7,-3, or -1 (n = 4 to 6 per group). During the period following hepatectomy, mean arterial blood pressure was maintained above 80 mm Hg and hypoglycaemia was prevented. Severity of hepatic encephalopathy was assessed by clinical grading and EEG spectral analysis, together with determination of blood ammonia and plasma amino acid concentrations, and "survival" time. Histological examination of the spleen and lungs was performed after sacrifice. Intrasplenic hepatocyte transplantation resulted in a significant improvement in clinical grading in all transplanted groups (p 0.05), HTX at Day -3: 19.7 +/- 3.7 h vs. 6.5 +/- 0.3 h (p ammonia concentrations after total hepatectomy (p spleens after sacrifice showed clusters of hepatocytes in the red pulp. Hepatocytes present in the spleen for 3 and 7 days showed bile accumulation and spots of beginning necrosis. The present data show that in a hard model of complete liver failure in portacaval shunted rats, intrasplenic hepatocyte transplantation is able to prolong "survival" time significantly 2- to 3-fold. The relevance of this observation for human application is discussed.

  3. The Effect and Mechanism of Tamoxifen-Induced Hepatocyte Steatosis in Vitro

    Directory of Open Access Journals (Sweden)

    Fei Zhao

    2014-03-01

    Full Text Available The aim of this study was to determine the effect and mechanism of tamoxifen (TAM-induced steatosis in vitro. HepG 2 (Human hepatocellular liver carcinoma cell line cells were treated with different concentrations of TAM for 72 h. Steatosis of hepatocytes was determined after Oil Red O staining and measurement of triglyceride (TG concentration. The expressions of genes in the TG homeostasis pathway, including sterol regulatory element-binding protein-1c (SREBP-1c, peroxisome proliferator-activated receptor γ (PPARγ, CCAAT/enhancer-binding protein α (C/EBPα, fatty acid synthase (FAS, acetyl-CoA carboxylase (ACC, stearoyl-CoA desaturase (SCD, carnitine palmitoyltransferase 1 (CPT1 and microsomal triglyceride transfer protein (MTP, were examined using quantitative real-time PCR and Western blot analysis. Cell proliferation was examined using the cell counting kit-8 (CCK-8 assay. We found that hepatocytes treated with TAM had: (1 induced hepatocyte steatosis and increased hepatocyte TG; (2 upregulation of SREBP-1c, FAS, ACC, SCD and MTP mRNA expressions (300%, 600%, 70%, 130% and 160%, respectively; (3 corresponding upregulation of protein expression; and (4 no difference in HepG 2 cell proliferation. Our results suggest that TAM can induce hepatocyte steatosis in vitro and that the enhancement of fatty acid synthesis through the upregulations of SREBP-1c and its downstream target genes (FAS, ACC and SCD may be the key mechanism of TAM-induced hepatocyte steatosis.

  4. Two persistent organic pollutants which act through different xenosensors (alpha-endosulfan and 2,3,7,8 tetrachlorodibenzo-p-dioxin) interact in a mixture and downregulate multiple genes involved in human hepatocyte lipid and glucose metabolism.

    Science.gov (United States)

    Ambolet-Camoit, Ariane; Ottolenghi, Chris; Leblanc, Alix; Kim, Min Ji; Letourneur, Franck; Jacques, Sébastien; Cagnard, Nicolas; Guguen-Guillouzo, Christiane; Barouki, Robert; Aggerbeck, Martine

    2015-09-01

    Individuals, typically, are exposed to mixtures of environmental xenobiotics affecting multiple organs and acting through different xenosensors and pathways in species and cell-type specific manners. 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and α-endosulfan are Persistent Organic Pollutants (POPs) and endocrine disruptors which act through different xenosensors and accumulate in the liver. Our objective in this HEALS study was to investigate the effects of the mixture of these POPs on gene expression in a human-derived hepatocyte cell line, HepaRG. We found that, in spite of having largely uncorrelated effects, TCDD and α-endosulfan, when mixed, alter the expression of genes. The combined effects of the mixture of the POPs significantly altered the expression of 100 genes (42 up- and 58 down-regulated) whereas the same concentration of either POP alone did not alter significantly the expression of these genes. For 32 other genes, selective inhibitory crosstalk between TCDD and α-endosulfan was observed. One of the POPs inhibited the effect, on gene expression, of the other in the mixture although, when used alone, that POP did not affect expression. The expression of another 82 genes was significantly altered (up- or down-regulated) by a single POP. The addition of the second POP either increased, in the same direction, the effect on gene expression or had no further effect. At low concentrations (0.2 nM TCDD and 1 μM α-endosulfan), the POPs still had significant effects and the levels of expression of the corresponding proteins were found to be affected for some genes. Particularly striking was the 80-90% inhibition, by the mixture, of the expression of a number of genes of several hepatic intermediary metabolic pathways (glycerolipid metabolism, FXR/RXR activation, glycolysis/gluconeogenesis, retinoid and bile acid biosynthesis), whereas each pollutant alone had only a moderate effect. Copyright © 2015 Elsevier B.V. and Société Française de Biochimie

  5. Effects of bisphenol A-related diphenylalkanes on vitellogenin production in male carp (Cyprinus carpio)