Glutathione: an intracellular and extracellular protective agent in Salmonella typhimurium and Escherichia coli

International Nuclear Information System (INIS)

Owens, R.A.

1986-01-01

Levels of glutathione, were measured in several aerobically grown strains of Salmonella typhimurium and Escherichia coli. External accumulation of GSH was inhibited by 30 mM NaN 3 . Thus, GSH export may be energy dependent. Greater than 50% of the glutathione detected in the media was in the reduced form. Since the oxidized glutathione in the media could be accounted for by oxidation during aerobic incubation as well as in sample processing, the glutathione was predominantly exported in the reduced form. Extracellular glutathione was detected in log phase cultures of 2 out of 2 E. coli strains and 6 of 8 Salmonella strains tested. Two-dimensional paper chromatography of supernatants from cultures labelled with Na 2 35 SO 4 confirmed the presence of GSH and revealed five other sulfur-containing compounds in the media of Salmonella and E. coli cultures. Since media from cultures of an E. coli GSH - strain contained compounds with identical R/sub f/'s, the five unidentified compounds were not derivatives of GSH. The addition of 26 μM GSH to cultures of TA1534 partially protected the bacteria from the toxic effects of 54 μM N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). When MNNG was preincubated with equimolar GSH, the mutagenicity of the MNNG was neutralized. The addition of micromolar GSH to cultures and E. coli GSH - strain protected the cells from growth inhibition by micromolar concentrations of mercuric chloride, silver nitrate, cisplatin, cadmium chloride, and iodoacetamide. The data presented demonstrate that micromolar concentrations of external GSH can significantly shorten the recovery time of cells after exposure to toxic agents in the environment

Pyridine nucleotide cycling and control of intracellular redox state in relation to poly (ADP-ribose) polymerase activity and nuclear localization of glutathione during exponential growth of Arabidopsis cells in culture.

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Pellny, Till K; Locato, Vittoria; Vivancos, Pedro Diaz; Markovic, Jelena; De Gara, Laura; Pallardó, Federico V; Foyer, Christine H

2009-05-01

Pyridine nucleotides, ascorbate and glutathione are major redox metabolites in plant cells, with specific roles in cellular redox homeostasis and the regulation of the cell cycle. However, the regulation of these metabolite pools during exponential growth and their precise functions in the cell cycle remain to be characterized. The present analysis of the abundance of ascorbate, glutathione, and pyridine nucleotides during exponential growth of Arabidopsis cells in culture provides evidence for the differential regulation of each of these redox pools. Ascorbate was most abundant early in the growth cycle, but glutathione was low at this point. The cellular ascorbate to dehydroascorbate and reduced glutathione (GSH) to glutathione disulphide ratios were high and constant but the pyridine nucleotide pools were largely oxidized over the period of exponential growth and only became more reduced once growth had ceased. The glutathione pool increased in parallel with poly (ADP-ribose) polymerase (PARP) activities and with increases in the abundance of PARP1 and PARP2 mRNAs at a time of high cell cycle activity as indicated by transcriptome information. Marked changes in the intracellular partitioning of GSH between the cytoplasm and nucleus were observed. Extension of the exponential growth phase by dilution or changing the media led to increases in the glutathione and nicotinamide adenine dinucleotide, oxidized form (NAD)-plus-nicotinamide adenine dinucleotide, reduced form (NADH) pools and to higher NAD/NADH ratios but the nicotinamide adenine dinucleotide phosphate, oxidized form (NADP)-plus-nicotinamide adenine dinucleotide phosphate, reduced form (NADPH) pool sizes, and NAPD/NADPH ratios were much less affected. The ascorbate, glutathione, and pyridine nucleotide pools and PARP activity decreased before the exponential growth phase ended. We conclude that there are marked changes in intracellular redox state during the growth cycle but that redox homeostasis is

The depletion of nuclear glutathione impairs cell proliferation in 3t3 fibroblasts.

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Jelena Markovic

2009-07-01

Full Text Available Glutathione is considered essential for survival in mammalian cells and yeast but not in prokaryotic cells. The presence of a nuclear pool of glutathione has been demonstrated but its role in cellular proliferation and differentiation is still a matter of debate.We have studied proliferation of 3T3 fibroblasts for a period of 5 days. Cells were treated with two well known depleting agents, diethyl maleate (DEM and buthionine sulfoximine (BSO, and the cellular and nuclear glutathione levels were assessed by analytical and confocal microscopic techniques, respectively. Both agents decreased total cellular glutathione although depletion by BSO was more sustained. However, the nuclear glutathione pool resisted depletion by BSO but not with DEM. Interestingly, cell proliferation was impaired by DEM, but not by BSO. Treating the cells simultaneously with DEM and with glutathione ethyl ester to restore intracellular GSH levels completely prevented the effects of DEM on cell proliferation.Our results demonstrate the importance of nuclear glutathione in the control of cell proliferation in 3T3 fibroblasts and suggest that a reduced nuclear environment is necessary for cells to progress in the cell cycle.

Effect of cystamine on rat tissue GSH level and glutathione reductase activity

International Nuclear Information System (INIS)

Kovarova, H.; Pulpanova, J.

1979-01-01

Reduced glutathione (GSH) level and glutathione reductase activity were determined by means of the spectrophotometric method in various rat tissues after i.p. administration of cystamine (50 mg/kg and 20 mg/kg). GSH amount dropped in the spleen and kidney at 10 and 20 min; following this interval, an increase of GSH level was observed in the liver at 20-30 min, in the spleen and kidney at 60 min after the treatment with a radioprotective cystamine dose (50 mg/kg). The changes in GSH level induced by a non-radioprotective cystamine dose (20 mg/kg) had an opposite tendency. The activity of glutathione reductase was decreased in all tissues studied. As to the mechanism of the radioprotective action, both the inactivation of glutathione reductase activity and the changes in GSH level seem to be the factors contributing to the radioprotective effect of cystamine by strengthening the cellular radioresistance. (orig.) 891 MG/orig. 892 RKD [de

A novel plant glutathione S-transferase/peroxidase suppresses Bax lethality in yeast

DEFF Research Database (Denmark)

Kampranis, S C; Damianova, R; Atallah, M

2000-01-01

The mammalian inducer of apoptosis Bax is lethal when expressed in yeast and plant cells. To identify potential inhibitors of Bax in plants we transformed yeast cells expressing Bax with a tomato cDNA library and we selected for cells surviving after the induction of Bax. This genetic screen allows...... for the identification of plant genes, which inhibit either directly or indirectly the lethal phenotype of Bax. Using this method a number of cDNA clones were isolated, the more potent of which encodes a protein homologous to the class theta glutathione S-transferases. This Bax-inhibiting (BI) protein was expressed...... in Escherichia coli and found to possess glutathione S-transferase (GST) and weak glutathione peroxidase (GPX) activity. Expression of Bax in yeast decreases the intracellular levels of total glutathione, causes a substantial reduction of total cellular phospholipids, diminishes the mitochondrial membrane...

The Incomplete Glutathione Puzzle: Just Guessing at Numbers and Figures?

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Deponte, Marcel

2017-11-20

Glutathione metabolism is comparable to a jigsaw puzzle with too many pieces. It is supposed to comprise (i) the reduction of disulfides, hydroperoxides, sulfenic acids, and nitrosothiols, (ii) the detoxification of aldehydes, xenobiotics, and heavy metals, and (iii) the synthesis of eicosanoids, steroids, and iron-sulfur clusters. In addition, glutathione affects oxidative protein folding and redox signaling. Here, I try to provide an overview on the relevance of glutathione-dependent pathways with an emphasis on quantitative data. Recent Advances: Intracellular redox measurements reveal that the cytosol, the nucleus, and mitochondria contain very little glutathione disulfide and that oxidative challenges are rapidly counterbalanced. Genetic approaches suggest that iron metabolism is the centerpiece of the glutathione puzzle in yeast. Furthermore, recent biochemical studies provide novel insights on glutathione transport processes and uncoupling mechanisms. Which parts of the glutathione puzzle are most relevant? Does this explain the high intracellular concentrations of reduced glutathione? How can iron-sulfur cluster biogenesis, oxidative protein folding, or redox signaling occur at high glutathione concentrations? Answers to these questions not only seem to depend on the organism, cell type, and subcellular compartment but also on different ideologies among researchers. A rational approach to compare the relevance of glutathione-dependent pathways is to combine genetic and quantitative kinetic data. However, there are still many missing pieces and too little is known about the compartment-specific repertoire and concentration of numerous metabolites, substrates, enzymes, and transporters as well as rate constants and enzyme kinetic patterns. Gathering this information might require the development of novel tools but is crucial to address potential kinetic competitions and to decipher uncoupling mechanisms to solve the glutathione puzzle. Antioxid. Redox Signal

The influence of oxygen on the induction of radiation damage in DNA in mammalian cells after sensitization by intracellular glutathione depletion

International Nuclear Information System (INIS)

Schans, G.P. van der; Vos, O.; Roos-Verheij, W.S.D.; Lohman, P.H.M.

1986-05-01

Treatment of mammalian cells with buthionine sulphoximine (BSO) or diethyl maleate (DEM) results in a decrease in the intracellular GSH (glutathione) and NPSH (non-protein-bound SH) levels. The effect of depletion of GSH and NPSH on radiosensitivity was studied in relation to the concentration of oxygen during irradiation. Single- and double-strand DNA breaks (ssb and dsb) and cell killing were used as criteria for radiation damage. Under aerobic conditions, BSO and DEM treatment gave a small sensitization of 10-20% for the 3 types of radiation damage. Also under severely hypoxic conditions (0.01 μM oxygen in the medium) the sensitizing effect of both compounds on the induction of ssb and dsb and on cell killing was small (0-30%). At somewhat higher concentrations of oxygen (0.5-10 μM) however, the sensitization amounted to about 90% for the induction of ssb and dsb and about 50% for cell killing. These results strengthen the widely accepted idea that intracellular SH-compounds compete with oxygen and other electron-affinic radiosensitizers with respect to reaction with radiation-induced damage, thus preventing the fixation of DNA damages by oxygen. These results imply that the extent to which SH-compounds affect the radiosensitivity of cells in vivo depends strongly on the local concentration of oxygen. (Auth.)

Intracellular drug delivery nanocarriers of glutathione-responsive degradable block copolymers having pendant disulfide linkages.

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Khorsand, Behnoush; Lapointe, Gabriel; Brett, Christopher; Oh, Jung Kwon

2013-06-10

Self-assembled micelles of amphiphilic block copolymers (ABPs) with stimuli-responsive degradation (SRD) properties have a great promise as nanotherapeutics exhibiting enhanced release of encapsulated therapeutics into targeted cells. Here, thiol-responsive degradable micelles based on a new ABP consisting of a pendant disulfide-labeled methacrylate polymer block (PHMssEt) and a hydrophilic poly(ethylene oxide) (PEO) block were investigated as effective intracellular nanocarriers of anticancer drugs. In response to glutathione (GSH) as a cellular trigger, the cleavage of pendant disulfide linkages in hydrophobic PHMssEt blocks of micellar cores caused the destabilization of self-assembled micelles due to change in hydrophobic/hydrophilic balance. Such GSH-triggered micellar destabilization changed their size distribution with an appearance of large aggregates and led to enhanced release of encapsulated anticancer drugs. Cell culture results from flow cytometry and confocal laser scanning microscopy for cellular uptake as well as cell viability measurements for high anticancer efficacy suggest that new GSH-responsive degradable PEO-b-PHMssEt micelles offer versatility in multifunctional drug delivery applications.

Radiobiological studies with a series of human cell lines of varying glutathione content

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Astor, M.B. (Columbia Univ., New York (USA). Radiological Research Lab.)

1984-08-01

The effect of decreased levels of intracellular glutathione (GSH) on the radiosensitivity of aerated and hypoxic cells was studied using human skin fibroblasts obtained from patients affected with the inborn error of metabolism, 5-oxoprolinuria. The oxygen enhancement ratios (OER) were determined for four cell lines obtained from a single family, SR and SUR (heterozygous parents) and GM3877 and GM3878 (affected homozygous siblings). Glutathione values ranged from 7.4 to 130% of control values. Only GM3877 with a GSH value 7.4% of control exhibited a reduced OER of 1.9 compared with a control value of 3. These results suggest that a reduction in OER is observed only when GSH levels reach extremely low values.

The role of BCL-2 and glutathione in an antioxidant pathway to prevent radiation-induced apoptosis

International Nuclear Information System (INIS)

Vlachaki, Maria T.; Meyn, Raymond E.

1997-01-01

Objective: The expression of the bcl-2 gene has been associated with resistance to radiation induced apoptosis. There is evidence that the bcl-2 protein acts in the antioxidant pathways to block the effects of reactive oxygen spieces that mediate apoptosis possibly by increasing the levels of intracellular glutathione. Our hypothesis is that pretreatment of radiation-sensitive cells, known to lack bcl-2 expression, with antioxidants will reduce radiation-induced apoptosis. For this purpose, the apoptotic response to radiation and the intracellular levels of glutathione were tested before and after pretreatment with antioxidants in two murine lymphoma cell lines, a radiation resistant-bcl-2 expressing (Ly-ar) line and a radiation sensitive (Ly-as) line. Methods and Materials: Ly-ar and Ly-as cells were irradiated at 0,1,2,3 and 4 hours before collection. The intracellular levels of reduced (GSH) and oxidized (GSSG) glutathione were determined by the use of the fluorescent dye ophthalaldehyde. Ly-as cells were pretreated with dihydrolipoic acid and lipoamide for 1 hour before irradiation. Apoptosis response was measured by the DNA fragmentation assay. The radiation dose was 2.5 Gy. Results: After irradiation, the apoptotic rate of Ly-ar and Ly-as cells is 11-19% and 66-87% respectively. Ly-ar cells have higher intracellular GSH and GSSG levels compared to Ly-as cells by 69.9% and 91.9% respectively and the GSH/GSSG ratio in Ly-ar and Ly-as cells is 17.09 and 15.09 respectively (a difference of 13.25%). GSH levels do not change during the first three hours after irradiation; however there is a 46% reduction at four hours after irradiation, a time at which the Ly-as cells have already fragmented their DNA. Pretreatment of cells with dihydrolipoic acid or lipoamide at concentrations of 4mM and 2mM respectively was toxic and resulted in cell death in the absence of irradiation. Conclusions: GSH and GSSG levels are elevated in radiation-resistant murine lymphoma cells

In vitro Effects of Four Native Brazilian Medicinal Plants in CYP3A4 mRNA Gene Expression, Glutathione Levels, and P-Glycoprotein Activity.

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Mazzari, Andre L D A; Milton, Flora; Frangos, Samantha; Carvalho, Ana C B; Silveira, Dâmaris; de Assis Rocha Neves, Francisco; Prieto, Jose M

2016-01-01

Erythrina mulungu Benth. (Fabaceae), Cordia verbenacea A. DC. (Boraginaceae), Solanum paniculatum L. (Solanaceae) and Lippia sidoides Cham. (Verbenaceae) are medicinal plant species native to Brazil shortlisted by the Brazilian National Health System for future clinical use. However, nothing is known about their effects in metabolic and transporter proteins, which could potentially lead to herb-drug interactions (HDI). In this work, we assess non-toxic concentrations (100 μg/mL) of the plant infusions for their in vitro ability to modulate CYP3A4 mRNA gene expression and intracellular glutathione levels in HepG2 cells, as well as P-glycoprotein (P-gp) activity in vincristine-resistant Caco-2 cells (Caco-2 VCR). Their mechanisms of action were further studied by measuring the activation of human pregnane X receptor (hPXR) in transiently co-transfected HeLa cells and the inhibition of γ-glutamyl transferase (GGT) in HepG2 cells. Our results show that P-gp activity was not affected in any case and that only Solanum paniculatum was able to significantly change CYP3A4 mRNA gene expression (twofold decrease, p effect upon hPXR (EC50 = 0.38 mg/mL). Total intracellular glutathione levels were significantly depleted by exposure to Solanum paniculatum (-44%, p Cordia verbenacea (-47%, p activity (-48%, p active pharmacovigilance is recommended for the other three species, especially in the case of Solanum paniculatum.

Radiobiological studies with a series of human cell lines of varying glutathione content

International Nuclear Information System (INIS)

Astor, M.B.

1984-01-01

The effect of decreased levels of intracellular glutathione (GSH) on the radiosensitivity of aerated and hypoxic cells was studied using human skin fibroblasts obtained from patients affected with the inborn error of metabolism, 5-oxoprolinuria. The oxygen enhancement ratios (OER) were determined for four cell lines obtained from a single family, SR and SUR (heterozygous parents) and GM3877 and GM3878 (affected homozygous siblings). Glutathione values ranged from 7.4 to 130% of control values. Only GM3877 with a GSH value 7.4% of control exhibited a reduced OER of 1.9 compared with a control value of 3. These results suggest that a reduction in OER is observed only when GSH levels reach extremely low values. (author)

Organophosphorus insecticides chlorpyrifos and diazinon and oxidative stress in neuronal cells in a genetic model of glutathione deficiency

International Nuclear Information System (INIS)

Giordano, Gennaro; Afsharinejad, Zhara; Guizzetti, Marina; Vitalone, Annabella; Kavanagh, Terrance J.; Costa, Lucio G.

2007-01-01

Over the past several years evidence has been accumulating from in vivo animal studies, observations in humans, and in vitro studies, that organophosphorus (OP) insecticides may induce oxidative stress. Such effects may contribute to some of the toxic manifestations of OPs, particularly upon chronic or developmental exposures. The aim of this study was to investigate the role of oxidative stress in the neurotoxicity of two commonly used OPs, chlorpyrifos (CPF) and diazinon (DZ), their oxygen analogs (CPO and DZO), and their 'inactive' metabolites (TCP and IMP), in neuronal cells from a genetic model of glutathione deficiency. Cerebellar granule neurons from wild type mice (Gclm +/+) and mice lacking the modifier subunit of glutamate cysteine ligase (Gclm -/-), the first and limiting step in the synthesis of glutathione (GSH), were utilized. The latter display very low levels of GSH and are more susceptible to the toxicity of agents that increase oxidative stress. CPO and DZO were the most cytotoxic compounds, followed by CPF and DZ, while TCP and IMP displayed lower toxicity. Toxicity was significantly higher (10- to 25-fold) in neurons from Gclm (-/-) mice, and was antagonized by various antioxidants. Depletion of GSH from Gclm (+/+) neurons significantly increased their sensitivity to OP toxicity. OPs increased intracellular levels of reactive oxygen species and lipid peroxidation and in both cases the effects were greater in neurons from Gclm (-/-) mice. OPs did not alter intracellular levels of GSH, but significantly increased those of oxidized glutathione (GSSG). Cytotoxicity was not antagonized by cholinergic antagonists, but was decreased by the calcium chelator BAPTA-AM. These studies indicate that cytotoxicity of OPs involves generation of reactive oxygen species and is modulated by intracellular GSH, and suggest that it may involve disturbances in intracellular homeostasis of calcium

Glutathione level and its relation to radiation therapy in patients with cancer of uterine cervix

International Nuclear Information System (INIS)

Mukundan, H.; Bahadur, A.K.; Kumar, A.; Sardana, S.; Naik, S.L.D.; Ray, A.; Sharma, B.K.

1999-01-01

Glutathione functions as an important antioxidant in the destruction of hydrogen peroxide and lipid peroxides by providing substrate for the glutathione peroxidase and also promotes the ascorbic acid. Glutathione plays a vital role in detoxification of xenobiotics, carcinogens, free radicals and maintenance of immune functions. The study was aimed to determine plasma glutathione as well as erythrocyte glutathione and glutathione peroxidase in patients with invasive cervical carcinoma (n=30) before initiation and after completion of radiotherapy and subsequently, at the time of first three monthly follow-up visit. The levels of plasma glutathione, erythrocyte glutathione and glutathione peroxidase activity were found to be lower in all cervical cancer patients as compared to age matched normal control women. The study indicates a change in antioxidant status in relation with the glutathione system among patients with invasive carcinoma of the uterine cervix. This study also demonstrates the effect of radiation therapy on this antioxidant system. (author)

Glutathione (GSH Production as Protective Adaptation Against Light Regime Radiation of Symbiodinium Natural Population

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Moh Muhaemin

2017-08-01

Full Text Available Glutathione (GSH, as a wide range of low molecular weight, which found in marine microalgae and event bacteria, are essential to prevent photooxidation and productivity loss from these Radical Oxigen Species (ROS. Symbiodinium, endo-symbiont of corals, were exposed with different UV radiation combined with irradiance treatments to explore biomass specific initial response. Intracellular glutahione was observed as potential adaptive response of Symbiodinium population under environmental specific stress. The result showed that GSH production increased significantly with increasing irradiance and/or UV levels. GSH concentration was fluctuated among populations exposed by different irradiance treatments, but not effected by UV and irradiance exposure. GSH production as a response of UV exposure was higher than irradiance treatments. Both these high correlative fluctuation of intracellular GSH production and the presence of both treatments indicated protective specific adaptation of Symbiodinium under specific environmental stress, respectively.   Keywords: zooxanthellae, irradiance, glutathione (GSH, corals, Fungia

Insecticide resistance and glutathione S-transferases in mosquitoes ...

African Journals Online (AJOL)

Mosquito glutathione S-transferases (GSTs) have received considerable attention in the last 20 years because of their role in insecticide metabolism producing resistance. Many different compounds, including toxic xenobiotics and reactive products of intracellular processes such as lipid peroxidation, act as GST substrates.

Semen Quality of Post-Thawed Local Ram’s in Tris-Egg Yolk Extender with Different Glutathione Level

Science.gov (United States)

Solihati, N.; Rasad, S. D.; Setiawan, R.; Foziah, E. N.; Wigiyanti, E. T.

2018-02-01

The aims of this research were to find out the effect of glutathione level on semen quality of local ram and to find out the glutathione level that provide the best quality of local ram semen. This research use Completely Randomized Design (CRD) with five treatments of glutathione level (0 mM, 4 mM, 5 mM, 6 mM and 7mM) and was repeated for 10 times. Data were analyzed using analysis of varians (ANOVA) and differences between treatment was analyzed using Duncan test. The parameter were semen quality consist of motility, intact plasma membrane (IPM), abnormality, and recovery rate. Result of this research showed that glutathione level significantly (pegg yolk tris extender provide the best quality of local ram semen.

Activation of glutathione peroxidase via Nrf1 mediates genistein's protection against oxidative endothelial cell injury

International Nuclear Information System (INIS)

Hernandez-Montes, Eva; Pollard, Susan E.; Vauzour, David; Jofre-Montseny, Laia; Rota, Cristina; Rimbach, Gerald; Weinberg, Peter D.; Spencer, Jeremy P.E.

2006-01-01

Cellular actions of isoflavones may mediate the beneficial health effects associated with high soy consumption. We have investigated protection by genistein and daidzein against oxidative stress-induced endothelial injury. Genistein but not daidzein protected endothelial cells from damage induced by oxidative stress. This protection was accompanied by decreases in intracellular glutathione levels that could be explained by the generation of glutathionyl conjugates of the oxidised genistein metabolite, 5,7,3',4'-tetrahydroxyisoflavone. Both isoflavones evoked increased protein expression of γ-glutamylcysteine synthetase-heavy subunit (γ-GCS-HS) and increased cytosolic accumulation and nuclear translocation of Nrf2. However, only genistein led to increases in the cytosolic accumulation and nuclear translocation of Nrf1 and the increased expression of and activity of glutathione peroxidase. These results suggest that genistein-induced protective effects depend primarily on the activation of glutathione peroxidase mediated by Nrf1 activation, and not on Nrf2 activation or increases in glutathione synthesis

Different roles of glutathione in copper and zinc chelation in Brassica napus roots.

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Zlobin, Ilya E; Kartashov, Alexander V; Shpakovski, George V

2017-09-01

We investigated the specific features of copper and zinc excess action on the roots of canola (Brassica napus L.) plants. Copper rapidly accumulated in canola root cells and reached saturation during several hours of treatment, whereas the root zinc content increased relatively slowly. Excessive copper and zinc entry inside the cell resulted in significant cell damage, as evidenced by alterations in plasmalemma permeability and decreases in cellular enzymatic activity. Zinc excess specifically damaged root hair cells, which correlated with a pronounced elevation of their labile zinc level. In vitro, we showed that reduced glutathione (GSH) readily reacted with copper ions to form complexes with blocked sulfhydryl groups. In contrast, zinc ions were ineffective as glutathione blockers, and glutathione molecules did not lose their specific chemical activity in the presence of Zn 2+ ions. The effect of copper and zinc excess on the glutathione pool in canola root cells was analysed by a combination of biochemical determination of total and oxidized glutathione contents and fluorescent staining of free reduced glutathione with monochlorobimane dye. Excess copper led to dose-dependent diminution of free reduced glutathione contents in the root cells, which could not be explained by the loss of total cellular glutathione or its oxidation. In contrast, we observed little effect of much higher intracellular zinc concentrations on the free reduced glutathione content. We concluded that GSH plays an important role in copper excess, but not zinc excess chelation, in canola root cells. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Glutathione attenuates uranyl toxicity in Lactococcus lactis

International Nuclear Information System (INIS)

Fahmy, Karim; Oertel, Jana; Solioz, M.

2017-01-01

We investigated the role of intracellular glutathione (GSH), which in a large number of taxa plays a role in the protection against the toxicity of heavy metals. Anaerobically grown Lactococcus lactis containing an inducible GSH synthesis pathway was used as a model organism allowing the study of GSH-dependent uranyl detoxification without interference from additional reactive oxygen species. Microcalorimetric measurements of the metabolic heat showed that intracellular GSH attenuates the toxicity of uranium at a concentration in the range of 10-150 μM. Isothermal titration calorimetry revealed the endothermic binding of U(VI) to the carboxyl group(s) of GSH. The data indicate that the primary detoxifying mechanism is the intracellular sequestration of carboxyl-coordinated U(VI) into an insoluble complex with GSH.

Glutathione attenuates uranyl toxicity in Lactococcus lactis

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Fahmy, Karim; Oertel, Jana [Helmholtz-Zentrum Dresden-Rossendorf e.V., Dresden (Germany). Biophysics; Obeid, M. [Technische Univ. Dresden (Germany); Solioz, M. [Bern Univ. (Switzerland)

2017-06-01

We investigated the role of intracellular glutathione (GSH), which in a large number of taxa plays a role in the protection against the toxicity of heavy metals. Anaerobically grown Lactococcus lactis containing an inducible GSH synthesis pathway was used as a model organism allowing the study of GSH-dependent uranyl detoxification without interference from additional reactive oxygen species. Microcalorimetric measurements of the metabolic heat showed that intracellular GSH attenuates the toxicity of uranium at a concentration in the range of 10-150 μM. Isothermal titration calorimetry revealed the endothermic binding of U(VI) to the carboxyl group(s) of GSH. The data indicate that the primary detoxifying mechanism is the intracellular sequestration of carboxyl-coordinated U(VI) into an insoluble complex with GSH.

Induction of glutathione synthesis in human hepatocytes by acute and chronic arsenic exposure: Differential roles of mitogen-activated protein kinases

International Nuclear Information System (INIS)

Hou, Yongyong; Wang, Yi; Wang, Huihui; Xu, Yuanyuan

2014-01-01

Highlights: • Arsenic exposure increased intracellular levels of glutathione. • Mitogen-activated protein kinases were involved in glutathione homeostasis. • ERK contributed to glutathione synthesis during acute arsenic exposure. • Glutathione synthesis was regulated by p38 at least in part independent of NRF2 during chronic arsenic exposure. - Abstract: Glutathione (GSH) is a vital component of antioxidant defense which protects cells from toxic insults. Previously we found intracellular GSH was involved in cell resistance against arsenic-induced cytotoxicity. However, molecular mechanisms of GSH homeostasis during arsenic exposure are largely undefined. Here, we investigated roles of mitogen-activated protein kinases (MAPKs) in GSH synthesis pathway with two arsenic exposure strategies by using Chang human hepatocytes. In one strategy, acute arsenic exposure (20 μM, 24 h) was applied, as MAPK signaling is generally considered to be transient. In the other one, chronic arsenic exposure (500 nM, 20 weeks) was applied, which mimicked the general human exposure to arsenic. We found that acute arsenic exposure activated extracellular signal-regulated 1/2 kinases (ERK1/2) and c-Jun N-terminal kinase (JNK) in parallel with increased transcription and nuclear translocation of factor-erythroid 2-related factor 2 (NRF2) and enhanced expression of γ-glutamyl cysteine ligase catalytic subunit (GCLC), resulting in elevated intracellular GSH levels. Specific ERK inhibitor abolished arsenic-induced NRF2 nuclear translocation and GSH synthesis. During chronic arsenic exposure which induced a malignant cellular phenotype, continuous p38 activation and NRF2 nuclear translocation were observed with enhanced GSH synthesis. Specific p38 inhibitor attenuated arsenic-enhanced GSH synthesis without changing NRF2 nuclear translocation. Taken together, our results indicate MAPK pathways play an important role in cellular GSH homeostasis in response to arsenic. However, the

Expression of Glutathione Peroxidase and Glutathione Reductase and Level of Free Radical Processes under Toxic Hepatitis in Rats

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Igor Y. Iskusnykh

2013-01-01

Full Text Available Correlation between intensity of free radical processes estimated by biochemiluminesce parameters, content of lipoperoxidation products, and changes of glutathione peroxidase (GP, EC 1.11.1.9 and glutathione reductase (GR, EC 1.6.4.2 activities at rats liver injury, after 12, 36, 70, 96, 110, and 125 hours & tetrachloromethane administration have been investigated. The histological examination of the liver sections of rats showed that prominent hepatocytes with marked vacuolisation and inflammatory cells which were arranged around the necrotic tissue are more at 96 h after exposure to CCl4. Moreover maximum increase in GR and GP activities, 2.1 and 2.5 times, respectively, was observed at 96 h after exposure to CCl4, what coincided with the maximum of free radical oxidation processes. Using a combination of reverse transcription and real-time polymerase chain reaction, expression of the glutathione peroxidase and glutathione reductase genes (Gpx1 and Gsr was analyzed by the determination of their respective mRNAs in the rat liver tissue under toxic hepatitis conditions. The analyses of Gpx1 and Gsr expression revealed that the transcript levels increased in 2.5- and 3.0-folds, respectively. Western blot analysis revealed that the amounts of hepatic Gpx1 and Gsr proteins increased considerably after CCl4 administration. It can be proposed that the overexpression of these enzymes could be a mechanism of enhancement of hepatocytes tolerance to oxidative stress.

Glutathione oxidation in response to intracellular H2O2: Key but overlapping roles for dehydroascorbate reductases.

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Rahantaniaina, Marie-Sylviane; Li, Shengchun; Chatel-Innocenti, Gilles; Tuzet, Andrée; Mhamdi, Amna; Vanacker, Hélène; Noctor, Graham

2017-08-03

Glutathione is a pivotal molecule in oxidative stress, during which it is potentially oxidized by several pathways linked to H 2 O 2 detoxification. We have investigated the response and functional importance of 3 potential routes for glutathione oxidation pathways mediated by glutathione S-transferases (GST), glutaredoxin-dependent peroxiredoxins (PRXII), and dehydroascorbate reductases (DHAR) in Arabidopsis during oxidative stress. Loss-of-function gstU8, gstU24, gstF8, prxIIE and prxIIF mutants as well as double gstU8 gstU24, gstU8 gstF8, gstU24 gstF8, prxIIE prxIIF mutants were obtained. No mutant lines showed marked changes in their phenotype and glutathione profiles in comparison to the wild-type plants in either optimal conditions or oxidative stress triggered by catalase inhibition. By contrast, multiple loss of DHAR functions markedly decreased glutathione oxidation triggered by catalase deficiency. To assess whether this effect was mediated directly by loss of DHAR enzyme activity, or more indirectly by upregulation of other enzymes involved in glutathione and ascorbate recycling, we measured expression of glutathione reductase (GR) and expression and activity of monodehydroascorbate reductases (MDHAR). No evidence was obtained that either GRs or MDHARs were upregulated in plants lacking DHAR function. Hence, interplay between different DHARs appears to be necessary to couple ascorbate and glutathione pools and to allow glutathione-related signaling during enhanced H 2 O 2 metabolism.

Upregulation of cellular glutathione levels in human ABCB5- and murine Abcb5-transfected cells.

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Kondo, Shingo; Hongama, Keita; Hanaya, Kengo; Yoshida, Ryota; Kawanobe, Takaaki; Katayama, Kazuhiro; Noguchi, Kohji; Sugimoto, Yoshikazu

2015-12-15

Previously, we have demonstrated that human ABCB5 is a full-sized ATP-binding cassette transporter that shares strong homology with ABCB1/P-glycoprotein. ABCB5-transfected cells showed resistance to taxanes and anthracyclines. Herein, we further screened ABCB5 substrates, and explored the mechanism of resistance. Sensitivity of the cells to test compounds was evaluated using cell growth inhibition assay. Cellular levels of buthionine sulfoximine (BSO), glutathione and amino acids were measured using HPLC and an enzyme-based assay. Cellular and vesicular transport of glutathione was evaluated by a radiolabeled substrate. Expression levels of glutathione-metabolizing enzymes were assessed by RT-PCR. Human ABCB5-transfected 293/B5-11 cells and murine Abcb5-transfected 293/mb5-8 cells showed 6.5- and 14-fold higher resistance to BSO than the mock-transfected 293/mock cells, respectively. BSO is an inhibitor of gamma-glutamylcysteine ligase (GCL), which is a key enzyme of glutathione synthesis. 293/B5-11 and 293/mb5-8 cells also showed resistance to methionine sulfoximine, another GCL inhibitor. A cellular uptake experiment revealed that BSO accumulation in 293/B5-11 and 293/mb5-8 cells was similar to that in 293/mock cells, suggesting that BSO is not an ABCB5 substrate. The cellular glutathione content in 293/B5-11 and 293/mb5-8 cells was significantly higher than that in 293/mock cells. Evaluation of the BSO effect on the cellular glutathione content showed that compared with 293/mock cells the BSO concentration required for a 50 % reduction in glutathione content in 293/B5-11 and 293/mb5-8 cells was approximately 2- to 3-fold higher. This result suggests that the BSO resistance of the ABCB5- and Abcb5-transfected cells can be attributed to the reduced effect of BSO on the transfectants. Cellular and vesicular transport assays showed that the transport of radiolabeled glutathione in 293/B5-11 cells was similar to that in 293/mock cells. The mRNA expression of genes

Labor Augmentation with Oxytocin Decreases Glutathione Level

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Naomi Schneid-Kofman

2009-01-01

Full Text Available Objective. To compare oxidative stress following spontaneous vaginal delivery with that induced by Oxytocin augmented delivery. Methods. 98 women recruited prior to labor. 57 delivered spontaneously, while 41 received Oxytocin for augmentation of labor. Complicated deliveries and high-risk pregnancies were excluded. Informed consent was documented. Arterial cord blood gases, levels of Hematocrit, Hemoglobin, and Bilirubin were studied. Glutathione (GSH concentration was measured by a spectroscopic method. Plasma and red blood cell (RBC levels of Malondialdehyde indicated lipid peroxidation. RBC uptake of phenol red denoted cell penetrability. SPSS data analysis was used. Results. Cord blood GSH was significantly lower in the Oxytocin group (2.3±0.55 mM versus 2.55±0.55 mM, =.01. No differences were found in plasma or RBC levels of MDA or in uptake of Phenol red between the groups. Conclusion. Lower GSH levels following Oxytocin augmentation indicate an oxidative stress, though selected measures of oxidative stress demonstrate no cell damage.

Combined radiation-protective and radiation-sensitizing agents. III. Radiosensitization by misonidazole as a function of concentrations of endogenous glutathione or exogenous thiols

International Nuclear Information System (INIS)

Koch, C.J.; Stobbe, C.C.; Baer, K.A.

1986-01-01

Radiosensitization of V79 Chinese hamster fibroblasts by 0.5 mM misonidazole is a smooth function of endogenous glutathione (GSH) levels as modulated upwards by pre-incubation in medium containing cysteamine, or downwards by pre-incubation in medium containing buthionine sulfoximine. The enhancement ratio (radiation sensitivity in nitrogen/radiation sensitivity in nitrogen +/- sensitizer or thiol) varies from 1.3 at 12 mM to 2.25 at less than 0.1 mM endogenous GSH. The enhanced radiosensitivity of thiol-depleted hypoxic cells is reversed when exogenous thiols are added, and for equivalent ER, the exogenous thiol concentrations are much lower than the endogenous GSH concentrations. Measurement of intracellular drug concentrations amplified rather than diminished the above discrepancy, since intracellular concentrations of cysteamine were lower and glutathione much lower than the extracellular concentrations. Three possible explanations are addressed: an external membrane component of damage is involved, long-range protection to DNA target radicals is possible from outside the cell (e.g., donation of electrons), and (c) endogenous glutathione is not in a free or exchangeable state (e.g., bound)

Correction of glutathione deficiency in the lower respiratory tract of HIV seropositive individuals by glutathione aerosol treatment.

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Holroyd, K J; Buhl, R; Borok, Z; Roum, J H; Bokser, A D; Grimes, G J; Czerski, D; Cantin, A M; Crystal, R G

1993-10-01

Concentrations of glutathione, a ubiquitous tripeptide with immune enhancing and antioxidant properties, are decreased in the blood and lung epithelial lining fluid of human immunodeficiency virus (HIV) seropositive individuals. Since the lung is the most common site of infection in those who progress to AIDS it is rational to consider whether it is possible to safely augment glutathione levels in the epithelial lining fluid of HIV seropositive individuals, thus potentially improving local host defence. Purified reduced glutathione was delivered by aerosol to HIV seropositive individuals (n = 14) and the glutathione levels in lung epithelial lining fluid were compared before and at one, two, and three hours after aerosol administration. Before treatment total glutathione concentrations in the epithelial lining fluid were approximately 60% of controls. After three days of twice daily doses each of 600 mg reduced glutathione, total glutathione levels in the epithelial lining fluid increased and remained in the normal range for at least three hours after treatment. Strikingly, even though > 95% of the glutathione in the aerosol was in its reduced form, the percentage of oxidised glutathione in epithelial lining fluid increased from 5% before treatment to about 40% three hours after treatment, probably reflecting the use of glutathione as an antioxidant in vivo. No adverse effects were observed. It is feasible and safe to use aerosolised reduced glutathione to augment the deficient glutathione levels of the lower respiratory tract of HIV seropositive individuals. It is rational to evaluate further the efficacy of this tripeptide in improving host defence in HIV seropositive individuals.

In vitro effects of four native Brazilian medicinal plants in CYP3A4 mRNA gene expression, glutathione levels and P-glycoprotein activity.

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Andre Luis Dias Araujo Mazzari

2016-08-01

Full Text Available Erythrina mulungu Benth. (Fabaceae, Cordia verbenacea A. DC. (Boraginaceae, Solanum paniculatum L. (Solanaceae and Lippia sidoides Cham. (Verbenaceae are medicinal plants species native to Brazil shortlisted by the Brazilian National Health System for future clinical use. However, nothing is known about their effects in metabolic and transporter proteins, which could potentially lead to herb-drug interactions (HDI. In this work we assess non-toxic concentrations (100μg/mL of their infusions for their in vitro ability to modulate CYP3A4 mRNA gene expression and intracellular glutathione levels in HepG2 cells, as well as P-glycoprotein (P-gp activity in vincristine-resistant Caco-2 cells (Caco-2 VCR. Their mechanisms of action were further studied by measuring the activation of human pregnane X receptor (hPXR in transiently co-transfected HeLa cells and the inhibition of γ-glutamyl transferase (GGT in HepG2 cells. Our results show that P-gp activity was not affected in any case and that only Solanum paniculatum was able to significantly change CYP3A4 mRNA gene expression (two-fold decrease, p<0.05, this being correlated with an antagonist effect upon hPXR (EC50 = 0.38mg/mL. Total intracellular glutathione levels were significantly depleted by exposure to Solanum paniculatum (-44%, p<0.001, Lippia sidoides (-12%, p<0.05 and Cordia verbenacea (-47%, p<0.001. The later plant extract was able to decrease GGT activity (-48%, p<0.01. In conclusion, this preclinical study shows that the administration of some of these herbal medicines may be able to cause disturbances to metabolic mechanisms in vitro. Although Erythrina mulungu appears safe in our tests, active pharmacovigilance is recommended for the other three species, especially in the case of Solanum paniculatum.

Survival curves of irradiated glutathione-deficient human fibroblasts: indication of a reduced enhancement of radiosensitivity by oxygen and misonidazole

International Nuclear Information System (INIS)

Midander, J.; Deschavanne, P.J.; Malaise, E.P.; Revesz, L.

1982-01-01

Fibroblasts derived from a patient with 5-oxoprolinuria are genetically deficient in glutathione synthetase. This deficiency causes a dramatic decrease in intracellular glutathione (GSH) level. The radiosensitivity of GSH deficient cells (GSH) was studied in vitro using colony forming ability as an endpoint. Cells with normal GSH level, obtained from the healthy brother of the patient, were used as controls. When irradiated in 95% air-5% CO 2 , GSH - cells are slightly but significantly more radiosensitive than GSH + controls (dose modifying factor (DMF) of 1.2). When irradiated in argon, the survival curve of GSH - cells indicates an oxygen enhancement ratio (OER) of 1.5 when compared to the curve obtained in oxic conditions. The OER of control cells in the same conditions is 2.9. In comparison to results obtained in air, 100% oxygen moderately increases the radiosensitivity of GSH + cells (DMF 1,23), while it has a very low effect on GSH - cells (DMF 1.06). These results suggest that intracellular GSH plays an essential protective role in hypoxia, its effect is reduced in air and practically disappears in 100% oxygen. When cells are incubated with 8 mM misonidazole 2 hours before irradiation, the drug has a much greater sensitizing effect on GSH + cells (DMF 2.33) than on GSH - cells (DMF 1.55). The results demonstrate that intracellular GSH level plays a major role in the response of hypoxic cells, irradiated either alone or in the presence of misonidazole

The use of drugs which deplete intracellular glutathione in hypoxic cell radiosensitization

International Nuclear Information System (INIS)

Bump, E.A.; Yu, N.Y.; Brown, J.M.

1982-01-01

Diethylmaleate (DEM) is a thiol-biding reagent with specificity toward glutathione. Treatment of chinese hamster ovary (CHO) cells in vitro with 2 x 10 -4 M DEM for one hour results in a decrease in glutathione content to less than 5% of control, without cytotoxicity. This treatment results in dose-modifying sensitization to radiation under hypoxic conditions, with no effect on the shoulder of the radiation survival curve. No effect on the radiation sensitivity of oxygenated cells was seen. DEM pretreatment enhances the radiosensitization of hypoxic cells by misonidazole, as well. Similar results were obtained in vivo with EMT6 tumors in BALB/c mice. Analysis of DNA damage by the alkaline elution assay indicates that DEM enhances radiation-induced single-strand breaks, but does not significantly affect repair, while diamide and N-ethylmaleimide inhibit repair, in addition to enhancing radiation-induced single-strand breaks

Low levels of glutathione are sufficient for survival of keratinocytes after UV irradiation and for healing of mouse skin wounds.

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Telorack, Michèle; Abplanalp, Jeannette; Werner, Sabine

2016-08-01

Reduced levels of the cellular antioxidant glutathione are associated with premature skin aging, cancer and impaired wound healing, but the in vivo functions of glutathione in the skin remain largely unknown. Therefore, we analyzed mice lacking the modifier subunit of the glutamate cysteine ligase (Gclm), the enzyme that catalyzes the rate-limiting step of glutathione biosynthesis. Glutathione levels in the skin of these mice were reduced by 70 %. However, neither skin development and homeostasis, nor UVA- or UVB-induced apoptosis in the epidermis were affected. Histomorphometric analysis of excisional wounds did not reveal wound healing abnormalities in young Gclm-deficient mice, while the area of hyperproliferative epithelium as well as keratinocyte proliferation were affected in aged mice. These findings suggest that low levels of glutathione are sufficient for wound repair in young mice, but become rate-limiting upon aging.

Antioxidant role of glutathione S-transferases: 4-Hydroxynonenal, a key molecule in stress-mediated signaling

International Nuclear Information System (INIS)

Singhal, Sharad S.; Singh, Sharda P.; Singhal, Preeti; Horne, David; Singhal, Jyotsana; Awasthi, Sanjay

2015-01-01

determinants of the intracellular concentration of 4HNE. • Higher concentrations of 4HNE promote apoptosis whereas lower promote proliferation. • Stress-mediated signaling can be modulated by the α-class glutathione S-transferase. • Genotoxic effect of 4HNE may be ameliorated by modulating the cellular GSH levels. • RLIP76 (RalBP1) mediates ATP-dependent transport of GSH-conjugate of 4HNE (GSHNE).

Astro research fellowship: the role of bcl-2 and glutathione in an antioxidant pathway to prevent radiation-induced apoptosis

International Nuclear Information System (INIS)

Vlachaki, Maria T.; Meyn, Raymond E.

1998-01-01

Purpose: The expression of the bcl-2 proto-oncogene has been associated with resistance to radiation-induced apoptosis. There is evidence that the bcl-2 protein acts in an antioxidant pathway to block the effects of reactive oxygen species that mediate apoptosis possibly by increasing the levels of intracellular glutathione. Our hypothesis is that pretreatment of radiation-sensitive cells, known to lack bcl-2 expression, with antioxidants will reduce radiation-induced apoptosis. For this purpose, the apoptotic response to radiation and the intracellular levels of GSH were tested before and after pretreatment with antioxidants in two murine lymphoma cell lines, a radiation-resistant, bcl-2- expressing (LY-ar) line and a radiation-sensitive, non-bcl-2-expressing (LY-as) line. Methods and Materials: LY-ar and LY-as cells were irradiated at 0,1,2,3, and 4 hours before collection. The intracellular levels of reduced (GSH) and oxidized (GSSG) glutathione were determined by the use of the fluorescent dye o-phthalaldehyde. LY-as cells were treated with GSH ethyl-ester for 1 and 2 hours after irradiation. Apoptotic response was measured by the DNA fragmentation assay. The radiation dose was 2.5 Gy. Results: After irradiation, the apoptotic rate of LY-ar and LY-as cells was 10-20% and 50-70% respectively. LY-ar cells had higher intracellular GSH and GSSG levels compared to LY-as cells by 69.9% and 91.9% respectively and the GSH/GSSG ratio in LY-ar and LY-as cells was 15.09 and 17.09 respectively. GSH levels did not change during the first 2 hours after irradiation; however, there was a 49% and 84% reduction at 3 and 4 hours after irradiation, respectively, times at which the LY-as cells have already fragmented their DNA. Treatment of LY-as cells with GSH ethyl-ester at a concentration of 7 mM for 1 and 2 hours resulted in 70% and 231% increases in the intracellular GSH levels respectively. Treatment of LY-as cells with GSH ethyl-ester for 1 and 2 hours also conferred a 25

Glutathione in Cancer Cell Death

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Ortega, Angel L. [Department of Physiology, Faculty of Medicine and Odontology, University of Valencia, 17 Av. Blasco Ibanez, 46010 Valencia (Spain); Mena, Salvador [Green Molecular SL, Pol. Ind. La Coma-Parc Cientific, 46190 Paterna, Valencia (Spain); Estrela, Jose M., E-mail: jose.m.estrela@uv.es [Department of Physiology, Faculty of Medicine and Odontology, University of Valencia, 17 Av. Blasco Ibanez, 46010 Valencia (Spain)

2011-03-11

Glutathione (L-γ-glutamyl-L-cysteinyl-glycine; GSH) in cancer cells is particularly relevant in the regulation of carcinogenic mechanisms; sensitivity against cytotoxic drugs, ionizing radiations, and some cytokines; DNA synthesis; and cell proliferation and death. The intracellular thiol redox state (controlled by GSH) is one of the endogenous effectors involved in regulating the mitochondrial permeability transition pore complex and, in consequence, thiol oxidation can be a causal factor in the mitochondrion-based mechanism that leads to cell death. Nevertheless GSH depletion is a common feature not only of apoptosis but also of other types of cell death. Indeed rates of GSH synthesis and fluxes regulate its levels in cellular compartments, and potentially influence switches among different mechanisms of death. How changes in gene expression, post-translational modifications of proteins, and signaling cascades are implicated will be discussed. Furthermore, this review will finally analyze whether GSH depletion may facilitate cancer cell death under in vivo conditions, and how this can be applied to cancer therapy.

Shikonin protects dopaminergic cell line PC12 against 6-hydroxydopamine-mediated neurotoxicity via both glutathione-dependent and independent pathways and by inhibiting apoptosis.

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Esmaeilzadeh, Emran; Gardaneh, Mossa; Gharib, Ehsan; Sabouni, Farzaneh

2013-08-01

We have investigated the mechanism of shikonin function on protection of dopaminergic neurons against 6-OHDA-induced neurotoxicity. Treatment of rat pheochromocytoma cell line PC12 by serial dilutions of shikonin determined 10 μM of the compound as its optimum concentration for protection saving nearly 70 % of the cells against toxicity. Reverse transcription-PCR analysis of shikonin-treated cells showed threefold increase in mRNA levels of glutathione peroxidase-1 (GPX-1) as a representative component of the intracellular anti-oxidant defense system. To elucidate shikonin-GPX1 relationships and maximize protection, we transduced PC12 cells using recombinant lentivirus vectors that harbored GPX-1 coding sequence. This change upregulated GPX-1 expression, increased peroxidase activity and made neuronal cells resistant to 6-OHDA-mediated toxicity. More importantly, addition of shikonin to GPX1-overexpressing PC12 cells augmented GPX-1 protein content by eightfold leading to fivefold increase of enzymatic activity, 91 % cell survival against neurotoxicity and concomitant increases in intracellular glutathione (GSH) levels. Depletion of intracellular GSH rendered all cell groups highly susceptible to toxicity; however, shikonin was capable of partially saving them. Subsequently, GSH-independent superoxide dismutase mRNA was found upregulated by shikonin. As signs of apoptosis inhibition, the compound upregulated Bcl-2, downregulated Bax, and prevented cell nuclei from undergoing morphological changes typical of apoptosis. Also, a co-staining method demonstrated GPX-1 overexpression significantly increases the percent of live cells that is maximized by shikonin treatment. Our data indicate that shikonin as an antioxidant compound protects dopaminergic neurons against 6-OHDA toxicity and enhances their survival via both glutathione-dependent and direct anti-apoptotic pathways.

Acrolein toxicity involves oxidative stress caused by glutathione depletion in the yeast Saccharomyces cerevisiae.

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Kwolek-Mirek, M; Bednarska, S; Bartosz, G; Biliński, T

2009-08-01

Exposure of yeast cells to allyl alcohol results in intracellular production of acrolein. The toxicity of so formed acrolein involves oxidative stress, as (1) strains deficient in antioxidant defense are hypersensitive to allyl alcohol, (2) exposure to allyl alcohol increases the level of thiobarbituric-acid-reactive substances and decreases glutathione level in the cells, (3) hypoxic and anoxic atmosphere and antioxidants protect against allyl alcohol toxicity, and (4) allyl alcohol causes activation of Yap1p. No increased formation of reactive oxygen species was detected in cells exposed to allyl alcohol, so oxidative stress is due to depletion of cellular thiols and thus alteration in the redox state of yeast cells.

Estrogen-induced disruption of intracellular iron metabolism leads to oxidative stress, membrane damage, and cell cycle arrest in MCF-7 cells.

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Bajbouj, Khuloud; Shafarin, Jasmin; Abdalla, Maher Y; Ahmad, Iman M; Hamad, Mawieh

2017-10-01

It is well established that several forms of cancer associate with significant iron overload. Recent studies have suggested that estrogen (E2) disrupts intracellular iron homeostasis by reducing hepcidin synthesis and maintaining ferroportin integrity. Here, the ability of E2 to alter intracellular iron status and cell growth potential was investigated in MCF-7 cells treated with increasing concentrations of E2. Treated cells were assessed for intracellular iron status, the expression of key proteins involved in iron metabolism, oxidative stress, cell survival, growth, and apoptosis. E2 treatment resulted in a significant reduction in hepcidin expression and a significant increase in hypoxia-inducible factor 1 alpha, ferroportin, transferrin receptor, and ferritin expression; a transient decrease in labile iron pool; and a significant increase in total intracellular iron content mainly at 20 nM/48 h E2 dose. Treated cells also showed increased total glutathione and oxidized glutathione levels, increased superoxide dismutase activity, and increased hemoxygenase 1 expression. Treatment with E2 at 20 nM for 48 h resulted in a significant reduction in cell growth (0.35/1 migration rate) and decreased cell survival (iron metabolism and precipitates adverse effects concerning cell viability, membrane integrity, and growth potential.

Glutathione.

Science.gov (United States)

Noctor, Graham; Queval, Guillaume; Mhamdi, Amna; Chaouch, Sejir; Foyer, Christine H

2011-01-01

Glutathione is a simple sulfur compound composed of three amino acids and the major non-protein thiol in many organisms, including plants. The functions of glutathione are manifold but notably include redox-homeostatic buffering. Glutathione status is modulated by oxidants as well as by nutritional and other factors, and can influence protein structure and activity through changes in thiol-disulfide balance. For these reasons, glutathione is a transducer that integrates environmental information into the cellular network. While the mechanistic details of this function remain to be fully elucidated, accumulating evidence points to important roles for glutathione and glutathione-dependent proteins in phytohormone signaling and in defense against biotic stress. Work in Arabidopsis is beginning to identify the processes that govern glutathione status and that link it to signaling pathways. As well as providing an overview of the components that regulate glutathione homeostasis (synthesis, degradation, transport, and redox turnover), the present discussion considers the roles of this metabolite in physiological processes such as light signaling, cell death, and defense against microbial pathogen and herbivores.

In Vivo Monitoring of pH, Redox Status, and Glutathione Using L-Band EPR for Assessment of Therapeutic Effectiveness in Solid Tumors

Science.gov (United States)

Bobko, Andrey A.; Eubank, Timothy D.; Voorhees, Jeffrey L.; Efimova, Olga V.; Kirilyuk, Igor A.; Petryakov, Sergey; Trofimiov, Dmitrii G.; Marsh, Clay B.; Zweier, Jay L.; Grigor’ev, Igor A.; Samouilov, Alexandre; Khramtsov, Valery V.

2011-01-01

Approach for in vivo real-time assessment of tumor tissue extracellular pH (pHe), redox, and intracellular glutathione based on L-band EPR spectroscopy using dual function pH and redox nitroxide probe and disulfide nitroxide biradical, is described. These parameters were monitored in PyMT mice bearing breast cancer tumors during treatment with granulocyte macrophage colony-stimulating factor. It was observed that tumor pHe is about 0.4 pH units lower than that in normal mammary gland tissue. Treatment with granulocyte macrophage colony-stimulating factor decreased the value of pHe by 0.3 units compared with PBS control treatment. Tumor tissue reducing capacity and intracellular glutathione were elevated compared with normal mammary gland tissue. Granulocyte macrophage colony-stimulating factor treatment resulted in a decrease of the tumor tissue reducing capacity and intracellular glutathione content. In addition to spectroscopic studies, pHe mapping was performed using recently proposed variable frequency proton–electron double-resonance imaging. The pH mapping superimposed with MRI image supports probe localization in mammary gland/tumor tissue, shows high heterogeneity of tumor tissue pHe and a difference of about 0.4 pH units between average pHe values in tumor and normal mammary gland. In summary, the developed multifunctional approach allows for in vivo, noninvasive pHe, extracellular redox, and intracellular glutathione content monitoring during investigation of various therapeutic strategies for solid tumors. Magn Reson Med 000:000–000, 2011. PMID:22113626

From glutathione transferase to pore in a CLIC

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Cromer, B A; Morton, C J; Parker, M W; 10.1007/s00249-002-0219-1

2002-01-01

Many plasma membrane chloride channels have been cloned and characterized in great detail. In contrast, very little is known about intracellular chloride channels. Members of a novel class of such channels, called the CLICs (chloride intracellular channels), have been identified over the last few years. A striking feature of the CLIC family of ion channels is that they can exist in a water- soluble state as well as a membrane-bound state. A major step forward in understanding the functioning of these channels has been the recent crystal structure determination of one family member, CLIC1. The structure confirms that CLICs are members of the glutathione S- transferase superfamily and provides clues as to how CLICs can insert into membranes to form chloride channels. (69 refs).

Intracellular glutathione status regulates mouse bone marrow monocyte-derived macrophage differentiation and phagocytic activity

International Nuclear Information System (INIS)

Kim, Jin-Man; Kim, Hyunsoo; Kwon, Soon Bok; Lee, Soo Young; Chung, Sung-Chang; Jeong, Dae-Won; Min, Byung-Moo

2004-01-01

Although a redox shift can regulate the development of cells, including proliferation, differentiation, and survival, the role of the glutathione (GSH) redox status in macrophage differentiation remains unclear. In order to elucidate the role of a redox shift, macrophage-like cells were differentiated from the bone marrow-derived monocytes that were treated with a macrophage colony stimulating factor (M-CSF or CSF-1) for 3 days. The macrophagic cells were characterized by a time-dependent increase in three major symptoms: the number of phagocytic cells, the number of adherent cells, and the mRNA expression of c-fms, a M-CSF receptor that is one of the macrophage-specific markers and mediates development signals. Upon M-CSF-driven macrophage differentiation, the GSH/GSSG ratio was significantly lower on day 1 than that observed on day 0 but was constant on days 1-3. To assess the effect of the GSH-depleted and -repleted status on the differentiation and phagocytosis of the macrophages, GSH depletion by BSO, a specific inhibitor of the de novo GSH synthesis, inhibited the formation of the adherent macrophagic cells by the down-regulation of c-fms, but did not affect the phagocytic activity of the macrophages. To the contrary, GSH repletion by the addition of NAC, which is a GSH precursor, or reduced GSH in media had no effect on macrophage differentiation, and led to a decrease in the phagocytic activity. Furthermore, we observed that there is checkpoint that is capable of releasing from the inhibition of the formation of the adherent macrophagic cells according to GSH depletion by BSO. Summarizing, these results indicate that the intracellular GSH status plays an important role in the differentiation and phagocytosis of macrophages

Human gastric signet ring carcinoma (KATO-III) cell apoptosis induced by Vitex agnus-castus fruit extract through intracellular oxidative stress.

Science.gov (United States)

Ohyama, Kunio; Akaike, Takenori; Imai, Masahiko; Toyoda, Hiroo; Hirobe, Chieko; Bessho, Toshio

2005-07-01

We have previously reported that an ethanol extract of the dried ripe fruit of Vitex agnus-castus (Vitex) displays cytotoxic activity against certain kinds of human cancer cell line resulting in the induction of apoptosis. In this paper, we investigate the molecular mechanism of apoptosis induced by Vitex using a human gastric signet ring carcinoma cell line, KATO-III. DNA fragmentation was observed in Vitex-treated KATO-III cells in a time- and dose-dependent manner. DNA fragmentation was accompanied by the following phenomena: elevation in the level of hemeoxygenase-1 protein and thioredoxin reductase mRNA; repression of Mn-superoxide dismutase and catalase mRNAs; release of cytochrome c from mitochondria into the cytosol; activation of caspases-8, -9 and -3; decrease in the level of Bcl-2, Bcl-XL and Bid protein; increase in the level of Bad protein. The intracellular oxidized state, measured using 2',7'-dichlorofluorescin diacetate, increased after Vitex treatment. While the amount of intracellular GSH decreased significantly after treatment with Vitex, the level of GSSG was unaffected. Furthermore, no significant perturbation in the amount of proteins/mRNAs related to glutathione metabolism could be detected. These apoptotic alterations induced by exposure to Vitex were blocked by the presence of an anti-oxidative reagent, N-acetyl-l-cysteine, or the addition of exogenous GSH. Our results demonstrate that intracellular oxidative stress and mitochondrial membrane damage is responsible for Vitex-induced apoptosis, which may be mediated by a diminution of reduced type glutathione within the cell.

The influence of single application of paracetamol and/or N-acetylcysteine on rats in subchronic exposition to trichloroethylene vapours. II. Effect on hepatic glutathione level

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Danuta Plewka

2012-09-01

Full Text Available Background: Feature of modern existing hazards both environmental and occupational is cumulative exposure often leading to unexpected response of the organism resulting, among other things, in interactions with cytochrome P450 system involved in biotransformation of trichloroethylene and paracetamol. Hepatotoxity of paracetamol is closely connected with hepatic glutathione level. „In therapy of acute paracetamol poisoning application of N-acetylcysteine as a factor, which protects GSH level in cells, is recommended.” Materials and method: Tests were performed on rats which were treated with trichloroethylene, paracetamol and/or N-acetylcysteine. In rat liver total level of glutathione was determined i.e. reduced and oxidized form. Results: Paracetamol just after completion of the exposure affected the glutathione level. Trichloroethylene throughout the period of observation stimulated growth of glutathione level in liver. N-acetylcysteine didn’t have any influence on the level of investigated tripeptyde. Conclusions: N-acetylcysteine removes negative effect of paracetamol especially when it’s applied with 2-hour delay. After exposure for trichloethylene immediate application of N-acetylcysteine caused noticeable lowering of glutathione level. Cumulative exposure for three xenobiotics had positive influence for glutathione level in rat liver.

Glutathione Redox System in β-Thalassemia/Hb E Patients

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Ruchaneekorn W. Kalpravidh

2013-01-01

Full Text Available β-thalassemia/Hb E is known to cause oxidative stress induced by iron overload. The glutathione system is the major endogenous antioxidant that protects animal cells from oxidative damage. This study aimed to determine the effect of disease state and splenectomy on redox status expressed by whole blood glutathione (GSH/glutathione disulfide (GSSG and also to evaluate glutathione-related responses to oxidation in β-thalassemia/Hb E patients. Twenty-seven normal subjects and 25 β-thalassemia/Hb E patients were recruited and blood was collected. The GSH/GSSG ratio, activities of glutathione-related enzymes, hematological parameters, and serum ferritin levels were determined in individuals. Patients had high iron-induced oxidative stress, shown as significantly increased serum ferritin, a decreased GSH/GSSG ratio, and increased activities of glutathione-related enzymes. Splenectomy increased serum ferritin levels and decreased GSH levels concomitant with unchanged glutathione-related enzyme activities. The redox ratio had a positive correlation with hemoglobin levels and negative correlation with levels of serum ferritin. The glutathione system may be the body’s first-line defense used against oxidative stress and to maintain redox homeostasis in thalassemic patients based on the significant correlations between the GSH/GSSH ratio and degree of anemia or body iron stores.

Nanotoxicity of pure silica mediated through oxidant generation rather than glutathione depletion in human lung epithelial cells.

Science.gov (United States)

Akhtar, Mohd Javed; Ahamed, Maqusood; Kumar, Sudhir; Siddiqui, Huma; Patil, Govil; Ashquin, Mohd; Ahmad, Iqbal

2010-10-09

Though, oxidative stress has been implicated in silica nanoparticles induced toxicity both in vitro and in vivo, but no similarities exist regarding dose-response relationship. This discrepancy may, partly, be due to associated impurities of trace metals that may present in varying amounts. Here, cytotoxicity and oxidative stress parameters of two sizes (10 nm and 80 nm) of pure silica nanoparticles was determined in human lung epithelial cells (A549 cells). Both sizes of silica nanoparticles induced dose-dependent cytotoxicity as measured by MTT [3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide] and lactate dehydrogenase (LDH) assays. Silica nanoparticles were also found to induce oxidative stress in dose-dependent manner indicated by induction of reactive oxygen species (ROS) generation, and membrane lipid peroxidation (LPO). However, both sizes of silica nanoparticles had little effect on intracellular glutathione (GSH) level and the activities of glutathione metabolizing enzymes; glutathione reductase (GR) and glutathione peroxidase (GPx). Buthionine-[S,R]-sulfoximine (BSO) plus silica nanoparticles did not result in significant GSH depletion than that caused by BSO alone nor N-acetyl cysteine (NAC) afforded significant protection from ROS and LPO induced by silica nanoparticles. The rather unaltered level of GSH is also supported by finding no appreciable alteration in the level of GR and GPx. Our data suggest that the silica nanoparticles exert toxicity in A549 cells through the oxidant generation (ROS and LPO) rather than the depletion of GSH. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

Involvement of human glutathione S-transferase isoenzymes in the conjugation of cyclophosphamide metabolites with glutathione

NARCIS (Netherlands)

Dirven, H.A.A.M.; Ommen, B. van; Bladeren, P.J. van

1994-01-01

Alkylating agents can be detoxified by conjugation with glutathione (GSH). One of the physiological significances of this lies in the observation that cancer cells resistant to the cytotoxic effects of alkylating agents have higher levels of GSH and high glutathione S-transferase (GST) activity.

Glutathione synthesis and homeostasis in isolated type II alveolar cells

International Nuclear Information System (INIS)

Saito, K.; Warshaw, J.B.; Prough, R.A.

1986-01-01

After isolation of Type II cells from neonatal rat lung, the glutathione (GSH) levels in these cells were greatly depressed. The total glutathione content could be increased 5-fold within 12-24 h by incubating the cells in media containing sulfur amino acids. Similarly, the activity of γ-glutamyltranspeptidase was low immediately after isolation, but was increased 2-fold during the first 24 h culture. Addition of either GSH or GSSG to the culture media increased the GSH content of Type II cells 2-2.5-fold. Buthionine sulfoximine and NaF prevented this replenishment of GSH during 24 h culture. When the rates of de novo synthesis of GSH and GSSG from 35 S-cysteine were measured, the amounts of newly formed GSH decreased to 80% in the presence of GSH or GSSG. This suggests that exogenous GSH/GSSG can be taken up by the Type II cells to replenish the intracellular pool of GSH. Methionine was not as effective as cysteine in the synthesis of GSH. These results suggest that GSH levels in the isolated Type II cell can be maintained by de novo synthesis or uptake of exogenous GSH. Most of the GSH synthesized from cysteine, however, was excreted into the media of the cultured cells indicative of a potential role for the type II cell in export of the non-protein thiol

Glutathione enzyme and selenoprotein polymorphisms associate with mercury biomarker levels in Michigan dental professionals

Energy Technology Data Exchange (ETDEWEB)

Goodrich, Jaclyn M.; Wang, Yi [Department of Environmental Health Sciences, University of Michigan School of Public Health, 1415 Washington Heights, Ann Arbor, MI 48109 (United States); Gillespie, Brenda [Department of Biostatistics, University of Michigan School of Public Health, 1415 Washington Heights, Ann Arbor, MI 48109 (United States); Werner, Robert [Department of Environmental Health Sciences, University of Michigan School of Public Health, 1415 Washington Heights, Ann Arbor, MI 48109 (United States); Department of Physical Medicine and Rehabilitation, University of Michigan, 325 E. Eisenhower Parkway Suite 100, Ann Arbor, MI 48108 (United States); Franzblau, Alfred [Department of Environmental Health Sciences, University of Michigan School of Public Health, 1415 Washington Heights, Ann Arbor, MI 48109 (United States); Basu, Niladri, E-mail: niladri@umich.edu [Department of Environmental Health Sciences, University of Michigan School of Public Health, 1415 Washington Heights, Ann Arbor, MI 48109 (United States)

2011-12-15

Mercury is a potent toxicant of concern to both the general public and occupationally exposed workers (e.g., dentists). Recent studies suggest that several genes mediating the toxicokinetics of mercury are polymorphic in humans and may influence inter-individual variability in mercury accumulation. This work hypothesizes that polymorphisms in key glutathione synthesizing enzyme, glutathione s-transferase, and selenoprotein genes underlie inter-individual differences in mercury body burden as assessed by analytical mercury measurement in urine and hair, biomarkers of elemental mercury and methylmercury, respectively. Urine and hair samples were collected from a population of dental professionals (n = 515), and total mercury content was measured. Average urine (1.06 {+-} 1.24 ug/L) and hair mercury levels (0.49 {+-} 0.63 ug/g) were similar to national U.S. population averages. Taqman assays were used to genotype DNA from buccal swab samples at 15 polymorphic sites in genes implicated in mercury metabolism. Linear regression modeling assessed the ability of polymorphisms to modify the relationship between mercury biomarker levels and exposure sources (e.g., amalgams, fish consumption). Five polymorphisms were significantly associated with urine mercury levels (GSTT1 deletion), hair mercury levels (GSTP1-105, GSTP1-114, GSS 5 Prime ), or both (SEPP1 3 Prime UTR). Overall, this study suggests that polymorphisms in selenoproteins and glutathione-related genes may influence elimination of mercury in the urine and hair or mercury retention following exposures to elemental mercury (via dental amalgams) and methylmercury (via fish consumption). -- Highlights: Black-Right-Pointing-Pointer We explore the influence of 15 polymorphisms on urine and hair Hg levels. Black-Right-Pointing-Pointer Urine and hair Hg levels in dental professionals were similar to the US population. Black-Right-Pointing-Pointer GSTT1 and SEPP1 polymorphisms associated with urine Hg levels. Black

Glutathione enzyme and selenoprotein polymorphisms associate with mercury biomarker levels in Michigan dental professionals

International Nuclear Information System (INIS)

Goodrich, Jaclyn M.; Wang, Yi; Gillespie, Brenda; Werner, Robert; Franzblau, Alfred; Basu, Niladri

2011-01-01

Mercury is a potent toxicant of concern to both the general public and occupationally exposed workers (e.g., dentists). Recent studies suggest that several genes mediating the toxicokinetics of mercury are polymorphic in humans and may influence inter-individual variability in mercury accumulation. This work hypothesizes that polymorphisms in key glutathione synthesizing enzyme, glutathione s-transferase, and selenoprotein genes underlie inter-individual differences in mercury body burden as assessed by analytical mercury measurement in urine and hair, biomarkers of elemental mercury and methylmercury, respectively. Urine and hair samples were collected from a population of dental professionals (n = 515), and total mercury content was measured. Average urine (1.06 ± 1.24 ug/L) and hair mercury levels (0.49 ± 0.63 ug/g) were similar to national U.S. population averages. Taqman assays were used to genotype DNA from buccal swab samples at 15 polymorphic sites in genes implicated in mercury metabolism. Linear regression modeling assessed the ability of polymorphisms to modify the relationship between mercury biomarker levels and exposure sources (e.g., amalgams, fish consumption). Five polymorphisms were significantly associated with urine mercury levels (GSTT1 deletion), hair mercury levels (GSTP1-105, GSTP1-114, GSS 5′), or both (SEPP1 3′UTR). Overall, this study suggests that polymorphisms in selenoproteins and glutathione-related genes may influence elimination of mercury in the urine and hair or mercury retention following exposures to elemental mercury (via dental amalgams) and methylmercury (via fish consumption). -- Highlights: ► We explore the influence of 15 polymorphisms on urine and hair Hg levels. ► Urine and hair Hg levels in dental professionals were similar to the US population. ► GSTT1 and SEPP1 polymorphisms associated with urine Hg levels. ► Accumulation of Hg in hair following exposure from fish was modified by genotype. ► GSTP1, GSS

Corneal endothelial glutathione after photodynamic change

International Nuclear Information System (INIS)

Hull, D.S.; Riley, M.V.; Csukas, S.; Green, K.

1982-01-01

Rabbit corneal endothelial cells perfused with 5 X 10(-6)M rose bengal and exposed to incandescent light demonstrated no alteration of either total of or percent oxidized glutathione after 1 hr. Addition of 5400 U/ml catalase to the perfusing solution had no effect on total glutathione levels but caused a marked reduction in percent oxidized glutathione in corneas exposed to light as well as in those not exposed to light. Substitution of sucrose for glucose in the perfusing solution had no effect on total or percent oxidized glutathione. Perfusion of rabbit corneal endothelium with 0.5 mM chlorpromazine and exposure to ultraviolet (UV) light resulted in no change in total glutathione content. A marked reduction in percent oxidized glutathione occurred, however, in corneas perfused with 0.5 mM chlorpromazine both in the presence and absence of UV light. It is concluded that photodynamically induced swelling of corneas is not the result of a failure of the glutathione redox system

Effect of Nonsurgical Periodontal Therapy on Crevicular Fluid and Serum Glutathione Peroxidase Levels

Directory of Open Access Journals (Sweden)

Swati Pradeep Patel

2012-01-01

Full Text Available Background: Plasma glutathione peroxidase (eGPx is an important selenium containing antioxidant in human defense against oxidative stress. While crevicular fluid (GCF eGPx levels and its association with periodontal disease is well documented, there is no data on correlation of GCF and serum eGPx levels in chronic periodontitis. Hence this study was undertaken to further probe into the role of oxidative stress in periodontal diseases and effect of nonsurgical periodontal therapy (NSPT by correlating GCF and serum levels of eGPx.

The diverse roles of glutathione-associated cell resistance against hypericin photodynamic therapy

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Theodossis A. Theodossiou

2017-08-01

Full Text Available The diverse responses of different cancers to treatments such as photodynamic therapy of cancer (PDT have fueled a growing need for reliable predictive markers for treatment outcome. In the present work we have studied the differential response of two phenotypically and genotypically different breast adenocarcinoma cell lines, MCF7 and MDA-MB-231, to hypericin PDT (HYP-PDT. MDA-MB-231 cells were 70% more sensitive to HYP PDT than MCF7 cells at LD50. MCF7 were found to express a substantially higher level of glutathione peroxidase (GPX4 than MDA-MB-231, while MDA-MB-231 differentially expressed glutathione-S-transferase (GSTP1, mainly used for xenobiotic detoxification. Eighty % reduction of intracellular glutathione (GSH by buthionine sulfoximine (BSO, largely enhanced the sensitivity of the GSTP1 expressing MDA-MB-231 cells to HYP-PDT, but not in MCF7 cells. Further inhibition of the GSH reduction however by carmustine (BCNU resulted in an enhanced sensitivity of MCF7 to HYP-PDT. HYP loading studies suggested that HYP can be a substrate of GSTP for GSH conjugation as BSO enhanced the cellular HYP accumulation by 20% in MDA-MB-231 cells, but not in MCF7 cells. Studies in solutions showed that L-cysteine can bind the GSTP substrate CDNB in the absence of GSTP. This means that the GSTP-lacking MCF7 may use L-cysteine for xenobiotic detoxification, especially during GSH synthesis inhibition, which leads to L-cysteine build-up. This was confirmed by the lowered accumulation of HYP in both cell lines in the presence of BSO and the L-cysteine source NAC. NAC reduced the sensitivity of MCF7, but not MDA-MB-231, cells to HYP PDT which is in accordance with the antioxidant effects of L-cysteine and its potential as a GSTP substrate. As a conclusion we have herein shown that the different GSH based cell defense mechanisms can be utilized as predictive markers for the outcome of PDT and as a guide for selecting optimal combination strategies. Keywords

JS-K, a glutathione/glutathione S-transferase-activated nitric oxide releasing prodrug inhibits androgen receptor and WNT-signaling in prostate cancer cells.

Science.gov (United States)

Laschak, Martin; Spindler, Klaus-Dieter; Schrader, Andres J; Hessenauer, Andrea; Streicher, Wolfgang; Schrader, Mark; Cronauer, Marcus V

2012-03-30

Nitric oxide (NO) and its oxidative reaction products have been repeatedly shown to block steroid receptor function via nitrosation of zinc finger structures in the DNA-binding domain (DBD). In consequence NO-donors could be of special interest for the treatment of deregulated androgen receptor(AR)-signaling in castration resistant prostate cancer (CRPC). Prostate cancer (PCa) cells were treated with JS-K, a diazeniumdiolate derivate capable of generating large amounts of intracellular NO following activation by glutathione S-transferase. Generation of NO was determined indirectly by the detection of nitrate in tissue culture medium or by immunodetection of nitrotyrosine in the cytoplasm. Effects of JS-K on intracellular AR-levels were determined by western blotting. AR-dimerization was analyzed by mammalian two hybrid assay, nuclear translocation of the AR was visualized in PCa cells transfected with a green fluorescent AR-Eos fusion protein using fluorescence microscopy. Modulation of AR- and WNT-signalling by JS-K was investigated using reporter gene assays. Tumor cell proliferation following JS-K treatment was measured by MTT-Assay. The NO-releasing compound JS-K was shown to inhibit AR-mediated reporter gene activity in 22Rv1 CRPC cells. Inhibition of AR signaling was neither due to an inhibition of nuclear import nor to a reduction in AR-dimerization. In contrast to previously tested NO-donors, JS-K was able to reduce the intracellular concentration of functional AR. This could be attributed to the generation of extremely high intracellular levels of the free radical NO as demonstrated indirectly by high levels of nitrotyrosine in JS-K treated cells. Moreover, JS-K diminished WNT-signaling in AR-positive 22Rv1 cells. In line with these observations, castration resistant 22Rv1 cells were found to be more susceptible to the growth inhibitory effects of JS-K than the androgen dependent LNCaP which do not exhibit an active WNT-signaling pathway. Our results

In Vitro Reactivity of Carboxylic Acid-CoA Thioesters with Glutathione

DEFF Research Database (Denmark)

Sidenius, Ulrik; Skonberg, Christian; Olsen, Jørgen

2004-01-01

was to investigate whether a correlation could be found between the structure of acyl-CoA thioesters and their reactivities toward the tripeptide, glutathione (ç- Glu-Cys-Gly).  The  acyl-CoA  thioesters  of  eight  carboxylic  acids  (ibuprofen,  clofibric  acid, indomethacin,  fenbufen,  tolmetin,  salicylic  acid......The chemical reactivity of acyl-CoA thioesters toward nucleophiles has been demonstrated in several recent studies. Thus, intracellularly formed acyl-CoAs of xenobiotic carboxylic acids may react covalently with endogenous proteins and potentially lead to adverse effects. The purpose of this study......,  2-phenoxypropionic  acid,  and  (4-chloro-2-methyl-phenoxy)acetic  acid  (MCPA))  were  synthesized,  and  each  acyl-CoA  (0.5  mM)  was incubated with glutathione (5.0 mM) in 0.1 M potassium phosphate (pH 7.4, 37 °C). All of the acyl-CoAs reacted with glutathione to form the respective acyl...

Intracellular NAD(H) levels control motility and invasion of glioma cells.

NARCIS (Netherlands)

Horssen, R. van; Willemse, M.P.; Haeger, A.; Attanasio, F.; Guneri, T.; Schwab, A.; Stock, C.M.; Buccione, R.; Fransen, J.A.M.; Wieringa, B.

2013-01-01

Oncogenic transformation involves reprogramming of cell metabolism, whereby steady-state levels of intracellular NAD(+) and NADH can undergo dramatic changes while ATP concentration is generally well maintained. Altered expression of nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting

Cell internalizable and intracellularly degradable cationic polyurethane micelles as a potential platform for efficient imaging and drug delivery.

Science.gov (United States)

Ding, Mingming; Zeng, Xin; He, Xueling; Li, Jiehua; Tan, Hong; Fu, Qiang

2014-08-11

A cell internalizable and intracellularly degradable micellar system, assembled from multiblock polyurethanes bearing cell-penetrating gemini quaternary ammonium pendent groups in the side chain and redox-responsive disulfide linkages throughout the backbone, was developed for potential magnetic resonance imaging (MRI) and drug delivery. The nanocarrier is featured as a typical "cleavable core-internalizable shell-protective corona" architecture, which exhibits small size, positive surface charge, high loading capacity, and reduction-triggered destabilization. Furthermore, it can rapidly enter tumor cells and release its cargo in response to an intracellular level of glutathione, resulting in enhanced drug efficacy in vitro. The magnetic micelles loaded with superparamagnetic iron oxide (SPIO) nanoparticles demonstrate excellent MRI contrast enhancement, with T2 relaxivity found to be affected by the morphology of SPIO-clustering inside the micelle core. The multifunctional carrier with good cytocompatibility and nontoxic degradation products can serve as a promising theranostic candidate for efficient intracellular delivery of anticancer drugs and real-time monitoring of therapeutic effect.

Bioavailable Concentrations of Delphinidin and Its Metabolite, Gallic Acid, Induce Antioxidant Protection Associated with Increased Intracellular Glutathione in Cultured Endothelial Cells

Science.gov (United States)

Goszcz, Katarzyna; Deakin, Sherine J.; Duthie, Garry G.; Stewart, Derek

2017-01-01

Despite limited bioavailability and rapid degradation, dietary anthocyanins are antioxidants with cardiovascular benefits. This study tested the hypothesis that the antioxidant protection conferred by the anthocyanin, delphinidin, is mediated by modulation of endogenous antioxidant defences, driven by its degradation product, gallic acid. Delphinidin was found to degrade rapidly (t1/2 ~ 30 min), generating gallic acid as a major degradation product. Both delphinidin and gallic acid generated oxygen-centred radicals at high (100 μM) concentrations in vitro. In a cultured human umbilical vein endothelial cell model of oxidative stress, the antioxidant protective effects of both delphinidin and gallic acid displayed a hormesic profile; 100 μM concentrations of both were cytotoxic, but relatively low concentrations (100 nM–1 μM) protected the cells and were associated with increased intracellular glutathione. We conclude that delphinidin is intrinsically unstable and unlikely to confer any direct antioxidant activity in vivo yet it offered antioxidant protection to cells at low concentrations. This paradox might be explained by the ability of the degradation product, gallic acid, to confer benefit. The findings are important in understanding the mode of protection conferred by anthocyanins and reinforce the necessity to conduct in vitro experiments at biologically relevant concentrations. PMID:29081896

[Effect of UV-radiation on the level of ascorbic acid, SH-groups, and activity of glutathione reductase in the eye lens].

Science.gov (United States)

Byshneva, L N; Senchuk, V V

2002-01-01

The effect of UV radiation in vitro on the level of ascorbate, SH-groups and glutathione reductase activity in the soluble fraction of bovine eye lens was studied. UV-Irradiation increased NADPH-oxidoreductase activity, the level of ascorbate oxidation and decreased the content of SH-groups and activity of glutathione reductase. Significant activation of the NADPH-oxidoreductase activity in the presence of ascorbate and Cu2+ was observed after UV-irradiation. It is suggested that ascorbate may play an important role in the UV-induced lens pathology.

Response of Glutathione and Glutathione S-transferase in Rice Seedlings Exposed to Cadmium Stress

Directory of Open Access Journals (Sweden)

Chun-hua ZHANG

2008-03-01

Full Text Available A hydroponic culture experiment was done to investigate the effect of Cd stress on glutathione content (GSH and glutathione S-transferase (GST, EC 2.5.1.18 activity in rice seedlings. The rice growth was severely inhibited when Cd level in the solution was higher than 10 mg/L. In rice shoots, GSH content and GST activity increased with the increasing Cd level, while in roots, GST was obviously inhibited by Cd treatments. Compared with shoots, the rice roots had higher GSH content and GST activity, indicating the ability of Cd detoxification was much higher in roots than in shoots. There was a significant correlation between Cd level and GSH content or GST activity, suggesting that both parameters may be used as biomarkers of Cd stress in rice.

Expression of inducible nitric oxide synthase in endotoxemic rat hepatocytes is dependent on the cellular glutathione status

NARCIS (Netherlands)

Vos, TA; van Goor, H; Tuyt, L; de Jager-Krikken, A; Leuvenink, R; Kuipers, F; Jansen, PLM; Moshage, H

The inducible nitric oxide synthase (iNOS) promoter contains nuclear factor kappa B (NF-kappa B) binding sites. NF-kappa B activation is determined, in part, by the intracellular redox status, The aim of this study was to determine the importance of the cellular glutathione status in relation to

The Severity of Autism Is Associated with Toxic Metal Body Burden and Red Blood Cell Glutathione Levels

Energy Technology Data Exchange (ETDEWEB)

Adams, J B; Mitchell, I J [Division of Basic Medical Sciences, Southwest College of Naturopathic Medicine, Tempe, AZ 85282 (United States); Baral, M; Bradstreet, J [Department of Pediatric Medicine, Southwest College of Naturopathic Medicine, Tempe, AZ 85282 (United States); Geis, E; Ingram, J; Hensley, A; Zappia, I; Gehn, E; Mitchell, K [Autism Research Institute, San Diego, CA 92116-2599 (United States); Newmark, S [Center for Integrative Pediatric Medicine, Tucson, AZ 85711 (United States); Rubin, R A [Department of Mathematics, Whittier College, Whittier, CA 90601-4413 (United States); Bradstreet, J [International Child Development Resource Center, Phoenix, AZ (United States); El-Dahrn, J M [Department of Pediatrics, Tulane University School of Medicine, New Orleans, LA 70112 (United States)

2009-07-01

This study investigated the relationship of children's autism symptoms with their toxic metal body burden and red blood cell (RBC) glutathione levels. In children ages 38 years, the severity of autism was assessed using four tools: ADOS, PDD-BI, ATEC, and SAS. Toxic metal body burden was assessed by measuring urinary excretion of toxic metals, both before and after oral dimercaptosuccinic acid (DMSA). Multiple positive correlations were found between the severity of autism and the urinary excretion of toxic metals. Variations in the severity of autism measurements could be explained, in part, by regression analyses of urinary excretion of toxic metals before and after DMSA and the level of RBC glutathione (adjusted R2 of 0.220.45, P<.005 in all cases). This study demonstrates a significant positive association between the severity of autism and the relative body burden of toxic metals.

The Severity of Autism Is Associated with Toxic Metal Body Burden and Red Blood Cell Glutathione Levels

International Nuclear Information System (INIS)

Adams, J.B.; Mitchell, I.J.; Baral, M.; Bradstreet, J.; Geis, E.; Ingram, J.; Hensley, A.; Zappia, I.; Gehn, E.; Mitchell, K.; Newmark, S.; Rubin, R.A.; Bradstreet, J.; El-Dahrn, J.M.

2009-01-01

This study investigated the relationship of children's autism symptoms with their toxic metal body burden and red blood cell (RBC) glutathione levels. In children ages 38 years, the severity of autism was assessed using four tools: ADOS, PDD-BI, ATEC, and SAS. Toxic metal body burden was assessed by measuring urinary excretion of toxic metals, both before and after oral dimercaptosuccinic acid (DMSA). Multiple positive correlations were found between the severity of autism and the urinary excretion of toxic metals. Variations in the severity of autism measurements could be explained, in part, by regression analyses of urinary excretion of toxic metals before and after DMSA and the level of RBC glutathione (adjusted R2 of 0.220.45, P<.005 in all cases). This study demonstrates a significant positive association between the severity of autism and the relative body burden of toxic metals.

Glutathione S-transferase P influences redox and migration pathways in bone marrow.

Directory of Open Access Journals (Sweden)

Jie Zhang

Full Text Available To interrogate why redox homeostasis and glutathione S-transferase P (GSTP are important in regulating bone marrow cell proliferation and migration, we isolated crude bone marrow, lineage negative and bone marrow derived-dendritic cells (BMDDCs from both wild type (WT and knockout (Gstp1/p2(-/- mice. Comparison of the two strains showed distinct thiol expression patterns. WT had higher baseline and reactive oxygen species-induced levels of S-glutathionylated proteins, some of which (sarco-endoplasmic reticulum Ca2(+-ATPase regulate Ca(2+ fluxes and subsequently influence proliferation and migration. Redox status is also a crucial determinant in the regulation of the chemokine system. CXCL12 chemotactic response was stronger in WT cells, with commensurate alterations in plasma membrane polarization/permeability and intracellular calcium fluxes; activities of the downstream kinases, ERK and Akt were also higher in WT. In addition, expression levels of the chemokine receptor CXCR4 and its associated phosphatase, SHP-2, were higher in WT. Inhibition of CXCR4 or SHP2 decreased the extent of CXCL12-induced migration in WT BMDDCs. The differential surface densities of CXCR4, SHP-2 and inositol trisphosphate receptor in WT and Gstp1/p2(-/- cells correlated with the differential CXCR4 functional activities, as measured by the extent of chemokine-induced directional migration and differences in intracellular signaling. These observed differences contribute to our understanding of how genetic ablation of GSTP causes different levels of myeloproliferation and migration [corrected

DIP and DIP + 2 as glutathione oxidants and radiation sensitizers in cultured Chinese hamster cells

International Nuclear Information System (INIS)

Harris, J.W.; Power, J.A.; Kosower, N.S.; Kosower, E.M.

1975-01-01

Two diamide analogues, diazene dicarboxylic acid bis (N'-methyl-piperazide) or DIP, and its bis-N'-methyl iodide salt, or DIP + 2, were tested for their ability to penetrate cultured Chinese hamster cells and oxidize intracellular glutathione. DIP penetrated the cells at a reasonable rate at 18 0 C, 160 nmoles being required to oxidize the endogenous glutathione of 2 x 10 6 cells, but it penetrated very slowly at 0 0 C. DIP + 2 did not effectively oxidize glutathione in Chinese hamster cells, possibly because it did not enter the cels. DIP became toxic after about 10 min of exposure, but its toxicity could be moderated by using anoxic conditions. DIP, but not DIP + 2, sensitized anoxic Chinese hamster cells to X-radiation by a factor of 1.5, an effect that was due entirely to removal of the shoulder from the survival curve. (author)

The interplay between sulphur and selenium metabolism influences the intracellular redox balance in Saccharomyces cerevisiae

DEFF Research Database (Denmark)

Mapelli, Valeria; Hillestrøm, Peter René; Patil, Kalpesh

2012-01-01

oxidative stress response is active when yeast actively metabolizes Se, and this response is linked to the generation of intracellular redox imbalance. The redox imbalance derives from a disproportionate ratio between the reduced and oxidized forms of glutathione and also from the influence of Se metabolism...

Subcellular distribution of glutathione and cysteine in cyanobacteria.

Science.gov (United States)

Zechmann, Bernd; Tomasić, Ana; Horvat, Lucija; Fulgosi, Hrvoje

2010-10-01

Glutathione plays numerous important functions in eukaryotic and prokaryotic cells. Whereas it can be found in virtually all eukaryotic cells, its production in prokaryotes is restricted to cyanobacteria and proteobacteria and a few strains of gram-positive bacteria. In bacteria, it is involved in the protection against reactive oxygen species (ROS), osmotic shock, acidic conditions, toxic chemicals, and heavy metals. Glutathione synthesis in bacteria takes place in two steps out of cysteine, glutamate, and glycine. Cysteine is the limiting factor for glutathione biosynthesis which can be especially crucial for cyanobacteria, which rely on both the sufficient sulfur supply from the growth media and on the protection of glutathione against ROS that are produced during photosynthesis. In this study, we report a method that allows detection and visualization of the subcellular distribution of glutathione in Synechocystis sp. This method is based on immunogold cytochemistry with glutathione and cysteine antisera and computer-supported transmission electron microscopy. Labeling of glutathione and cysteine was restricted to the cytosol and interthylakoidal spaces. Glutathione and cysteine could not be detected in carboxysomes, cyanophycin granules, cell walls, intrathylakoidal spaces, periplasm, and vacuoles. The accuracy of the glutathione and cysteine labeling is supported by two observations. First, preadsorption of the antiglutathione and anticysteine antisera with glutathione and cysteine, respectively, reduced the density of the gold particles to background levels. Second, labeling of glutathione and cysteine was strongly decreased by 98.5% and 100%, respectively, in Synechocystis sp. cells grown on media without sulfur. This study indicates a strong similarity of the subcellular distribution of glutathione and cysteine in cyanobacteria and plastids of plants and provides a deeper insight into glutathione metabolism in bacteria.

Nrf2 activation ameliorates cytotoxic effects of arsenic trioxide in acute promyelocytic leukemia cells through increased glutathione levels and arsenic efflux from cells

Energy Technology Data Exchange (ETDEWEB)

Nishimoto, Shoichi; Suzuki, Toshihiro; Koike, Shin [Department of Analytical Biochemistry, Meiji Pharmaceutical University, 2-522-1 Noshio, Kiyose, Tokyo 204-8588 (Japan); Yuan, Bo; Takagi, Norio [Department of Applied Biochemistry, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, Hachioji, Tokyo 192-0392 (Japan); Ogasawara, Yuki, E-mail: yo@my-pharm.ac.jp [Department of Analytical Biochemistry, Meiji Pharmaceutical University, 2-522-1 Noshio, Kiyose, Tokyo 204-8588 (Japan)

2016-08-15

Carnosic acid (CA), a phenolic diterpene isolated from Rosmarinus officinalis, has been shown to activate nuclear transcription factor E2-related factor 2 (Nrf2), which plays a central role in cytoprotective responses to oxidative and electrophilic stress. Recently, the Nrf2-Kelch ECH associating protein 1 (Keap1) pathway has been associated with cancer drug resistance attributable to modulation of the expression and activation of antioxidant and detoxification enzymes. However, the exact mechanisms by which Nrf2 activation results in chemoresistance are insufficiently understood to date. This study investigated the mechanisms by which the cytotoxic effects of arsenic trioxide (ATO), an anticancer drug, were decreased in acute promyelocytic leukemia cells treated with CA, a typical activator of Nrf2 used to stimulate the Nrf2/Keap1 system. Our findings suggest that arsenic is non-enzymatically incorporated into NB4 cells and forms complexes that are dependent on intracellular glutathione (GSH) concentrations. In addition, the arsenic complexes are recognized as substrates by multidrug resistance proteins and subsequently excreted from the cells. Therefore, Nrf2-associated activation of the GSH biosynthetic pathway, followed by increased levels of intracellular GSH, are key mechanisms underlying accelerated arsenic efflux and attenuation of the cytotoxic effects of ATO. - Highlights: • Nrf2 activation attenuates the effect of arsenic trioxide to acute promyelocytic leukemia cells. • The sensitivity of arsenic trioxide to NB4 cells was dependent on efflux rate of arsenic. • Activation of the GSH biosynthesis is essential in Nrf2-regulated responses for arsenic efflux.

Plasma interleukin-6 levels, glutathione peroxidase and isoprostane in obese women before and after weight loss. Association with cardiovascular risk factors.

Science.gov (United States)

Bougoulia, Maria; Triantos, Athanassios; Koliakos, George

2006-01-01

To evaluate the levels of Interleukin-6 (IL-6), glutathione peroxidase and isoprostane in obese women and their association with markers of cardiovascular risk factors before and after weight loss. 36 healthy obese women of reproductive age (group A: age (mean+/-SD) 35.4+/-9.2 years, Body Mass Index (BMI) 38.5+/-7 kg/m2) and 30 healthy, normal weight women (group B: age mean+/-SD 34.9+/-7.4 y., BMI 24+/-1.1 kg/m2) were included in the study. Glucose tolerance was normal in all participating women. Il-6, glutathione peroxidase and isoprostane, C-Reactive Protein (CRP), insulin, fasting plasma glucose, HOMA-IR as well as the lipid profile were evaluated. Body weight, BMI, Waist to Hip ratio (W/H) ratio, Waist Circumference (WC), %free fat mass and the %fat mass were also measured. A hypo-caloric diet was prescribed for the obese women and all participants were re-examined after six months. In obese women after weight loss, anthropometric obesity markers (BMI, W/H ratio), %fat, lipid profile, insulin levels and inflammation indices such as IL-6 and CRP, the oxidative stress index isoprostane, as well as glutathione peroxidase were significantly ameliorated. The levels of serum glutathione peroxidase activity were negatively correlated with IL-6 levels and were significantly increased after weight reduction. In obese women there was an association between IL-6 levels and the values of %fat, %free fat mass, insulin and HOMA-IR before and after weight loss. Weight loss is related to reduction of oxidative stress and inflammation; this beneficial effect could possibly be translated into reduction of cardiovascular risk in obese individuals.

Intracellular calcium levels can regulate Importin-dependent nuclear import

International Nuclear Information System (INIS)

Kaur, Gurpreet; Ly-Huynh, Jennifer D.; Jans, David A.

2014-01-01

Highlights: • High intracellular calcium inhibits Impα/β1- or Impβ1-dependent nuclear protein import. • The effect of Ca 2+ on nuclear import does not relate to changes in the nuclear pore. • High intracellular calcium can result in mislocalisation of Impβ1, Ran and RCC1. - Abstract: We previously showed that increased intracellular calcium can modulate Importin (Imp)β1-dependent nuclear import of SRY-related chromatin remodeling proteins. Here we extend this work to show for the first time that high intracellular calcium inhibits Impα/β1- or Impβ1-dependent nuclear protein import generally. The basis of this relates to the mislocalisation of the transport factors Impβ1 and Ran, which show significantly higher nuclear localization in contrast to various other factors, and RCC1, which shows altered subnuclear localisation. The results here establish for the first time that intracellular calcium modulates conventional nuclear import through direct effects on the nuclear transport machinery

Intracellular calcium levels can regulate Importin-dependent nuclear import

Energy Technology Data Exchange (ETDEWEB)

Kaur, Gurpreet; Ly-Huynh, Jennifer D.; Jans, David A., E-mail: David.Jans@monash.edu

2014-07-18

Highlights: • High intracellular calcium inhibits Impα/β1- or Impβ1-dependent nuclear protein import. • The effect of Ca{sup 2+} on nuclear import does not relate to changes in the nuclear pore. • High intracellular calcium can result in mislocalisation of Impβ1, Ran and RCC1. - Abstract: We previously showed that increased intracellular calcium can modulate Importin (Imp)β1-dependent nuclear import of SRY-related chromatin remodeling proteins. Here we extend this work to show for the first time that high intracellular calcium inhibits Impα/β1- or Impβ1-dependent nuclear protein import generally. The basis of this relates to the mislocalisation of the transport factors Impβ1 and Ran, which show significantly higher nuclear localization in contrast to various other factors, and RCC1, which shows altered subnuclear localisation. The results here establish for the first time that intracellular calcium modulates conventional nuclear import through direct effects on the nuclear transport machinery.

Beta-amyloidolysis and glutathione in Alzheimer's disease

Directory of Open Access Journals (Sweden)

Lasierra-Cirujeda J

2013-04-01

Full Text Available J Lasierra-Cirujeda,1 P Coronel,2 MJ Aza,3 M Gimeno2 1CM Hematológico SC, Logroño, La Rioja, Spain; 2Tedec-Meiji Farma, SA, Alcalá de Henares, Madrid, Spain; 3Pharmaceutical Act, Ministry of Health, Regional Government, La Rioja, Spain Abstract: In this review, we hypothesized the importance of the interaction between the brain glutathione (GSH system, the proteolytic tissue plasminogen activator (t-PA/plasminogen/plasmin system, regulated by plasminogen activator inhibitor (PAI-1, and neuroserpin in the pathogenesis of Alzheimer's disease. The histopathological characteristic hallmark that gives personality to the diagnosis of Alzheimer's disease is the accumulation of neurofibroid tangles located intracellularly in the brain, such as the protein tau and extracellular senile plaques made primarily of amyloidal substance. These formations of complex etiology are intimately related to GSH, brain protective antioxidants, and the proteolytic system, in which t-PA plays a key role. There is scientific evidence that suggests a relationship between aging, a number of neurodegenerative disorders, and the excessive production of reactive oxygen species and accompanying decreased brain proteolysis. The plasminogen system in the brain is an essential proteolytic mechanism that effectively degrades amyloid peptides ("beta-amyloidolysis" through action of the plasmin, and this physiologic process may be considered to be a means of prevention of neurodegenerative disorders. In parallel to the decrease in GSH levels seen in aging, there is also a decrease in plasmin brain activity and a progressive decrease of t-PA activity, caused by a decrease in the expression of the t-PA together with an increase of the PAI-1 levels, which rise to an increment in the production of amyloid peptides and a lesser clearance of them. Better knowledge of the GSH mechanism and cerebral proteolysis will allow us to hypothesize about therapeutic practices. Keywords: glutathione

Increased Zn/Glutathione Levels and Higher Superoxide Dismutase-1 Activity as Biomarkers of Oxidative Stress in Women with Long-Term Dental Amalgam Fillings: Correlation between Mercury/Aluminium Levels (in Hair) and Antioxidant Systems in Plasma

Science.gov (United States)

Cabaña-Muñoz, María Eugenia; Parmigiani-Izquierdo, José María; Bravo-González, Luis Alberto; Kyung, Hee-Moon; Merino, José Joaquín

2015-01-01

Background The induction of oxidative stress by Hg can affect antioxidant enzymes. However, epidemiological studies have failed to establish clear association between dental fillings presence and health problems. Objectives To determine whether heavy metals (in hair), antioxidant enzymes (SOD-1) and glutathione levels could be affected by the chronic presence of heavy metals in women who had dental amalgam fillings. Materials and Methods 55 hair samples (42 females with amalgam fillings and 13 female control subjects) were obtained. All subjects (mean age 44 years) who had dental amalgam filling for more than 10 years (average 15 years). Certain metals were quantified by ICP-MS (Mass Spectrophotometry) in hair (μg/g: Al, Hg, Ba, Ag, Sb, As, Be, Bi, Cd, Pb, Pt, Tl, Th, U, Ni, Sn, Ti) and SOD-1 and Glutathione (reduced form) levels in plasma. Data were compared with controls without amalgams, and analyzed to identify any significant relation between metals and the total number of amalgam fillings, comparing those with four or less (n = 27) with those with more than four (n = 15). As no significant differences were detected, the two groups were pooled (Amlgam; n = 42). Findings Hg, Ag, Al and Ba were higher in the amalgam group but without significant differences for most of the heavy metals analyzed. Increased SOD-1 activity and glutathione levels (reduced form) were observed in the amalgam group. Aluminum (Al) correlated with glutathione levels while Hg levels correlated with SOD-1. The observed Al/glutathione and Hg/SOD-1 correlation could be adaptive responses against the chronic presence of mercury. Conclusions Hg, Ag, Al and Ba levels increased in women who had dental amalgam fillings for long periods. Al correlated with glutathione, and Hg with SOD-1. SOD-1 may be a possible biomarker for assessing chronic Hg toxicity. PMID:26076368

Alterations in glutathione levels and apoptotic regulators are associated with acquisition of arsenic trioxide resistance in multiple myeloma.

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Shannon M Matulis

Full Text Available Arsenic trioxide (ATO has been tested in relapsed/refractory multiple myeloma with limited success. In order to better understand drug mechanism and resistance pathways in myeloma we generated an ATO-resistant cell line, 8226/S-ATOR05, with an IC50 that is 2-3-fold higher than control cell lines and significantly higher than clinically achievable concentrations. Interestingly we found two parallel pathways governing resistance to ATO in 8226/S-ATOR05, and the relevance of these pathways appears to be linked to the concentration of ATO used. We found changes in the expression of Bcl-2 family proteins Bfl-1 and Noxa as well as an increase in cellular glutathione (GSH levels. At low, clinically achievable concentrations, resistance was primarily associated with an increase in expression of the anti-apoptotic protein Bfl-1 and a decrease in expression of the pro-apoptotic protein Noxa. However, as the concentration of ATO increased, elevated levels of intracellular GSH in 8226/S-ATOR05 became the primary mechanism of ATO resistance. Removal of arsenic selection resulted in a loss of the resistance phenotype, with cells becoming sensitive to high concentrations of ATO within 7 days following drug removal, indicating changes associated with high level resistance (elevated GSH are dependent upon the presence of arsenic. Conversely, not until 50 days without arsenic did cells once again become sensitive to clinically relevant doses of ATO, coinciding with a decrease in the expression of Bfl-1. In addition we found cross-resistance to melphalan and doxorubicin in 8226/S-ATOR05, suggesting ATO-resistance pathways may also be involved in resistance to other chemotherapeutic agents used in the treatment of multiple myeloma.

Glutathione peroxidases of the potato cyst nematode Globodera Rostochiensis.

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Jones, J T; Reavy, B; Smant, G; Prior, A E

2004-01-07

We report the cloning and characterisation of full-length DNAs complementary to RNA (cDNAs) encoding two glutathione peroxidases (GpXs) from a plant parasitic nematode, the potato cyst nematode (PCN) Globodera rostochiensis. One protein has a functional signal peptide that targets the protein for secretion from animal cells while the other is predicted to be intracellular. Both genes are expressed in all parasite stages tested. The mRNA encoding the intracellular GpX is present throughout the nematode second stage juvenile and is particularly abundant in metabolically active tissues including the genital primordia. The mRNA encoding the secreted GpX is restricted to the hypodermis, the outermost cellular layer of the nematode, a location from which it is likely to be secreted to the parasite surface. Biochemical studies confirmed the secreted protein as a functional GpX and showed that, like secreted GpXs of other parasitic nematodes, it does not metabolise hydrogen peroxide but has a preference for larger hydroperoxide substrates. The intracellular protein is likely to have a role in metabolism of active oxygen species derived from internal body metabolism while the secreted protein may protect the parasite from host defences. Other functional roles for this protein are discussed.

Analysis of MTHFR, CBS, Glutathione, Taurine, and Hydrogen Sulfide Levels in Retinas of Hyperhomocysteinemic Mice.

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Cui, Xuezhi; Navneet, Soumya; Wang, Jing; Roon, Penny; Chen, Wei; Xian, Ming; Smith, Sylvia B

2017-04-01

Hyperhomocysteinemia (Hhcy) is implicated in certain retinal neurovascular diseases, although whether it is causative remains uncertain. In isolated ganglion cells (GCs), mild Hhcy induces profound death, whereas retinal phenotypes in Hhcy mice caused by mutations in remethylation (methylene tetrahydrofolatereductase [Mthfr+/-]) or transsulfuration pathways (cystathionine β-synthase [Cbs+/-]) demonstrate mild GC loss and mild vasculopathy. The current work investigated compensation in vivo of one pathway for the other, and, because the transsulfuration pathway yields cysteine necessary for formation of glutathione (GSH), taurine, and hydrogen sulfide (H2S), they were analyzed also. Retinas isolated from wild-type (WT), Mthfr+/-, and Cbs+/- mice (12 and 22 weeks) were analyzed for methylene tetrahydrofolate reductase (MTHFR), cystathionine-β-synthase (CBS), and cystathionase (CTH) RNA/protein levels. Retinas were evaluated for levels of reduced:oxidized GSH (GSH:GSSG), Slc7a11 (xCT), taurine, taurine transporter (TAUT), and H2S. Aside from decreased CBS RNA/protein levels in Cbs+/- retinas, there were minimal alterations in remethylation/transsulfuration pathways in the two mutant mice strains. Glutathione and taurine levels in Mthfr+/- and Cbs+/- retinas were similar to WT, which may be due to robust levels of xCT and TAUT in mutant retinas. Interestingly, levels of H2S were markedly increased in retinas of Mthfr+/- and Cbs+/- mice compared with WT. Ganglion cell loss and vasculopathy observed in Mthfr+/- and Cbs+/- mouse retinas may be milder than expected, not because of compensatory increases of enzymes in remethylation/transsulfuration pathways, but because downstream transsulfuration pathway products GSH, taurine, and H2S are maintained at robust levels. Elevation of H2S is particularly intriguing owing to neuroprotective properties reported for this gasotransmitter.

Gelsolin-Cu/ZnSOD interaction alters intracellular reactive oxygen species levels to promote cancer cell invasion

KAUST Repository

Tochhawng, Lalchhandami

2016-07-07

The actin-binding protein, gelsolin, is a well known regulator of cancer cell invasion. However, the mechanisms by which gelsolin promotes invasion are not well established. As reactive oxygen species (ROS) have been shown to promote cancer cell invasion, we investigated on the hypothesis that gelsolin-induced changes in ROS levels may mediate the invasive capacity of colon cancer cells. Herein, we show that increased gelsolin enhances the invasive capacity of colon cancer cells, and this is mediated via gelsolin\\'s effects in elevating intracellular superoxide (O2 .-) levels. We also provide evidence for a novel physical interaction between gelsolin and Cu/ZnSOD, that inhibits the enzymatic activity of Cu/ZnSOD, thereby resulting in a sustained elevation of intracellular O2 .-. Using microarray data of human colorectal cancer tissues from Gene Omnibus, we found that gelsolin gene expression positively correlates with urokinase plasminogen activator (uPA), an important matrix-degrading protease invovled in cancer invasion. Consistent with the in vivo evidence, we show that increased levels of O2 .- induced by gelsolin overexpression triggers the secretion of uPA. We further observed reduction in invasion and intracellular O2 .- levels in colon cancer cells, as a consequence of gelsolin knockdown using two different siRNAs. In these cells, concurrent repression of Cu/ ZnSOD restored intracellular O2 .- levels and rescued invasive capacity. Our study therefore identified gelsolin as a novel regulator of intracellular O2 .- in cancer cells via interacting with Cu/ZnSOD and inhibiting its enzymatic activity. Taken together, these findings provide insight into a novel function of gelsolin in promoting tumor invasion by directly impacting the cellular redox milieu.

The glutathione cycle: Glutathione metabolism beyond the γ-glutamyl cycle.

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Bachhawat, Anand Kumar; Yadav, Shambhu

2018-04-17

Glutathione was discovered in 1888, over 125 years ago. Since then, our understanding of various functions and metabolism of this important molecule has grown over these years. But it is only now, in the last decade, that a somewhat complete picture of its metabolism has emerged. Glutathione metabolism has till now been largely depicted and understood by the γ-glutamyl cycle that was proposed in 1970. However, new findings and knowledge particularly on the transport and degradation of glutathione have revealed that many aspects of the γ-glutamyl cycle are incorrect. Despite this, an integrated critical analysis of the cycle has never been undertaken and this has led to the cycle and its errors perpetuating in the literature. This review takes a careful look at the γ-glutamyl cycle and its shortcomings and presents a "glutathione cycle" that captures the current understanding of glutathione metabolism. © 2018 IUBMB Life, 2018. © 2018 International Union of Biochemistry and Molecular Biology.

Antioxidant role of glutathione S-transferases: 4-Hydroxynonenal, a key molecule in stress-mediated signaling.

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Singhal, Sharad S; Singh, Sharda P; Singhal, Preeti; Horne, David; Singhal, Jyotsana; Awasthi, Sanjay

2015-12-15

4-Hydroxy-2-trans-nonenal (4HNE), one of the major end products of lipid peroxidation (LPO), has been shown to induce apoptosis in a variety of cell lines. It appears to modulate signaling processes in more than one way because it has been suggested to have a role in signaling for differentiation and proliferation. It has been known that glutathione S-transferases (GSTs) can reduce lipid hydroperoxides through their Se-independent glutathione-peroxidase activity and that these enzymes can also detoxify LPO end-products such as 4HNE. Available evidence from earlier studies together with results of recent studies in our laboratories strongly suggests that LPO products, particularly hydroperoxides and 4HNE, are involved in the mechanisms of stress-mediated signaling and that it can be modulated by the alpha-class GSTs through the regulation of the intracellular concentrations of 4HNE. We demonstrate that 4HNE induced apoptosis in various cell lines is accompanied with c-Jun-N-terminal kinase (JNK) and caspase-3 activation. Cells exposed to mild, transient heat or oxidative stress acquire the capacity to exclude intracellular 4HNE at a faster rate by inducing GSTA4-4 which conjugates 4HNE to glutathione (GSH), and RLIP76 which mediates the ATP-dependent transport of the GSH-conjugate of 4HNE (GS-HNE). The balance between formation and exclusion promotes different cellular processes - higher concentrations of 4HNE promote apoptosis; whereas, lower concentrations promote proliferation. In this article, we provide a brief summary of the cellular effects of 4HNE, followed by a review of its GST-catalyzed detoxification, with an emphasis on the structural attributes that play an important role in the interactions with alpha-class GSTA4-4. Taken together, 4HNE is a key signaling molecule and that GSTs being determinants of its intracellular concentrations, can regulate stress-mediated signaling, are reviewed in this article. Copyright © 2015 Elsevier Inc. All rights

An anthocyanin/polyphenolic-rich fruit juice reduces oxidative DNA damage and increases glutathione level in healthy probands.

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Weisel, Tamara; Baum, Matthias; Eisenbrand, Gerhard; Dietrich, Helmut; Will, Frank; Stockis, Jean-Pierre; Kulling, Sabine; Rüfer, Corinna; Johannes, Christian; Janzowski, Christine

2006-04-01

Oxidative cell damage is involved in the pathogenesis of atherosclerosis, cancer, diabetes and other diseases. Uptake of fruit juice with especially high content of antioxidant flavonoids/polyphenols, might reduce oxidative cell damage. Therefore, an intervention study was performed with a red mixed berry juice [trolox equivalent antioxidative capacity (TEAC): 19.1 mmol/L trolox] and a corresponding polyphenol-depleted juice (polyphenols largely removed, TEAC 2.4 mmol/L trolox), serving as control. After a 3-week run-in period, 18 male probands daily consumed 700 mL juice, and 9 consumed control juice, in a 4-week intervention, followed by a 3-week wash-out. Samples were collected weekly to analyze DNA damage (comet assay), lipid peroxidation (plasma malondialdehyde: HPLC/fluorescence; urinary isoprostanes: GC-MS), blood glutathione (photometrically), DNA-binding activity of nuclear factor-kappaB (ELISA) and plasma carotenoid/alpha-tocopherol levels (HPLC-DAD). During intervention with the fruit juice, a decrease of oxidative DNA damage (p<5x10(-4)) and an increase of reduced glutathione (p<5x10(-4)) and of glutathione status (p<0.05) were observed, which returned to the run-in levels in the subsequent wash-out phase. The other biomarkers were not significantly modulated by the juice supplement. Intervention with the control juice did not result in reduction of oxidative damage. In conclusion, the fruit juice clearly reduces oxidative cell damage in healthy probands.

Effects of Ionizing Radiation and Glutathione Precursor on Antioxidant Enzyme and Cell Survival in Yeast

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Kim, Jinkyu; Roh, Changhyun; Ryu, Taeho; Park, Jiyoung [Korea Atomic Energy Research Institute, Daejeon (Korea, Republic of); Nili, Michael A. [Oxiage Cosmeceutical Research Institute, Virginia (United States)

2013-05-15

Cells react to such an induced oxidative stress through scavenging the generated reactive oxygen species to reduce oxidative damage. Antioxidant enzymes such as glutathione peroxidase, catalase, and superoxide dismutase are immediately triggered for reactive oxygen species. N-acetyl-L-cysteine (NAC), a precursor of glutathione, is one of the antioxidants. The effect of NAC as an antioxidant and/or a cell rescue agent was investigated in the present study. Glutathione (GSH) is the most abundant intracellular thiol, which involves in antioxidant defense via direct interaction with ROS or via activities of detoxication enzymes like glutathione peroxidases (GPx). NAC flowed in the cell is converted to cysteine by deacetylation, that is supplied to the depleted GSH by oxidative stress. NAC prevents the depletion of GSH by radiation, increases the production of GSH, and improves enzymes activity such as GPx and alkaline phosphatase. Cell growth and survivorship and transcriptional level of glutathione gene are analyzed in two yeast strains exposed to combined treatment of NAC with gamma-rays. The effect of NAC on cell growth was measured during 72 hours. The cell growth was hampered by higher concentrations of NAC at stationary phase. NAC, however, didn't affect the cell division at the exponential phase. The survival of the cells decreased with radiation dose. The cell viability of the strain W303-1A was reduced significantly at the low dose (10 and 30 Gy). By comparison, the strain W303-1A was more sensitive to radiation with having a half lethal dose (LD{sub 50}) of about 20 Gy. The quantitative RT-PCR analysis showed that the transcriptional expression of antioxidant enzyme gene GPX1 increased after irradiation while the expression of the gene decreased by the combined treatment of NAC with 100 Gy radiation. The present study shows that NAC can directly scavenge ROS against oxidative stress in vivo. In conclusion, NAC can prevent radiation-induced oxidative

Effects of Ionizing Radiation and Glutathione Precursor on Antioxidant Enzyme and Cell Survival in Yeast

International Nuclear Information System (INIS)

Kim, Jinkyu; Roh, Changhyun; Ryu, Taeho; Park, Jiyoung; Nili, Michael A.

2013-01-01

Cells react to such an induced oxidative stress through scavenging the generated reactive oxygen species to reduce oxidative damage. Antioxidant enzymes such as glutathione peroxidase, catalase, and superoxide dismutase are immediately triggered for reactive oxygen species. N-acetyl-L-cysteine (NAC), a precursor of glutathione, is one of the antioxidants. The effect of NAC as an antioxidant and/or a cell rescue agent was investigated in the present study. Glutathione (GSH) is the most abundant intracellular thiol, which involves in antioxidant defense via direct interaction with ROS or via activities of detoxication enzymes like glutathione peroxidases (GPx). NAC flowed in the cell is converted to cysteine by deacetylation, that is supplied to the depleted GSH by oxidative stress. NAC prevents the depletion of GSH by radiation, increases the production of GSH, and improves enzymes activity such as GPx and alkaline phosphatase. Cell growth and survivorship and transcriptional level of glutathione gene are analyzed in two yeast strains exposed to combined treatment of NAC with gamma-rays. The effect of NAC on cell growth was measured during 72 hours. The cell growth was hampered by higher concentrations of NAC at stationary phase. NAC, however, didn't affect the cell division at the exponential phase. The survival of the cells decreased with radiation dose. The cell viability of the strain W303-1A was reduced significantly at the low dose (10 and 30 Gy). By comparison, the strain W303-1A was more sensitive to radiation with having a half lethal dose (LD 50 ) of about 20 Gy. The quantitative RT-PCR analysis showed that the transcriptional expression of antioxidant enzyme gene GPX1 increased after irradiation while the expression of the gene decreased by the combined treatment of NAC with 100 Gy radiation. The present study shows that NAC can directly scavenge ROS against oxidative stress in vivo. In conclusion, NAC can prevent radiation-induced oxidative stress by

Targeting Aberrant Glutathione Metabolism to Eradicate Human Acute Myelogenous Leukemia Cells*

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Pei, Shanshan; Minhajuddin, Mohammad; Callahan, Kevin P.; Balys, Marlene; Ashton, John M.; Neering, Sarah J.; Lagadinou, Eleni D.; Corbett, Cheryl; Ye, Haobin; Liesveld, Jane L.; O'Dwyer, Kristen M.; Li, Zheng; Shi, Lei; Greninger, Patricia; Settleman, Jeffrey; Benes, Cyril; Hagen, Fred K.; Munger, Joshua; Crooks, Peter A.; Becker, Michael W.; Jordan, Craig T.

2013-01-01

The development of strategies to eradicate primary human acute myelogenous leukemia (AML) cells is a major challenge to the leukemia research field. In particular, primitive leukemia cells, often termed leukemia stem cells, are typically refractory to many forms of therapy. To investigate improved strategies for targeting of human AML cells we compared the molecular mechanisms regulating oxidative state in primitive (CD34+) leukemic versus normal specimens. Our data indicate that CD34+ AML cells have elevated expression of multiple glutathione pathway regulatory proteins, presumably as a mechanism to compensate for increased oxidative stress in leukemic cells. Consistent with this observation, CD34+ AML cells have lower levels of reduced glutathione and increased levels of oxidized glutathione compared with normal CD34+ cells. These findings led us to hypothesize that AML cells will be hypersensitive to inhibition of glutathione metabolism. To test this premise, we identified compounds such as parthenolide (PTL) or piperlongumine that induce almost complete glutathione depletion and severe cell death in CD34+ AML cells. Importantly, these compounds only induce limited and transient glutathione depletion as well as significantly less toxicity in normal CD34+ cells. We further determined that PTL perturbs glutathione homeostasis by a multifactorial mechanism, which includes inhibiting key glutathione metabolic enzymes (GCLC and GPX1), as well as direct depletion of glutathione. These findings demonstrate that primitive leukemia cells are uniquely sensitive to agents that target aberrant glutathione metabolism, an intrinsic property of primary human AML cells. PMID:24089526

Endoplasmic reticulum transport of glutathione by Sec61 is regulated by Ero1 and Bip

DEFF Research Database (Denmark)

Ponsero, Alise J.; Igbaria, Aeid; Darch, Maxwell A.

2017-01-01

In the endoplasmic reticulum (ER), Ero1 catalyzes disulfide bond formation and promotes glutathione (GSH) oxidation to GSSG. Since GSSG cannot be reduced in the ER, maintenance of the ER glutathione redox state and levels likely depends on ER glutathione import and GSSG export. We used quantitative...... oxidation through Ero1 reductive activation, which inhibits glutathione import in a negative regulatory loop. During ER stress, transport is activated by UPR-dependent Ero1 induction, and cytosolic glutathione levels increase. Thus, the ER redox poise is tuned by reciprocal control of glutathione import...... by reduction, causing Bip oxidation and inhibition of glutathione transport. Coupling of glutathione ER import to Ero1 activation provides a basis for glutathione ER redox poise maintenance....

Depletion of liver glutathione levels in rats: a potential confound of nose-only inhalation.

Science.gov (United States)

Fechter, Laurence D; Nelson-Miller, Alisa; Gearhart, Caroline

2008-07-01

Nose-only inhalation exposure chambers offer key advantages to whole-body systems, particularly when aerosol or mixed aerosol-vapor exposures are used. Specifically, nose-only chambers provide enhanced control over the route of exposure and dose by minimizing the deposition of particles either on the subjects skin/fur or on surfaces of a whole-body exposure system. In the current series of experiments, liver, brain, and lung total glutathione (GSH) levels were assessed following either nose-only or whole-body exposures to either jet fuel or to clean, filtered air. The data were compared to untreated control subjects. Acute nose-only inhalation exposures of rats resulted in a significant depletion of liver GSH levels both in subjects that were exposed to clean, filtered air as well as those exposed to JP-8 jet fuel and to a synthetic jet fuel. Glutathione levels were not altered in lung or brain tissue. Whole-body inhalation exposure had no effect on GSH levels in any tissue for any of the treatment groups. A second experiment demonstrated that the loss of GSH did not occur if rats were anaesthetized prior to and during nose-only exposure to clean, filtered air or to mixed hydrocarbons. These data appear to be consistent with studies demonstrating depletion in liver GSH levels among rats subjected to restraint stress. Finally, the depletion of GSH that was observed in liver following a single acute exposure was reduced following five daily exposures to clean, filtered air, suggesting the possibility of habituation to restraint in the nose-only exposure chamber. The finding that placement in a nose-only exposure chamber per se yields liver GSH depletion raises the possibility of an interaction between this mode of toxicant exposure and the toxicological effects of certain inhaled test substances.

Control of intracellular heme levels: Heme transporters and heme oxygenases

OpenAIRE

Khan, Anwar A.; Quigley, John G.

2011-01-01

Heme serves as a co-factor in proteins involved in fundamental biological processes including oxidative metabolism, oxygen storage and transport, signal transduction and drug metabolism. In addition, heme is important for systemic iron homeostasis in mammals. Heme has important regulatory roles in cell biology, yet excessive levels of intracellular heme are toxic; thus, mechanisms have evolved to control the acquisition, synthesis, catabolism and expulsion of cellular heme. Recently, a number...

Plaque formation reduction with glutathione monoester in mice fed on atherogenic diet

International Nuclear Information System (INIS)

Iqbal, M.; Mehboobali, N.; Pervez, S.

2006-01-01

To determine the role of glutathione monoester on reducing the development of plaque formation in an animal model. Twenty-four Balb/c mice were divided into 3 equal groups. First group was fed on atherogenic diet alone, while the second group received atherogenic diet plus twice weekly injections of glutathione monoester. The third group was fed on normal diet for mice. After one year, the animals were sacrificed. Blood was analyzed for lipid levels, while liver, kidney, spleen, heart and aorta were removed to study morphological changes. Results: In the groups of mice receiving atherogenic diet (with and without glutathione monoesters), there was significant increase in levels of total cholesterol (p=0.011) and LDL cholesterol (p=0.001) compared to levels of these lipids in mice on normal diet. However, a significant decrease in levels of triglycerides (p=0.01) was observed in the group receiving atherogenic diet along with glutathione monoester. Supplementation with glutathione monoester had the most pronounced effect only on triglyceride levels. Atherosclerotic plaques were seen in heart and/or aorta of mice receiving atherogenic diet. However, such plaques were either totally absent or if seen in an animal, were extremely small and diffuse in the group receiving glutathione monoester along with atherogenic diet. Mice on normal diet had no evidence of any plaque formation. Cholesterol granuloma was seen in liver of mice on atherogenic diet alone. In mice receiving atherogenic diet plus glutathione monoester, no cholesterol granuloma was found in liver. There were no remarkable morphological changes in spleen and kidney in the three groups of mice. Glutathione monoester appears to inhibit or reduce the development of plaque formation in mice. (author)

Hepatocyte Hyperproliferation upon Liver-Specific Co-disruption of Thioredoxin-1, Thioredoxin Reductase-1, and Glutathione Reductase

Directory of Open Access Journals (Sweden)

Justin R. Prigge

2017-06-01

Full Text Available Energetic nutrients are oxidized to sustain high intracellular NADPH/NADP+ ratios. NADPH-dependent reduction of thioredoxin-1 (Trx1 disulfide and glutathione disulfide by thioredoxin reductase-1 (TrxR1 and glutathione reductase (Gsr, respectively, fuels antioxidant systems and deoxyribonucleotide synthesis. Mouse livers lacking both TrxR1 and Gsr sustain these essential activities using an NADPH-independent methionine-consuming pathway; however, it remains unclear how this reducing power is distributed. Here, we show that liver-specific co-disruption of the genes encoding Trx1, TrxR1, and Gsr (triple-null causes dramatic hepatocyte hyperproliferation. Thus, even in the absence of Trx1, methionine-fueled glutathione production supports hepatocyte S phase deoxyribonucleotide production. Also, Trx1 in the absence of TrxR1 provides a survival advantage to cells under hyperglycemic stress, suggesting that glutathione, likely via glutaredoxins, can reduce Trx1 disulfide in vivo. In triple-null livers like in many cancers, deoxyribonucleotide synthesis places a critical yet relatively low-volume demand on these reductase systems, thereby favoring high hepatocyte turnover over sustained hepatocyte integrity.

Effect of glutathione on phytochelatin synthesis in tomato cells. [Lycopersicon esculentum

Energy Technology Data Exchange (ETDEWEB)

Mendum, M.L.; Gupta, S.C.; Goldsbrough, P.B. (Purdue Univ., West Lafayette, IN (USA))

1990-06-01

Growth of cell suspension cultures of tomato, Lycopersicon esculentum Mill. cv VFNT-Cherry, in the presence of cadmium is inhibited by buthionine sulfoximine, an inhibitor of glutathione synthesis. Cell growth and phytochelatin synthesis are restored to cells treated with buthionine sulfoximine by the addition of glutathione to the medium. Glutathione stimulates the accumulation of phytochelatins in cadmium treated cells, indicating that availability of glutathione can limit synthesis of these peptides. Exogenous glutathione causes a disproportionate increase in the level of smaller phytochelatins, notably ({gamma}-Glu-Cys){sub 2}-Gly. In the presence of buthionine sulfoximine and glutathione, phytochelatins that are produced upon exposure to cadmium incorporate little ({sup 35}S)cysteine, indicating that these peptides are probably not synthesized by sequential addition of cysteine and glutamate to glutathione.

Levels of glutathione and 2,3-diphosphoglycerate in the red blood cells of Australian Aborigines.

Science.gov (United States)

Agar, N S

1980-01-01

There were no significant differences in packed cell volume (PCV) and red cell 2,3-diphosphoglycerate (2,3-DPG) levels in Australian Aborigines and Caucasians. A highly significant negative correlation was found between PCV and 2,3-DPG in both Aborigines (r = 0.251; n = 231) and Caucasians (r = 0.435; n = 227). Levels of reduced glutathione (GSH) in the red blood cells of Aborigines were significantly lower (P < 0.001) compared to those of Caucasians. There was a significant negative correlation between PCV and GSH in both the groups; (Aborigines r = -0.637, n = 115; Caucasians r = 0.388, n = 111).

Electrochemical monitoring of intracellular enzyme activity of single living mammalian cells by using a double-mediator system

International Nuclear Information System (INIS)

Matsumae, Yoshiharu; Takahashi, Yasufumi; Ino, Kosuke; Shiku, Hitoshi; Matsue, Tomokazu

2014-01-01

Graphical abstract: NAD(P)H:quinone oxidoreductase (NQO) activity of single HeLa cells were evaluated by using the menadione–ferrocyanide double mediator system combined with scanning electrochemical microscopy (SECM). - Highlights: • NAD(P)H:quinone oxidoreductase activity of single cells were evaluated with SECM. • Fe(CN) 6 3− /menadione concentrations were optimized for long-term SECM monitoring. • Menadione affect the intracellular levels of reactive oxygen species and GSH. • At 100 μM menadione, the Fe(CN) 6 3− generation rate decreased rapidly within 30 min. - Abstract: We evaluated the intracellular NAD(P)H:quinone oxidoreductase (NQO) activity of single HeLa cells by using the menadione–ferrocyanide double-mediator system combined with scanning electrochemical microscopy (SECM). The double-mediator system was used to amplify the current response from the intracellular NQO activity and to reduce menadione-induced cell damage. The electron shuttle between the electrode and menadione was mediated by the ferrocyanide/ferricyanide redox couple. Generation of ferrocyanide was observed immediately after the addition of a lower concentration (10 μM) of menadione. The ferrocyanide generation rate was constant for 120 min. At a higher menadione concentration (100 μM), the ferrocyanide generation rate decreased within 30 min because of the cytotoxic effect of menadione. We also investigated the relationship between intracellular reactive oxygen species or glutathione levels and exposure to different menadione concentrations to determine the optimal condition for SECM with minimal invasiveness. The present study clearly demonstrates that SECM is useful for the analysis of intracellular enzymatic activities in single cells with a double-mediator system

Electrochemical monitoring of intracellular enzyme activity of single living mammalian cells by using a double-mediator system

Energy Technology Data Exchange (ETDEWEB)

Matsumae, Yoshiharu [Graduate School of Environmental Studies, Tohoku University, Aramaki 6-6-11-605, Aoba, Sendai 980-8579 (Japan); Takahashi, Yasufumi [Advanced Institute for Materials Research, Tohoku University, Katahira 2-1-1, Aoba, Sendai 980-8577 (Japan); Ino, Kosuke [Graduate School of Environmental Studies, Tohoku University, Aramaki 6-6-11-605, Aoba, Sendai 980-8579 (Japan); Shiku, Hitoshi, E-mail: shiku@bioinfo.che.tohoku.ac.jp [Graduate School of Environmental Studies, Tohoku University, Aramaki 6-6-11-605, Aoba, Sendai 980-8579 (Japan); Matsue, Tomokazu, E-mail: matsue@bioinfo.che.tohoku.ac.jp [Graduate School of Environmental Studies, Tohoku University, Aramaki 6-6-11-605, Aoba, Sendai 980-8579 (Japan); Advanced Institute for Materials Research, Tohoku University, Katahira 2-1-1, Aoba, Sendai 980-8577 (Japan)

2014-09-09

Graphical abstract: NAD(P)H:quinone oxidoreductase (NQO) activity of single HeLa cells were evaluated by using the menadione–ferrocyanide double mediator system combined with scanning electrochemical microscopy (SECM). - Highlights: • NAD(P)H:quinone oxidoreductase activity of single cells were evaluated with SECM. • Fe(CN){sub 6}{sup 3−}/menadione concentrations were optimized for long-term SECM monitoring. • Menadione affect the intracellular levels of reactive oxygen species and GSH. • At 100 μM menadione, the Fe(CN){sub 6}{sup 3−} generation rate decreased rapidly within 30 min. - Abstract: We evaluated the intracellular NAD(P)H:quinone oxidoreductase (NQO) activity of single HeLa cells by using the menadione–ferrocyanide double-mediator system combined with scanning electrochemical microscopy (SECM). The double-mediator system was used to amplify the current response from the intracellular NQO activity and to reduce menadione-induced cell damage. The electron shuttle between the electrode and menadione was mediated by the ferrocyanide/ferricyanide redox couple. Generation of ferrocyanide was observed immediately after the addition of a lower concentration (10 μM) of menadione. The ferrocyanide generation rate was constant for 120 min. At a higher menadione concentration (100 μM), the ferrocyanide generation rate decreased within 30 min because of the cytotoxic effect of menadione. We also investigated the relationship between intracellular reactive oxygen species or glutathione levels and exposure to different menadione concentrations to determine the optimal condition for SECM with minimal invasiveness. The present study clearly demonstrates that SECM is useful for the analysis of intracellular enzymatic activities in single cells with a double-mediator system.

The relationship between the violet pigment PP-V production and intracellular ammonium level in Penicillium purpurogenum.

Science.gov (United States)

Kojima, Ryo; Arai, Teppei; Matsufuji, Hiroshi; Kasumi, Takafumi; Watanabe, Taisuke; Ogihara, Jun

2016-12-01

Penicillium purpurogenum is the fungus that produces an azaphilone pigment. However, details about the pigment biosynthesis pathway are unknown. The violet pigment PP-V is the one of the main pigments biosynthesized by this fungus. This pigment contains an amino group in a pyran ring as its core structure. We focused on this pigment and examined the relationship between intracellular ammonium concentration and pigment production using glutamine as a nitrogen source. The intracellular ammonium level decreased about 1.5-fold in conditions favoring PP-V production. Moreover, P. purpurogenum was transferred to medium in which it commonly produces the related pigment PP-O after cultivating it in the presence or absence of glutamine to investigate whether this fungus biosynthesizes PP-V using surplus ammonium in cells. Only mycelia cultured in medium containing 10 mM glutamine produced the violet pigment, and simultaneously intracellular ammonium levels decreased under this condition. From comparisons of the amount of PP-V that was secreted with quantity of surplus intracellular ammonium, it is suggested that P. purpurogenum maintains ammonium homeostasis by excreting waste ammonium as PP-V.

Novel interaction of diethyldithiocarbamate with the glutathione/glutathione peroxidase system

International Nuclear Information System (INIS)

Kumar, K.S.; Sancho, A.M.; Weiss, J.F.

1986-01-01

Diethyldithiocarbamate (DDC) exhibits a variety of pharmacologic activities, including both radioprotective and sensitizing properties. Since the glutathione/glutathione peroxidase system may be a significant factor in determining radiation sensitivity, the potential mechanisms of action of DDC in relation to this system were examined in vitro. The interaction of DDC with reduced glutathione (GSH) was tested using a simple system based on the reduction of cytochrome c. When DDC (0.005 mM) was incubated with GSH (0.5 mM), the reduction of cytochrome c was eightfold greater than that expected from an additive effect of DDC and GSH. GSH could be replaced by oxidized glutathione and glutathione reductase. Cytochrome c reduced by DDC was oxidized by mitochondria. The interaction of DDC with both the hexosemonophosphate shunt pathway and the mitochondrial respiratory chain suggests the possibility of linking these two pathways through DDC. Oxidation of DDC by peroxide and reversal by GSH indicated that the drug can engage in a cyclic reaction with peroxide and GSH. This was confirmed when DDC was used in the assay system for glutathione peroxidase (GSHPx) without GSHPx. DDC at a concentration of 0.25 mM was more active than 0.01 unit of pure GSHPx in eliminating peroxide, and much more active than the other sulfhydryl compounds tested. These studies indicate that DDC can supplement GSHPx activity or substitute for it in detoxifying peroxides, and suggests a unique role in the chemical modification of radiation sensitivity

Positive correlation between decreased cellular uptake, NADPH-glutathione reductase activity and adriamycin resistance in Ehrlich ascites tumor lines.

Science.gov (United States)

Scheulen, M E; Hoensch, H; Kappus, H; Seeber, S; Schmidt, C G

1987-01-01

From a wild type strain of Ehrlich ascites tumor (EATWT) sublines resistant to daunorubicin (EATDNM), etoposide (EATETO), and cisplatinum (EATCIS) have been developed in vivo. Increase in survival and cure rate caused by adriamycin (doxorubicin) have been determined in female NMRI mice which were inoculated i.p. with EAT cells. Adriamycin concentrations causing 50% inhibition of 3H-thymidine (ICT) and 3H-uridine incorporation (ICU) and intracellular adriamycin steady-state concentrations (SSC) were measured in vitro. Adriamycin resistance increased and SSC decreased in the following sequence: EATWT - EATCIS - EATDNM - EATETO. When ICT and ICU were corrected for intracellular adriamycin concentrations in consideration of the different SSC (ICTc, ICUc), ICTc and ICUc still varied up to the 3.2 fold in EATCIS, EATDNM and EATETO in comparison to EATWT. Thus, in addition to different SSC other factors must be responsible for adriamycin resistance. Therefore, enzymes which may play a role in the cytotoxicity related to adriamycin metabolism (NADPH-cytochrome P-450 reductase, NADPH-glutathione reductase, NADP-glucose-6-phosphate dehydrogenase, NADP-isocitrate dehydrogenase) were measured. In contrast to the other parameters determined, NADPH-glutathione reductase was significantly (p less than 0.01) increased up to the 3.2 fold parallel to adriamycin resistance as determined by increase in life span, cure rate, ICTc, and ICUc, respectively. It is concluded that high activities of NADPH-glutathione reductase may contribute to an increase in adriamycin resistance of malignant tumors.

Glutathione--linking cell proliferation to oxidative stress.

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Diaz-Vivancos, Pedro; de Simone, Ambra; Kiddle, Guy; Foyer, Christine H

2015-12-01

The multifaceted functions of reduced glutathione (gamma-glutamyl-cysteinyl-glycine; GSH) continue to fascinate plants and animal scientists, not least because of the dynamic relationships between GSH and reactive oxygen species (ROS) that underpin reduction/oxidation (redox) regulation and signalling. Here we consider the respective roles of ROS and GSH in the regulation of plant growth, with a particular focus on regulation of the plant cell cycle. Glutathione is discussed not only as a crucial low molecular weight redox buffer that shields nuclear processes against oxidative challenge but also a flexible regulator of genetic and epigenetic functions. The intracellular compartmentalization of GSH during the cell cycle is remarkably consistent in plants and animals. Moreover, measurements of in vivo glutathione redox potentials reveal that the cellular environment is much more reducing than predicted from GSH/GSSG ratios measured in tissue extracts. The redox potential of the cytosol and nuclei of non-dividing plant cells is about -300 mV. This relatively low redox potential maintained even in cells experiencing oxidative stress by a number of mechanisms including vacuolar sequestration of GSSG. We propose that regulated ROS production linked to glutathione-mediated signalling events are the hallmark of viable cells within a changing and challenging environment. The concept that the cell cycle in animals is subject to redox controls is well established but little is known about how ROS and GSH regulate this process in plants. However, it is increasingly likely that redox controls exist in plants, although possibly through different pathways. Moreover, redox-regulated proteins that function in cell cycle checkpoints remain to be identified in plants. While GSH-responsive genes have now been identified, the mechanisms that mediate and regulate protein glutathionylation in plants remain poorly defined. The nuclear GSH pool provides an appropriate redox environment

Combining reduced glutathione and ascorbic acid has supplementary beneficial effects on boar sperm cryotolerance.

Science.gov (United States)

Giaretta, Elisa; Estrada, Efrén; Bucci, Diego; Spinaci, Marcella; Rodríguez-Gil, Joan E; Yeste, Marc

2015-02-01

The main aim of this work was to evaluate how supplementing freezing and thawing media with reduced glutathione (GSH) and l-ascorbic acid (AA) affected the quality parameters of frozen-thawed boar spermatozoa. With this purpose, semen samples of 12 ejaculates coming from 12 boars were used. Each ejaculate was split into seven aliquots to which 5 mM of GSH and 100 μM of AA were added separately or together at two different steps of freeze-thawing. Various sperm parameters (levels of free cysteine residues in sperm nucleoproteins, sperm viability, acrosome membrane integrity, intracellular peroxide and superoxide levels [ROS], and total and progressive motility) were evaluated before freezing and at 30 and 240 minutes after thawing. Both GSH and AA significantly improved boar sperm cryotolerance when they were separately added to freezing and thawing media. However, the highest improvement was recorded when both freezing and thawing media were supplemented with 5 mM of GSH plus 100 μM of AA. This improvement was observed in sperm viability and acrosome integrity, sperm motility, and nucleoprotein structure. Although ROS levels were not much increased by freeze-thawing procedures, the addition of GSH and AA to both freezing and thawing extenders significantly decreased intracellular peroxide levels and had no impact on superoxide levels. According to our results, we can conclude that supplementation of freezing and thawing media with both GSH and AA has a combined, beneficial effect on frozen-thawed boar sperm, which is greater than that obtained with the separate addition of either GSH or AA. Copyright © 2015 Elsevier Inc. All rights reserved.

Self-Assembly of Multi-nanozymes to Mimic an Intracellular Antioxidant Defense System.

Science.gov (United States)

Huang, Yanyan; Liu, Zhen; Liu, Chaoqun; Ju, Enguo; Zhang, Yan; Ren, Jinsong; Qu, Xiaogang

2016-06-01

In this work, for the first time, we constructed a novel multi-nanozymes cooperative platform to mimic intracellular antioxidant enzyme-based defense system. V2 O5 nanowire served as a glutathione peroxidase (GPx) mimic while MnO2 nanoparticle was used to mimic superoxide dismutase (SOD) and catalase (CAT). Dopamine was used as a linker to achieve the assembling of the nanomaterials. The obtained V2 O5 @pDA@MnO2 nanocomposite could serve as one multi-nanozyme model to mimic intracellular antioxidant enzyme-based defense procedure in which, for example SOD, CAT, and GPx co-participate. In addition, through assembling with dopamine, the hybrid nanocomposites provided synergistic antioxidative effect. Importantly, both in vitro and in vivo experiments demonstrated that our biocompatible system exhibited excellent intracellular reactive oxygen species (ROS) removal ability to protect cell components against oxidative stress, showing its potential application in inflammation therapy. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Chronic lithium treatment increased intracellular S100ß levels in rat primary neuronal culture.

Directory of Open Access Journals (Sweden)

Masoumeh Emamghoreishi

2015-02-01

Full Text Available S100ß a neurotrophic factor mainly released by astrocytes, has been implicated in the pathogenesis of bipolar disorder. Thus, lithium may exert its neuroprotective effects to some extent through S100ß. Furthermore, the possible effects of lithium on astrocytes as well as on interactions between neurons and astrocytes as a part of its mechanisms of actions are unknown. This study was undertaken to determine the effect of lithium on S100β in neurons, astrocytes and a mixture of neurons and astrocytes. Rat primary astrocyte, neuronal and mixed neuro-astroglia cultures were prepared from cortices of 18-day's embryos. Cell cultures were exposed to lithium (1mM or vehicle for 1day (acute or 7 days (chronic. RT-PCR and ELISA determined S100β mRNA and intra- and extracellular protein levels. Chronic lithium treatment significantly increased intracellular S100β in neuronal and neuro-astroglia cultures in comparison to control cultures (P<0.05. Acute and chronic lithium treatments exerted no significant effects on intracellular S100β protein levels in astrocytes, and extracellular S100β protein levels in three studied cultures as compared to control cultures. Acute and chronic lithium treatments did not significantly alter S100β mRNA levels in three studied cultures, compared to control cultures. Chronic lithium treatment increased intracellular S100ß protein levels in a cell-type specific manner which may favor its neuroprotective action. The findings of this study suggest that lithium may exert its neuroprotective action, at least partly, by increasing neuronal S100ß level, with no effect on astrocytes or interaction between neurons and astrocytes.

Altered glycogen metabolism in cultured astrocytes from mice with chronic glutathione deficit; relevance for neuroenergetics in schizophrenia.

OpenAIRE

Lavoie, S.; Allaman, I.; Petit, J.M.; Do, K.Q.; Magistretti, P.J.

2011-01-01

Neurodegenerative and psychiatric disorders including Alzheimer's, Parkinson's or Huntington's diseases and schizophrenia have been associated with a deficit in glutathione (GSH). In particular, a polymorphism in the gene of glutamate cysteine ligase modulatory subunit (GCLM) is associated with schizophrenia. GSH is the most important intracellular antioxidant and is necessary for the removal of reactive by-products generated by the utilization of glucose for energy supply. Furthermore, gluco...

Histochemical Localization of Glutathione Dependent NBT-Reductase in Mouse Skin

Institute of Scientific and Technical Information of China (English)

无

2001-01-01

Objective　Localization of the glutathione dependent Nitroblue tetrazolium (NBT) reductase in fresh frozen sections of mouse skin and possible dependence of NBT reductase on tissue thiol levels has been investigated. Methods　The fresh frozen tissue sections (8m thickness) were prepared and incubated in medium containing NBT, reduced glutathione (GSH) and phosphate buffer. The staining for GSH was performed with mercury orange. Results　 The activity of the NBT-reductase in mouse skin has been found to be localized in the areas rich in glutathione and actively proliferating area of the skin. Conclusion　The activity of the NBT-reductase seems to be dependent on the glutathione contents.

Hepatic and erythrocytic glutathione peroxidase activity in liver diseases.

Science.gov (United States)

Cordero, R; Ortiz, A; Hernández, R; López, V; Gómez, M M; Mena, P

1996-09-01

Hepatic and erythrocytic glutathione peroxidase activity, together with malondialdehyde levels, were determined as indicators of peroxidation in 83 patients from whom liver biopsies had been taken for diagnostic purposes. On histological study, the patients were classified into groups as minimal changes (including normal liver), steatosis, alcoholic hepatitis, hepatic cirrhosis, light to moderately active chronic hepatitis, and severe chronic active hepatitis. The glutathione peroxidase activity in erythrocytes showed no significant changes in any liver disease group. In the hepatic study, an increased activity was observed in steatosis with respect to the minimal changes group, this increased activity induced by the toxic agent in the initial stages of the alcoholic hepatic disease declining as the hepatic damage progressed. There was a negative correlation between the levels of hepatic malondialdehyde and hepatic glutathione peroxidase in subjects with minimal changes. This suggested the existence of an oxidative equilibrium in this group. This equilibrium is broken in the liver disease groups as was manifest in a positive correlation between malondialdehyde and glutathione peroxidase activity.

Fragrance chemicals lyral and lilial decrease viability of HaCat cells' by increasing free radical production and lowering intracellular ATP level: protection by antioxidants.

Science.gov (United States)

Usta, Julnar; Hachem, Yassmine; El-Rifai, Omar; Bou-Moughlabey, Yolla; Echtay, Karim; Griffiths, David; Nakkash-Chmaisse, Hania; Makki, Rajaa Fakhoury

2013-02-01

We investigate in this study the biochemical effects on cells in culture of two commonly used fragrance chemicals: lyral and lilial. Whereas both chemicals exerted a significant effect on primary keratinocyte(s), HaCat cells, no effect was obtained with any of HepG2, Hek293, Caco2, NIH3T3, and MCF7 cells. Lyral and lilial: (a) decreased the viability of HaCat cells with a 50% cell death at 100 and 60 nM respectively; (b) decreased significantly in a dose dependant manner the intracellular ATP level following 12-h of treatment; (c) inhibited complexes I and II of electron transport chain in liver sub-mitochondrial particles; and (d) increased reactive oxygen species generation that was reversed by N-acetyl cysteine and trolox and the natural antioxidant lipoic acid, without influencing the level of free and/or oxidized glutathione. Lipoic acid protected HaCat cells against the decrease in viability induced by either compound. Dehydrogenation of lyral and lilial produce α,β-unsaturated aldehydes, that reacts with lipoic acid requiring proteins resulting in their inhibition. We propose lyral and lilial as toxic to mitochondria that have a direct effect on electron transport chain, increase ROS production, derange mitochondrial membrane potential, and decrease cellular ATP level, leading thus to cell death. Copyright © 2012 Elsevier Ltd. All rights reserved.

Methyl vinyl ketone, a toxic ingredient in cigarette smoke extract, modifies glutathione in mouse melanoma cells.

Science.gov (United States)

Horiyama, Shizuyo; Takahashi, Yuta; Hatai, Mayuko; Honda, Chie; Suwa, Kiyoko; Ichikawa, Atsushi; Yoshikawa, Noriko; Nakamura, Kazuki; Kunitomo, Masaru; Date, Sachiko; Masujima, Tsutomu; Takayama, Mitsuo

2014-01-01

Cigarette smoke contains many harmful chemicals, which contribute to the pathogenesis of smoking-related diseases such as chronic obstructive pulmonary disease, cancer and cardiovascular disease. The cytotoxicity of cigarette smoke is well documented, but the definitive mechanism behind its toxicity remains unknown. Ingredients in cigarette smoke are known to deplete intracellular glutathione (GSH), the most abundant cellular thiol antioxidant, and to cause oxidative stress. In the present study, we investigated the mechanism of cigarette smoke extract (CSE)-induced cytotoxicity in B16-BL6 mouse melanoma (B16-BL6) cells using liquid chromatography-tandem mass spectrometry. CSE and ingredients in cigarette smoke, methyl vinyl ketone (MVK) and crotonaldehyde (CA), reduced cell viability in a concentration-dependent manner. Also, CSE and the ingredients (m/z 70, each) irreversibly reacted with GSH (m/z 308) to form GSH adducts (m/z 378) in cells and considerably decreased cellular GSH levels at concentrations that do not cause cell death. Mass spectral data showed that the major product formed in cells exposed to CSE was the GSH-MVK adduct via Michael-addition and was not the GSH-CA adduct. These results indicate that MVK included in CSE reacts with GSH in cells to form the GSH-MVK adduct, and thus a possible reason for CSE-induced cytotoxicity is a decrease in intracellular GSH levels.

ENDURANCE TRAINING AND GLUTATHIONE-DEPENDENT ANTIOXIDANT DEFENSE MECHANISM IN HEART OF THE DIABETIC RATS

Directory of Open Access Journals (Sweden)

Mustafa Atalay

2003-06-01

Full Text Available Regular physical exercise beneficially influences cardiac antioxidant defenses in normal rats. The aim of this study was to test whether endurance training can strengthen glutathione-dependent antioxidant defense mechanism and decrease lipid peroxidation in heart of the streptozotocin-induced diabetic rats. Redox status of glutathione in blood of diabetic rats in response to training and acute exercise was also examined. Eight weeks of treadmill training increased the endurance in streptozotocin-induced diabetic rats. It did not affect glutathione level in heart tissue at rest and also after exercise. On the other hand, endurance training decreased glutathione peroxidase activity in heart, while glutathione reductase and glutathione S-transferase activities were not affected either by acute exhaustive exercise or endurance training. Reduced and oxidized glutathione levels in blood were not affected by either training or acute exercise. Conjugated dienes levels in heart tissue were increased by acute exhaustive exercise and also 8 weeks treadmill training. Longer duration of exhaustion in trained group may have contributed to the increased conjugated dienes levels in heart after acute exercise. Our results suggest that endurance type exercise may make heart more susceptible to oxidative stress. Therefore it may be wise to combine aerobic exercise with insulin treatment to prevent its adverse effects on antioxidant defense in heart in patients with diabetes mellitus

Sodium nitroprusside induces autophagic cell death in glutathione-depleted osteoblasts.

Science.gov (United States)

Son, Min Jeong; Lee, Seong-Beom; Byun, Yu Jeong; Lee, Hwa Ok; Kim, Ho-Shik; Kwon, Oh-Joo; Jeong, Seong-Whan

2010-01-01

Previous studies reported that high levels of nitric oxide (NO) induce apoptotic cell death in osteoblasts. We examined molecular mechanisms of cytotoxic injury induced by sodium nitroprusside (SNP), a NO donor, in both glutathione (GSH)-depleted and control U2-OS osteoblasts. Cell viability was reduced by much lower effective concentrations of SNP in GSH-depleted cells compared to normal cells. The data suggest that the level of intracellular GSH is critical in SNP-induced cell death processes of osteoblasts. The level of oxidative stress due to SNP treatments doubled in GSH-depleted cells when measured with fluorochrome H2DCFDA. Pretreatment with the NO scavenger PTIO preserved the viability of cells treated with SNP. Viability of cells treated with SNP was recovered by pretreatment with Wortmannin, an autophagy inhibitor, but not by pretreatment with zVAD-fmk, a pan-specific caspase inhibitor. Large increases of LC3-II were shown by immunoblot analysis of the SNP-treated cells, and the increase was blocked by pretreatment with PTIO or Wortmannin; this implies that under GSH-depleted conditions SNP induces different molecular signaling that lead to autophagic cell death. The ultrastructural morphology of SNP-treated cells in transmission electron microscopy showed numerous autophagic vacuoles. These data suggest NO produces oxidative stress and cellular damage that culminate in autophagic cell death of GSH-depleted osteoblasts. Copyright 2010 Wiley Periodicals, Inc.

Loss of Selenium-Binding Protein 1 Decreases Sensitivity to Clastogens and Intracellular Selenium Content in HeLa Cells.

Science.gov (United States)

Zhao, Changhui; Zeng, Huawei; Wu, Ryan T Y; Cheng, Wen-Hsing

2016-01-01

Selenium-binding protein 1 (SBP1) is not a selenoprotein but structurally binds selenium. Loss of SBP1 during carcinogenesis usually predicts poor prognosis. Because genome instability is a hallmark of cancer, we hypothesize that SBP1 sequesters cellular selenium and sensitizes cancer cells to DNA-damaging agents. To test this hypothesis, we knocked down SBP1 expression in HeLa cervical cancer cells by employing a short hairpin RNA (shRNA) approach. Reduced sensitivity to hydrogen peroxide, paraquat and camptothecin, reactive oxygen species content, and intracellular retention of selenium after selenomethionine treatment were observed in SBP1 shRNA HeLa cells. Results from Western analyses showed that treatment of HeLa cells with selenomethionine resulted in increased SBP1 protein expression in a dose-dependent manner. Knockdown of SBP1 rendered HeLa cells increased expression of glutathione peroxidase-1 but not glutathione peroxidase-4 protein levels and accelerated migration from a wound. Altogether, SBP1 retains supplemental selenium and sensitizes HeLa cancer cells to clastogens, suggesting a new cancer treatment strategy by sequestering selenium through SBP1.

Cyanobacteria perceive nitrogen status by sensing intracellular 2-oxoglutarate levels.

Science.gov (United States)

Muro-Pastor, M I; Reyes, J C; Florencio, F J

2001-10-12

The regulatory circuits that control nitrogen metabolism are relatively well known in several bacterial model groups. However, much less is understood about how the nitrogen status of the cell is perceived in vivo. In cyanobacteria, the transcription factor NtcA is required for regulation (activation or repression) of an extensive number of genes involved in nitrogen metabolism. In contrast, how NtcA activity is regulated is largely unknown. Assimilation of ammonium by most microorganisms occurs through the sequential action of two enzymes: glutamine synthetase (GS) and glutamate synthase. Interestingly, regulation of the expression of NtcA-dependent genes in the cyanobacterium Synechocystis sp. PCC 6803 is altered in mutants with modified levels of GS activity. Two types of mutants were analyzed: glnA null mutants that lack GS type I and gif mutants unable to inactivate GS in the presence of ammonium. Changes in the intracellular pools of 19 different amino acids and the keto acid 2-oxoglutarate were recorded in wild-type and mutant strains under different nitrogen conditions. Our data strongly indicate that the nitrogen status in cyanobacteria is perceived as changes in the intracellular 2-oxoglutarate pool.

Ebselen exerts antifungal activity by regulating glutathione (GSH) and reactive oxygen species (ROS) production in fungal cells.

Science.gov (United States)

Thangamani, Shankar; Eldesouky, Hassan E; Mohammad, Haroon; Pascuzzi, Pete E; Avramova, Larisa; Hazbun, Tony R; Seleem, Mohamed N

2017-01-01

Ebselen, an organoselenium compound and a clinically safe molecule has been reported to possess potent antifungal activity, but its antifungal mechanism of action and in vivo antifungal activity remain unclear. The antifungal effect of ebselen was tested against Candida albicans, C. glabrata, C. tropicalis, C. parapsilosis, Cryptococcus neoformans, and C. gattii clinical isolates. Chemogenomic profiling and biochemical assays were employed to identify the antifungal target of ebselen. Ebselen's antifungal activity in vivo was investigated in a Caenorhabditis elegans animal model. Ebselen exhibits potent antifungal activity against both Candida spp. and Cryptococcus spp., at concentrations ranging from 0.5 to 2μg/ml. Ebselen rapidly eradicates a high fungal inoculum within 2h of treatment. Investigation of the drug's antifungal mechanism of action indicates that ebselen depletes intracellular glutathione (GSH) levels, leading to increased production of reactive oxygen species (ROS), and thereby disturbs the redox homeostasis in fungal cells. Examination of ebselen's in vivo antifungal activity in two Caenorhabditis elegans models of infection demonstrate that ebselen is superior to conventional antifungal drugs (fluconazole, flucytosine and amphotericin) in reducing Candida and Cryptococcus fungal load. Ebselen possesses potent antifungal activity against clinically relevant isolates of both Candida and Cryptococcus by regulating GSH and ROS production. The potent in vivo antifungal activity of ebselen supports further investigation for repurposing it for use as an antifungal agent. The present study shows that ebselen targets glutathione and also support that glutathione as a potential target for antifungal drug development. Copyright © 2016 Elsevier B.V. All rights reserved.

Optimization of Reduced Glutathione Production by a Lactobacillus plantarum Isolate Using Plackett-Burman and Box-Behnken Designs.

Science.gov (United States)

Al-Madboly, Lamiaa A; Khedr, Eman G; Ali, Safaa M

2017-01-01

In this work, we aim to optimize the production of reduced glutathione (GSH) synthesized intracellularly by a food-grade microorganism through a statistical approach. Using a colorimetric method, 25 Lactobacillus plantarum isolates were screened in an attempt to find a GSH-producing strain. It was found that 36% of the tested isolates showed positive result. Isolate (L 7 ) was found to produce 152.61 μM glutathione per gram which was the highest amount produced intracellularly. Accordingly, the later isolate was selected for the optimization process using Plackett-Burman and Box-Behnken designs. Temperature, amino acids, and urea were found to be the most significant independent variables. Following data analysis, the composition of the optimized medium was De Man-Sharp-Rogosa broth as a basal medium supplemented with NaCl (5%), H 2 O 2 (0.05%), sodium dodecyl sulfate (0.05%), amino acids (0.0281%), and urea (0.192%). The pH of the medium was adjusted to 8 and incubated for 24 h at 40°C. The GSH amount was increased by 10-fold (851%) using the optimized medium. Hence, our optimization design estimated the biotechnological potential of L. plantarum (L 7 ) for the production of GSH in the industry.

Optimization of Reduced Glutathione Production by a Lactobacillus plantarum Isolate Using Plackett–Burman and Box–Behnken Designs

Science.gov (United States)

Al-Madboly, Lamiaa A.; Khedr, Eman G.; Ali, Safaa M.

2017-01-01

In this work, we aim to optimize the production of reduced glutathione (GSH) synthesized intracellularly by a food-grade microorganism through a statistical approach. Using a colorimetric method, 25 Lactobacillus plantarum isolates were screened in an attempt to find a GSH-producing strain. It was found that 36% of the tested isolates showed positive result. Isolate (L7) was found to produce 152.61 μM glutathione per gram which was the highest amount produced intracellularly. Accordingly, the later isolate was selected for the optimization process using Plackett–Burman and Box–Behnken designs. Temperature, amino acids, and urea were found to be the most significant independent variables. Following data analysis, the composition of the optimized medium was De Man-Sharp-Rogosa broth as a basal medium supplemented with NaCl (5%), H2O2 (0.05%), sodium dodecyl sulfate (0.05%), amino acids (0.0281%), and urea (0.192%). The pH of the medium was adjusted to 8 and incubated for 24 h at 40°C. The GSH amount was increased by 10-fold (851%) using the optimized medium. Hence, our optimization design estimated the biotechnological potential of L. plantarum (L7) for the production of GSH in the industry. PMID:28536556

The influence of heroin abuse on glutathione-dependent enzymes in human brain.

Science.gov (United States)

Gutowicz, Marzena; Kaźmierczak, Beata; Barańczyk-Kuźma, Anna

2011-01-01

Heroin is an illicit narcotic abused by millions of people worldwide. In our earlier studies we have shown that heroin intoxication changes the antioxidant status in human brain. In the present work we continued our studies by estimating the effect of heroin abuse on reduced glutathione (GSH) and enzymes related to this cofactor, such as glutathione S-transferase detoxifying electrophilics (GST) and organic peroxides (as Se-independent glutathione peroxidase-GSHPx), and Se-dependent glutathione peroxidase (Se-GSHPx) specific mainly for hydrogen peroxide. Studies were conducted on human brains obtained from autopsy of 9 heroin abusers and 8 controls. The level of GSH and the activity of glutathione-related enzymes were determined spectrophotometrically. The expression of GST pi on mRNA and protein level was studied by RT-PCR and Western blotting, respectively. The results indicated significant increase of GST and GSHPx activities, unchanged Se-GSHPx activity, and decreased level of GSH in frontal, temporal, parietal and occipital cortex, brain stem, hippocampus, and white matter of heroin abusers. GST pi expression was increased on both mRNA and protein levels, however the increase was lower in brain stem than in other regions. Heroin affects all regions of human brain, and especially brain stem. Its intoxication leads to an increase of organic rather then inorganic peroxides in various brain regions. Glutathione S-transferase plays an important role during heroin intoxication, however its protective effect is lower in brain stem than in brain cortex or hippocampus. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

Glutathione in plants: an integrated overview.

Science.gov (United States)

Noctor, Graham; Mhamdi, Amna; Chaouch, Sejir; Han, Yi; Neukermans, Jenny; Marquez-Garcia, Belen; Queval, Guillaume; Foyer, Christine H

2012-02-01

Plants cannot survive without glutathione (γ-glutamylcysteinylglycine) or γ-glutamylcysteine-containing homologues. The reasons why this small molecule is indispensable are not fully understood, but it can be inferred that glutathione has functions in plant development that cannot be performed by other thiols or antioxidants. The known functions of glutathione include roles in biosynthetic pathways, detoxification, antioxidant biochemistry and redox homeostasis. Glutathione can interact in multiple ways with proteins through thiol-disulphide exchange and related processes. Its strategic position between oxidants such as reactive oxygen species and cellular reductants makes the glutathione system perfectly configured for signalling functions. Recent years have witnessed considerable progress in understanding glutathione synthesis, degradation and transport, particularly in relation to cellular redox homeostasis and related signalling under optimal and stress conditions. Here we outline the key recent advances and discuss how alterations in glutathione status, such as those observed during stress, may participate in signal transduction cascades. The discussion highlights some of the issues surrounding the regulation of glutathione contents, the control of glutathione redox potential, and how the functions of glutathione and other thiols are integrated to fine-tune photorespiratory and respiratory metabolism and to modulate phytohormone signalling pathways through appropriate modification of sensitive protein cysteine residues. © 2011 Blackwell Publishing Ltd.

Keeping the intracellular vitamin C at a physiologically relevant level in endothelial cell culture

DEFF Research Database (Denmark)

Frikke-Schmidt, Henriette Rønne; Lykkesfeldt, Jens

2010-01-01

It is generally accepted that the addition of vitamin C to cell culture medium improves cell growth. However, once added, the vitamin C concentration declines rapidly. This situation differs from the in vivo environment where the endothelium is constantly supplied with ascorbate from the blood....... With a focus on intracellular vitamin C, we simulated constant supply of ascorbate by the hourly addition of freshly prepared medium containing 75 lM ascorbate and subsequently compared it with more practical regimens using combinations of ascorbate and 2-phosphoascorbate. We found that a single supplement...... of ascorbate and 2-phosphoascorbate adequately maintains intracellular vitamin C at physiological levels for up to 72 h....

1-Methyl-4-phenylpyridinium-induced alterations of glutathione status in immortalized rat dopaminergic neurons

International Nuclear Information System (INIS)

Drechsel, Derek A.; Liang, L.-P.; Patel, Manisha

2007-01-01

Decreased glutathione levels associated with increased oxidative stress are a hallmark of numerous neurodegenerative diseases, including Parkinson's disease. GSH is an important molecule that serves as an anti-oxidant and is also a major determinant of cellular redox environment. Previous studies have demonstrated that neurotoxins can cause changes in reduced and oxidized GSH levels; however, information regarding steady state levels remains unexplored. The goal of this study was to characterize changes in cellular GSH levels and its regulatory enzymes in a dopaminergic cell line (N27) following treatment with the Parkinsonian toxin, 1-methyl-4-phenylpyridinium (MPP + ). Cellular GSH levels were initially significantly decreased 12 h after treatment, but subsequently recovered to values greater than controls by 24 h. However, oxidized glutathione (GSSG) levels were increased 24 h following treatment, concomitant with a decrease in GSH/GSSG ratio prior to cell death. In accordance with these changes, ROS levels were also increased, confirming the presence of oxidative stress. Decreased enzymatic activities of glutathione reductase and glutamate-cysteine ligase by 20-25% were observed at early time points and partly account for changes in GSH levels after MPP + exposure. Additionally, glutathione peroxidase activity was increased 24 h following treatment. MPP + treatment was not associated with increased efflux of glutathione to the medium. These data further elucidate the mechanisms underlying GSH depletion in response to the Parkinsonian toxin, MPP +

Glutathione-binding site of a bombyx mori theta-class glutathione transferase.

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M D Tofazzal Hossain

Full Text Available The glutathione transferase (GST superfamily plays key roles in the detoxification of various xenobiotics. Here, we report the isolation and characterization of a silkworm protein belonging to a previously reported theta-class GST family. The enzyme (bmGSTT catalyzes the reaction of glutathione with 1-chloro-2,4-dinitrobenzene, 1,2-epoxy-3-(4-nitrophenoxy-propane, and 4-nitrophenethyl bromide. Mutagenesis of highly conserved residues in the catalytic site revealed that Glu66 and Ser67 are important for enzymatic function. These results provide insights into the catalysis of glutathione conjugation in silkworm by bmGSTT and into the metabolism of exogenous chemical agents.

Cellular glutathione prevents cytolethality of monomethylarsonic acid

International Nuclear Information System (INIS)

Sakurai, Teruaki; Kojima, Chikara; Ochiai, Masayuki; Ohta, Takami; Sakurai, Masumi H.; Waalkes, Michael P.; Fujiwara, Kitao

2004-01-01

Inorganic arsenicals are clearly toxicants and carcinogens in humans. In mammals, including humans, inorganic arsenic often undergoes methylation, forming compounds such as monomethylarsonic acid (MMAs V ) and dimethylarsinic acid (DMAs V ). However, much less information is available on the in vitro toxic potential or mechanisms of these methylated arsenicals, especially MMAs V . We studied the molecular mechanisms of in vitro cytolethality of MMAs V using a rat liver epithelial cell line (TRL 1215). MMAs V was not cytotoxic in TRL 1215 cells even at concentrations exceeding 10 mM, but it became weakly cytotoxic and induced both necrotic and apoptotic cell death when cellular reduced glutathione (GSH) was depleted with the glutathione synthase inhibitor, L-buthionine-[S,R]-sulfoximine (BSO), or the glutathione reductase inhibitor, carmustine. Similar results were observed in the other mammalian cells, such as human skin TIG-112 cells, chimpanzee skin CRT-1609 cells, and mouse metallothionein (MT) positive and MT negative embryonic cells. Ethacrynic acid (EA), an inhibitor of glutathione S-transferase (GST) that catalyses GSH-substrate conjugation, also enhanced the cytolethality of MMAs V , but aminooxyacetic acid (AOAA), an inhibitor of β-lyase that catalyses the final breakdown of GSH-substrate conjugates, had no effect. Both the cellular GSH levels and the cellular GST activity were increased by the exposure to MMAs V in TRL 1215 cells. On the other hand, the addition of exogenous extracellular GSH enhanced the cytolethality of MMAs V , although cellular GSH levels actually prevented the cytolethality of combined MMAs V and exogenous GSH. These findings indicate that human arsenic metabolite MMAs V is not a highly toxic compound in mammalian cells, and the level of cellular GSH is critical to its eventual toxic effects

Yi-gan san restores behavioral alterations and a decrease of brain glutathione level in a mouse model of schizophrenia.

Science.gov (United States)

Makinodan, Manabu; Yamauchi, Takahira; Tatsumi, Kouko; Okuda, Hiroaki; Noriyama, Yoshinobu; Sadamatsu, Miyuki; Kishimoto, Toshifumi; Wanaka, Akio

2009-01-01

The traditional Chinese herbal medicine yi-gan san has been used to cure neuropsychological disorders. Schizophrenia can be one of the target diseases of yi-gan san. We aimed at evaluating the possible use of yi-gan san in improving the schizophrenic symptoms of an animal model. Yi-gan san or distilled water was administered to mice born from pregnant mice injected with polyinosinic-polycytidilic acid or phosphate buffered saline. The former is a model of schizophrenia based on the epidemiological data that maternal infection leads to psychotic disorders including schizophrenia in the offspring. Prepulse inhibition and sensitivity to methamphetamine in open field tests were analyzed and the total glutathione content of whole brains was measured. Yi-gan san reversed the decrease in prepulse inhibition, hypersensitivity to methamphetamine and cognitive deficits found in the model mice to the level of control mice. Total glutathione content in whole brains was reduced in the model mice but was restored to normal levels by yi-gan san treatment. These results suggest that yi-gan san may have ameliorating effects on the pathological symptoms of schizophrenia.

Overexpression of the transcription factor Yap1 modifies intracellular redox conditions and enhances recombinant protein secretion

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Marizela Delic

2014-10-01

Full Text Available Oxidative folding of secretory proteins in the endoplasmic reticulum (ER is a redox active process, which also impacts the redox conditions in the cytosol. As the transcription factor Yap1 is involved in the transcriptional response to oxidative stress, we investigate its role upon the production of secretory proteins, using the yeast Pichia pastoris as model, and report a novel important role of Yap1 during oxidative protein folding. Yap1 is needed for the detoxification of reactive oxygen species (ROS caused by increased oxidative protein folding. Constitutive co-overexpression of PpYAP1 leads to increased levels of secreted recombinant protein, while a lowered Yap1 function leads to accumulation of ROS and strong flocculation. Transcriptional analysis revealed that more than 150 genes were affected by overexpression of YAP1, in particular genes coding for antioxidant enzymes or involved in oxidation-reduction processes. By monitoring intracellular redox conditions within the cytosol and the ER using redox-sensitive roGFP1 variants, we could show that overexpression of YAP1 restores cellular redox conditions of protein-secreting P. pastoris by reoxidizing the cytosolic redox state to the levels of the wild type. These alterations are also reflected by increased levels of oxidized intracellular glutathione (GSSG in the YAP1 co-overexpressing strain. Taken together, these data indicate a strong impact of intracellular redox balance on the secretion of (recombinant proteins without affecting protein folding per se. Re-establishing suitable redox conditions by tuning the antioxidant capacity of the cell reduces metabolic load and cell stress caused by high oxidative protein folding load, thereby increasing the secretion capacity.

Catalysis of Silver catfish Major Hepatic Glutathione Transferase proceeds via rapid equilibrium sequential random Mechanism

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Ayodele O. Kolawole

Full Text Available Fish hepatic glutathione transferases are connected with the elimination of intracellular pollutants and detoxification of organic micro-pollutants in their aquatic ecosystem. The two-substrate steady state kinetic mechanism of Silver catfish (Synodontis eupterus major hepatic glutathione transferases purified to apparent homogeneity was explored. The enzyme was dimeric enzyme with a monomeric size of 25.6 kDa. Initial-velocity studies and Product inhibition patterns by methyl glutathione and chloride with respect to GSH-CDNB; GSH-ρ-nitrophenylacetate; and GSH-Ethacrynic acid all conforms to a rapid equilibrium sequential random Bi Bi kinetic mechanism rather than steady state sequential random Bi Bi kinetic. α was 2.96 ± 0.35 for the model. The pH profile of Vmax/KM (with saturating 1-chloro-2,4-dinitrobenzene and variable GSH concentrations showed apparent pKa value of 6.88 and 9.86. Inhibition studies as a function of inhibitor concentration show that the enzyme is a homodimer and near neutral GST. The enzyme poorly conjugates 4-hydroxylnonenal and cumene hydroperoxide and may not be involved in oxidative stress protection. The seGST is unique and overwhelmingly shows characteristics similar to those of homodimeric class Pi GSTs, as was indicated by its kinetic mechanism, substrate specificity and inhibition studies. The rate- limiting step, probably the product release, of the reaction is viscosity-dependent and is consequential if macro-viscosogen or micro-viscosogen. Keywords: Silver catfish, Glutathione transferase, Steady-state, Kinetic mechanism, Inhibition

In vitro antileishmanial activity of fisetin flavonoid via inhibition of glutathione biosynthesis and arginase activity in Leishmania infantum.

Science.gov (United States)

Adinehbeigi, Keivan; Razi Jalali, Mohammad Hossein; Shahriari, Ali; Bahrami, Somayeh

2017-06-01

With the increasing emergence of drug resistant Leishmania sp. in recent years, combination therapy has been considered as a useful way to treat and control of Leishmaniasis. The present study was designed to evaluate the antileishmanial effects of the fisetin alone and combination of fisetin plus Meglumine antimoniate (Fi-MA) against Leishmania infantum. The IC50 values for fisetin were obtained 0.283 and 0.102 μM against promastigotes and amastigote forms, respectively. Meglumine antimoniate (MA, Glucantime) as control drug also revealed IC50 values of 0.247 and 0.105 μM for promastigotes and amastigotes of L. infantum, respectively. In order to determine the mode of action of fisetin and Meglumine antimoniate (MA, Glucantime), the activities of arginase (ARG), catalase (CAT), glutathione peroxidase (GPx) and superoxide dismutase (SOD) were measured. Moreover, intracellular glutathione (GSH) and nitric oxide (NO) levels in L. infantum-infected macrophages and L. infantum promastigotes which were treated with IC50 concentrations of fisetin, MA and Fi-MA were investigated. Our results showed that MA decreased CAT and SOD activity and increased NO levels in L. infantum-infected macrophages. In promastigotes, MA inhibited parasite SOD activity and reduced parasite NO production. The decreased levels of most of the antioxidant enzymes, accompanying by the raised level of NO in treated macrophages with MA, were observed to regain their normal profiles due to Fi-MA treatment. Furthermore, fisetin could prevent the growth of promastigotes by inhibition of ARG activity and reduction of GSH levels and NO production. In conclusion, these findings showed that fisetin improves MA side effects.

The role of glutathione in DNA damage by potassium bromate in vitro.

Science.gov (United States)

Parsons, J L; Chipman, J K

2000-07-01

We have investigated the role of reduced glutathione (GSH) in the genetic toxicity of the rodent renal carcinogen potassium bromate (KBrO(3)). A statistically significant increase in the concentration of 8-oxodeoxyguanosine (8-oxodG) relative to deoxyguanosine was measured following incubation of calf thymus DNA with KBrO(3) and GSH or N-acetylcysteine (NACys). This was dependent on these thiols and was associated with the loss of GSH and production of oxidized glutathione. A short-lived (potassium chlorate (KClO(3)) or potassium iodate (KIO(3)) were used instead of KBrO(3), though GSH depletion also occurred with KIO(3), but not with KClO(3). Other reductants and thiols in combination with KBrO(3) did not cause a significant increase in DNA oxidation. DNA strand breakage was also induced by KBrO(3) in human white blood cells (5 mM) and rat kidney epithelial cells (NRK-52E, 1.5 mM). This was associated with an apparent small depletion of thiols in NRK-52E cells at 15 min and with an elevation of 8-oxodG at a delayed time of 24 h. Depletion of intra-cellular GSH by diethylmaleate in human lymphocytes decreased the amount of strand breakage induced by KBrO(3). Extracellular GSH, however, protected against DNA strand breakage by KBrO(3), possibly due to the inability of the reactive product to enter the cell. In contrast, membrane-permeant NACys enhanced KBrO(3)-induced DNA strand breakage in these cells. DNA damage by KBrO(3) is therefore largely dependent on access to intracellular GSH.

Intracellular chromium localization and cell physiological response in the unicellular alga Micrasterias

Science.gov (United States)

Volland, Stefanie; Lütz, Cornelius; Michalke, Bernhard; Lütz-Meindl, Ursula

2012-01-01

Various contaminants like metals and heavy metals are constantly released into the environment by anthropogenic activities. The heavy metal chromium has a wide industrial use and exists in two stable oxidation states: trivalent and hexavalent. Chromium can cause harm to cell metabolism and development, when it is taken up by plants instead of necessary micronutrients such as for example iron. The uptake of Cr VI into plant cells has been reported to be an active process via carriers of essential anions, while the cation Cr III seems to be taken up inactively. Micrasterias denticulata, an unicellular green alga of the family Desmidiaceae is a well-studied cell biological model organism. Cr III and VI had inhibiting effects on its cell development, while cell division rates were only impaired by Cr VI. Transmission electron microscopy (TEM) revealed ultrastructural changes such as increased vacuolization, condensed cytoplasm and dark precipitations in the cell wall after 3 weeks of Cr VI treatment. Electron energy loss spectroscopy (EELS) and electron spectroscopic imaging (ESI) were applied to measure intracellular chromium distribution. Chromium was only detected after 3 weeks of 10 μM Cr VI treatment in electron dense precipitations found in bag-like structures along the inner side of the cell walls together with iron and elevated levels of oxygen, pointing toward an accumulation respectively extrusion of chromium in form of an iron–oxygen compound. Atomic emission spectroscopy (EMS) revealed that Micrasterias cells are able to accumulate considerable amounts of chromium and iron. During chromium treatment the Cr:Fe ratio shifted in favor of chromium, which implied that chromium may be taken up instead of iron. Significant and rapid increase of ROS production within the first 5 min of treatment confirms an active Cr VI uptake. SOD and CAT activity after Cr VI treatment did not show a response, while the glutathione pool determined by immuno-TEM decreased

Intracellular chromium localization and cell physiological response in the unicellular alga Micrasterias

Energy Technology Data Exchange (ETDEWEB)

Volland, Stefanie, E-mail: Stefanie.Volland@stud.sbg.ac.at [Plant Physiology Division, Cell Biology Department, University of Salzburg, Hellbrunnerstr 34, 5020 Salzburg (Austria); Luetz, Cornelius, E-mail: cornelius.luetz@uibk.ac.at [Institute of Botany, University of Innsbruck, Sternwartestrasse 15, 6020 Innsbruck (Austria); Michalke, Bernhard, E-mail: bernhard.michalke@helmholtz-muenchen.de [Helmholtz Zentrum Muenchen, German Research Centre for Environmental Health, Institute of Ecological Chemistry, Ingolstaedter Landstrasse 1, 85764 Neuherberg (Germany); Luetz-Meindl, Ursula, E-mail: ursula.luetz-meindl@sbg.ac.at [Plant Physiology Division, Cell Biology Department, University of Salzburg, Hellbrunnerstr 34, 5020 Salzburg (Austria)

2012-03-15

Various contaminants like metals and heavy metals are constantly released into the environment by anthropogenic activities. The heavy metal chromium has a wide industrial use and exists in two stable oxidation states: trivalent and hexavalent. Chromium can cause harm to cell metabolism and development, when it is taken up by plants instead of necessary micronutrients such as for example iron. The uptake of Cr VI into plant cells has been reported to be an active process via carriers of essential anions, while the cation Cr III seems to be taken up inactively. Micrasterias denticulata, an unicellular green alga of the family Desmidiaceae is a well-studied cell biological model organism. Cr III and VI had inhibiting effects on its cell development, while cell division rates were only impaired by Cr VI. Transmission electron microscopy (TEM) revealed ultrastructural changes such as increased vacuolization, condensed cytoplasm and dark precipitations in the cell wall after 3 weeks of Cr VI treatment. Electron energy loss spectroscopy (EELS) and electron spectroscopic imaging (ESI) were applied to measure intracellular chromium distribution. Chromium was only detected after 3 weeks of 10 {mu}M Cr VI treatment in electron dense precipitations found in bag-like structures along the inner side of the cell walls together with iron and elevated levels of oxygen, pointing toward an accumulation respectively extrusion of chromium in form of an iron-oxygen compound. Atomic emission spectroscopy (EMS) revealed that Micrasterias cells are able to accumulate considerable amounts of chromium and iron. During chromium treatment the Cr:Fe ratio shifted in favor of chromium, which implied that chromium may be taken up instead of iron. Significant and rapid increase of ROS production within the first 5 min of treatment confirms an active Cr VI uptake. SOD and CAT activity after Cr VI treatment did not show a response, while the glutathione pool determined by immuno-TEM decreased

Intracellular chromium localization and cell physiological response in the unicellular alga Micrasterias

International Nuclear Information System (INIS)

Volland, Stefanie; Lütz, Cornelius; Michalke, Bernhard; Lütz-Meindl, Ursula

2012-01-01

Various contaminants like metals and heavy metals are constantly released into the environment by anthropogenic activities. The heavy metal chromium has a wide industrial use and exists in two stable oxidation states: trivalent and hexavalent. Chromium can cause harm to cell metabolism and development, when it is taken up by plants instead of necessary micronutrients such as for example iron. The uptake of Cr VI into plant cells has been reported to be an active process via carriers of essential anions, while the cation Cr III seems to be taken up inactively. Micrasterias denticulata, an unicellular green alga of the family Desmidiaceae is a well-studied cell biological model organism. Cr III and VI had inhibiting effects on its cell development, while cell division rates were only impaired by Cr VI. Transmission electron microscopy (TEM) revealed ultrastructural changes such as increased vacuolization, condensed cytoplasm and dark precipitations in the cell wall after 3 weeks of Cr VI treatment. Electron energy loss spectroscopy (EELS) and electron spectroscopic imaging (ESI) were applied to measure intracellular chromium distribution. Chromium was only detected after 3 weeks of 10 μM Cr VI treatment in electron dense precipitations found in bag-like structures along the inner side of the cell walls together with iron and elevated levels of oxygen, pointing toward an accumulation respectively extrusion of chromium in form of an iron–oxygen compound. Atomic emission spectroscopy (EMS) revealed that Micrasterias cells are able to accumulate considerable amounts of chromium and iron. During chromium treatment the Cr:Fe ratio shifted in favor of chromium, which implied that chromium may be taken up instead of iron. Significant and rapid increase of ROS production within the first 5 min of treatment confirms an active Cr VI uptake. SOD and CAT activity after Cr VI treatment did not show a response, while the glutathione pool determined by immuno-TEM decreased

Human glutathione transferases catalyzing the bioactivation of anticancer thiopurine prodrugs.

Science.gov (United States)

Eklund, Birgitta I; Gunnarsdottir, Sjofn; Elfarra, Adnan A; Mannervik, Bengt

2007-06-01

cis-6-(2-Acetylvinylthio)purine (cAVTP) and trans-6-(2-acetylvinylthio)guanine (tAVTG) are thiopurine prodrugs provisionally inactivated by an alpha,beta-unsaturated substituent on the sulfur of the parental thiopurines 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG). The active thiopurines are liberated intracellularly by glutathione (GSH) in reactions catalyzed by glutathione transferases (GSTs) (EC 2.5.1.18). Catalytic activities of 13 human GSTs representing seven distinct classes of soluble GSTs have been determined. The bioactivation of cAVTP and tAVTG occurs via a transient addition of GSH to the activated double bond of the S-substituent of the prodrug, followed by elimination of the thiopurine. The first of these consecutive reactions is rate-limiting for thiopurine release, but GST-activation of this first addition is shifting the rate limitation to the subsequent elimination. Highly active GSTs reveal the transient intermediate, which is detectable by UV spectroscopy and HPLC analysis. LC/MS analysis of the reaction products demonstrates that the primary GSH conjugate, 4-glutathionylbuten-2-one, can react with a second GSH molecule to form the 4-(bis-glutathionyl)butan-2-one. GST M1-1 and GST A4-4 were the most efficient enzymes with tAVTG, and GST M1-1 and GST M2-2 had highest activity with cAVTP. The highly efficient GST M1-1 is polymorphic and is absent in approximately half of the human population. GST P1-1, which is overexpressed in many cancer cells, had no detectable activity with cAVTP and only minor activity with tAVTG. Other GST-activated prodrugs have targeted GST P1-1-expressing cancer cells. Tumors expressing high levels of GST M1-1 or GST A4-4 can be predicted to be particularly vulnerable to chemotherapy with cAVTP or tAVTG.

Glutathione Preservation during Storage of Rat Lenses in Optisol-GS and Castor Oil

DEFF Research Database (Denmark)

Holm, Thomas; Brøgger-Jensen, Martin Rocho; Johnson, Leif

2013-01-01

Glutathione concentration in the lens decreases in aging and cataractous lenses, providing a marker for tissue condition. Experimental procedures requiring unfrozen lenses from donor banks rely on transportation in storage medium, affecting lens homeostasis and alterations in glutathione levels. ....... The aim of the study was to examine the effects of Optisol-GS and castor oil on lens condition, determined from their ability to maintain glutathione concentrations....

Glutathione oxidation correlates with one-lung ventilation time and PO2/FiO2 ratio during pulmonary lobectomy.

Science.gov (United States)

García-de-la-Asunción, José; García-Del-Olmo, Eva; Galan, Genaro; Guijarro, Ricardo; Martí, Francisco; Badenes, Rafael; Perez-Griera, Jaume; Duca, Alejandro; Delgado, Carlos; Carbonell, Jose; Belda, Javier

2016-09-01

During lung lobectomy, the operated lung completely collapses with simultaneous hypoxic pulmonary vasoconstriction, followed by expansion and reperfusion. Here, we investigated glutathione oxidation and lipoperoxidation in patients undergoing lung lobectomy, during one-lung ventilation (OLV) and after resuming two-lung ventilation (TLV), and examined the relationship with OLV duration. We performed a single-centre, observational, prospective study in 32 patients undergoing lung lobectomy. Blood samples were collected at five time-points: T0, pre-operatively; T1, during OLV, 5 minutes before resuming TLV; and T2, T3, and T4, respectively, 5, 60, and 180 minutes after resuming TLV. Samples were tested for reduced glutathione (GSH), oxidized glutathione (GSSG), glutathione redox potential, and malondialdehyde (MDA). GSSG and MDA blood levels increased at T1, and increased further at T2. OLV duration directly correlated with marker levels at T1 and T2. Blood levels of GSH and glutathione redox potential decreased at T1-T3. GSSG, oxidized glutathione/total glutathione ratio, and MDA levels were inversely correlated with arterial blood PO2/FiO2 at T1 and T2. During lung lobectomy and OLV, glutathione oxidation, and lipoperoxidation marker blood levels increase, with further increases after resuming TLV. Oxidative stress degree was directly correlated with OLV duration, and inversely correlated with arterial blood PO2/FiO2.

Carbon black nanoparticles promote endothelial activation and lipid accumulation in macrophages independently of intracellular ROS production

DEFF Research Database (Denmark)

Cao, Yi; Roursgaard, Martin; Danielsen, Pernille Høgh

2014-01-01

, the concentrations of CB to induce lipid accumulation were lower than the concentrations to promote intracellular ROS production in THP-1a cells. In conclusion, exposure to nano-sized CB induced endothelial dysfunction and foam cell formation, which was not dependent on intracellular ROS production....... and WST-1 assays, especially in THP-1 and THP-1a cells. The CB exposure decreased the glutathione (GSH) content in THP-1 and THP-1a cells, whereas GSH was increased in HUVECs. The reactive oxygen species (ROS) production was increased in all cell types after CB exposure. A reduction of the intracellular...... GSH concentration by buthionine sulfoximine (BSO) pre-treatment further increased the CB-induced ROS production in THP-1 cells and HUVECs. The expression of adhesion molecules ICAM-1 and VCAM-1, but not adhesion of THP-1 to HUVECs or culture dishes, was elevated by CB exposure, whereas these effects...

Reaction between nitracrine and glutathione: implications for hypoxic cell radiosensitization and cytotoxicity

International Nuclear Information System (INIS)

Wilson, W.R.; Anderson, R.F.

1989-01-01

Nitracrine (NC) is an electron affinic DNA intercalating agent and a potent hypoxia-selective cytotoxin and radiosensitizer in cell culture. Although NC is too cytotoxic and too rapidly metabolized to provide hypoxic cell radiosensitization in tumors, it is of mechanistic interest as an example of a DNA affinic radiosensitizer. We have observed a rapid chemical reaction between NC and reduced glutathione (GSH), which suggests that the observed potent in vitro cytotoxicity and radiosensitization might be dependent on thiol depletion by the large extracellular reservoir of drug. However, no GSH depletion was observed under conditions providing radiosensitization or rapid cell killing, and prior depletion of GSH by buthionine sulphoximine had no effect on cytotoxicity or formation of macromolecular adducts. Further, the intracellular reaction of NC with GSH is slower than predicted on the basis of the measured second order rate constant and the total intracellular concentrations of both species. The results are consistent with a role for DNA binding in protecting NC from reaction with GSH, and in improving the efficiency with which reduced electrophilic metabolites react with DNA in preference to GSH

Relationship between oxidizable fatty acid content and level of antioxidant glutathione peroxidases in marine fish

Science.gov (United States)

Grim, Jeffrey M.; Hyndman, Kelly A.; Kriska, Tamas; Girotti, Albert W.; Crockett, Elizabeth L.

2011-01-01

SUMMARY Biological membranes can be protected from lipid peroxidation by antioxidant enzymes including catalase (CAT) and selenium-dependent glutathione peroxidases 1 and 4 (GPx1 and GPx4). Unlike GPx1, GPx4 can directly detoxify lipid hydroperoxides in membranes without prior action of phospholipase A2. We hypothesized that (1) GPx4 is enhanced in species that contain elevated levels of highly oxidizable polyunsaturated fatty acids (PUFA) and (2) activities of antioxidant enzymes are prioritized to meet species-specific oxidative stresses. In this study we examined (i) activities of the oxidative enzyme citrate synthase (CS) and antioxidant (CAT, GPx1 and GPx4) enzymes, (ii) GPx4 protein expression, and (iii) phospholipid composition in livers of five species of marine fish (Myxine glutinosa, Petromyzon marinus, Squalus acanthias, Fundulus heteroclitus and Myoxocephalus octodecemspinosus) that contain a range of PUFA. GPx4 activity was, on average, 5.8 times higher in F. heteroclitus and S. acanthias than in the other three marine fish species sampled. Similarly, activities of CAT and GPx1 were highest in S. acanthias and F. heteroclitus, respectively. GPx4 activity for all species correlates with membrane unsaturation, as well as oxidative activity as indicated by CS. These data support our hypothesis that GPx4 level in marine fish is a function, at least in part, of high PUFA content in these animals. GPx1 activity was also correlated with membrane unsaturation, indicating that marine species partition resources among glutathione-dependent defenses for protection from the initial oxidative insult (e.g. H2O2) and to repair damaged lipids within biological membranes. PMID:22031739

Balneotherapy and platelet glutathione metabolism in type II diabetic patients

Science.gov (United States)

Ohtsuka, Yoshinori; Yabunaka, Noriyuki; Watanabe, Ichiro; Noro, Hiroshi; Agishi, Yuko

1996-09-01

Effects of balneotherapy on platelet glutathione metabolism were investigated in 12 type II (non-insulin-dependent) diabetic patients. Levels of the reduced form of glutathione (GSH) on admission were well correlated with those of fasting plasma glucose (FPG; r=0.692, Pbalneotherapy, the mean level of GSH showed no changes; however, in well-controlled patients (FPG 150 mg/dl), the value decreased ( Pbalneotherapy, the activity increased in 5 patients, decreased in 3 patients and showed no changes (alteration within ±3%) in all the other patients. From these findings in diabetic patients we concluded: (1) platelet GSH synthesis appeared to be induced in response to oxidative stress; (2) lowered GPX activities indicated that the antioxidative defense system was impaired; and (3) platelet glutathione metabolism was partially improved by 4 weeks balneotherapy, an effect thought to be dependent on the control status of plasma glucose levels. It is suggested that balneotherapy is beneficial for patients whose platelet antioxidative defense system is damaged, such as those with diabetes mellitus and coronary heart disease.

Yi-Gan San Restores Behavioral Alterations and a Decrease of Brain Glutathione Level in a Mouse Model of Schizophrenia

Directory of Open Access Journals (Sweden)

Manabu Makinodan M.D.

2009-01-01

Full Text Available The traditional Chinese herbal medicine yi-gan san has been used to cure neuropsychological disorders. Schizophrenia can be one of the target diseases of yi-gan san. We aimed at evaluating the possible use of yi-gan san in improving the schizophrenic symptoms of an animal model. Yi-gan san or distilled water was administered to mice born from pregnant mice injected with polyinosinic-polycytidilic acid or phosphate buffered saline. The former is a model of schizophrenia based on the epidemiological data that maternal infection leads to psychotic disorders including schizophrenia in the offspring. Prepulse inhibition and sensitivity to methamphetamine in open field tests were analyzed and the total glutathione content of whole brains was measured. Yi-gan san reversed the decrease in prepulse inhibition, hypersensitivity to methamphetamine and cognitive deficits found in the model mice to the level of control mice. Total glutathione content in whole brains was reduced in the model mice but was restored to normal levels by yi-gan san treatment. These results suggest that yi-gan san may have ameliorating effects on the pathological symptoms of schizophrenia.

Changes of reduced glutathion, glutathion reductase, and glutathione peroxidase after radiation in guinea pigs

International Nuclear Information System (INIS)

Erden, M.; Bor, N.M.

1984-01-01

In this series of experiments the protective action of reduced glutathion due to ionizing radiation has been studied. In the experimental group 18 guinea pigs were exposed to successive radiations of 150 rad 3 or 4 days apart. Total dose given amounted to 750 rad which is the LD50 for guinea pigs. Blood samples were taken 30 min after each exposure. The control series were sham radiated but otherwise treated identically. The cells of the removed blood samples were separated by centrifugation and were subjected to the reduced glutathion stability test. GSSGR, GPer, and LDH enzyme activities were also measured of which the latter served as a marked enzyme. It was found that LDH did not show any alteration after radiation. The reduced glutathion stability test showed a consistent but minor reduction (P greater than 0.05), in the experimental group. GSSGR enzyme activity on the other hand was reduced significantly (from 176.48 +/- 11.32 to 41.34 +/- 1.17 IU/ml of packed erythrocytes, P less than 0.001) in the same group. GPer activity showed a consistent but minor elevation during the early phase of the experimental group. It was later increased significantly beginning after 600 rad total radiation on the fourth session (P less than 0.050)

The effect of EGb 761 on retinal lipid peroxidation and glutathione peroxidase level in experimental lens induced uveitis.

Science.gov (United States)

Bilgihan, A; Aricioğlu, A; Bilgihan, K; Onol, M; Hasanreisoğlu, B; Türközkan, N

1994-01-01

An acute lens-induced necrotizing intraocular inflammation was produced in pigmented guinea pigs. Treatment of these animals by 100 mg/kg/day EGb 761 a free oxygen radical scavenger for 10 days, reduced retinal lipid peroxidation (p > 0.05) and increased the retinal glutathione peroxidase level (p > 0.05). Although not significantly, these findings suggest that EGb 761 could be combined with other antiinflammatory drugs and may be beneficial in the treatment of uveitis.

Docosahexaenoic acid prevents paraquat-induced reactive oxygen species production in dopaminergic neurons via enhancement of glutathione homeostasis

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Lee, Hyoung Jun; Han, Jeongsu; Jang, Yunseon; Kim, Soo Jeong; Park, Ji Hoon; Seo, Kang Sik [Department of Biochemistry, College of Medicine, Chungnam National University, Daejeon (Korea, Republic of); Jeong, Soyeon; Shin, Soyeon; Lim, Kyu [Department of Biochemistry, College of Medicine, Chungnam National University, Daejeon (Korea, Republic of); Infection Signaling Network Research Center, Chungnam National University, Daejeon (Korea, Republic of); Heo, Jun Young, E-mail: junyoung3@gmail.com [Brainscience Institute, Chungnam National University, Daejeon (Korea, Republic of); Kweon, Gi Ryang, E-mail: mitochondria@cnu.ac.kr [Department of Biochemistry, College of Medicine, Chungnam National University, Daejeon (Korea, Republic of); Infection Signaling Network Research Center, Chungnam National University, Daejeon (Korea, Republic of)

2015-01-30

Highlights: • DHA prevents PQ-induced dopaminergic neuronal loss via decreasing of excessive ROS. • DHA increases GR and GCLm derivate GSH pool by enhancement of Nrf2 expression. • Protective mechanism is removal of PQ-induced ROS via DHA-dependent GSH pool. • DHA may be a good preventive strategy for Parkinson’s disease (PD) therapy. - Abstract: Omega-3 polyunsaturated fatty acid levels are reduced in the substantia nigra area in Parkinson’s disease patients and animal models, implicating docosahexaenoic acid (DHA) as a potential treatment for preventing Parkinson’s disease and suggesting the need for investigations into how DHA might protect against neurotoxin-induced dopaminergic neuron loss. The herbicide paraquat (PQ) induces dopaminergic neuron loss through the excessive production of reactive oxygen species (ROS). We found that treatment of dopaminergic SN4741 cells with PQ reduced cell viability in a dose-dependent manner, but pretreatment with DHA ameliorated the toxic effect of PQ. To determine the toxic mechanism of PQ, we measured intracellular ROS content in different organelles with specific dyes. As expected, all types of ROS were increased by PQ treatment, but DHA pretreatment selectively decreased cytosolic hydrogen peroxide content. Furthermore, DHA treatment-induced increases in glutathione reductase and glutamate cysteine ligase modifier subunit (GCLm) mRNA expression were positively correlated with glutathione (GSH) content. Consistent with this increase in GCLm mRNA levels, Western blot analysis revealed that DHA pretreatment increased nuclear factor-erythroid 2 related factor 2 (Nrf2) protein levels. These findings indicate that DHA prevents PQ-induced neuronal cell loss by enhancing Nrf2-regulated GSH homeostasis.

Docosahexaenoic acid prevents paraquat-induced reactive oxygen species production in dopaminergic neurons via enhancement of glutathione homeostasis

International Nuclear Information System (INIS)

Lee, Hyoung Jun; Han, Jeongsu; Jang, Yunseon; Kim, Soo Jeong; Park, Ji Hoon; Seo, Kang Sik; Jeong, Soyeon; Shin, Soyeon; Lim, Kyu; Heo, Jun Young; Kweon, Gi Ryang

2015-01-01

Highlights: • DHA prevents PQ-induced dopaminergic neuronal loss via decreasing of excessive ROS. • DHA increases GR and GCLm derivate GSH pool by enhancement of Nrf2 expression. • Protective mechanism is removal of PQ-induced ROS via DHA-dependent GSH pool. • DHA may be a good preventive strategy for Parkinson’s disease (PD) therapy. - Abstract: Omega-3 polyunsaturated fatty acid levels are reduced in the substantia nigra area in Parkinson’s disease patients and animal models, implicating docosahexaenoic acid (DHA) as a potential treatment for preventing Parkinson’s disease and suggesting the need for investigations into how DHA might protect against neurotoxin-induced dopaminergic neuron loss. The herbicide paraquat (PQ) induces dopaminergic neuron loss through the excessive production of reactive oxygen species (ROS). We found that treatment of dopaminergic SN4741 cells with PQ reduced cell viability in a dose-dependent manner, but pretreatment with DHA ameliorated the toxic effect of PQ. To determine the toxic mechanism of PQ, we measured intracellular ROS content in different organelles with specific dyes. As expected, all types of ROS were increased by PQ treatment, but DHA pretreatment selectively decreased cytosolic hydrogen peroxide content. Furthermore, DHA treatment-induced increases in glutathione reductase and glutamate cysteine ligase modifier subunit (GCLm) mRNA expression were positively correlated with glutathione (GSH) content. Consistent with this increase in GCLm mRNA levels, Western blot analysis revealed that DHA pretreatment increased nuclear factor-erythroid 2 related factor 2 (Nrf2) protein levels. These findings indicate that DHA prevents PQ-induced neuronal cell loss by enhancing Nrf2-regulated GSH homeostasis

Epalrestat increases glutathione, thioredoxin, and heme oxygenase-1 by stimulating Nrf2 pathway in endothelial cells

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Kaori Yama

2015-04-01

Full Text Available Epalrestat (EPS is the only aldose reductase inhibitor that is currently available for the treatment of diabetic neuropathy. Recently, we found that EPS at near-plasma concentration increases the intracellular levels of glutathione (GSH in rat Schwann cells. GSH plays a crucial role in protecting endothelial cells from oxidative stress, thereby preventing vascular diseases. Here we show that EPS increases GSH levels in not only Schwann cells but also endothelial cells. Treatment of bovine aortic endothelial cells (BAECs, an in vitro model of the vascular endothelium, with EPS caused a dramatic increase in intracellular GSH levels. This was concomitant with the up-regulation of glutamate cysteine ligase, an enzyme catalyzing the first and rate-limiting step in de novo GSH synthesis. Moreover, EPS stimulated the expression of thioredoxin and heme oxygenase-1, which have important redox regulatory functions in endothelial cells. Nuclear factor erythroid 2-related factor 2 (Nrf2 is a key transcription factor that regulates the expression of antioxidant genes. EPS increased nuclear Nrf2 levels in BAECs. Nrf2 knockdown by siRNA suppressed the EPS-induced glutamate cysteine ligase, thioredoxin-1, and heme oxygenase-1 expression. Interestingly, LY294002, an inhibitor of phosphatidylinositol 3-kinase, abolished the EPS-stimulated GSH synthesis, suggesting that the kinase is associated with Nrf2 activation induced by EPS. Furthermore, EPS reduced the cytotoxicity induced by H2O2 and tert-butylhydroperoxide, indicating that EPS plays a role in protecting cells from oxidative stress. Taken together, the results provide evidence that EPS exerts new beneficial effects on endothelial cells by increasing GSH, thioredoxin, and heme oxygenase-1 levels through the activation of Nrf2. We suggest that EPS has the potential to prevent several vascular diseases caused by oxidative stress.

The influence of royal jelly and human interferon-alpha (HuIFN-αN3) on proliferation, glutathione level and lipid peroxidation in human colorectal adenocarcinoma cells in vitro.

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Filipič, Bratko; Gradišnik, Lidija; Rihar, Klemen; Šooš, Eugen; Pereyra, Adriana; Potokar, Jana

2015-12-01

Among royal jelly's (RJ) various biological activities, its possible antitumour activity deserves particular attention. The purpose of this study was to investigate the influence of RJ, its bioactive component 10-hydroxy-2-decenoic acid (10- HDA), and human interferon-alpha (HuIFN-αN3) on the proliferation of human colorectal adenocarcinoma cells (CaCo- 2), and ascertain their effect on intracellular glutathione (GSH) level and lipid peroxidation. We studied the antiproliferative (AP) activity of RJ [(0.1 g/10 mL phosphate buffer saline (PBS)], HuIFN-αN3 (1000 I.U. mL⁻¹), 10-HDA at 100.0 μmol L⁻¹, and their different combinations, in the ratio 1:1, 1:2, and 2:1 on CaCo-2 cells. The GSH level was measured by glutathione assay. The lipid peroxidation was measured by malondialdehyde (MDA) assay. Single RJ had a low AP activity: 2.0 (0.5 mg mL⁻¹). HuIFN-αN3 had an AP activity of 2.5 (208.33 I.U. mL⁻¹), while 10-HDA had an AP activity of 1.5 (37.5 μmol mL⁻¹). The highest AP activity of 3.8 was obtained when RJ and HuIFN-αN3 were applied at the ratio 2:1. In that combination the level of GSH was 24.9±2.4 nmol g⁻³ of proteins (vs. 70.2±3.2 nmol g⁻³ in the control) and the level of MDA was 72.3±3.1 nmol g⁻³ (vs. 23.6±9.1 nmol g⁻³ in the control). It is generally assumed that 10-HDA, an important constituent of RJ, together with HuIFN-αN3, is responsible for the inhibition of CaCo-2 cells proliferation in vitro. In our study, however, RJ and HuIFN-αN3 applied at 2:1 decreased the level of GSH the most and significantly increased lipid peroxidation via MDA in CaCo-2 cells. Future studies should show whether these GSH- and MDA-related activities of RJ, HuIFN-αN3, 10-HDA, and their combinations may decrease the tumorigenicity index and tumorigenic potential of various tumour cells in vitro.

Oxidative stress protection and glutathione metabolism in response to hydrogen peroxide and menadione in riboflavinogenic fungus Ashbya gossypii.

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Kavitha, S; Chandra, T S

2014-11-01

Ashbya gossypii is a plant pathogen and a natural overproducer of riboflavin and is used for industrial riboflavin production. A few literature reports depict a link between riboflavin overproduction and stress in this fungus. However, the stress protection mechanisms and glutathione metabolism are not much explored in A. gossypii. In the present study, an increase in the activity of catalase and superoxide dismutase was observed in response to hydrogen peroxide and menadione. The lipid peroxide and membrane lipid peroxide levels were increased by H2O2 and menadione, indicating oxidative damage. The glutathione metabolism was altered with a significant increase in oxidized glutathione (GSSG), glutathione peroxidase (GPX), glutathione S transferase (GST), and glutathione reductase (GR) and a decrease in reduced glutathione (GSH) levels in the presence of H2O2 and menadione. Expression of the genes involved in stress mechanism was analyzed in response to the stressors by semiquantitative RT-PCR. The messenger RNA (mRNA) levels of CTT1, SOD1, GSH1, YAP1, and RIB3 were increased by H2O2 and menadione, indicating the effect of stress at the transcriptional level. A preliminary bioinformatics study for the presence of stress response elements (STRE)/Yap response elements (YRE) depicted that the glutathione metabolic genes, stress genes, and the RIB genes hosted either STRE/YRE, which may enable induction of these genes during stress.

Metformin induces an intracellular reductive state that protects oesophageal squamous cell carcinoma cells against cisplatin but not copper-bis(thiosemicarbazones)

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Damelin, Leonard Howard; Jivan, Rupal; Veale, Robin Bruce; Rousseau, Amanda Louise; Mavri-Damelin, Demetra

2014-01-01

Oesophageal squamous cell carcinoma (OSCC) is a highly aggressive carcinoma with a poor survival rate. One of the most commonly used chemotherapeutic drugs, cisplatin, displays varied and often poor efficacy in vivo. Therefore, alternative, cost-effective and more efficacious treatments are required. Metformin has been previously shown to reduce proliferative rates in various carcinoma cell lines. We report for the first time, the effect of metformin on OSCC cell proliferation and show that it antagonises cisplatin-induced but not copper-bis(thiosemicarbazone)-induced cytotoxicity in OSCC cells. Cell proliferation and stage of the cell cycle were quantified by trypan blue counts and flow cytometry, respectively. All cytotoxicity measurements were made using the tetrazolium based MTT assay. Metabolic alterations to cells were determined as follows: glycolysis via a lactate dehydrogenase assay, reducing equivalents by MTT reduction and reduced intracellular thiols by monobromobimane-thiol fluorescence, and glutathione depletion using buthionine sulfoximine. Inductively coupled plasma mass spectrometry was used to quantify cisplatin-DNA adduct formation. Metformin was found to reduce cell proliferation significantly in all OSCC cell lines, with an accumulation of cells in G0/G1 phase of the cell cycle. However, metformin significantly protected OSCC cells against cisplatin toxicity. Our results indicate that a major mechanism of metformin-induced cisplatin resistance results from a significant increase in glycolysis, intracellular NAD(P)H levels with a concomitant increase in reduced intracellular thiols, leading to decreased cisplatin-DNA adduct formation. The glutathione synthesis inhibitor buthionine sulfoximine significantly ablated the protective effect of metformin. We subsequently show that the copper-bis(thiosemicarbazones), Cu-ATSM and Cu-GTSM, which are trapped in cells under reducing conditions, cause significant OSCC cytotoxicity, both alone and in

[The activity of glutathione antioxidant system at melaksen and valdoxan action under experimental hyperthyroidism in rats].

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Gorbenko, M V; Popova, T N; Shul'gin, K K; Popov, S S

2013-01-01

Investigation of glutathione antioxidant system activity and diene conjugates content in rats liver and blood serum at the influence of melaksen and valdoxan under experimental hyperthyroidism (EG) has been revealed. It has been established that the activities of glutathione reductase (GR), glutathione peroxidase (GP) and glutathione transferase (GT), growing at pathological conditions, change to the side of control value at these substunces introduction. Reduced glutathione content (GSH) at melaxen and valdoxan action increased compared with values under the pathology, that, obviously, could be associated with a reduction of its spending on the detoxication of free radical oxidation (FRO) toxic products. Diene conjugates level in rats liver and blood serum, increasing at experimental hyperthyroidism conditions, under introduction of melatonin level correcting drugs, also approached to the control meaning. Results of the study indicate on positive effect of melaxen and valdoxan on free radical homeostasis, that appears to be accompanied by decrease of load on the glutathione antioxidant system in comparison with the pathology.

Single-cell intracellular nano-pH probes†

OpenAIRE

Özel, Rıfat Emrah; Lohith, Akshar; Mak, Wai Han; Pourmand, Nader

2015-01-01

Within a large clonal population, such as cancerous tumor entities, cells are not identical, and the differences between intracellular pH levels of individual cells may be important indicators of heterogeneity that could be relevant in clinical practice, especially in personalized medicine. Therefore, the detection of the intracellular pH at the single-cell level is of great importance to identify and study outlier cells. However, quantitative and real-time measurements of the intracellular p...

The antitumour activity of alkylating agents is not correlated with the levels of glutathione, glutathione transferase and O6-alkylguanine-DNA-alkyltransferase of human tumour xenografts. EORTC SPG and PAMM Groups.

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D'Incalci, M; Bonfanti, M; Pifferi, A; Mascellani, E; Tagliabue, G; Berger, D; Fiebig, H H

1998-10-01

Twenty-three human xenografts, including five colon, five gastric, nine lung (three small cell lung cancer) and four breast carcinomas, were investigated for their sensitivity to nitrosoureas, dacarbazine (DTIC), cyclophosphamide (CTX) and cisplatin (DDP). In 12 cases, at least one of the drugs produced complete or partial remission, in 2, a minor regression was observed and in the other 9, treatment was ineffective. The level of sensitivity to each drug, using a score from 1 to 5, was correlated to three biochemical parameters reported to be involved in resistance to alkylating agents: glutathione (GSH), glutathione transferase (GST) and O6-alkylguanine-DNA-alkyltransferase (AGT). A wide variability was found in these parameters in the xenografts investigated. No correlation was found between any of the three parameters and sensitivity to the drugs used or between sensitivity to one drug and to any of the other drugs tested. These results illustrate the complexity of the question of resistance to alkylating agents and indicate that, at least in xenografts, the biochemical parameters examined are not predictive of response to alkylating agents.

Timing of developmental reduction in epithelial glutathione redox potential is associated with increased epithelial proliferation in the immature murine intestine.

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Reid, Graham K; Berardinelli, Andrew J; Ray, Laurie; Jackson, Arena R; Neish, Andrew S; Hansen, Jason M; Denning, Patricia W

2017-08-01

BackgroundThe intracellular redox potential of the glutathione (GSH)/glutathione disulfide (GSSG) couple regulates cellular processes. In vitro studies indicate that a reduced GSH/GSSG redox potential favors proliferation, whereas a more oxidized redox potential favors differentiation. Intestinal growth depends upon an appropriate balance between the two. However, how the ontogeny of intestinal epithelial cellular (IEC) GSH/GSSG redox regulates these processes in the developing intestine has not been fully characterized in vivo.MethodsOntogeny of intestinal GSH redox potential and growth were measured in neonatal mice.ResultsWe show that IEC GSH/GSSG redox potential becomes increasingly reduced (primarily driven by increased GSH concentration) over the first 3 weeks of life. Increased intracellular GSH has been shown to drive proliferation through increased poly-ADP-ribose polymerase (PARP) activity. We show that increasing IEC poly-ADP-ribose chains can be measured over the first 3 weeks of life, indicating an increase in IEC PARP activity. These changes are accompanied by increased intestinal growth and IEC proliferation as assessed by villus height/crypt depth, intestinal length, and Ki67 staining.ConclusionUnderstanding how IEC GSH/GSSG redox potential is developmentally regulated may provide insight into how premature human intestinal redox states can be manipulated to optimize intestinal growth and adaptation.

Chemical form of selenium affects its uptake, transport, and glutathione peroxidase activity in the human intestinal Caco-2 cell model.

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Zeng, Huawei; Jackson, Matthew I; Cheng, Wen-Hsing; Combs, Gerald F

2011-11-01

Determining the effect of selenium (Se) chemical form on uptake, transport, and glutathione peroxidase activity in human intestinal cells is critical to assess Se bioavailability at nutritional doses. In this study, we found that two sources of L-selenomethionine (SeMet) and Se-enriched yeast each increased intracellular Se content more effectively than selenite or methylselenocysteine (SeMSC) in the human intestinal Caco-2 cell model. Interestingly, SeMSC, SeMet, and digested Se-enriched yeast were transported at comparable efficacy from the apical to basolateral sides, each being about 3-fold that of selenite. In addition, these forms of Se, whether before or after traversing from apical side to basolateral side, did not change the potential to support glutathione peroxidase (GPx) activity. Although selenoprotein P has been postulated to be a key Se transport protein, its intracellular expression did not differ when selenite, SeMSC, SeMet, or digested Se-enriched yeast was added to serum-contained media. Taken together, our data show, for the first time, that the chemical form of Se at nutritional doses can affect the absorptive (apical to basolateral side) efficacy and retention of Se by intestinal cells; but that, these effects are not directly correlated to the potential to support GPx activity.

Intracellular calcium levels determine differential modulation of allosteric interactions within G protein-coupled receptor heteromers.

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Navarro, Gemma; Aguinaga, David; Moreno, Estefania; Hradsky, Johannes; Reddy, Pasham P; Cortés, Antoni; Mallol, Josefa; Casadó, Vicent; Mikhaylova, Marina; Kreutz, Michael R; Lluís, Carme; Canela, Enric I; McCormick, Peter J; Ferré, Sergi

2014-11-20

The pharmacological significance of the adenosine A2A receptor (A2AR)-dopamine D2 receptor (D2R) heteromer is well established and it is being considered as an important target for the treatment of Parkinson’s disease and other neuropsychiatric disorders. However, the physiological factors that control its distinctive biochemical properties are still unknown. We demonstrate that different intracellular Ca2+ levels exert a differential modulation of A2AR-D2R heteromer-mediated adenylyl-cyclase and MAPK signaling in striatal cells. This depends on the ability of low and high Ca2+ levels to promote a selective interaction of the heteromer with the neuronal Ca2+-binding proteins NCS-1 and calneuron-1, respectively. These Ca2+-binding proteins differentially modulate allosteric interactions within the A2AR-D2R heteromer, which constitutes a unique cellular device that integrates extracellular (adenosine and dopamine) and intracellular (Ca+2) signals to produce a specific functional response.

Understanding the degradation of ascorbic acid and glutathione in relation to the levels of oxidative stress biomarkers in broccoli (Brassica oleracea L. italica cv. Bellstar) during storage and mechanical processing.

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Raseetha, Siva; Leong, Sze Ying; Burritt, David John; Oey, Indrawati

2013-06-01

The purpose of this research was to understand the degradation of ascorbic acid and glutathione content in broccoli florets (Brassica oleracea L. italica cv. Bellstar) during prolonged storage and subsequent mechanical processing. The initial content of total ascorbic acid and glutathione in broccoli florets averaged at 5.18 ± 0.23 and 0.70 ± 0.03 μmol/g fresh weight, respectively. Results showed that the content of ascorbic acid and glutathione in broccoli degraded during storage at 23°C, for at least 4.5-fold after 6 days of storage. On each day of storage, broccoli florets were mechanically processed, but the content of total ascorbic acid and glutathione was not significantly affected. When the mechanically processed broccoli florets were further incubated for up to 6h, the amount of ascorbic acid was greatly reduced as compared to glutathione. To obtain an in-depth understanding on the degradation of ascorbic acid and glutathione, the activity of enzymes involved in plant antioxidative system via ascorbate-glutathione cycle, as a response towards oxidative stress that took place during storage was determined in this study. The content of total ascorbic acid and glutathione in broccoli florets before and after mechanical processing were found to decrease concurrently with the activity of ascorbic acid peroxidase and glutathione reductase over the experimental storage duration. Meanwhile, the effect of oxidative stress on the content of ascorbic acid and glutathione was apparent during the 6h of incubation after mechanical processing. This phenomenon was demonstrated by the level of oxidative stress biomarkers examined, in which the formation of lipid peroxides, protein carbonyls and DNA oxidised products was positively associated with the degradation of total ascorbic acid and glutathione. Copyright © 2012 Elsevier Ltd. All rights reserved.

Correction of glutathione metabolism in the liver of albino rats affected by low radiation doses

International Nuclear Information System (INIS)

Moiseenok, A.G.; Slyshenkov, V.S.; Khomich, T.I.; Zimatkina, T.I.; Kanunnikova, N.P.

1997-01-01

The levels of total glutathione GSH, GSSG and the activities of glutathione reductase and glutathione peroxidase were studied in the liver of adult albino rats subjected to 3-fold external γ-irradiation throughout 2 weeks at the overall dose of 0.75 Gy after 15 h, 2 and 5 days from the last irradiation. Some animals were injected intraperitoneally with the pantothenate containing complex > 3 times on days 1-3 before the irradiation. The radiation related decrease of GSH, GSH/GSSG and the total glutathione level was prevented by the prophylactic administration of the complex and probably at the expense of the activation of the G-SH biosynthesis and/or transport in the liver by the CoA biosynthetic precursor. (author)

Implications of Glutathione Levels in the Plasmodium berghei Response to Chloroquine and Artemisinin.

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Joel Vega-Rodríguez

Full Text Available Malaria is one of the most devastating parasitic diseases worldwide. Plasmodium drug resistance remains a major challenge to malaria control and has led to the re-emergence of the disease. Chloroquine (CQ and artemisinin (ART are thought to exert their anti-malarial activity inducing cytotoxicity in the parasite by blocking heme degradation (for CQ and increasing oxidative stress. Besides the contribution of the CQ resistance transporter (PfCRT and the multidrug resistant gene (pfmdr, CQ resistance has also been associated with increased parasite glutathione (GSH levels. ART resistance was recently shown to be associated with mutations in the K13-propeller protein. To analyze the role of GSH levels in CQ and ART resistance, we generated transgenic Plasmodium berghei parasites either deficient in or overexpressing the gamma-glutamylcysteine synthetase gene (pbggcs encoding the rate-limiting enzyme in GSH biosynthesis. These lines produce either lower (pbggcs-ko or higher (pbggcs-oe levels of GSH than wild type parasites. In addition, GSH levels were determined in P. berghei parasites resistant to CQ and mefloquine (MQ. Increased GSH levels were detected in both, CQ and MQ resistant parasites, when compared to the parental sensitive clone. Sensitivity to CQ and ART remained unaltered in both pgggcs-ko and pbggcs-oe parasites when tested in a 4 days drug suppressive assay. However, recrudescence assays after the parasites have been exposed to a sub-lethal dose of ART showed that parasites with low levels of GSH are more sensitive to ART treatment. These results suggest that GSH levels influence Plasmodium berghei response to ART treatment.

The effect of aluminium-stress and exogenous spermidine on chlorophyll degradation, glutathione reductase activity and the photosystem II D1 protein gene (psbA) transcript level in lichen Xanthoria parietina.

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Sen, Gulseren; Eryilmaz, Isil Ezgi; Ozakca, Dilek

2014-02-01

In this study, the effects of short-term aluminium toxicity and the application of spermidine on the lichen Xanthoria parietina were investigated at the physiological and transcriptional levels. Our results suggest that aluminium stress leads to physiological processes in a dose-dependent manner through differences in lipid peroxidation rate, chlorophyll content and glutathione reductase (EC 1.6.4.2) activity in aluminium and spermidine treated samples. The expression of the photosystem II D1 protein (psbA) gene was quantified using semi-quantitative RT-PCR. Increased glutathione reductase activity and psbA mRNA transcript levels were observed in the X. parietina thalli that were treated with spermidine before aluminium-stress. The results showed that the application of spermidine could mitigate aluminium-induced lipid peroxidation and chlorophyll degradation on lichen X. parietina thalli through an increase in psbA transcript levels and activity of glutathione reductase (GR) enzymes. Copyright © 2013 Elsevier Ltd. All rights reserved.

A regulatory review for products containing glutathione

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Nur Hidayah Abd Rahim

2016-01-01

Full Text Available Glutathione is a potent antioxidant as well as has important role for DNA synthesis and repair, protein synthesis, amino acid transport, and enzyme activation. Besides this, Glutathione products are now mainly selling as whitening agent which are mainly marketing through social media (Facebook and different websites. Information is not available whether glutathione product are following the regulatory guidelines of National Pharmaceutical Control Bureau of Malaysia (NPCB for selling, advertisement and promotion. This review was carried out by extracting information about glutathione from scientific database using PubMed, Cochrane Library and Embase. Analysis of the available information, case example of glutathione products showed that a brand of glutathione (Glutacaps HQ did not show the product's registration number from NPCB, and also did not show the name, address, contact number of the advertiser, and even not found the name of the manufacture. Without providing the above mentioned information, the product is selling and promoting through social media (fb which is not allowed by the NPCB guidelines part 4.14. So far, only two clinical trials were conducted on glutathione supplementation for 4 weeks duration. There was no serious or systematic adverse effects reported in clinical trials. As the two clinic trials resulted contradictory outcomes, further studies needed for conformation of the clinic benefits of glutathione. Otherwise, random use of glutathione may be risk for the health of the people. Besides, the marketer mainly promoting glutathione as the skin whitening beauty product instead of using as health supplement, it may cause additional and serious risk to the users as the manufacturer not providing sufficient information about the product, its registration number, manufacturing company, etc.

Gelsolin-Cu/ZnSOD interaction alters intracellular reactive oxygen species levels to promote cancer cell invasion

KAUST Repository

Tochhawng, Lalchhandami; Deng, Shuo; Ganesan, Pugalenthi; Kumar, Alan Prem; Lim, Kiat Hon; Yang, Henry; Hooi, Shing Chuan; Goh, Yaw Chong; Maciver, Sutherland K.; Pervaiz, Shazib; Yap, Celestial T.

2016-01-01

, and this is mediated via gelsolin's effects in elevating intracellular superoxide (O2 .-) levels. We also provide evidence for a novel physical interaction between gelsolin and Cu/ZnSOD, that inhibits the enzymatic activity of Cu/ZnSOD, thereby resulting in a sustained

Evaluation of intracellular anion superoxide level, heat shock protein A2 and protamine positive spermatozoa percentages in teratoasthenozoospermia

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Parvin Sabeti

2017-09-01

Full Text Available Background: Teratoasthenozoospermia (TA is a severe form of male infertility with no clear etiology. Objective: To compare the level of intracellular anion superoxide (O2–, heat shock protein A2 (HSPA2 and protamine deficiencies in ejaculated spermatozoa between teratoasthenozoospermic and normozoospermic men. Materials and Methods: In this case- control study, semen samples of 20 infertile men, with TA (with normal morphology lower than 4%_ and total motility lower than 40% as the case group and 20 normozoospermic fertile men as the control group were evaluated for intracellular O2 – and HSPA2 by flow cytometry and protamine deficiency by Chromomycin A3 (CMA3 test. Results: The rate of CMA3+ spermatozoa in the case group was higher than controls (p=0.001. The percentages of HSPA2+ spermatozoa in the cases were significantly lower than controls (p=0.001. Also, intracellular O2 – levels in the case group were significantly higher than controls (p=0.001 and had positive correlations with sperm apoptosis (r=0.79, p=0.01 and CMA3 positive sperm (r=0.76, p=0.01, but negative correlations with normal morphology (r=-0.81, p=0.01 and motility (r=-0.81, p=0.01. There was no significant correlation between intracellular O2 – and HSPA2 in the case group (r=0.041, p=0.79. Conclusion: We suggest that the increase in intracellular O2 –, decrease in spermatozoa HSPA2+, and high percentages of spermatozoa with immature chromatin might be considered as etiologies of infertility in TA patients

Arabidopsis GLUTATHIONE REDUCTASE1 plays a crucial role in leaf responses to intracellular hydrogen peroxide and in ensuring appropriate gene expression through both salicylic acid and jasmonic acid signaling pathways.

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Mhamdi, Amna; Hager, Jutta; Chaouch, Sejir; Queval, Guillaume; Han, Yi; Taconnat, Ludivine; Saindrenan, Patrick; Gouia, Houda; Issakidis-Bourguet, Emmanuelle; Renou, Jean-Pierre; Noctor, Graham

2010-07-01

Glutathione is a major cellular thiol that is maintained in the reduced state by glutathione reductase (GR), which is encoded by two genes in Arabidopsis (Arabidopsis thaliana; GR1 and GR2). This study addressed the role of GR1 in hydrogen peroxide (H(2)O(2)) responses through a combined genetic, transcriptomic, and redox profiling approach. To identify the potential role of changes in glutathione status in H(2)O(2) signaling, gr1 mutants, which show a constitutive increase in oxidized glutathione (GSSG), were compared with a catalase-deficient background (cat2), in which GSSG accumulation is conditionally driven by H(2)O(2). Parallel transcriptomics analysis of gr1 and cat2 identified overlapping gene expression profiles that in both lines were dependent on growth daylength. Overlapping genes included phytohormone-associated genes, in particular implicating glutathione oxidation state in the regulation of jasmonic acid signaling. Direct analysis of H(2)O(2)-glutathione interactions in cat2 gr1 double mutants established that GR1-dependent glutathione status is required for multiple responses to increased H(2)O(2) availability, including limitation of lesion formation, accumulation of salicylic acid, induction of pathogenesis-related genes, and signaling through jasmonic acid pathways. Modulation of these responses in cat2 gr1 was linked to dramatic GSSG accumulation and modified expression of specific glutaredoxins and glutathione S-transferases, but there is little or no evidence of generalized oxidative stress or changes in thioredoxin-associated gene expression. We conclude that GR1 plays a crucial role in daylength-dependent redox signaling and that this function cannot be replaced by the second Arabidopsis GR gene or by thiol systems such as the thioredoxin system.

Ajoene restored behavioral patterns and liver glutathione level in morphine treated C57BL6 mice.

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Yun, Jaesuk; Oliynyk, Sergiy; Lee, Yeonju; Kim, Jieun; Yun, Kyunghwa; Jeon, Raok; Ryu, Jae-Ha; Oh, Seikwan

2017-01-01

Oxidative stress exacerbates drug dependence induced by administration of opiate analgesics such as morphine-induced tolerance and physical dependence associated with the reduction in hepatic glutathione (GSH) level. Ajoene obtained from garlic (Allium sativum L.) has been reported for anti-tumorigenic, anti-oxidative and neuroprotective properties, however, little is known about its effect on morphine-induced dependence. Therefore, this study aimed at the effect of ajoene on physical and/or psychological dependence and liver GSH content in morphine-treated mice. Conditioned place preference (CPP) test and measurement of morphine withdrawal syndrome were performed in C57BL6 mice for behavioral experiments. Thereafter, mice were sacrificed for measurement of serum and liver GSH levels. Ajoene restored CPP and naloxone-precipitated jumping behavior in mice exposed to morphine. Moreover, the reduced level of liver GSH content in morphine treated mice was back to normal after ajoene administration. Taken together, ajoene improved behavioral patterns in mice exposed to morphine suggesting its potential therapeutic benefit against morphine-induced dependence.

[Establishment of oxygen and glucose deprive model of in vitro cultured hippocampal neuron and effect of ligustrazine on intracellular Ca+ level in model neurons].

Science.gov (United States)

Wan, Hai-tong; Wang, Yu; Yang, Jie-hong

2007-03-01

To establish the oxygen and glucose deprive (OGD) model in cultured hippocampal neuron and study the effect of ligustrazine on intracellular Ca2+ level in the model neurons. The OGD model was established in cultured hippocampal neuron, and the intracellular Ca2+ level in it was detected by laser scanning confocal microscope (LSCM). The OGD model was successfully established in cultured hippocampal neurons; the intracellular Ca2+ level in the OGD model group was significantly higher than that in the blank control group (P neuron, which could be antagonized by ligustrazine, indicating that ligustrazine has a protective effect on hippocampal neuron from hypoxic-ischemic injury.

Heat and exercise acclimation increases intracellular levels of Hsp72 and inhibits exercise-induced increase in intracellular and plasma Hsp72 in humans.

Science.gov (United States)

Magalhães, Flávio de Castro; Amorim, Fabiano Trigueiro; Passos, Renata L Freitas; Fonseca, Michele Atalla; Oliveira, Kenya Paula Moreira; Lima, Milene Rodrigues Malheiros; Guimarães, Juliana Bohen; Ferreira-Júnior, João Batista; Martini, Angelo R P; Lima, Nilo R V; Soares, Danusa Dias; Oliveira, Edilamar Menezes; Rodrigues, Luiz Oswaldo Carneiro

2010-11-01

In order to verify the effects of heat and exercise acclimation (HA) on resting and exercise-induced expression of plasma and leukocyte heat shock protein 72 (Hsp72) in humans, nine healthy young male volunteers (25.0 ± 0.7 years; 80.5 ± 2.0 kg; 180 ± 2 cm, mean ± SE) exercised for 60 min in a hot, dry environment (40 ± 0°C and 45 ± 0% relative humidity) for 11 days. The protocol consisted of running on a treadmill using a controlled hyperthermia technique in which the work rate was adjusted to elevate the rectal temperature by 1°C in 30 min and maintain it elevated for another 30 min. Before and after the HA, the volunteers performed a heat stress test (HST) at 50% of their individual maximal power output for 90 min in the same environment. Blood was drawn before (REST), immediately after (POST) and 1 h after (1 h POST) HST, and plasma and leukocytes were separated and stored. Subjects showed expected adaptations to HA: reduced exercise rectal and mean skin temperatures and heart rate, and augmented sweat rate and exercise tolerance. In HST1, plasma Hsp72 increased from REST to POST and then returned to resting values 1 h POST (REST: 1.11 ± 0.07, POST: 1.48 ± 0.10, 1 h POST: 1.22 ± 0.11 ng mL(-1); p  0.05). HA increased resting levels of intracellular Hsp72 (HST1: 1 ± 0.02 and HST2: 4.2 ± 1.2 density units, p  0.05). Regression analysis showed that the lower the pre-exercise expression of intracellular Hsp72, the higher the exercise-induced increase (R = -0.85, p < 0.05). In conclusion, HA increased resting leukocyte Hsp72 levels and inhibited exercise-induced expression. This intracellular adaptation probably induces thermotolerance. In addition, the non-increase in plasma Hsp72 after HA may be related to lower stress at the cellular level in the acclimated individuals.

Intracellular response to process optimization and impact on productivity and product aggregates for a high-titer CHO cell process.

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Handlogten, Michael W; Lee-O'Brien, Allison; Roy, Gargi; Levitskaya, Sophia V; Venkat, Raghavan; Singh, Shailendra; Ahuja, Sanjeev

2018-01-01

A key goal in process development for antibodies is to increase productivity while maintaining or improving product quality. During process development of an antibody, titers were increased from 4 to 10 g/L while simultaneously decreasing aggregates. Process development involved optimization of media and feed formulations, feed strategy, and process parameters including pH and temperature. To better understand how CHO cells respond to process changes, the changes were implemented in a stepwise manner. The first change was an optimization of the feed formulation, the second was an optimization of the medium, and the third was an optimization of process parameters. Multiple process outputs were evaluated including cell growth, osmolality, lactate production, ammonium concentration, antibody production, and aggregate levels. Additionally, detailed assessment of oxygen uptake, nutrient and amino acid consumption, extracellular and intracellular redox environment, oxidative stress, activation of the unfolded protein response (UPR) pathway, protein disulfide isomerase (PDI) expression, and heavy and light chain mRNA expression provided an in-depth understanding of the cellular response to process changes. The results demonstrate that mRNA expression and UPR activation were unaffected by process changes, and that increased PDI expression and optimized nutrient supplementation are required for higher productivity processes. Furthermore, our findings demonstrate the role of extra- and intracellular redox environment on productivity and antibody aggregation. Processes using the optimized medium, with increased concentrations of redox modifying agents, had the highest overall specific productivity, reduced aggregate levels, and helped cells better withstand the high levels of oxidative stress associated with increased productivity. Specific productivities of different processes positively correlated to average intracellular values of total glutathione. Additionally

Control of intracellular heme levels: Heme transporters and Heme oxygenases

Science.gov (United States)

Khan, Anwar A.; Quigley, John G.

2011-01-01

Heme serves as a co-factor in proteins involved in fundamental biological processes including oxidative metabolism, oxygen storage and transport, signal transduction and drug metabolism. In addition, heme is important for systemic iron homeostasis in mammals. Heme has important regulatory roles in cell biology, yet excessive levels of intracellular heme are toxic; thus, mechanisms have evolved to control the acquisition, synthesis, catabolism and expulsion of cellular heme. Recently, a number of transporters of heme and heme synthesis intermediates have been described. Here we review aspects of heme metabolism and discuss our current understanding of heme transporters, with emphasis on the function of the cell-surface heme exporter, FLVCR. Knockdown of Flvcr in mice leads to both defective erythropoiesis and disturbed systemic iron homeostasis, underscoring the critical role of heme transporters in mammalian physiology. PMID:21238504

Determination of glutathione and glutathione disulfide in biological samples: an in-depth review.

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Monostori, Péter; Wittmann, Gyula; Karg, Eszter; Túri, Sándor

2009-10-15

Glutathione (GSH) is a thiol-containing tripeptide, which plays central roles in the defence against oxidative damage and in signaling pathways. Upon oxidation, GSH is transformed to glutathione disulfide (GSSG). The concentrations of GSH and GSSG and their molar ratio are indicators of cell functionality and oxidative stress. Assessment of redox homeostasis in various clinical states and medical applications for restoration of the glutathione status are of growing importance. This review is intended to provide a state-of-the-art overview of issues relating to sample pretreatment and choices for the separation and detection of GSH and GSSG. High-performance liquid chromatography, capillary electrophoresis and gas chromatography (as techniques with a separation step) with photometric, fluorimetric, electrochemical and mass spectrometric detection are discussed, stress being laid on novel approaches.

Effect of supplementation with methionine and different fat sources on the glutathione redox system of growing chickens.

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Németh, Katalin; Mézes, M; Gaál, T; Bartos, A; Balogh, K; Husvéth, F

2004-01-01

The effect of supplementary methionine and fats of different saturation levels on the glutathione redox system of growing broiler cockerels was studied. The diet of three groups of chicks was supplemented with corn germ oil, beef tallow and fish oil at the levels of 30 g/kg and 50 g/kg of feed, respectively. The diet of further three groups was supplemented with methionine (5 g/kg of feed) in addition to the different fat sources. Control chicks were fed with a compound feed without methionine and fat supplementation. Reduced glutathione (GSH) and glutathione disulphide (GSSG) content as well as glutathione peroxidase activity in the liver were determined and GSH/GSSG ratio was calculated at day old and then at one and three weeks of age. Our results indicate that supplementary methionine stimulates both the synthesis of the glutathione redox system and glutathione peroxidase activity in growing chickens in the first period of postnatal life, when the risk of lipid peroxidation is high due to feeding unsaturated fats in the diet.

Is Glutathione the Major Cellular Target of Cisplatin?

DEFF Research Database (Denmark)

Kasherman, Yonit; Stürup, Stefan; gibson, dan

2009-01-01

Cisplatin is an anticancer drug whose efficacy is limited because tumors develop resistance to the drug. Resistant cells often have elevated levels of cellular glutathione (GSH), believed to be the major cellular target of cisplatin that inactivates the drug by binding to it irreversibly, forming...

CHANGES IN THE GLUTATHIONE SYSTEM IN P19 EMBRYONAL CARCINOMA CELLS UNDER HYPOXIC CONDITIONS

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D. S. Orlov

2015-01-01

Full Text Available Introduction. According to modern perceptions, tumor growth, along with oxidative stress formation, is accompanied by hypoxia. Nowadays studying the regulation of cellular molecular system functioning by conformational changes in proteins appears to be a topical issue. Research goal was to evaluate the state of the glutathione system and the level of protein glutathionylation in P19 embryonal carcinoma (EC cells under hypoxic conditions.Material and methods. P19 EC cells (mouse embryonal carcinoma cultured under normoxic and hypox-ic conditions served the research material.The concentration of total, oxidized, reduced and protein-bound glutathione, the reduced to oxidized thiol ratio as well as glutathione peroxidase and glutathione reductase activity were determined by spectropho-tometry.Results. Glutathione imbalance was accompanied by a decrease in P19 EC cell redox status under hypox-ic conditions against the backdrop of a rise in protein-bound glutathione.Conclusions. As a result of the conducted study oxidative stress formation was identified when modeling hypoxia in P19 embryonal carcinoma cells. The rise in the concentration of protein-bound glutathione may indicate the role of protein glutathionylation in regulation of P19 cell metabolism and functions un-der hypoxia. 

Glutathione regulation of redox-sensitive signals in tumor necrosis factor-α-induced vascular endothelial dysfunction

International Nuclear Information System (INIS)

Tsou, T.-C.; Yeh, S.C.; Tsai, F.-Y.; Chen, J.-W.; Chiang, H.-C.

2007-01-01

We investigated the regulatory role of glutathione in tumor necrosis factor-alpha (TNF-α)-induced vascular endothelial dysfunction as evaluated by using vascular endothelial adhesion molecule expression and monocyte-endothelial monolayer binding. Since TNF-α induces various biological effects on vascular cells, TNF-α dosage could be a determinant factor directing vascular cells into different biological fates. Based on the adhesion molecule expression patterns responding to different TNF-α concentrations, we adopted the lower TNF-α (0.2 ng/ml) to rule out the possible involvement of other TNF-α-induced biological effects. Inhibition of glutathione synthesis by L-buthionine-(S,R)-sulfoximine (BSO) resulted in down-regulations of the TNF-α-induced adhesion molecule expression and monocyte-endothelial monolayer binding. BSO attenuated the TNF-α-induced nuclear factor-kappaB (NF-κB) activation, however, with no detectable effect on AP-1 and its related mitogen-activated protein kinases (MAPKs). Deletion of an AP-1 binding site in intercellular adhesion molecule-1 (ICAM-1) promoter totally abolished its constitutive promoter activity and its responsiveness to TNF-α. Inhibition of ERK, JNK, or NF-κB attenuates TNF-α-induced ICAM-1 promoter activation and monocyte-endothelial monolayer binding. Our study indicates that TNF-α induces adhesion molecule expression and monocyte-endothelial monolayer binding mainly via activation of NF-κB in a glutathione-sensitive manner. We also demonstrated that intracellular glutathione does not modulate the activation of MAPKs and/or their downstream AP-1 induced by lower TNF-α. Although AP-1 activation by the lower TNF-α was not detected in our systems, we could not rule out the possible involvement of transiently activated MAPKs/AP-1 in the regulation of TNF-α-induced adhesion molecule expression

Glutathione system in Wolfram syndrome 1‑deficient mice.

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Porosk, Rando; Kilk, Kalle; Mahlapuu, Riina; Terasmaa, Anton; Soomets, Ursel

2017-11-01

Wolfram syndrome 1 (WS) is a rare neurodegenerative disease that is caused by mutations in the Wolfram syndrome 1 (WFS1) gene, which encodes the endoplasmic reticulum (ER) glycoprotein wolframin. The pathophysiology of WS is ER stress, which is generally considered to induce oxidative stress. As WS has a well‑defined monogenetic origin and a model for chronic ER stress, the present study aimed to characterize how glutathione (GSH), a major intracellular antioxidant, was related to the disease and its progression. The concentration of GSH and the activities of reduction/oxidation system enzymes GSH peroxidase and GSH reductase were measured in Wfs1‑deficient mice. The GSH content was lower in most of the studied tissues, and the activities of antioxidative enzymes varied between the heart, kidneys and liver tissues. The results indicated that GSH may be needed for ER stress control; however, chronic ER stress from the genetic syndrome eventually depletes the cellular GSH pool and leads to increased oxidative stress.

High performance liquid chromatographic assay for the quantitation of total glutathione in plasma

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Abukhalaf, Imad K.; Silvestrov, Natalia A.; Menter, Julian M.; von Deutsch, Daniel A.; Bayorh, Mohamed A.; Socci, Robin R.; Ganafa, Agaba A.

2002-01-01

A simple and widely used homocysteine HPLC procedure was applied for the HPLC identification and quantitation of glutathione in plasma. The method, which utilizes SBDF as a derivatizing agent utilizes only 50 microl of sample volume. Linear quantitative response curve was generated for glutathione over a concentration range of 0.3125-62.50 micromol/l. Linear regression analysis of the standard curve exhibited correlation coefficient of 0.999. Limit of detection (LOD) and limit of quantitation (LOQ) values were 5.0 and 15 pmol, respectively. Glutathione recovery using this method was nearly complete (above 96%). Intra-assay and inter-assay precision studies reflected a high level of reliability and reproducibility of the method. The applicability of the method for the quantitation of glutathione was demonstrated successfully using human and rat plasma samples.

Chrysin enhances doxorubicin-induced cytotoxicity in human lung epithelial cancer cell lines: The role of glutathione

Energy Technology Data Exchange (ETDEWEB)

Brechbuhl, Heather M. [Pediatrics, National Jewish Health, Denver, Colorado (United States); Kachadourian, Remy; Min, Elysia [Department of Medicine, National Jewish Health, Denver, Colorado (United States); Chan, Daniel [Medical Oncology, University of Colorado Denver Health Sciences Center (United States); Day, Brian J., E-mail: dayb@njhealth.org [Department of Medicine, University of Colorado Denver Health Sciences Center (United States); Immunology, University of Colorado Denver Health Sciences Center (United States); Pharmaceutical Sciences, University of Colorado Denver Health Sciences Center (United States); Department of Medicine, National Jewish Health, Denver, Colorado (United States)

2012-01-01

We hypothesized that flavonoid-induced glutathione (GSH) efflux through multi-drug resistance proteins (MRPs) and subsequent intracellular GSH depletion is a viable mechanism to sensitize cancer cells to chemotherapies. This concept was demonstrated using chrysin (5–25 μM) induced GSH efflux in human non-small cell lung cancer lines exposed to the chemotherapeutic agent, doxorubicin (DOX). Treatment with chrysin resulted in significant and sustained intracellular GSH depletion and the GSH enzyme network in the four cancer cell types was predictive of the severity of chrysin induced intracellular GSH depletion. Gene expression data indicated a positive correlation between basal MRP1, MRP3 and MRP5 expression and total GSH efflux before and after chrysin exposure. Co-treating the cells for 72 h with chrysin (5–30 μM) and DOX (0.025–3.0 μM) significantly enhanced the sensitivity of the cells to DOX as compared to 72-hour DOX alone treatment in all four cell lines. The maximum decrease in the IC{sub 50} values of cells treated with DOX alone compared to co-treatment with chrysin and DOX was 43% in A549 cells, 47% in H157 and H1975 cells and 78% in H460 cells. Chrysin worked synergistically with DOX to induce cancer cell death. This approach could allow for use of lower concentrations and/or sensitize cancer cells to drugs that are typically resistant to therapy. -- Graphical abstract: Possible mechanisms by which chrysin enhances doxorubicin-induced toxicity in cancer cells. Highlights: ► Chyrsin sustains a significant depletion of GSH levels in lung cancer cells. ► Chyrsin synergistically potentiates doxorubicin-induced cancer cell cytotoxicity. ► Cancer cell sensitivity correlated with GSH and MRP gene network expression. ► This approach could allow for lower side effects and targeting resistant tumors.

Oxidative Stress Markers and Genetic Polymorphisms of Glutathione ...

African Journals Online (AJOL)

Hence, we evaluated the serum levels of oxidative stress markers and investigated genetic polymorphisms of glutathione S-transferase associated with autism. Materials and Methods: Forty-two children clinically diagnosed with ASD using the Diagnostic and Statistical Manual for Mental Disorders (DSM-5) criteria and a ...

The crucial protective role of glutathione against tienilic acid hepatotoxicity in rats

International Nuclear Information System (INIS)

Nishiya, Takayoshi; Mori, Kazuhiko; Hattori, Chiharu; Kai, Kiyonori; Kataoka, Hiroko; Masubuchi, Noriko; Jindo, Toshimasa; Manabe, Sunao

2008-01-01

To investigate the hepatotoxic potential of tienilic acid in vivo, we administered a single oral dose of tienilic acid to Sprague-Dawley rats and performed general clinicopathological examinations and hepatic gene expression analysis using Affymetrix microarrays. No change in the serum transaminases was noted at up to 1000 mg/kg, although slight elevation of the serum bile acid and bilirubin, and very mild hepatotoxic changes in morphology were observed. In contrast to the marginal clinicopathological changes, marked upregulation of the genes involved in glutathione biosynthesis [glutathione synthetase and glutamate-cysteine ligase (Gcl)], oxidative stress response [heme oxygenase-1 and NAD(P)H dehydrogenase quinone 1] and phase II drug metabolism (glutathione S-transferase and UDP glycosyltransferase 1A6) were noted after 3 or 6 h post-dosing. The hepatic reduced glutathione level decreased at 3-6 h, and then increased at 24 or 48 h, indicating that the upregulation of NF-E2-related factor 2 (Nrf2)-regulated gene and the late increase in hepatic glutathione are protective responses against the oxidative and/or electrophilic stresses caused by tienilic acid. In a subsequent experiment, tienilic acid in combination with L-buthionine-(S,R)-sulfoximine (BSO), an inhibitor of Gcl caused marked elevation of serum alanine aminotransferase (ALT) with extensive centrilobular hepatocyte necrosis, whereas BSO alone showed no hepatotoxicity. The elevation of ALT by this combination was observed at the same dose levels of tienilic acid as the upregulation of the Nrf2-regulated genes by tienilic acid alone. In conclusion, these results suggest that the impairment of glutathione biosynthesis may play a critical role in the development of tienilic acid hepatotoxicity through extensive oxidative and/or electrophilic stresses

Differential inhibitory response to telcagepant on αCGRP induced vasorelaxation and intracellular Ca(2+) levels in the perfused and non-perfused isolated rat middle cerebral artery

DEFF Research Database (Denmark)

Erdling, André; Sheykhzade, Majid; Edvinsson, Lars

2017-01-01

BACKGROUND: Calcitonin gene-related peptide (CGRP) is one of the most potent endogenous vasodilators identified to date. The present study elucidates the differential interaction of CGRP, its receptor and the effect of the CGRP-receptor antagonist telcagepant on intracellular Ca(2+) -levels...... and tension in rat middle cerebral arteries (MCA) by pressurized arteriography, FURA-2/wire myography and immunohistochemistry. METHODS: A pressurized arteriograph system was used to evaluate changes in MCA tension when subjected to CGRP and/or telcagepant. Intracellular calcium levels were evaluated using......, while abluminal telcagepant inhibited the relaxation (10(-6) M). Using the FURA-2 method in combination with wire myography we observed that αCGRP reduced intracellular calcium levels and in parallel the vascular tone. Telcagepant (10(-6) M) inhibited both vasorelaxation and drop in intracellular...

Glutathione role in gallium induced toxicity

African Journals Online (AJOL)

Asim

2012-01-26

GSH) present in tissues. It is very important and interesting to study the reaction of gallium nitrate and glutathione as biomarker of glutathione role in detoxification and conjugation in whole blood components (plasma and ...

Glutathione preservation during storage of rat lenses in optisol-GS and castor oil.

Science.gov (United States)

Holm, Thomas; Brøgger-Jensen, Martin Rocho; Johnson, Leif; Kessel, Line

2013-01-01

Glutathione concentration in the lens decreases in aging and cataractous lenses, providing a marker for tissue condition. Experimental procedures requiring unfrozen lenses from donor banks rely on transportation in storage medium, affecting lens homeostasis and alterations in glutathione levels. The aim of the study was to examine the effects of Optisol-GS and castor oil on lens condition, determined from their ability to maintain glutathione concentrations. Rat lenses were stored in the two types of storage media at varying time intervals up to 3 days. Glutathione concentration was afterwards determined in an enzymatic detection assay, specific for both reduced and oxidized forms. Lenses removed immediately after death exhibited a glutathione concentration of 4.70±0.29 mM. In vitro stored lenses in Optisol-GS lost glutathione quickly, ending with a concentration of 0.60±0.34 mM after 3 days while castor oil stored lenses exhibited a slower decline and ended at 3 times the concentration. A group of lenses were additionally stored under post mortem conditions within the host for 6 hours before its removal. Total glutathione after 6 hours was similar to that of lenses removed immediately after death, but with altered GSH and GSSG concentrations. Subsequent storage of these lenses in media showed changes similar to those in the first series of experiments, albeit to a lesser degree. It was determined that storage in Optisol-GS resulted in a higher loss of glutathione than lenses stored in castor oil. Storage for more than 12 hours reduced glutathione to half its original concentration, and was considered unusable after 24 hours.

Glutathione preservation during storage of rat lenses in optisol-GS and castor oil.

Directory of Open Access Journals (Sweden)

Thomas Holm

Full Text Available BACKGROUND: Glutathione concentration in the lens decreases in aging and cataractous lenses, providing a marker for tissue condition. Experimental procedures requiring unfrozen lenses from donor banks rely on transportation in storage medium, affecting lens homeostasis and alterations in glutathione levels. The aim of the study was to examine the effects of Optisol-GS and castor oil on lens condition, determined from their ability to maintain glutathione concentrations. METHODOLOGY/PRINCIPAL FINDINGS: Rat lenses were stored in the two types of storage media at varying time intervals up to 3 days. Glutathione concentration was afterwards determined in an enzymatic detection assay, specific for both reduced and oxidized forms. Lenses removed immediately after death exhibited a glutathione concentration of 4.70±0.29 mM. In vitro stored lenses in Optisol-GS lost glutathione quickly, ending with a concentration of 0.60±0.34 mM after 3 days while castor oil stored lenses exhibited a slower decline and ended at 3 times the concentration. A group of lenses were additionally stored under post mortem conditions within the host for 6 hours before its removal. Total glutathione after 6 hours was similar to that of lenses removed immediately after death, but with altered GSH and GSSG concentrations. Subsequent storage of these lenses in media showed changes similar to those in the first series of experiments, albeit to a lesser degree. CONCLUSIONS/SIGNIFICANCE: It was determined that storage in Optisol-GS resulted in a higher loss of glutathione than lenses stored in castor oil. Storage for more than 12 hours reduced glutathione to half its original concentration, and was considered unusable after 24 hours.

Transient light-induced intracellular oxidation revealed by redox biosensor

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Kolossov, Vladimir L., E-mail: viadimer@illinois.edu [Institute for Genomic Biology, University of Illinois at Urbana-Champaign, 1206 W. Gregory Drive, Urbana, IL 61801 (United States); Beaudoin, Jessica N. [Institute for Genomic Biology, University of Illinois at Urbana-Champaign, 1206 W. Gregory Drive, Urbana, IL 61801 (United States); Department of Animal Sciences, University of Illinois at Urbana-Champaign, 1207 W. Gregory Drive, Urbana, IL 61801 (United States); Hanafin, William P. [Institute for Genomic Biology, University of Illinois at Urbana-Champaign, 1206 W. Gregory Drive, Urbana, IL 61801 (United States); DiLiberto, Stephen J. [Institute for Genomic Biology, University of Illinois at Urbana-Champaign, 1206 W. Gregory Drive, Urbana, IL 61801 (United States); Department of Animal Sciences, University of Illinois at Urbana-Champaign, 1207 W. Gregory Drive, Urbana, IL 61801 (United States); Kenis, Paul J.A. [Institute for Genomic Biology, University of Illinois at Urbana-Champaign, 1206 W. Gregory Drive, Urbana, IL 61801 (United States); Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, 600 S. Mathews Avenue, Urbana, IL 61801 (United States); Rex Gaskins, H. [Institute for Genomic Biology, University of Illinois at Urbana-Champaign, 1206 W. Gregory Drive, Urbana, IL 61801 (United States); Department of Animal Sciences, University of Illinois at Urbana-Champaign, 1207 W. Gregory Drive, Urbana, IL 61801 (United States); Department of Pathobiology, University of Illinois at Urbana-Champaign, 2001 S. Lincoln Avenue, Urbana, IL 61801 (United States); Division of Nutritional Sciences, University of Illinois at Urbana-Champaign, 905 S. Goodwin Avenue, Urbana, IL 61801 (United States)

2013-10-04

Highlights: •Time-resolved live cell imaging revealed light-induced oxidation. •Only the roGFP probe fused with glutaredoxin reveals photooxidation. •The transient oxidation is rapidly reduced by the cytosolic antioxidant system. •Intracellular photooxidation is media-dependent. •Oxidation is triggered exclusively by exposure to short wavelength excitation. -- Abstract: We have implemented a ratiometric, genetically encoded redox-sensitive green fluorescent protein fused to human glutaredoxin (Grx1-roGFP2) to monitor real time intracellular glutathione redox potentials of mammalian cells. This probe enabled detection of media-dependent oxidation of the cytosol triggered by short wavelength excitation. The transient nature of light-induced oxidation was revealed by time-lapse live cell imaging when time intervals of less than 30 s were implemented. In contrast, transient ROS generation was not observed with the parental roGFP2 probe without Grx1, which exhibits slower thiol-disulfide exchange. These data demonstrate that the enhanced sensitivity of the Grx1-roGFP2 fusion protein enables the detection of short-lived ROS in living cells. The superior sensitivity of Grx1-roGFP2, however, also enhances responsiveness to environmental cues introducing a greater likelihood of false positive results during image acquisition.

Transient light-induced intracellular oxidation revealed by redox biosensor

International Nuclear Information System (INIS)

Kolossov, Vladimir L.; Beaudoin, Jessica N.; Hanafin, William P.; DiLiberto, Stephen J.; Kenis, Paul J.A.; Rex Gaskins, H.

2013-01-01

Highlights: •Time-resolved live cell imaging revealed light-induced oxidation. •Only the roGFP probe fused with glutaredoxin reveals photooxidation. •The transient oxidation is rapidly reduced by the cytosolic antioxidant system. •Intracellular photooxidation is media-dependent. •Oxidation is triggered exclusively by exposure to short wavelength excitation. -- Abstract: We have implemented a ratiometric, genetically encoded redox-sensitive green fluorescent protein fused to human glutaredoxin (Grx1-roGFP2) to monitor real time intracellular glutathione redox potentials of mammalian cells. This probe enabled detection of media-dependent oxidation of the cytosol triggered by short wavelength excitation. The transient nature of light-induced oxidation was revealed by time-lapse live cell imaging when time intervals of less than 30 s were implemented. In contrast, transient ROS generation was not observed with the parental roGFP2 probe without Grx1, which exhibits slower thiol-disulfide exchange. These data demonstrate that the enhanced sensitivity of the Grx1-roGFP2 fusion protein enables the detection of short-lived ROS in living cells. The superior sensitivity of Grx1-roGFP2, however, also enhances responsiveness to environmental cues introducing a greater likelihood of false positive results during image acquisition

Variable Levels of Glutathione S-Transferases Are Responsible for the Differential Tolerance to Metolachlor between Maize (Zea mays) Shoots and Roots.

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Li, Dongzhi; Xu, Li; Pang, Sen; Liu, Zhiqian; Wang, Kai; Wang, Chengju

2017-01-11

Glutathione S-transferases (GSTs) play important roles in herbicide tolerance. However, studies on GST function in herbicide tolerance among plant tissues are still lacking. To explore the mechanism of metolachlor tolerance difference between maize shoots and roots, the effects of metolachlor on growth, GST activity, and the expression of the entire GST gene family were investigated. It was found that this differential tolerance to metolachlor was correlated with contrasting GST activity between the two tissues and can be eliminated by a GST inhibitor. An in vitro metolachlor-glutathione conjugation assay confirmed that the transformation of metolachlor is 2-fold faster in roots than in shoots. The expression analysis of the GST gene family revealed that most GST genes are expressed much higher in roots than shoots, both in control and in metolachlor-treated plants. Taken together, higher level expression of most GST genes, leading to higher GST activity and faster herbicide transformation, appears to be responsible for the higher tolerance to metolachlor of maize roots than shoots.

Effects of Combined Low Glutathione with Mild Oxidative and Low Phosphorus Stress on the Metabolism of Arabidopsis thaliana

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Atsushi Fukushima

2017-08-01

Full Text Available Plants possess highly sensitive mechanisms that monitor environmental stress levels for a dose-dependent fine-tuning of their growth and development. Differences in plant responses to severe and mild abiotic stresses have been recognized. Although many studies have revealed that glutathione can contribute to plant tolerance to various environmental stresses, little is known about the relationship between glutathione and mild abiotic stress, especially the effect of stress-induced altered glutathione levels on the metabolism. Here, we applied a systems biology approach to identify key pathways involved in the gene-to-metabolite networks perturbed by low glutathione content under mild abiotic stress in Arabidopsis thaliana. We used glutathione synthesis mutants (cad2-1 and pad2-1 and plants overexpressing the gene encoding γ-glutamylcysteine synthetase, the first enzyme of the glutathione biosynthetic pathway. The plants were exposed to two mild stress conditions—oxidative stress elicited by methyl viologen and stress induced by the limited availability of phosphate. We observed that the mutants and transgenic plants showed similar shoot growth as that of the wild-type plants under mild abiotic stress. We then selected the synthesis mutants and performed multi-platform metabolomics and microarray experiments to evaluate the possible effects on the overall metabolome and the transcriptome. As a common oxidative stress response, several flavonoids that we assessed showed overaccumulation, whereas the mild phosphate stress resulted in increased levels of specific kaempferol- and quercetin-glycosides. Remarkably, in addition to a significant increased level of sugar, osmolytes, and lipids as mild oxidative stress-responsive metabolites, short-chain aliphatic glucosinolates over-accumulated in the mutants, whereas the level of long-chain aliphatic glucosinolates and specific lipids decreased. Coordinated gene expressions related to glucosinolate and

Alterations of energy metabolism and glutathione levels of HL-60 cells induced by methacrylates present in composite resins.

Science.gov (United States)

Nocca, G; De Palma, F; Minucci, A; De Sole, P; Martorana, G E; Callà, C; Morlacchi, C; Gozzo, M L; Gambarini, G; Chimenti, C; Giardina, B; Lupi, A

2007-03-01

Methacrylic compounds such as 2-hydroxyethyl methacrylate (HEMA), triethylene glycol dimethacrylate (TEGDMA) and bisphenol A glycerolate (1 glycerol/phenol) dimethacrylate (Bis-GMA) are largely present in auto- or photopolymerizable composite resins. Since the polymerization reaction is never complete, these molecules are released into the oral cavity tissues and biological fluids where they could cause local adverse effects. The aim of this work was to verify the hypothesis that the biological effects of HEMA, TEGDMA and Bis-GMA - at a non-cytotoxic concentration - depend on the interaction with mitochondria and exert consequent alterations of energy metabolism, GSH levels and the related pathways in human promyelocytic cell line (HL-60). The biological effects of methacrylic monomers were determined by analyzing the following parameters: GSH concentration, glucose-6-phosphate dehydrogenase (G6PDH) and glutathione reductase (GR) activity, oxygen and glucose consumption and lactate production along with cell differentiation and proliferation. All monomers induced both cellular differentiation and decrease in oxygen consumption. Cells treated with TEGDMA and Bis-GMA showed a significant enhancement of glucose consumption and lactate production. TEGDMA and HEMA induced GSH depletion stimulating G6PDH and GR activity. All the monomers under study affect the metabolism of HL-60 cells and show differentiating activity. Since alterations in cellular metabolism occurred at compound concentrations well below cytotoxic levels, the changes in energy metabolism and glutathione redox balance could be considered as potential mechanisms for inducing clinical and sub-clinical adverse effects and thus providing useful parameters when testing biocompatibility of dental materials.

Identification and characterization of a selenium-dependent glutathione peroxidase in Setaria cervi

International Nuclear Information System (INIS)

Singh, Anchal; Rathaur, Sushma

2005-01-01

Setaria cervi a bovine filarial parasite secretes selenium glutathione peroxidase during in vitro cultivation. A significant amount of enzyme activity was detected in the somatic extract of different developmental stages of the parasite. Among different stages, microfilariae showed a higher level of selenium glutathione peroxidase activity followed by males then females. However, when the activity was compared in excretory secretory products of these stages males showed higher activity than microfilariae and female worms. The enzyme was purified from female somatic extract using a combination of glutathione agarose and gel filtration chromatography, which migrated as a single band of molecular mass ∼20 kDa. Selenium content of purified enzyme was estimated by atomic absorption spectroscopy and found to be 3.5 ng selenium/μg of protein. Further, inhibition of enzyme activity by potassium cyanide suggested the presence of selenium at the active site of enzyme. This is the first report of identification of selenium glutathione peroxidase from any filarial parasite

Inhibitory effect of glutathione on oxidative liver injury induced by dengue virus serotype 2 infections in mice.

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Juan Wang

Full Text Available The pathogenesis of dengue virus (DV infection has not been completely defined and change of redox status mediated by depletion of glutathione (GSH in host cell is a common result of viral infection. Our previous study has demonstrated that DV serotype 2 (DV2 infection alters host intracellular GSH levels, and exogenous GSH inhibits viral production by modulating the activity of NF-κB in HepG2 cells. GSH is the most powerful intracellular antioxidant and involved in viral infections. Thus, this study was to investigate whether DV2 infection can induce alteration in redox balance and effect of GSH on the disease in HepG2 xenografts SCID mice. Our results revealed that mice infected with DV2 showed alterations in oxidative stress by increasing the level of malondialdehyde (MDA, an end product of lipid peroxidation, and GSSG/GSH ratio. DV2-infected mice also showed a decrease in the activity of catalase (CAT and total superoxide dismutase (T-SOD in the serum and/or observed organs, especially the liver. Moreover, DV2 infection resulted in elevated serum levels of the cytokines tumor necrosis factor-α and interlukin-6 and obvious histopathological changes in the liver. The administration of exogenous GSH significantly reversed all of the aforementioned pathological changes and prevented significant liver damage. Furthermore, in vitro treatment of HepG2 cells with antioxidants such as GSH inhibited viral entry as well as the production of reactive oxygen species in HepG2 cells. These results suggest that GSH prevents DV2-induced oxidative stress and liver injury in mice by inhibiting proinflammatory cytokine production, and GSH and may be a promising therapeutic agent for prevention of oxidative liver damage during DV infection.

Multidrug-resistance-associated protein plays a protective role in menadione-induced oxidative stress in endothelial cells.

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Takahashi, Kyohei; Shibata, Tomohito; Oba, Tatsuya; Ishikawa, Tetsuya; Yoshikawa, Masahito; Tatsunami, Ryosuke; Takahashi, Kazuhiko; Tampo, Yoshiko

2009-02-13

Menadione, a redox-cycling quinone known to cause oxidative stress, binds to reduced glutathione (GSH) to form glutathione S-conjugate. Glutathione S-conjugates efflux is often mediated by multidrug-resistance-associated protein (MRP). We investigated the effect of a transporter inhibitor, MK571 (3-[[3-[2-(7-chloroquinolin-2-yl)vinyl]phenyl]-(2-dimethylcarbamoylethylsulfanyl)methylsulfanyl] propionic acid), on menadione-induced oxidative stress in bovine aortic endothelial cells (BAECs). BAECs were treated with menadione and MK571, and cell viability was measured. Modulation of intracellular GSH levels was performed with buthionine sulfoximine and GSH ethyl ester treatments. Intracellular superoxide was estimated by dihydroethidium oxidation using fluorescence microscopy or flow cytometry. Expression of MRP was determined by flow cytometry using phycoerythrin-conjugated anti-MRP monoclonal antibody. Intracellular GSH depletion by buthionine sulfoximine promoted the loss of viability of BAECs exposed to menadione. Exogenous GSH, which does not permeate the cell membrane, or GSH ethyl ester protected BAECs against the loss of viability induced by menadione. The results suggest that GSH binds to menadione outside the cells as well as inside. Pretreatment of BAECs with MK571 dramatically increased intracellular levels of superoxide generated from menadione, indicating that menadione may accumulate in the intracellular milieu. Finally, we found that MK571 aggravated menadione-induced toxicity in BAECs and that MRP levels were increased in menadione-treated cells. We conclude that MRP plays a vital role in protecting BAECs against menadione-induced oxidative stress, presumably due to its ability to transport glutathione S-conjugate.

Phg1/TM9 proteins control intracellular killing of bacteria by determining cellular levels of the Kil1 sulfotransferase in Dictyostelium.

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Marion Le Coadic

Full Text Available Dictyostelium discoideum has largely been used to study phagocytosis and intracellular killing of bacteria. Previous studies have shown that Phg1A, Kil1 and Kil2 proteins are necessary for efficient intracellular killing of Klebsiella bacteria. Here we show that in phg1a KO cells, cellular levels of lysosomal glycosidases and lysozyme are decreased, and lysosomal pH is increased. Surprisingly, overexpression of Kil1 restores efficient killing in phg1a KO cells without correcting these lysosomal anomalies. Conversely, kil1 KO cells are defective for killing, but their enzymatic content and lysosomal pH are indistinguishable from WT cells. The killing defect of phg1a KO cells can be accounted for by the observation that in these cells the stability and the cellular amount of Kil1 are markedly reduced. Since Kil1 is the only sulfotransferase characterized in Dictyostelium, an (unidentified sulfated factor, defective in both phg1a and kil1 KO cells, may play a key role in intracellular killing of Klebsiella bacteria. In addition, Phg1B plays a redundant role with Phg1A in controlling cellular amounts of Kil1 and intracellular killing. Finally, cellular levels of Kil1 are unaffected in kil2 KO cells, and Kil1 overexpression does not correct the killing defect of kil2 KO cells, suggesting that Kil2 plays a distinct role in intracellular killing.

Elevated oxidized glutathione in cystinotic proximal tubular epithelial cells.

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Wilmer, Martijn J G; de Graaf-Hess, Adriana; Blom, Henk J; Dijkman, Henry B P M; Monnens, Leo A; van den Heuvel, Lambertus P; Levtchenko, Elena N

2005-11-18

Cystinosis, the most frequent cause of inborn Fanconi syndrome, is characterized by the lysosomal cystine accumulation, caused by mutations in the CTNS gene. To elucidate the pathogenesis of cystinosis, we cultured proximal tubular cells from urine of cystinotic patients (n = 9) and healthy controls (n = 9), followed by immortalization with human papilloma virus (HPV E6/E7). Obtained cell lines displayed basolateral polarization, alkaline phosphatase activity, and presence of aminopeptidase N (CD-13) and megalin, confirming their proximal tubular origin. Cystinotic cell lines exhibited elevated cystine levels (0.86 +/- 0.95 nmol/mg versus 0.09 +/- 0.01 nmol/mg protein in controls, p = 0.03). Oxidized glutathione was elevated in cystinotic cells (1.16 +/- 0.83 nmol/mg versus 0.29 +/- 0.18 nmol/mg protein, p = 0.04), while total glutathione, free cysteine, and ATP contents were normal in these cells. In conclusion, elevated oxidized glutathione in cystinotic proximal tubular epithelial cell lines suggests increased oxidative stress, which may contribute to tubular dysfunction in cystinosis.

Beta-carotene reduces oxidative stress, improves glutathione metabolism and modifies antioxidant defense systems in lead-exposed workers

International Nuclear Information System (INIS)

Kasperczyk, Sławomir; Dobrakowski, Michał; Kasperczyk, Janusz; Ostałowska, Alina; Zalejska-Fiolka, Jolanta; Birkner, Ewa

2014-01-01

The aim of this study was to determine whether beta-carotene administration reduces oxidative stress and influences antioxidant, mainly glutathione-related, defense systems in workers chronically exposed to lead. The population consisted of two randomly divided groups of healthy male volunteers exposed to lead. Workers in the first group (reference group) were not administered any antioxidants, while workers in the second group (CAR group) were treated orally with 10 mg of beta-carotene once a day for 12 weeks. Biochemical analysis included measuring markers of lead-exposure and oxidative stress in addition to the levels and activities of selected antioxidants. After treatment, levels of malondialdehyde, lipid hydroperoxides and lipofuscin significantly decreased compared with the reference group. However, the level of glutathione significantly increased compared with the baseline. Treatment with beta-carotene also resulted in significantly decreased glutathione peroxidase activity compared with the reference group, while the activities of other glutathione-related enzymes and of superoxide dismutase were not significantly changed. However, the activities of glucose-6-phosphate dehydrogenase and catalase, as well as the level of alpha-tocopherol, were significantly higher after treatment compared with the baseline. Despite controversy over the antioxidant properties of beta-carotene in vivo, our findings showed reduced oxidative stress after beta-carotene supplementation in chronic lead poisoning. - Highlights: • Beta-carotene reduces oxidative stress in lead-exposed workers. • Beta-carotene elevates glutathione level in lead-exposed workers. • Beta-carotene administration could be beneficial in lead poisoning

Beta-carotene reduces oxidative stress, improves glutathione metabolism and modifies antioxidant defense systems in lead-exposed workers

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Kasperczyk, Sławomir, E-mail: kaslav@mp.pl [Dept. of Biochemistry, School of Medicine with the Division of Dentistry, Medical University of Silesia, ul. Jordana 19, 41-808 Zabrze (Poland); Dobrakowski, Michał [Dept. of Biochemistry, School of Medicine with the Division of Dentistry, Medical University of Silesia, ul. Jordana 19, 41-808 Zabrze (Poland); Kasperczyk, Janusz [Dept. of Environmental Medicine and Epidemiology, School of Medicine with the Division of Dentistry, Medical University of Silesia, ul. Jordana 19, 41-808 Zabrze (Poland); Ostałowska, Alina; Zalejska-Fiolka, Jolanta; Birkner, Ewa [Dept. of Biochemistry, School of Medicine with the Division of Dentistry, Medical University of Silesia, ul. Jordana 19, 41-808 Zabrze (Poland)

2014-10-01

The aim of this study was to determine whether beta-carotene administration reduces oxidative stress and influences antioxidant, mainly glutathione-related, defense systems in workers chronically exposed to lead. The population consisted of two randomly divided groups of healthy male volunteers exposed to lead. Workers in the first group (reference group) were not administered any antioxidants, while workers in the second group (CAR group) were treated orally with 10 mg of beta-carotene once a day for 12 weeks. Biochemical analysis included measuring markers of lead-exposure and oxidative stress in addition to the levels and activities of selected antioxidants. After treatment, levels of malondialdehyde, lipid hydroperoxides and lipofuscin significantly decreased compared with the reference group. However, the level of glutathione significantly increased compared with the baseline. Treatment with beta-carotene also resulted in significantly decreased glutathione peroxidase activity compared with the reference group, while the activities of other glutathione-related enzymes and of superoxide dismutase were not significantly changed. However, the activities of glucose-6-phosphate dehydrogenase and catalase, as well as the level of alpha-tocopherol, were significantly higher after treatment compared with the baseline. Despite controversy over the antioxidant properties of beta-carotene in vivo, our findings showed reduced oxidative stress after beta-carotene supplementation in chronic lead poisoning. - Highlights: • Beta-carotene reduces oxidative stress in lead-exposed workers. • Beta-carotene elevates glutathione level in lead-exposed workers. • Beta-carotene administration could be beneficial in lead poisoning.

In vitro Effects of Four Native Brazilian Medicinal Plants in CYP3A4 mRNA Gene Expression, Glutathione Levels, and P-Glycoprotein Activity

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Mazzari, Andre L. D. A.; Milton, Flora; Frangos, Samantha; Carvalho, Ana C. B.; Silveira, Dâmaris; de Assis Rocha Neves, Francisco; Prieto, Jose M.

2016-01-01

Erythrina mulungu Benth. (Fabaceae), Cordia verbenacea A. DC. (Boraginaceae), Solanum paniculatum L. (Solanaceae) and Lippia sidoides Cham. (Verbenaceae) are medicinal plant species native to Brazil shortlisted by the Brazilian National Health System for future clinical use. However, nothing is known about their effects in metabolic and transporter proteins, which could potentially lead to herb-drug interactions (HDI). In this work, we assess non-toxic concentrations (100 μg/mL) of the plant infusions for their in vitro ability to modulate CYP3A4 mRNA gene expression and intracellular glutathione levels in HepG2 cells, as well as P-glycoprotein (P-gp) activity in vincristine-resistant Caco-2 cells (Caco-2 VCR). Their mechanisms of action were further studied by measuring the activation of human pregnane X receptor (hPXR) in transiently co-transfected HeLa cells and the inhibition of γ-glutamyl transferase (GGT) in HepG2 cells. Our results show that P-gp activity was not affected in any case and that only Solanum paniculatum was able to significantly change CYP3A4 mRNA gene expression (twofold decrease, p Cordia verbenacea (-47%, p < 0.001). The latter plant extract was able to decrease GGT activity (-48%, p < 0.01). In conclusion, this preclinical study shows that the administration of some of these herbal medicines may be able to cause disturbances to metabolic mechanisms in vitro. Although Erythrina mulungu appears safe in our tests, active pharmacovigilance is recommended for the other three species, especially in the case of Solanum paniculatum. PMID:27594838

The change of intracellular NAD level at the process of fusarium sambucinum growth and development

International Nuclear Information System (INIS)

Gulyamova, T.G.; Ehshtukhtarova, M.Kh.; Umarova, G.D.; Kerbalaeva, A.M.; Khalmuradov, A.G.

1996-01-01

Alterations of intracellular NAD(Nicotinamide-Adenine Dinucleotide) level have been studied in the process of growth and development of Fusanium sambucinum, selected earlier as a potential NAD producer. It was established that essential fluctuations of NAD concentration are dependent on growth phase, morphological cell type and DNA biosynthesis, that allowed to propose a real linkage between coenzyme pool and replicative activity of cells. (author). 7 refs., 2 figs

Glutathione-responsive core cross-linked micelles for controlled cabazitaxel delivery

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Han, Xiaoxiong; Gong, Feirong; Sun, Jing; Li, Yueqi; Liu, XiaoFei; Chen, Dan; Liu, Jianwen; Shen, Yaling

2018-02-01

Stimulus-responsive polymeric micelles (PMs) have recently received attention due to the controlled delivery of drug or gene for application in cancer diagnosis and treatment. In this work, novel glutathione-responsive PMs were prepared to encapsulate hydrophobic antineoplastic drug, cabazitaxel (CTX), to improve its solubility and toxicity. These CTX-loaded micelles core cross-linked by disulfide bonds (DCL-CTX micelles) were prepared by a novel copolymer, lipoic acid grafted mPEG-PLA. These micelles had regular spherical shape, homogeneous diameter of 18.97 ± 0.23 nm, and a narrow size distribution. The DCL-CTX micelles showed high encapsulation efficiency of 98.65 ± 1.77%, and the aqueous solubility of CTX was improved by a factor of 1:1200. In vitro release investigation showed that DCL-CTX micelles were stable in the medium without glutathione (GSH), whereas the micelles had burst CTX release in the medium with 10 mM GSH. Cell uptake results implied that DCL-CTX micelles were internalized into MCF-7 cells through clathrin-mediated endocytosis and released cargo more effectively than Jevtana (commercially available CTX) owing to GSH-stimulated degradation. In MTT assay against MCF-7 cells, these micelles inhibited tumor cell proliferation more effectively than Jevtana due to their GSH-responsive CTX release. All results revealed the potency of GSH-responsive DCL-CTX micelles for stable delivery in blood circulation and for intracellular GSH-trigged release of CTX. Therefore, DCL-CTX micelles show potential as safe and effective CTX delivery carriers and as a cancer chemotherapy formulation.

The potential role of the antioxidant and detoxification properties of glutathione in autism spectrum disorders: a systematic review and meta-analysis

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2012-01-01

Background Glutathione has a wide range of functions; it is an endogenous anti-oxidant and plays a key role in the maintenance of intracellular redox balance and detoxification of xenobiotics. Several studies have indicated that children with autism spectrum disorders may have altered glutathione metabolism which could play a key role in the condition. Methods A systematic literature review and meta-analysis was conducted of studies examining metabolites, interventions and/or genes of the glutathione metabolism pathways i.e. the γ-glutamyl cycle and trans-sulphuration pathway in autism spectrum disorders. Results Thirty nine studies were included in the review comprising an in vitro study, thirty two metabolite and/or co-factor studies, six intervention studies and six studies with genetic data as well as eight studies examining enzyme activity. Conclusions The review found evidence for the involvement of the γ-glutamyl cycle and trans-sulphuration pathway in autistic disorder is sufficiently consistent, particularly with respect to the glutathione redox ratio, to warrant further investigation to determine the significance in relation to clinical outcomes. Large, well designed intervention studies that link metabolites, cofactors and genes of the γ-glutamyl cycle and trans-sulphuration pathway with objective behavioural outcomes in children with autism spectrum disorders are required. Future risk factor analysis should include consideration of multiple nutritional status and metabolite biomarkers of pathways linked with the γ-glutamyl cycle and the interaction of genotype in relation to these factors. PMID:22524510

Glutathione Transferase GSTπ In Breast Tumors Evaluated By Three Techniques

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Rafael Molina

1993-01-01

Full Text Available The glutathione transferases are involved in intracellular detoxification reactions. One of these, GSTπ, is elevated in some breast cancer cells, particularly cells selected for resistance to anticancer agents. We evaluated GSTπ expression in 60 human breast tumors by three techniques, immunohistochemistry, Northern hybridization, and Western blot analysis. There was a significant positive correlation between the three methods, with complete concordance seen in 64% of the tumors. There was strong, inverse relationship between GSTπ expression and steroid receptor status with all of the techniques utili zed. [n addition, there was a trend toward higher GSTπ expression in poorly differentiated tumors, but no correlation was found between tumor GSTπ content and DNA ploidy or %S-phase. GSTπ expression was also detected in adjacent benign breast tissue as well as infiltrating lymphocytes; this expression may contribute to GSTπ measurements using either Northern hybridization or Western blot analysis. These re sults suggest that immunohistochemistry is the method of choice for measuring GSTπ in breast tumors.

Single-cell intracellular nano-pH probes†

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Özel, Rıfat Emrah; Lohith, Akshar; Mak, Wai Han; Pourmand, Nader

2016-01-01

Within a large clonal population, such as cancerous tumor entities, cells are not identical, and the differences between intracellular pH levels of individual cells may be important indicators of heterogeneity that could be relevant in clinical practice, especially in personalized medicine. Therefore, the detection of the intracellular pH at the single-cell level is of great importance to identify and study outlier cells. However, quantitative and real-time measurements of the intracellular pH of individual cells within a cell population is challenging with existing technologies, and there is a need to engineer new methodologies. In this paper, we discuss the use of nanopipette technology to overcome the limitations of intracellular pH measurements at the single-cell level. We have developed a nano-pH probe through physisorption of chitosan onto hydroxylated quartz nanopipettes with extremely small pore sizes (~100 nm). The dynamic pH range of the nano-pH probe was from 2.6 to 10.7 with a sensitivity of 0.09 units. We have performed single-cell intracellular pH measurements using non-cancerous and cancerous cell lines, including human fibroblasts, HeLa, MDA-MB-231 and MCF-7, with the pH nanoprobe. We have further demonstrated the real-time continuous single-cell pH measurement capability of the sensor, showing the cellular pH response to pharmaceutical manipulations. These findings suggest that the chitosan-functionalized nanopore is a powerful nano-tool for pH sensing at the single-cell level with high temporal and spatial resolution. PMID:27708772

Single-cell intracellular nano-pH probes.

Science.gov (United States)

Özel, Rıfat Emrah; Lohith, Akshar; Mak, Wai Han; Pourmand, Nader

2015-01-01

Within a large clonal population, such as cancerous tumor entities, cells are not identical, and the differences between intracellular pH levels of individual cells may be important indicators of heterogeneity that could be relevant in clinical practice, especially in personalized medicine. Therefore, the detection of the intracellular pH at the single-cell level is of great importance to identify and study outlier cells. However, quantitative and real-time measurements of the intracellular pH of individual cells within a cell population is challenging with existing technologies, and there is a need to engineer new methodologies. In this paper, we discuss the use of nanopipette technology to overcome the limitations of intracellular pH measurements at the single-cell level. We have developed a nano-pH probe through physisorption of chitosan onto hydroxylated quartz nanopipettes with extremely small pore sizes (~100 nm). The dynamic pH range of the nano-pH probe was from 2.6 to 10.7 with a sensitivity of 0.09 units. We have performed single-cell intracellular pH measurements using non-cancerous and cancerous cell lines, including human fibroblasts, HeLa, MDA-MB-231 and MCF-7, with the pH nanoprobe. We have further demonstrated the real-time continuous single-cell pH measurement capability of the sensor, showing the cellular pH response to pharmaceutical manipulations. These findings suggest that the chitosan-functionalized nanopore is a powerful nano-tool for pH sensing at the single-cell level with high temporal and spatial resolution.

Connexin43 hemichannels contributes to the disassembly of cell junctions through modulation of intracellular oxidative status

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Yuan Chi

2016-10-01

Full Text Available Connexin (Cx hemichannels regulate many cellular processes with little information available regarding their mechanisms. Given that many pathological factors that activate hemichannels also disrupts the integrity of cellular junctions, we speculated a potential participation of hemichannels in the regulation of cell junctions. Here we tested this hypothesis. Exposure of renal tubular epithelial cells to Ca2+-free medium led to disassembly of tight and adherens junctions, as indicated by the reduced level of ZO-1 and cadherin, disorganization of F-actin, and severe drop in transepithelial electric resistance. These changes were preceded by an activation of Cx43 hemichannels, as revealed by extracellular efflux of ATP and intracellular influx of Lucifer Yellow. Inhibition of hemichannels with chemical inhibitors or Cx43 siRNA greatly attenuated the disassembly of cell junctions. Further analysis using fetal fibroblasts derived from Cx43 wide-type (Cx43+/+, heterozygous (Cx43+/- and knockout (Cx43-/- littermates showed that Cx43-positive cells (Cx43+/+ exhibited more dramatic changes in cell shape, F-actin, and cadherin in response to Ca2+ depletion, as compared to Cx43-null cells (Cx43-/-. Consistently, these cells had higher level of protein carbonyl modification and phosphorylation, and much stronger activation of P38 and JNK. Hemichannel opening led to extracellular loss of the major antioxidant glutathione (GSH. Supplement of cells with exogenous GSH or inhibition of oxidative sensitive kinases largely prevented the above-mentioned changes. Taken together, our study indicates that Cx43 hemichannels promote the disassembly of cell junctions through regulation of intracellular oxidative status.

MR imaging of intracellular and extracellular deoxyhemoglobin

International Nuclear Information System (INIS)

Janick, P.A.; Grossman, R.I.; Asakura, T.

1989-01-01

MR imaging was performed on varying concentrations of intracellular and extracellular deoxyhemoglobin as well as varying proportions of deoxyhemoglobin and oxyhemoglobin in vitro at 1.5T with use of standard spin-echo and gradient-refocused spin sequences. This study indicates that susceptibility-induced T2 shortening occurs over a broad range of intracellular deoxyhemoglobin concentrations (maximal at hematocrits between 20% and 45%), reflecting diffusional effects at the cellular level. T2* gradient-echo imaging enhances the observed hypointensity in images of intracellular deoxyhemoglobin. The characteristic MR appearance of acute hemotomas can be modeled by the behavior of intracellular and extracellular deoxyhemoglobin and oxyhemoglobin

Measurement of glutathione-protein mixed disulfides

International Nuclear Information System (INIS)

Livesey, J.C.; Reed, D.J.

1984-01-01

The development of a sensitive and highly specific assay for the presence of mixed disulfides between protein thiol groups and endogenous thiols has been undertaken. Previous investigations on the concentrations of glutathione (GSH), glutathione disulfide (GSSG) and protein glutathione mixed disulfides (ProSSG) have been of limited usefulness because of the poor specificity of the assays used. Our assay for these forms of glutathione is based on high performance liquid chromatography (HPLC) and is an extension of an earlier method. After perchloric acid precipitation, the protein sample is washed with an organic solvent to fully denature the protein. Up to a 10-fold increase in GSH released from fetal bovine serum (FBS) protein has been found when the protein precipitate is washed with ethanol rather than ether, as earlier suggested. Similar effects have been observed with an as yet unidentified thiol which elutes in the chromatography system with a retention volume similar to cysteine

Antibacterial activity of exogenous glutathione and its synergism on antibiotics sensitize carbapenem-associated multidrug resistant clinical isolates of Acinetobacter baumannii.

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Alharbe, Roaa; Almansour, Ayidh; Kwon, Dong H

2017-10-01

A major clinical impact of A. baumannii is hospital-acquired infections including ventilator-associated pneumonia. The treatment of this pathogen is often difficult due to its innate and acquired resistance to almost all commercially available antibiotics. Infections with carbapenem-associated multidrug resistant A. baumannii is the most problematic. Glutathione is a tripeptide thiol-antioxidant and antibacterial activity of exogenous glutathione was reported in some bacteria. However, clinical relevance and molecular details of the antibacterial activity of glutathione are currently unclear. Seventy clinical isolates of A. baumannii including 63 carbapenem-associated multidrug resistant isolates and a type strain A. baumannii ATCC 19606 were used to determine minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). Fractional inhibitory concentration (FIC) and time-killing activity with meropenem and/or glutathione were also determined in the carbapenem-associated multidrug resistant isolates. In addition, the roles of exogenous glutathione in multidrug efflux pumps and β-lactamase production were examined. Levels of MIC and MBC were ranged from 10 to 15mM of exogenous glutathione. All tested carbapenem-associated multidrug resistant isolates were sensitized by all tested antibiotics in combination with subinhibitory concentrations of glutathione. FIC levels of glutathione with carbapenem (meropenem) were allcarbapenem-associated multidrug resistant isolates were killed by subinhibitory concentrations of both glutathione and meropenem at>2log10 within 12h, suggesting glutathione synergistically interacts with meropenem. The roles of multidrug efflux pumps and β-lactamase production were excluded for the glutathione-mediated antibiotic susceptibility. Overall results demonstrate that the antibacterial activity of glutathione is clinically relevant and its synergism on antibiotics sensitizes clinical isolates of A. baumannii regardless

Biatriosporin D displays anti-virulence activity through decreasing the intracellular cAMP levels

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Zhang, Ming; Chang, Wenqiang; Shi, Hongzhuo; Zhou, Yanhui; Zheng, Sha; Li, Ying; Li, Lin; Lou, Hongxiang, E-mail: louhongxiang@sdu.edu.cn

2017-05-01

Candidiasis has long been a serious human health problem, and novel antifungal approaches are greatly needed. During both superficial and systemic infection, C. albicans relies on a battery of virulence factors, such as adherence, filamentation, and biofilm formation. In this study, we found that a small phenolic compound, Biatriosporin D (BD), isolated from an endolichenic fungus, Biatriospora sp., displayed anti-virulence activity by inhibiting adhesion, hyphal morphogenesis and biofilm formation of C. albicans. Of note is the high efficacy of BD in preventing filamentation with a much lower dose than its MIC value. Furthermore, BD prolonged the survival of worms infected by C. albicans in vivo. Quantitative real-time PCR analysis, exogenous cAMP rescue experiments and intracellular cAMP measurements revealed that BD regulates the Ras1-cAMP-Efg1 pathway by reducing cAMP levels to inhibit the hyphal formation. Further investigation showed that BD could upregulate Dpp3 to synthesize much more farnesol, which could inhibit the activity of Cdc35 and reduce the generation of cAMP. Taken together, these findings indicate that BD stimulates the expression of Dpp3 to synthesize more farnesol that directly inhibits the Cdc35 activity, reducing intracellular cAMP and thereby disrupting the morphologic transition and attenuating the virulence of C. albicans. Our study uncovers the underlying mechanism of BD as a prodrug in fighting against pathogenic C. albicans and provides a potential application of BD in fighting clinically relevant fungal infections by targeting fungal virulence. - Highlights: • BD inhibits the filamentation of C. albicans in multiple hypha-inducing conditions. • BD can prolong the survival of nematodes infected by C. albicans. • BD stimulates the expression of Dpp3 to synthesize more farnesol. • BD reduces intracellular cAMP and regulates Ras1-cAMP-PKA pathway.

Biatriosporin D displays anti-virulence activity through decreasing the intracellular cAMP levels

International Nuclear Information System (INIS)

Zhang, Ming; Chang, Wenqiang; Shi, Hongzhuo; Zhou, Yanhui; Zheng, Sha; Li, Ying; Li, Lin; Lou, Hongxiang

2017-01-01

Candidiasis has long been a serious human health problem, and novel antifungal approaches are greatly needed. During both superficial and systemic infection, C. albicans relies on a battery of virulence factors, such as adherence, filamentation, and biofilm formation. In this study, we found that a small phenolic compound, Biatriosporin D (BD), isolated from an endolichenic fungus, Biatriospora sp., displayed anti-virulence activity by inhibiting adhesion, hyphal morphogenesis and biofilm formation of C. albicans. Of note is the high efficacy of BD in preventing filamentation with a much lower dose than its MIC value. Furthermore, BD prolonged the survival of worms infected by C. albicans in vivo. Quantitative real-time PCR analysis, exogenous cAMP rescue experiments and intracellular cAMP measurements revealed that BD regulates the Ras1-cAMP-Efg1 pathway by reducing cAMP levels to inhibit the hyphal formation. Further investigation showed that BD could upregulate Dpp3 to synthesize much more farnesol, which could inhibit the activity of Cdc35 and reduce the generation of cAMP. Taken together, these findings indicate that BD stimulates the expression of Dpp3 to synthesize more farnesol that directly inhibits the Cdc35 activity, reducing intracellular cAMP and thereby disrupting the morphologic transition and attenuating the virulence of C. albicans. Our study uncovers the underlying mechanism of BD as a prodrug in fighting against pathogenic C. albicans and provides a potential application of BD in fighting clinically relevant fungal infections by targeting fungal virulence. - Highlights: • BD inhibits the filamentation of C. albicans in multiple hypha-inducing conditions. • BD can prolong the survival of nematodes infected by C. albicans. • BD stimulates the expression of Dpp3 to synthesize more farnesol. • BD reduces intracellular cAMP and regulates Ras1-cAMP-PKA pathway.

Contribution of Fdh3 and Glr1 to Glutathione Redox State, Stress Adaptation and Virulence in Candida albicans.

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Anna T Tillmann

Full Text Available The major fungal pathogen of humans, Candida albicans, is exposed to reactive nitrogen and oxygen species following phagocytosis by host immune cells. In response to these toxins, this fungus activates potent anti-stress responses that include scavenging of reactive nitrosative and oxidative species via the glutathione system. Here we examine the differential roles of two glutathione recycling enzymes in redox homeostasis, stress adaptation and virulence in C. albicans: glutathione reductase (Glr1 and the S-nitrosoglutathione reductase (GSNOR, Fdh3. We show that the NADPH-dependent Glr1 recycles GSSG to GSH, is induced in response to oxidative stress and is required for resistance to macrophage killing. GLR1 deletion increases the sensitivity of C. albicans cells to H2O2, but not to formaldehyde or NO. In contrast, Fdh3 detoxifies GSNO to GSSG and NH3, and FDH3 inactivation delays NO adaptation and increases NO sensitivity. C. albicans fdh3⎔ cells are also sensitive to formaldehyde, suggesting that Fdh3 also contributes to formaldehyde detoxification. FDH3 is induced in response to nitrosative, oxidative and formaldehyde stress, and fdh3Δ cells are more sensitive to killing by macrophages. Both Glr1 and Fdh3 contribute to virulence in the Galleria mellonella and mouse models of systemic infection. We conclude that Glr1 and Fdh3 play differential roles during the adaptation of C. albicans cells to oxidative, nitrosative and formaldehyde stress, and hence during the colonisation of the host. Our findings emphasise the importance of the glutathione system and the maintenance of intracellular redox homeostasis in this major pathogen.

Impaired synthesis and antioxidant defense of glutathione in the cerebellum of autistic subjects: alterations in the activities and protein expression of glutathione-related enzymes.

Science.gov (United States)

Gu, Feng; Chauhan, Ved; Chauhan, Abha

2013-12-01

Autism is a neurodevelopmental disorder associated with social deficits and behavioral abnormalities. Recent evidence in autism suggests a deficit in glutathione (GSH), a major endogenous antioxidant. It is not known whether the synthesis, consumption, and/or regeneration of GSH is affected in autism. In the cerebellum tissues from autism (n=10) and age-matched control subjects (n=10), the activities of GSH-related enzymes glutathione peroxidase (GPx), glutathione-S-transferase (GST), glutathione reductase (GR), and glutamate cysteine ligase (GCL) involved in antioxidant defense, detoxification, GSH regeneration, and synthesis, respectively, were analyzed. GCL is a rate-limiting enzyme for GSH synthesis, and the relationship between its activity and the protein expression of its catalytic subunit GCLC and its modulatory subunit GCLM was also compared between the autistic and the control groups. Results showed that the activities of GPx and GST were significantly decreased in autism compared to that of the control group (Pautistic subjects showed lower GR activity than 95% confidence interval (CI) of the control group. GCL activity was also significantly reduced by 38.7% in the autistic group compared to the control group (P=0.023), and 8 of 10 autistic subjects had values below 95% CI of the control group. The ratio of protein levels of GCLC to GCLM in the autism group was significantly higher than that of the control group (P=0.022), and GCLM protein levels were reduced by 37.3% in the autistic group compared to the control group. A positive strong correlation was observed between GCL activity and protein levels of GCLM (r=0.887) and GCLC (r=0.799) subunits in control subjects but not in autistic subjects, suggesting that regulation of GCL activity is affected in autism. These results suggest that enzymes involved in GSH homeostasis have impaired activities in the cerebellum in autism, and lower GCL activity in autism may be related to decreased protein expression

Auranofin induces apoptosis and necrosis in HeLa cells via oxidative stress and glutathione depletion.

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You, Bo Ra; Shin, Hye Rim; Han, Bo Ram; Kim, Suhn Hee; Park, Woo Hyun

2015-02-01

Auranofin (Au), an inhibitor of thioredoxin reductase, is a known anti‑cancer drug. In the present study, the anti‑growth effect of Au on HeLa cervical cancer cells was examined in association with levels of reactive oxygen species (ROS) and glutathione (GSH). Au inhibited the growth of HeLa cells with an IC50 of ~2 µM at 24 h. This agent induced apoptosis and necrosis, accompanied by the cleavage of poly (ADP‑ribose) polymerase and loss of mitochondrial membrane potential. The pan‑caspase inhibitor, benzyloxycarbonyl‑Val‑Ala‑Asp‑fluoromethylketone, prevented apoptotic cell death and each of the assessed caspase inhibitors inhibited necrotic cell death induced by Au. With respect to the levels of ROS and GSH, Au increased intracellular O2•- in the HeLa cells and induced GSH depletion. The pan‑caspase inhibitor reduced the levels of O2•- and GSH depletion in Au‑treated HeLa cells. The antioxidant, N‑acetyl cysteine, not only attenuated apoptosis and necrosis in the Au‑treated HeLa cells, but also decreased the levels of O2•- and GSH depletion in the cells. By contrast, L‑buthionine sulfoximine, a GSH synthesis inhibitor, intensified cell death O2•- and GSH depletion in the Au‑treated HeLa cells. In conclusion, Au induced apoptosis and necrosis in HeLa cells via the induction of oxidative stress and the depletion of GSH.

In vitro neuroprotective potential of four medicinal plants against rotenone-induced toxicity in SH-SY5Y neuroblastoma cells.

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Seoposengwe, Keabetswe; van Tonder, Jacob John; Steenkamp, Vanessa

2013-12-12

Lannea schweinfurthii, Zanthoxylum capense, Scadoxus puniceus and Crinum bulbispermum are used traditionally to treat neurological disorders. The aim of this study was to evaluate the cytoprotective potential of the four plants, after induction of toxicity using rotenone, in SH-SY5Y neuroblastoma cells. Cytotoxicity of the plant extracts and rotenone was assessed using the sulforhodamine B (SRB) assay. Fluorometry was used to measure intracellular redox state (reactive oxygen species (ROS) and intracellular glutathione content), mitochondrial membrane potential (MMP) and caspase-3 activity, as a marker of apoptotic cell death. Of the tested plants, the methanol extract of Z. capense was the least cytotoxic; LC50 121.3 ± 6.97 μg/ml, while S. puniceus methanol extract was the most cytotoxic; LC50 20.75 ± 1.47 μg/ml. Rotenone reduced intracellular ROS levels after 24 h exposure. Pre-treating cells with S. puniceus and C. bulbispermum extracts reversed the effects of rotenone on intracellular ROS levels. Rotenone exposure also decreased intracellular glutathione levels, which was counteracted by pre-treatment with any one of the extracts. MMP was reduced by rotenone, which was neutralized by pre-treatment with C. bulbispermum ethyl acetate extract. All extracts inhibited rotenone-induced activation of caspase-3. The studied plants demonstrated anti-apoptotic activity and restored intracellular glutathione content following rotenone treatment, suggesting that they may possess neuroprotective properties.

Active biomonitoring of a subtropical river using glutathione-S ...

African Journals Online (AJOL)

Active biomonitoring of a subtropical river using glutathione-S-transferase (GST) and heat shock proteins (HSP 70) in. Oreochromis niloticusas surrogate biomarkers of metal contamination. Victor Kurauone Muposhi1, Beaven Utete1*, Idah Sithole-Niang2 and Stanley Mukangenyama2. 1Wildlife Ecology and Conservation, ...

Menadione stress in Saccharomyces cerevisiae strains deficient in the glutathione transferases.

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Castro, F A V; Herdeiro, R S; Panek, A D; Eleutherio, E C A; Pereira, M D

2007-02-01

Using S. cerevisiae as a eukaryotic cell model we have analyzed the involvement of both glutathione transferase isoforms, Gtt1 and Gtt2, in constitutive resistance and adaptive response to menadione, a quinone which can exert its toxicity as redox cycling and/or electrophiles. The detoxification properties, of these enzymes, have also been analyzed by the appearance of S-conjugates in the media. Direct exposure to menadione (20 mM/60 min) showed to be lethal for cells deficient on both Gtt1 and Gtt2 isoforms. However, after pre-treatment with a low menadione concentration, cells deficient in Gtt2 displayed reduced ability to acquire tolerance when compared with the control and the Gtt1 deficient strains. Analyzing the toxic effects of menadione we observed that the gtt2 mutant showed no reduction in lipid peroxidation levels. Moreover, measuring the levels of intracellular oxidation during menadione stress we have shown that the increase of this oxidative stress parameter was due to the capacity menadione possesses in generating reactive oxygen species (ROS) and that both GSH and Gtt2 isoform were required to enhance ROS production. Furthermore, the efflux of the menadione-GSH conjugate, which is related with detoxification of xenobiotic pathways, was not detected in the gtt2 mutant. Taken together, these results suggest that acquisition of tolerance against stress generated by menadione and the process of detoxification through S-conjugates are dependent upon Gtt2 activity. This assessment was corroborated by the increase of GTT2 expression, and not of GTT1, after menadione treatment.

Membrane accessibility of glutathione

DEFF Research Database (Denmark)

Garcia, Almudena; Eljack, N., D.; Sani, ND

2015-01-01

Regulation of the ion pumping activity of the Na(+),K(+)-ATPase is crucial to the survival of animal cells. Recent evidence has suggested that the activity of the enzyme could be controlled by glutathionylation of cysteine residue 45 of the β-subunit. Crystal structures so far available indicate...... that this cysteine is in a transmembrane domain of the protein. Here we have analysed via fluorescence and NMR spectroscopy as well as molecular dynamics simulations whether glutathione is able to penetrate into the interior of a lipid membrane. No evidence for any penetration of glutathione into the membrane...

Thiol-Disulfide Exchange between Glutaredoxin and Glutathione

DEFF Research Database (Denmark)

Iversen, Rasmus; Andersen, Peter Anders; Jensen, Kristine Steen

2010-01-01

Glutaredoxins are ubiquitous thiol-disulfide oxidoreductases which catalyze the reduction of glutathione-protein mixed disulfides. Belonging to the thioredoxin family, they contain a conserved active site CXXC motif. The N-proximal active site cysteine can form a mixed disulfide with glutathione ...... has been replaced with serine. The exchange reaction between the reduced protein and oxidized glutathione leading to formation of the mixed disulfide could readily be monitored by isothermal titration calorimetry (ITC) due to the enthalpic contributions from the noncovalent interactions...

Effect of Pistacia Atlantica Extract on Glutathione Peroxidase Tissue Levels and Total Oxidative Capacity of Liver and Plasma Lipid Profile of Rats

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Parvin Farzanegi

2013-11-01

Full Text Available Background: Exercise causes increased oxygen consumption, leaving cells exposed to oxidative stress. Antioxidants may have a protective effect by inhibiting lipid peroxidation. Thus, this study aims to examine the effect of Pistacia atlantica extract on glutathione peroxidase levels and total oxidative capacity of liver and plasma lipid profile of rats. Materials and Methods: In this experimental study, 28 female rats’ weight 155.8±2.7 grams were randomly and equally divided into 4 groups of exercise-saline, control-saline, exercise-mastic, and control-mastic. The exercise groups exercised for 8 weeks (5 days per week, 60 minutes daily, 25 meters per minute, on a zero degree slope. The rats received equal volumes of mastic and saline orally for 4 weeks. Blood and tissue samples were taken 72 hours after the last exercise session. Data were analyzed using one-way variance analysis (ANOVA.Results: Consumption of Pistacia atlantica extract together with endurance exercising for 8 weeks did not significantly affect glutathione peroxidase concentration, total oxidative capacity, LDL, triglyceride, or cholesterol, but significantly reduced HDL (p=0.002.Conclusion: Results showed that antioxidant and lipid profile levels were not affected by consumption of supplements and endurance exercising. However, further studies are required to assess the long term effects of this herbal extract.

Cystatins - Extra- and intracellular cysteine protease inhibitors: High-level secretion and uptake of cystatin C in human neuroblastoma cells

DEFF Research Database (Denmark)

Wallin, Hanna; Bjarnadottir, Maria; Vogel, Lotte

2010-01-01

signal peptides) for cellular export following translation. Results indicating existence of systems for significant internalisation of type 2 cystatins from the extracellular to intracellular compartments are reviewed. Data showing that human neuroblastoma cell lines generally secrete high levels...

Investigating the causes for decreased levels of glutathione in individuals with type II diabetes.

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Minette Lagman

Full Text Available Tuberculosis (TB remains an eminent global burden with one third of the world's population latently infected with Mycobacterium tuberculosis (M. tb. Individuals with compromised immune systems are especially vulnerable to M. tb infection. In fact, individuals with Type 2 Diabetes Mellitus (T2DM are two to three times more susceptible to TB than those without T2DM. In this study, we report that individuals with T2DM have lower levels of glutathione (GSH due to compromised levels of GSH synthesis and metabolism enzymes. Transforming growth factor beta (TGF-β, a cytokine that is known to decrease the expression of the catalytic subunit of glutamine-cysteine ligase (GCLC was found in increased levels in the plasma samples from individuals with T2DM, explaining the possible underlying mechanism that is responsible for decreased levels of GSH in individuals with T2DM. Moreover, increased levels of pro-inflammatory cytokines such as interleukin-6 (IL-6 and interleukin-17 (IL-17 were observed in plasma samples isolated from individuals with T2DM. Increased levels of IL-6 and IL-17 was accompanied by enhanced production of free radicals further indicating an alternative mechanism for the decreased levels of GSH in individuals with T2DM. Augmenting the levels of GSH in macrophages isolated from individuals with T2DM resulted in improved control of M. tb infection. Furthermore, cytokines that are responsible for controlling M. tb infection at the cellular and granuloma level such as tumor necrosis factor alpha (TNF-α, interleukin-1β (IL-1β, interleukin-2 (IL-2, interferon-gamma (IFN-γ, and interleukin-12 (IL-12, were found to be compromised in plasma samples isolated from individuals with T2DM. On the other hand, interleukin-10 (IL-10, an immunosuppressive cytokine was increased in plasma samples isolated from individuals with T2DM. Overall, these findings suggest that lower levels of GSH in individuals with T2DM lead to their increased susceptibility

Radioprotective effect of cysteamine in glutathione synthetase-deficient cells

International Nuclear Information System (INIS)

Deschavanne, P.J.; Debieu, D.; Malaise, E.P.; Midander, J.; Revesz, L.

1986-01-01

The radioprotective role of endogenous and exogenous thiols was investigated, with survival as the end-point, after radiation exposure of cells under oxic and hypoxic conditions. Human cell strains originating from a 5-oxoprolinuria patient and from a related control were used. Due to a genetic deficiency in glutathione synthetase, the level of free SH groups, and in particular that of glutathione, is decreased in 5-oxoprolinuria cells. The glutathione synthetase deficient cells have a reduced oxygen enhancement ratio (1.5) compared to control cells (2.7). The radiosensitivity was assessed for both cell strains in the presence of different concentrations of an exogenous radioprotector:cysteamine. At concentrations varying between 0.1 and 20 mM, cysteamine protected the two cell strains to the same extent when irradiated under oxic and hypoxic conditions. The protective effect of cysteamine was lower under hypoxia than under oxic conditions for both cell strains. Consequently, the oxygen enhancement ratio decreased for both cell strains when cysteamine concentration increased. These results suggest that cysteamine cannot replace endogenous thiols as far as they are implicated in the radiobiological oxygen effect. (author)

Human glutathione S-transferase-mediated glutathione conjugation of curcumin and efflux of these conjugates in Caco-2 cells

NARCIS (Netherlands)

Usta, M.; Wortelboer, H.M.; Vervoort, J.J.M.; Boersma, M.G.; Rietjens, I.M.C.M.; Bladeren, van P.J.; Cnubben, N.H.P.

2007-01-01

Curcumin, an alpha,beta-unsaturated carbonyl compound, reacts with glutathione, leading to the formation of two monoglutathionyl curcumin conjugates. In the present study, the structures of both glutathione conjugates of curcumin were identified by LC-MS and one- and two-dimensional H-1 NMR

Combined radiation-protective and radiation-sensitizing agents. IV. Measurement of intracellular protector concentrations

International Nuclear Information System (INIS)

Koch, C.J.; Stobbe, C.C.; Hettiaratchi, P.

1989-01-01

Radiosensitization of hypoxic V79 Chinese hamster cells by 0.5 mM misonidazole at approximately 0-4 degrees C is substantially enhanced by pretreating the cells overnight with 0.1 mM buthionine sulfoximine, which lowers the cellular glutathione content to 5% of control values (from 4 mM to approximately 0.2 mM). The enhanced sensitization is reversed by concentrations of exogenous cysteine that are much lower (0.02 mM) than the original glutathione content. Reduced Co-enzyme A affords reversal of the enhancing effect at concentrations of about 1 mM. Sodium ascorbate gives no protection at all even at concentrations of 2 mM. The intracellular concentration of the reducing agents was measured using a spin-through oil technique. There was no diffusion of Co-A (MW greater than 750) or ascorbate (excluded by charge) into the cells. In contrast, cysteine was rapidly concentrated by factors of 4-10, even at the low temperatures used. Extracellular ascorbate's inability to radioprotect argues against electron transfer across the cell membrane as a mechanism for radioprotection. This mechanism could have explained the ability of exogenous thiols to radioprotect in former studies using glutathione, and in the present studies using Co-A. The potential of cysteine to be concentrated by cells poses a problem in the interpretation of exogenous protection by non-diffusing thiols, since trace contamination by cysteine could lead to the actual protection observed. Cysteine could also be formed by exchange reactions of exogenous thiols with the disulfide of cysteine, present in all media formulations

HYPERTHERMIA, INTRACELLULAR FREE CALCIUM AND CALCIUM IONOPHORES

NARCIS (Netherlands)

STEGE, GJJ; WIERENGA, PK; KAMPINGA, HH; KONINGS, AWT

1993-01-01

It is shown that heat-induced increase of intracellular calcium does not correlate with hyperthermic cell killing. Six different cell lines were investigated; in four (EAT, HeLa S3, L5178Y-R and L5178Y-S) heat treatments killing 90% of the cells did not affect the levels of intracellular free

Quantitative detection of nitroxyl upon trapping with glutathione and labeling with a specific fluorogenic reagent.

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Johnson, Gail M; Chozinski, Tyler J; Salmon, Debra J; Moghaddam, Alan D; Chen, Hsin Chih; Miranda, Katrina M

2013-10-01

Donors of nitroxyl (HNO) have shown promise for treatment of stroke, heart failure, alcoholism and cancer. However, comparing the pharmacological capacities of various donors is difficult without first quantifying the amount of HNO released from each donor. Detection and quantitation of HNO has been complicated by the rapid self-consumption of HNO through irreversible dimerization, poor selectivity of trapping agents against other nitrogen oxides, and/or low sensitivity towards HNO. Here, an assay is described for the trapping of HNO by glutathione (GSH) followed by labeling of GSH with the fluorogenic agent, naphthalene-2,3-dicarboxaldehyde (NDA), and subsequent quantitation by fluorescence difference. The newly developed assay was used to validate the pH-dependence of HNO release from isopropylamine NONOate (IPA/NO), which is a dual donor of HNO and NO at physiological pH. Furthermore, varied assay conditions were utilized to suggest the ratios of the products of the reaction of GSH with HNO. At intracellular concentrations of GSH, the disulfide (GSSG) was the major product, but significant concentrations of glutathione sulfinamide (GS(O)NH₂) were also detected. This suggests that GS(O)NH₂, which is a selective biomarker of HNO, may be produced in concentrations that are amenable to in vivo analysis. Copyright © 2013 Elsevier Inc. All rights reserved.

Human glutathione S-transferase-mediated glutathione conjugation of curcumin and efflux of these conjugates in caco-2 cells

NARCIS (Netherlands)

Usta, M.; Wortelboer, H.M.; Vervoort, J.; Boersma, M.G.; Rietjens, I.M.C.M.; Bladeren, P.J. van; Cnubben, N.H.P.

2007-01-01

Curcumin, an α,β-unsaturated carbonyl compound, reacts with glutathione, leading to the formation of two monoglutathionyl curcumin conjugates. In the present study, the structures of both glutathione conjugates of curcumin were identified by LC-MS and one- and two-dimensional 1H NMR analysis, and

Determination of serum neuron specific enolase and glutathion S transferases levels in patients with acute cerebral infarction and its clinical significance

International Nuclear Information System (INIS)

Guo Jianyi; Lu Tianhe; Bao Yanmei

2002-01-01

Objective: To evaluate the variation of serum neuron specific enolase (NSE) and glutathion S transferases (GST) levels in patients with cerebral infarction and its clinical significance. Methods: The serum levels of NSE in cerebral infarction patients were determined with immunoradiometric assay (IRMA), and the serum level of GST were determined by enzyme immuno sandwich assay (ELISA). Results: Serum NSE levels linked in patients were significantly higher (p<0.01) and GST serum levels were significantly lower (p < 0.01) within 3 days after onset of disease than those at two weeks and those in the controls. There was a positive correlation between serum NSE levels and neurological deficit scores (p < 0.001) and a negative correlation with serum GST levels (p < 0.05). There was also a close relationship between the serum NSE levels and the volume of infarction (p < 0.001). Conclusion: There was a close relationship between the Serum levels of NSE, GST and clinical features of Patients in the early stage of cerebral infarction

Modeling HIV-1 intracellular replication: two simulation approaches

NARCIS (Netherlands)

Zarrabi, N.; Mancini, E.; Tay, J.; Shahand, S.; Sloot, P.M.A.

2010-01-01

Many mathematical and computational models have been developed to investigate the complexity of HIV dynamics, immune response and drug therapy. However, there are not many models which consider the dynamics of virus intracellular replication at a single level. We propose a model of HIV intracellular

3-Methylcholanthrene inhibits lymphocyte proliferation and increases intracellular calcium levels in common carp (Cyprinus carpio L)

International Nuclear Information System (INIS)

Reynaud, S.; Duchiron, C.; Deschaux, P.

2003-01-01

Polycyclic aromatic hydrocarbons (PAHs) are an important class of environmental pollutants that are known to be carcinogenic and immunotoxic. Many authors have focused on macrophage activities in fish exposed to PAHs. However, fewer studies have reported decrease in specific immunity in such fish. We investigated the intracellular mechanisms by which the 3-methylcholanthrene (3-MC) decreased lymphocyte proliferation in carp. T- and B-lymphocyte proliferation induced by Concanavalin A (Con A) and lipopolysaccharide (LPS) were inhibited by 3-MC (0.5-50 μM). 3-MC also produced a rapid and a sustained increase in intracellular calcium concentration ([Ca 2+ ] i ) (2 h minimum). However, the cytochrome P450 1A and Ah receptor inhibitor, α-naphtoflavone (a-NF), also inhibited lymphocyte proliferation and did not reverse the effects of 3-MC. Moreover, since a-NF and 3-MC increased [Ca 2+ ] i and inhibited lymphocyte proliferation it was possible that calcium release played a role in 3-MC-inhibited lymphocyte proliferation. The rise in [Ca 2+ ] i induced by 3-MC was potentiated by the inhibitor of the endoplasmic reticulum calcium ATPases, thapsigargin. Treating cells with 3-MC decreased calcium mobilization caused by thapsigargin. These results suggest that 3-MC acts on the endoplasmic reticulum, perhaps directly on calcium ATPases, to increase intracellular calcium levels in carp leucocytes

Metabolic cooperation of ascorbic acid and glutathione in normal and vitamin C-deficient ODS rats.

Science.gov (United States)

Wang, Y; Kashiba, M; Kasahara, E; Tsuchiya, M; Sato, E F; Utsumi, K; Inoue, M

2001-01-01

Although the coordination of various antioxidants is important for the protection of organisms from oxidative stress, dynamic aspects of the interaction of endogenous antioxidants in vivo remain to be elucidated. We studied the metabolic coordination of two naturally occurring water-soluble antioxidants, ascorbic acid (AA) and reduced glutathione (GSH), in liver, kidney and plasma of control and scurvy-prone osteogenic disorder Shionogi (ODS) rats that hereditarily lack the ability to synthesize AA. When supplemented with AA, its levels in liver and kidney of ODS rats increased to similar levels of those in control rats. Hepato-renal levels of glutathione were similar with the two animal groups except for the slight increase in its hepatic levels in AA-supplemented ODS rats. Administration of L-buthionine sulfoximine (BSO), a specific inhibitor of GSH synthesis, rapidly decreased the hepato-renal levels of glutathione in a biphasic manner, a rapid phase followed by a slower phase. Kinetic analysis revealed that glutathione turnover was enhanced significantly in liver mitochondria and renal cytosol of ODS rats. Administration of BSO significantly increased AA levels in the liver and kidney of control rats but decreased them in AA-supplemented ODS rats. Kinetic analysis revealed that AA is synthesized by control rat liver by some BSO-enhanced mechanism and the de novo synthesized AA is transferred to the kidney. Such a coordination of the metabolism of GSH and AA in liver and kidney is suppressed in AA-deficient ODS rats. These and other results suggest that the metabolism of AA and GSH forms a compensatory network by which oxidative stress can be decreased.

The influence of low dose irradiation on intracellular pH level, synthesizing activity and ATP level in cultured chinese fibroblasts

International Nuclear Information System (INIS)

Parkhomenko, I.M.; Perishvili, G.V.; Turovetskij, V.B.; Kudryashov, Yu.B.; Rubin, A.B.; Brovko, L.Yu.

1993-01-01

X-irradiation of Chinese fibroblasts with doses of 0.05-0.15 Gy was shown to cause intracellular pH (pH i ) changes: its diminishing during the first 40-60 min by 0.16-0.18 pH units, then the return to the control level 120 min after irradiation and, finally, the increase by 0.18-0.20 pH units. Simultaneously, the synthesizing activity of the cells changed in the same way. The ATP level changed in the opposite way: increased when pH fell and decreased when pH grew. It was shown that pH i changes were connected with the changes in Na + /H + -exchange system, and they seemed to be primary in the chain of the alterations observed

[Association between intracellular zinc levels and nutritional status in HIV-infected and uninfected children exposed to the virus].

Science.gov (United States)

Gómez G, Erika María; Maldonado C, María Elena; Rojas L, Mauricio; Posada J, Gladys

2015-01-01

Malnutrition, growth retardation and opportunistic infections outlast the metabolic, immune and gastrointestinal disorders produced by HIV. Zinc deficiency has been associated with deteriorating nutritional status, growth failure, and risk of infection. The aim of this study is to determine the association between zinc levels in peripheral blood mononuclear cells (PBMC) and the nutritional status of HIV-infected and uninfected children exposed to the virus. An analytical, observational, cross-sectional study was conducted on 17 infected and 17 exposed children, aged 2-10 years. Anthropometric measurements, clinical and nutritional history, 24h recall, measurement of physical activity, and zinc in PBMC by flow cytometry analysis were recorded. Height according to age, energy consumption and adequacy of energy, protein and dietary zinc were significantly higher in children exposed to the virus compared to those infected with HIV (P .05). However, the median levels of zinc in monocytes of infected patients was higher (218.6) compared to the control group (217.0). No association was found between zinc intake and levels of intracellular zinc. The deterioration of nutritional status and growth retardation in children were associated with HIV, but not with the levels of intracellular zinc. The dietary intake of this nutrient was not associated with levels of zinc in monocytes or CD4 + and CD4- lymphocytes. Copyright © 2015. Publicado por Elsevier España, S.L.U.

Effects of methylmercury exposure on glutathione metabolism, oxidative stress, and chromosomal damage in captive-reared common loon (Gavia immer) chicks

International Nuclear Information System (INIS)

Kenow, Kevin P.; Hoffman, David J.; Hines, Randy K.; Meyer, Michael W.; Bickham, John W.; Matson, Cole W.; Stebbins, Katie R.; Montagna, Paul; Elfessi, Abdulaziz

2008-01-01

We quantified the level of dietary mercury (Hg), delivered as methylmercury chloride (CH 3 HgCl), associated with negative effects on organ and plasma biochemistries related to glutathione (GSH) metabolism and oxidative stress, and chromosomal damage in captive-reared common loon (Gavia immer) chicks reared from hatch to 105 days. Mercury-associated effects related to oxidative stress and altered glutathione metabolism occurred at 1.2 μg Hg/g and 0.4 μg Hg/g, an ecologically relevant dietary mercury level, but not at 0.08 μg Hg/g. Among the variables that contributed most to dissimilarities in tissue chemistries between control and treatment groups were increased levels of oxidized glutathione (GSSG), GSH peroxidase, and the ratio of GSSG to GSH in brain tissue; increased levels of hepatic GSH; and decreased levels of hepatic glucose-6-phosphate dehydrogenase (G-6-PDH). Our results also suggest that chronic exposure to environmentally relevant dietary Hg levels did not result in statistically significant somatic chromosomal damage in common loon chicks. - Oxidative stress and altered glutathione metabolism were evident in common loon chicks exposed to ≥0.4 μg Hg as CH 3 HgCl per gram wet food intake

The antioxidant master glutathione and periodontal health

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Vivek Kumar Bains

2015-01-01

Full Text Available Glutathione, considered to be the master antioxidant (AO, is the most-important redox regulator that controls inflammatory processes, and thus damage to the periodontium. Periodontitis patients have reduced total AO capacity in whole saliva, and lower concentrations of reduced glutathione (GSH in serum and gingival crevicular fluid, and periodontal therapy restores the redox balance. Therapeutic considerations for the adjunctive use of glutathione in management of periodontitis, in limiting the tissue damage associated with oxidative stress, and enhancing wound healing cannot be underestimated, but need to be evaluated further through multi-centered randomized controlled trials.

Selective effects of whey protein concentrate on glutathione levels and apoptosis in rats with mammary tumors.

Science.gov (United States)

Cheng, Shih-Hsuan; Tseng, Yang-Ming; Wu, Szu-Hsien; Tsai, Shih-Meng; Tsai, Li-Yu

2017-09-01

Glutathione (GSH) plays an important role in antioxidant defense and regulation of apoptosis. GSH deficiency is related to many diseases, including cancer, and increased GSH levels in cancer cells are associated with chemotherapy resistance because of resistance to apoptosis. In this study, we investigated the effects of whey protein concentrate (WPC), a precursor of GSH, in rats with mammary tumors induced by treatment with 7,12-dimethylbenz(a)anthracene (DMBA). DMBA treatment results in cellular changes that mimic the initiation and promotion of carcinogenesis of breast tissue. We aimed to examine the possible preventive effects of diets containing whey protein on DMBA-induced mammary tumors in rats. The results indicate that WPC (0.334 g/kg) supplementation significantly increased the liver GSH levels by 92%, and were accompanied by low Bax/Bcl-2 ratio (from 5 to 3) and cleaved caspase-3/procaspase-3 ratio (from 2.4 to 1.2) in DMBA-treated rats. Furthermore, tumor GSH levels were decreased by 47% in WPC-supplemented rats, which resulted in increased Bax/Bcl-2 ratio (from 0.9 to 2) and cleaved caspase-3/procaspase-3 ratio (from 1.1 to 2.7). In conclusion, supplementation with WPC could selectively deplete tumor GSH levels and, therefore, WPC supplementation might be a promising strategy to overcome treatment resistance in cancer therapy. Copyright © 2017 Elsevier Ltd. All rights reserved.

Tenofovir-induced Fanconi syndrome and osteomalacia in two HIV-infected patients: Role of intracellular tenofovir diphosphate levels and review of the literature

NARCIS (Netherlands)

Haverkort, M.E.; van der Spek, B.W.; Lips, P.T.A.M.; Slieker, W.A.; ter Heine, R.; Huitema, A.D.; Bronsveld, W.

2011-01-01

We present 2 human immunodeficiency virus-infected patients with tenofovir disoproxil fumarate-induced Fanconi syndrome, leading to osteomalacia. Intracellular tenofovir diphosphate levels were measured in 1 patient and were found to be very high, with plasma tenofovir levels just slightly elevated.

GLUTATHIONE AND ANTIOXIDANT ENZYMES IN THE HEPATOPANCREAS OF CRAYFISH PROCAMBARUS CLARKII (GIRARD, 1852 OF LAKE TRASIMENO (ITALY

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ELIA A. C.

2006-01-01

Full Text Available Antioxidant parameters, such as total glutathione, glutathione S-transferase, glutathione peroxidase, glutathione reductase, glyoxalases, catalase, and some heavy metals such as, lead, cadmium and chromium were examined in hepatopancreas of both sexes of Procambarus clarkii collected seasonally from Lake Trasimeno, from winter 2002-2003 to autumn 2003. Heavy metals content in hepatopancreas in males and females of P. clarkii was low and did not vary through the sampling periods and between sexes. On the contrary, crayfish exhibited sex-dependent differences in levels of some enzyme activities and of total glutathione, and no apparent relationship was found between contaminant burdens and antioxidant indexes in hepatopancreas. Because measured metal concentrations were low, other factors, presumably, were involved in antioxidant variations in P. clarkii and these latter seemed to be affected more by biological and environmental factors, other than those related to pollutants body burdens.

Enhanced glutathione content allows the in vivo synthesis of fluorescent CdTe nanoparticles by Escherichia coli.

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Juan P Monrás

Full Text Available The vast application of fluorescent semiconductor nanoparticles (NPs or quantum dots (QDs has prompted the development of new, cheap and safer methods that allow generating QDs with improved biocompatibility. In this context, green or biological QDs production represents a still unexplored area. This work reports the intracellular CdTe QDs biosynthesis in bacteria. Escherichia coli overexpressing the gshA gene, involved in glutathione (GSH biosynthesis, was used to produce CdTe QDs. Cells exhibited higher reduced thiols, GSH and Cd/Te contents that allow generating fluorescent intracellular NP-like structures when exposed to CdCl(2 and K(2TeO(3. Fluorescence microscopy revealed that QDs-producing cells accumulate defined structures of various colors, suggesting the production of differently-sized NPs. Purified fluorescent NPs exhibited structural and spectroscopic properties characteristic of CdTe QDs, as size and absorption/emission spectra. Elemental analysis confirmed that biosynthesized QDs were formed by Cd and Te with Cd/Te ratios expected for CdTe QDs. Finally, fluorescent properties of QDs-producing cells, such as color and intensity, were improved by temperature control and the use of reducing buffers.

21 CFR 864.7375 - Glutathione reductase assay.

Science.gov (United States)

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Glutathione reductase assay. 864.7375 Section 864.7375 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7375 Glutathione...

Molecularly imprinted solid-phase extraction of glutathione from urine samples

International Nuclear Information System (INIS)

Song, Renyuan; Hu, Xiaoling; Guan, Ping; Li, Ji; Zhao, Na; Wang, Qiaoli

2014-01-01

Molecularly imprinted polymer (MIP) particles for glutathione were synthesized through iniferter-controlled living radical precipitation polymerization (IRPP) under ultraviolet radiation at ambient temperature. Static adsorption, solid-phase extraction, and high-performance liquid chromatography were carried out to evaluate the adsorption properties and selective recognition characteristics of the polymers for glutathione and its structural analogs. The obtained IRPP-MIP particles exhibited a regularly spherical shape, rapid binding kinetics, high imprinting factor, and high selectivity compared with the MIP particles prepared using traditional free-radical precipitation polymerization. The selective separation and enrichment of glutathione from the mixture of glycyl-glycine and glutathione disulfide could be achieved on the IRPP-MIP cartridge. The recoveries of glutathione, glycyl-glycine, and glutathione disulfide were 95.6% ± 3.65%, 29.5% ± 1.26%, and 49.9% ± 1.71%, respectively. The detection limit (S/N = 3) of glutathione was 0.5 mg·L −1 . The relative standard deviations (RSDs) for 10 replicate detections of 50 mg·L −1 of glutathione were 5.76%, and the linear range of the calibration curve was 0.5 mg·L −1 to 200 mg·L −1 under optimized conditions. The proposed approach was successfully applied to determine glutathione in spiked human urine samples with recoveries of 90.24% to 96.20% and RSDs of 0.48% to 5.67%. - Highlights: • Imprinted polymer particles were prepared by IRPP at ambient temperature. • High imprinting factor, high selectivity, and rapid binding kinetics were achieved. • Selective solid-phase extraction of glutathione from human urine samples

Mechanism-based biomarker gene sets for glutathione depletion-related hepatotoxicity in rats

International Nuclear Information System (INIS)

Gao Weihua; Mizukawa, Yumiko; Nakatsu, Noriyuki; Minowa, Yosuke; Yamada, Hiroshi; Ohno, Yasuo; Urushidani, Tetsuro

2010-01-01

Chemical-induced glutathione depletion is thought to be caused by two types of toxicological mechanisms: PHO-type glutathione depletion [glutathione conjugated with chemicals such as phorone (PHO) or diethyl maleate (DEM)], and BSO-type glutathione depletion [i.e., glutathione synthesis inhibited by chemicals such as L-buthionine-sulfoximine (BSO)]. In order to identify mechanism-based biomarker gene sets for glutathione depletion in rat liver, male SD rats were treated with various chemicals including PHO (40, 120 and 400 mg/kg), DEM (80, 240 and 800 mg/kg), BSO (150, 450 and 1500 mg/kg), and bromobenzene (BBZ, 10, 100 and 300 mg/kg). Liver samples were taken 3, 6, 9 and 24 h after administration and examined for hepatic glutathione content, physiological and pathological changes, and gene expression changes using Affymetrix GeneChip Arrays. To identify differentially expressed probe sets in response to glutathione depletion, we focused on the following two courses of events for the two types of mechanisms of glutathione depletion: a) gene expression changes occurring simultaneously in response to glutathione depletion, and b) gene expression changes after glutathione was depleted. The gene expression profiles of the identified probe sets for the two types of glutathione depletion differed markedly at times during and after glutathione depletion, whereas Srxn1 was markedly increased for both types as glutathione was depleted, suggesting that Srxn1 is a key molecule in oxidative stress related to glutathione. The extracted probe sets were refined and verified using various compounds including 13 additional positive or negative compounds, and they established two useful marker sets. One contained three probe sets (Akr7a3, Trib3 and Gstp1) that could detect conjugation-type glutathione depletors any time within 24 h after dosing, and the other contained 14 probe sets that could detect glutathione depletors by any mechanism. These two sets, with appropriate scoring

Ibuprofen administration attenuates serum TNF-α levels, hepatic glutathione depletion, hepatic apoptosis and mouse mortality after Fas stimulation

International Nuclear Information System (INIS)

Cazanave, Sophie; Vadrot, Nathalie; Tinel, Marina; Berson, Alain; Letteron, Philippe; Larosche, Isabelle; Descatoire, Veronique; Feldmann, Gerard; Robin, Marie-Anne; Pessayre, Dominique

2008-01-01

Fas stimulation recruits neutrophils and activates macrophages that secrete tumor necrosis factor-α (TNF-α), which aggravates Fas-mediated liver injury. To determine whether nonsteroidal anti-inflammatory drugs modify these processes, we challenged 24-hour-fasted mice with the agonistic Jo2 anti-Fas antibody (4 μg/mouse), and treated the animals 1 h later with saline or ibuprofen (250 mg/kg), a dual cyclooxygenase (COX)-1 and COX-2 inhibitor. Ibuprofen attenuated the Jo2-mediated recruitment/activation of myeloperoxidase-secreting neutrophils/macrophages in the liver, and attenuated the surge in serum TNF-α. Ibuprofen also minimized hepatic glutathione depletion, Bid truncation, caspase activation, outer mitochondrial membrane rupture, hepatocyte apoptosis and the increase in serum alanine aminotransferase (ALT) activity 5 h after Jo2 administration, to finally decrease mouse mortality at later times. The concomitant administration of pentoxifylline (decreasing TNF-α secretion) and infliximab (trapping TNF-α) likewise attenuated the Jo2-mediated increase in TNF-α, the decrease in hepatic glutathione, and the increase in serum ALT activity 5 h after Jo2 administration. The concomitant administration of the COX-1 inhibitor, SC-560 (10 mg/kg) and the COX-2 inhibitor, celecoxib (40 mg/kg) 1 h after Jo2 administration, also decreased liver injury 5 h after Jo2 administration. In contrast, SC-560 (10 mg/kg) or celecoxib (40 or 160 mg/kg) given alone had no significant protective effects. In conclusion, secondary TNF-α secretion plays an important role in Jo2-mediated glutathione depletion and liver injury. The combined inhibition of COX-1 and COX-2 by ibuprofen attenuates TNF-α secretion, glutathione depletion, mitochondrial alterations, hepatic apoptosis and mortality in Jo2-treated fasted mice

Binding of Glutathione to Enterovirus Capsids Is Essential for Virion Morphogenesis

Science.gov (United States)

Thibaut, Hendrik Jan; Thys, Bert; Canela, María-Dolores; Aguado, Leire; Wimmer, Eckard; Paul, Aniko; Pérez-Pérez, María-Jesús; van Kuppeveld, Frank J. M.; Neyts, Johan

2014-01-01

Enteroviruses (family of the Picornaviridae) cover a large group of medically important human pathogens for which no antiviral treatment is approved. Although these viruses have been extensively studied, some aspects of the viral life cycle, in particular morphogenesis, are yet poorly understood. We report the discovery of TP219 as a novel inhibitor of the replication of several enteroviruses, including coxsackievirus and poliovirus. We show that TP219 binds directly glutathione (GSH), thereby rapidly depleting intracellular GSH levels and that this interferes with virus morphogenesis without affecting viral RNA replication. The inhibitory effect on assembly was shown not to depend on an altered reducing environment. Using TP219, we show that GSH is an essential stabilizing cofactor during the transition of protomeric particles into pentameric particles. Sequential passaging of coxsackievirus B3 in the presence of low GSH-levels selected for GSH-independent mutants that harbored a surface-exposed methionine in VP1 at the interface between two protomers. In line with this observation, enteroviruses that already contained this surface-exposed methionine, such as EV71, did not rely on GSH for virus morphogenesis. Biochemical and microscopical analysis provided strong evidence for a direct interaction between GSH and wildtype VP1 and a role for this interaction in localizing assembly intermediates to replication sites. Consistently, the interaction between GSH and mutant VP1 was abolished resulting in a relocalization of the assembly intermediates to replication sites independent from GSH. This study thus reveals GSH as a novel stabilizing host factor essential for the production of infectious enterovirus progeny and provides new insights into the poorly understood process of morphogenesis. PMID:24722756

Binding of glutathione to enterovirus capsids is essential for virion morphogenesis.

Directory of Open Access Journals (Sweden)

Hendrik Jan Thibaut

2014-04-01

Full Text Available Enteroviruses (family of the Picornaviridae cover a large group of medically important human pathogens for which no antiviral treatment is approved. Although these viruses have been extensively studied, some aspects of the viral life cycle, in particular morphogenesis, are yet poorly understood. We report the discovery of TP219 as a novel inhibitor of the replication of several enteroviruses, including coxsackievirus and poliovirus. We show that TP219 binds directly glutathione (GSH, thereby rapidly depleting intracellular GSH levels and that this interferes with virus morphogenesis without affecting viral RNA replication. The inhibitory effect on assembly was shown not to depend on an altered reducing environment. Using TP219, we show that GSH is an essential stabilizing cofactor during the transition of protomeric particles into pentameric particles. Sequential passaging of coxsackievirus B3 in the presence of low GSH-levels selected for GSH-independent mutants that harbored a surface-exposed methionine in VP1 at the interface between two protomers. In line with this observation, enteroviruses that already contained this surface-exposed methionine, such as EV71, did not rely on GSH for virus morphogenesis. Biochemical and microscopical analysis provided strong evidence for a direct interaction between GSH and wildtype VP1 and a role for this interaction in localizing assembly intermediates to replication sites. Consistently, the interaction between GSH and mutant VP1 was abolished resulting in a relocalization of the assembly intermediates to replication sites independent from GSH. This study thus reveals GSH as a novel stabilizing host factor essential for the production of infectious enterovirus progeny and provides new insights into the poorly understood process of morphogenesis.

OXIDATIVE MODIFICATION OF PROTEINS AND GLUTATHIONE SYSTEM IN ADIPOCYTES UNDER DIABETES

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Ye. V. Shakhristova

2014-01-01

Full Text Available Currently, diabetes ranks third in relation to medical and social significance after cardiovascular diseases and cancer and is the leading cause of blindness; it greatly increases the risk of myocardial infarction, coronary heart disease, nephropathy and hypertension in patients with this disorder; therefore clinical and experimental studies aimed at investigation of diabetes emergence and development mechanisms are urgent.The aim of the study was to investigate the status of oxidative modification of proteins and glutathionedependent antioxidant defense system in adipocytes of rats with alloxan diabetes under conditions of oxidative stress.Material and methods. Development of type 1 diabetes was induced in rats by alloxan administration (90 mg/kg of body mass. Adipocytes were obtained from epididymal adipose tissue of rats. The level of carbonyl derivatives of proteins, oxidized tryptophan, bityrosine, general, reduced, oxygenated and protein-bound glutathione, as well as glutathione peroxidase activity in adipocytes of rats was determined.Results. In adipocytes of rats with alloxan diabetes, concentration of carbonyl derivatives of proteins, bityrosine and oxidized tryptophan increased on the background of redox-potential of glutathione system and glutathione peroxidase activity decrease.Conclusion. The obtained data indicate the activation of free-radical oxidation of proteins and reduction of antioxidant defense under conditions of oxidative stress in the adipose tissue of rats with alloxan diabetes; this process plays an important role in pathogenesis of diabetes and its complications development.

Mechanisms of radiosensitization and protection studied with glutathione-deficient human cell lines

International Nuclear Information System (INIS)

Revesz, L.; Edgren, M.

1982-01-01

Glutathione-deficient fibroblasts and lymphoblastoid cells, derived from patients with an inborn error of glutathione synthetase activity, and glutathione-proficient cells, derived from clinically healthy individuals, were used to investigate the importance of glutathione for radiosensitization by misonidazole. With single-strand DNA breaks as an end point, misonidazole as well as oxygen was found to lack any sensitizing effect on cells deficient in glutathione. The post-irradiation repair of single-strand breaks induced by hypoxic irradiation of misonidazole treated cells was found to be a great extent glutathione dependent, like the repair of breaks induced by oxic irradiation. Naturally occurring aminothiols in glutathione-deficient cells appeared to be in efficient as substitutes for glutatione. Artificial aminothiols, such as cysteamine or dithiothreitol, were found to effectively replace glutathione

Purification of human hepatic glutathione S-transferases and the development of a radioimmunoassay for their measurement in plasma

International Nuclear Information System (INIS)

Hayes, J.D.; Gilligan, D.; Beckett, G.J.

1983-01-01

A purification scheme is described for six human hepatic glutathione S-transferases from a single liver. Five of the transferases comprised Ya monomers and had a molecular mass of 44000. The remaining enzyme comprised Yb monomers and had a molecular mass of 47000. Data are presented demonstrating that there are at least two distinct Ya monomers. A radioimmunoassay has been developed that has sufficient precision and sensitivity to allow direct measurement of glutathione S-transferase concentrations in unextracted plasma. A comparison of aminotransferase and glutathione S-transferase levels, in three patients who had taken a paracetamol overdose, indicated that glutathione S-transferase measurements provided a far more sensitive index of hepatocellular integrity than the more conventional aminotransferase measurements. (Auth.)

Purification of human hepatic glutathione S-transferases and the development of a radioimmunoassay for their measurement in plasma

Energy Technology Data Exchange (ETDEWEB)

Hayes, J.D.; Gilligan, D.; Beckett, G.J. (Edinburgh Univ. (UK). Dept. of Clinical Chemistry); Chapman, B.J. (Royal Infirmary, Edinburgh (UK))

1983-10-31

A purification scheme is described for six human hepatic glutathione S-transferases from a single liver. Five of the transferases comprised Ya monomers and had a molecular mass of 44000. The remaining enzyme comprised Yb monomers and had a molecular mass of 47000. Data are presented demonstrating that there are at least two distinct Ya monomers. A radioimmunoassay has been developed that has sufficient precision and sensitivity to allow direct measurement of glutathione S-transferase concentrations in unextracted plasma. A comparison of aminotransferase and glutathione S-transferase levels, in three patients who had taken a paracetamol overdose, indicated that glutathione S-transferase measurements provided a far more sensitive index of hepatocellular integrity than the more conventional aminotransferase measurements.

Role of glutathione biosynthesis in endothelial dysfunction and fibrosis

Directory of Open Access Journals (Sweden)

Cristina Espinosa-Díez

2018-04-01

Full Text Available Glutathione (GSH biosynthesis is essential for cellular redox homeostasis and antioxidant defense. The rate-limiting step requires glutamate-cysteine ligase (GCL, which is composed of the catalytic (GCLc and the modulatory (GCLm subunits. To evaluate the contribution of GCLc to endothelial function we generated an endothelial-specific Gclc haplo-insufficient mouse model (Gclc e/+ mice. In murine lung endothelial cells (MLEC derived from these mice we observed a 50% reduction in GCLc levels compared to lung fibroblasts from the same mice. MLEC obtained from haplo-insufficient mice showed significant reduction in GSH levels as well as increased basal and stimulated ROS levels, reduced phosphorylation of eNOS (Ser 1177 and increased eNOS S-glutathionylation, compared to MLEC from wild type (WT mice. Studies in mesenteric arteries demonstrated impaired endothelium-dependent vasodilation in Gclc(e/+ male mice, which was corrected by pre-incubation with GSH-ethyl-ester and BH4. To study the contribution of endothelial GSH synthesis to renal fibrosis we employed the unilateral ureteral obstruction model in WT and Gclc(e/+ mice. We observed that obstructed kidneys from Gclc(e/+ mice exhibited increased deposition of fibrotic markers and reduced Nrf2 levels. We conclude that the preservation of endothelial GSH biosynthesis is not only critical for endothelial function but also in anti-fibrotic responses. Keywords: Glutamate-cysteine ligase, ROS, Glutathione, Endothelial dysfunction, Kidney Fibrosis

Glutathione, cell proliferation and differentiation | Ashtiani | African ...

African Journals Online (AJOL)

All organisms require an equivalent source for living. Reduced glutathione is the most abundant thiol containing protein in mammalian cells and organs. Glutathione was discovered by Hopkins in 1924 who published his findings in JBC. It is a three peptide containing glutamic acid, cystein and glycin and is found in reduced ...

Effects of reduced glutathion and vitamin c on cisplatin-induced ...

African Journals Online (AJOL)

glutathione peroxidase [GSHPx], catalase [CAT], glutathione reductase [GSHR] activities and gene expression, glutathione [GSH] content) and lipid peroxidation products (malondialdehyde, MDA) in rat liver tissue were measured. CDDP hepatotoxicity was manifested by an increase in serum ALT and AST, elevation of MDA ...

Effects of methylmercury exposure on glutathione metabolism, oxidative stress, and chromosomal damage in captive-reared common loon (Gavia immer) chicks

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Kenow, Kevin P. [U.S. Geological Survey, Upper Midwest Environmental Sciences Center, 2630 Fanta Reed Road, La Crosse, WI 54603 (United States)], E-mail: kkenow@usgs.gov; Hoffman, David J. [U.S. Geological Survey, Patuxent Wildlife Research Center, 10300 Baltimore Avenue, Beltsville, MD 20705 (United States)], E-mail: djhoffman@usgs.gov; Hines, Randy K. [U.S. Geological Survey, Upper Midwest Environmental Sciences Center, 2630 Fanta Reed Road, La Crosse, WI 54603 (United States)], E-mail: rkhines@usgs.gov; Meyer, Michael W. [Wisconsin Department of Natural Resources, 107 Sutliff Avenue, Rhinelander, WI 54501 (United States)], E-mail: michael.meyer@dnr.state.wi.us; Bickham, John W. [Center for the Environment and Department of Forestry and Natural Resources, Purdue University, West Lafayette, IN 47907 (United States)], E-mail: bickham@purdue.edu; Matson, Cole W. [Integrated Toxicology and Environmental Health Program, Duke University, Durham, NC 27708 (United States)], E-mail: matson@duke.edu; Stebbins, Katie R. [U.S. Geological Survey, Patuxent Wildlife Research Center, 10300 Baltimore Avenue, Beltsville, MD 20705 (United States); Montagna, Paul [Texas A and M University-Corpus Christi, Harte Research Institute, Corpus Christi, TX (United States)], E-mail: paul.montagna@tamucc.edu; Elfessi, Abdulaziz [University of Wisconsin-La Crosse, La Crosse, WI 54601 (United States)], E-mail: elfessi.abdu@uwlax.edu

2008-12-15

We quantified the level of dietary mercury (Hg), delivered as methylmercury chloride (CH{sub 3}HgCl), associated with negative effects on organ and plasma biochemistries related to glutathione (GSH) metabolism and oxidative stress, and chromosomal damage in captive-reared common loon (Gavia immer) chicks reared from hatch to 105 days. Mercury-associated effects related to oxidative stress and altered glutathione metabolism occurred at 1.2 {mu}g Hg/g and 0.4 {mu}g Hg/g, an ecologically relevant dietary mercury level, but not at 0.08 {mu}g Hg/g. Among the variables that contributed most to dissimilarities in tissue chemistries between control and treatment groups were increased levels of oxidized glutathione (GSSG), GSH peroxidase, and the ratio of GSSG to GSH in brain tissue; increased levels of hepatic GSH; and decreased levels of hepatic glucose-6-phosphate dehydrogenase (G-6-PDH). Our results also suggest that chronic exposure to environmentally relevant dietary Hg levels did not result in statistically significant somatic chromosomal damage in common loon chicks. - Oxidative stress and altered glutathione metabolism were evident in common loon chicks exposed to {>=}0.4 {mu}g Hg as CH{sub 3}HgCl per gram wet food intake.

Glutathione Metabolism and Parkinson’s Disease

OpenAIRE

Smeyne, Michelle; Smeyne, Richard Jay

2013-01-01

It has been established that oxidative stress, defined as the condition when the sum of free radicals in a cell exceeds the antioxidant capacity of the cell, contributes to the pathogenesis of Parkinson’s disease. Glutathione is a ubiquitous thiol tripeptide that acts alone, or in concert with enzymes within cells to reduce superoxide radicals, hydroxyl radicals and peroxynitrites. In this review, we examine the synthesis, metabolism and functional interactions of glutathione, and discuss how...

Application of superparamagnetic microspheres for affinity adsorption and purification of glutathione

International Nuclear Information System (INIS)

Wang Qiang; Guan Yueping; Yang Mingzhu

2012-01-01

The superparamagnetic poly-(MA–DVB) microspheres with micron size were synthesized by the modified suspension polymerization method. Adsorption of glutathione by magnetic poly-(MA–DVB) microspheres with IDA-copper was investigated. The effect of solution pH value, affinity adsorption and desorption of glutathione was studied. The results showed that the optimum pH value for glutathione adsorption was found at pH=3.5, the maximum capacity for glutathione of magnetic poly-(MA–DVB) microspheres was estimated at 42.4 mg/g by fitting the experimental data to the Langmuir equation. The adsorption equilibrium of glutathione was obtained in about 10 min and the adsorbed glutathione was desorbed from the magnetic microspheres in about 30 min using NaCl buffer solution. The magnetic microspheres could be repeatedly utilized for the affinity adsorption of glutathione. - Highlights: ► The magnetic microsphere with surface IDA–Cu groups was synthesized. ► The magnetic microspheres were applied for adsorption of GSH. ► The adsorption–desorption of glutathione was investigated. ► The maximum adsorption capacity of GSH was fitted at 42.4 mg/g.

Suppression of the increasing level of acetylcholine-stimulated intracellular Ca2+ in guinea pig airway smooth muscle cells by mabuterol.

Science.gov (United States)

Song, Xirui; Zhao, Chao; Dai, Cailing; Ren, Yanxin; An, Nan; Wen, Huimin; Pan, L I; Cheng, Maosheng; Zhang, Yuyang

2015-11-01

The present study aimed to establish an effective method for the in vitro culture of guinea pig airway smooth muscle (ASM) cells, and also investigate the suppressive effect of mabuterol hydrochloride (Mab) on the increased level of intracellular Ca 2+ in ASM cells induced with acetylcholine (Ach). Two different methods, i.e. with or without collagenase to pretreat tracheal tissues, were applied to the manufacture of ASM cells. Cell viability was determined with the 3-(4,5-dimethylthinazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Immunocytochemistry and immunofluorescence were used for the identification of ASM cells. Different concentration levels (10 -3 , 10 -4 , 10 -5 , 10 -6 and 10 -7 mmol/l) of Mab were administered 5 min before Ach (10 -4 M) treatment, respectively. The Ca 2+ fluorescent probe, Fura-2/AM or Fluo-3/AM were applied to the inspection of Ca 2+ fluorescent intensity with Varioskan Flash, immunocytometry systems and an inverted system microscope, respectively. The results showed that the fresh method, in which isolated tracheal tissues were previously treated with collagenase for 20 min, was more advantageous for the preparation of guinea pig ASM cells compared to when the enzyme was not used. The time for the ASM cells to initially migrate out of the 'tissue blocks' and the culture having to be generated due to the thick cell density was significantly less. On identification with immunocytochemistry or immunofluorescent staining, >95% of the cells were ASM cells. Mab (10 -3 -10 -7 mmol/l) significantly suppressed the elevation of intracellular Ca 2+ induced by Ach in a concentration-dependent manner. The inhibitory rates of intracellular Ca 2+ by different concentrations of Mab, from low to high, were 14.93, 24.73, 40.06, 48.54 and 57.13%, respectively, when Varioskan Flash was used for determination. In conclusion, this novel method has a shorter harvesting period for ASM cells. Mab can suppress the increasing level of intracellular Ca 2

Relations between metals (Zn, Pb, Cd and Cu) and glutathione-dependent detoxifying enzymes in spiders from a heavy metal pollution gradient

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Wilczek, Grazyna [Department of Animal Physiology and Ecotoxicology, University of Silesia, Bankowa 9, 40-007 Katowice (Poland); Babczynska, Agnieszka [Department of Animal Physiology and Ecotoxicology, University of Silesia, Bankowa 9, 40-007 Katowice (Poland); Augustyniak, Maria [Department of Animal Physiology and Ecotoxicology, University of Silesia, Bankowa 9, 40-007 Katowice (Poland); Migula, Pawel [Department of Animal Physiology and Ecotoxicology, University of Silesia, Bankowa 9, 40-007 Katowice (Poland)]. E-mail: migula@us.edu.pl

2004-12-01

We studied the relations between glutathione-dependent detoxifying enzymes and heavy metal burdens in the web-building spider Agelena labyrinthica (Agelenidae) and the wolf spider Pardosa lugubris (Lycosidae) from five meadow sites along a heavy metal pollution gradient. We assayed the activity of glutathione-S-transferase (GST) and glutathione peroxidases (GPOX, GSTPx), and glutathione (GSH) levels in both sexes. Except for GSH vs Pb content, we found significant correlations between GPOX and GSTPx activity and metal concentrations in females of A. labyrinthica. The highest activity of these enzymes measured in the web-building spiders was found in the individuals from the most polluted sites. In P. lugubris males significant correlations were found between GST and Pb and Zn concentrations, and between GPOX and GSTPx and the concentration of Cu. GST activity was higher in males collected from less polluted areas. Thus, detoxifying strategies against pollutants seemed to be sex-dependent. Actively hunting spiders had higher metal concentrations, maintaining lower activity of detoxifying enzymes and a lower glutathione level. - Capsule: Glutathione-linked enzyme activity in spiders from polluted areas depends on hunting strategy and sex.

Relations between metals (Zn, Pb, Cd and Cu) and glutathione-dependent detoxifying enzymes in spiders from a heavy metal pollution gradient

International Nuclear Information System (INIS)

Wilczek, Grazyna; Babczynska, Agnieszka; Augustyniak, Maria; Migula, Pawel

2004-01-01

We studied the relations between glutathione-dependent detoxifying enzymes and heavy metal burdens in the web-building spider Agelena labyrinthica (Agelenidae) and the wolf spider Pardosa lugubris (Lycosidae) from five meadow sites along a heavy metal pollution gradient. We assayed the activity of glutathione-S-transferase (GST) and glutathione peroxidases (GPOX, GSTPx), and glutathione (GSH) levels in both sexes. Except for GSH vs Pb content, we found significant correlations between GPOX and GSTPx activity and metal concentrations in females of A. labyrinthica. The highest activity of these enzymes measured in the web-building spiders was found in the individuals from the most polluted sites. In P. lugubris males significant correlations were found between GST and Pb and Zn concentrations, and between GPOX and GSTPx and the concentration of Cu. GST activity was higher in males collected from less polluted areas. Thus, detoxifying strategies against pollutants seemed to be sex-dependent. Actively hunting spiders had higher metal concentrations, maintaining lower activity of detoxifying enzymes and a lower glutathione level. - Capsule: Glutathione-linked enzyme activity in spiders from polluted areas depends on hunting strategy and sex

Effect of Vitamin C on Glutathione Peroxidase Activities in Pregnant ...

African Journals Online (AJOL)

Glutathione peroxidase is one of the most important antioxidant enzymes in humans. We studied the relationship between serum glutathione peroxidase activity and vitamin C ingestion during normal pregnancy in women attending antenatal clinic in the University of Ilorin Teaching Hospital, Ilorin. Glutathione peroxidase ...

Compartment specific importance of glutathione during abiotic and biotic stress

Directory of Open Access Journals (Sweden)

Bernd eZechmann

2014-10-01

Full Text Available The tripeptide thiol glutathione (γ-L-glutamyl-L-cysteinyl-glycine is the most important sulfur containing antioxidant in plants and essential for plant defense against abiotic and biotic stress conditions. It is involved in the detoxification of reactive oxygen species, redox signaling, the modulation of defense gene expression and important for the regulation of enzymatic activities. Even though changes in glutathione contents are well documented in plants and its roles in plant defense are well established, still too little is known about its compartment specific importance during abiotic and biotic stress conditions. Due to technical advances in the visualization of glutathione and the redox state of plants through microscopical methods some progress was made in the last few years in studying the importance of subcellular glutathione contents during stress conditions in plants. This review summarizes the data available on compartment specific importance of glutathione in the protection against abiotic and biotic stress conditions such as high light stress, exposure to cadmium, drought, and pathogen attack (Pseudomonas, Botrytis, Tobacco Mosaic Virus. The data will be discussed in connection with the subcellular accumulation of ROS during these conditions and glutathione synthesis which are both highly compartment specific (e.g. glutathione synthesis takes place in chloroplasts and the cytosol. Thus this review will reveal the compartment specific importance of glutathione during abiotic and biotic stress conditions.

Influence of TiO{sub 2} nanoparticles on cellular antioxidant defense and its involvement in genotoxicity in HepG2 cells

Energy Technology Data Exchange (ETDEWEB)

Petkovic, Jana; Zegura, Bojana; Filipic, Metka, E-mail: metka.filipic@nib.si [Department of Genetic Toxicology and Cancer Biology, National Institute of Biology, SI-1000 Ljubljana (Slovenia)

2011-07-06

We investigated the effects of two types of TiO{sub 2} nanoparticles (<25 nm anatase, TiO{sub 2}-An; <100 nm rutile, TiO{sub 2}-Ru) on cellular antioxidant defense in HepG2 cells. We previously showed that in HepG2 cells, TiO{sub 2} nanoparticles are not toxic, although they induce oxidative DNA damage, production of intracellular reactive oxygen species, and up-regulation of mRNA expression of DNA-damage-responsive genes (p53, p21, gadd45{alpha} and mdm2). In the present study, we measured changes in mRNA expression of several antioxidant enzymes: catalase, superoxide dismutase, glutathione peroxidase, nitric oxide synthase, glutathione reductase and glutamate-cysteine ligase. As reduced glutathione has a central role in cellular antioxidant defense, we determined the effects of TiO{sub 2} nanoparticles on changes in the intracellular glutathione content. To confirm a role for glutathione in protection against TiO{sub 2}-nanoparticle-induced DNA damage, we compared the extent of TiO{sub 2}-nanoparticle-induced DNA damage in HepG2 cells that were glutathione depleted with buthionine-(S,R)-sulfoximine pretreatment and in nonglutathione-depleted cells. Our data show that both types of TiO{sub 2} nanoparticles up-regulate mRNA expression of oxidative-stress-related genes, with TiO{sub 2}-Ru being a stronger inducer than TiO{sub 2}-An. Both types of TiO{sub 2} nanoparticles also induce dose-dependent increases in intracellular glutathione levels, and in glutathione-depleted cells, TiO{sub 2}-nanoparticle-induced DNA damage was significantly greater than in nonglutathione-depleted cells. Interestingly, the glutathione content and the extent of DNA damage were significantly higher in TiO{sub 2}-An- than TiO{sub 2}-Ru-exposed cells. Thus, we show that TiO{sub 2} nanoparticles cause activation of cellular antioxidant processes, and that intracellular glutathione has a critical role in defense against this TiO{sub 2}-nanoparticle-induced DNA damage.

Superoxide radical (O2-) reactivity with respect to glutathione

International Nuclear Information System (INIS)

Sekaki, A.; Gardes-Albert, M.; Ferradini, C.

1984-01-01

Influence of superoxide radicals formed during gamma irradiation of glutathione in aerated aqueous solutions is examined. Solutions are buffered at pH7 and contain sodium formate for capture of H and OH radicals which are transformed in COO - radicals and then O 2 - radicals. G value of glutathione disparition vs glutathione concentration are given with and without enzyme or catalase. Reaction mechanism are interpreted [fr

Effect of rosella ( Hibiscus sabdariffa L ) extract on glutathione-S ...

African Journals Online (AJOL)

Purpose: To determine the effect of rosella (Hibiscus sabdariffa L) extract on glutathione-S-trasferase (GST) activity and its hepatoprotective effect. Methods: A total of 25 rats were divided randomly into 5 groups (5 rats per group). Group I served as the baseline, group II was the negative control group, while groups III, IV and ...

Chronic aspartame intake causes changes in the trans-sulphuration pathway, glutathione depletion and liver damage in mice

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Isabela Finamor

2017-04-01

Full Text Available No-caloric sweeteners, such as aspartame, are widely used in various food and beverages to prevent the increasing rates of obesity and diabetes mellitus, acting as tools in helping control caloric intake. Aspartame is metabolized to phenylalanine, aspartic acid, and methanol. Our aim was to study the effect of chronic administration of aspartame on glutathione redox status and on the trans-sulphuration pathway in mouse liver. Mice were divided into three groups: control; treated daily with aspartame for 90 days; and treated with aspartame plus N-acetylcysteine (NAC. Chronic administration of aspartame increased plasma alanine aminotransferase (ALT and aspartate aminotransferase activities and caused liver injury as well as marked decreased hepatic levels of reduced glutathione (GSH, oxidized glutathione (GSSG, γ-glutamylcysteine ​​(γ-GC, and most metabolites of the trans-sulphuration pathway, such as cysteine, S-adenosylmethionine (SAM, and S-adenosylhomocysteine ​​(SAH. Aspartame also triggered a decrease in mRNA and protein levels of the catalytic subunit of glutamate cysteine ligase (GCLc and cystathionine γ-lyase, and in protein levels of methionine adenosyltransferase 1A and 2A. N-acetylcysteine prevented the aspartame-induced liver injury and the increase in plasma ALT activity as well as the decrease in GSH, γ-GC, cysteine, SAM and SAH levels and GCLc protein levels. In conclusion, chronic administration of aspartame caused marked hepatic GSH depletion, which should be ascribed to GCLc down-regulation and decreased cysteine levels. Aspartame triggered blockade of the trans-sulphuration pathway at two steps, cystathionine γ-lyase and methionine adenosyltransferases. NAC restored glutathione levels as well as the impairment of the trans-sulphuration pathway.

Chronic aspartame intake causes changes in the trans-sulphuration pathway, glutathione depletion and liver damage in mice.

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Finamor, Isabela; Pérez, Salvador; Bressan, Caroline A; Brenner, Carlos E; Rius-Pérez, Sergio; Brittes, Patricia C; Cheiran, Gabriele; Rocha, Maria I; da Veiga, Marcelo; Sastre, Juan; Pavanato, Maria A

2017-04-01

No-caloric sweeteners, such as aspartame, are widely used in various food and beverages to prevent the increasing rates of obesity and diabetes mellitus, acting as tools in helping control caloric intake. Aspartame is metabolized to phenylalanine, aspartic acid, and methanol. Our aim was to study the effect of chronic administration of aspartame on glutathione redox status and on the trans-sulphuration pathway in mouse liver. Mice were divided into three groups: control; treated daily with aspartame for 90 days; and treated with aspartame plus N-acetylcysteine (NAC). Chronic administration of aspartame increased plasma alanine aminotransferase (ALT) and aspartate aminotransferase activities and caused liver injury as well as marked decreased hepatic levels of reduced glutathione (GSH), oxidized glutathione (GSSG), γ-glutamylcysteine ​​(γ-GC), and most metabolites of the trans-sulphuration pathway, such as cysteine, S-adenosylmethionine (SAM), and S-adenosylhomocysteine ​​(SAH). Aspartame also triggered a decrease in mRNA and protein levels of the catalytic subunit of glutamate cysteine ligase (GCLc) and cystathionine γ-lyase, and in protein levels of methionine adenosyltransferase 1A and 2A. N-acetylcysteine prevented the aspartame-induced liver injury and the increase in plasma ALT activity as well as the decrease in GSH, γ-GC, cysteine, SAM and SAH levels and GCLc protein levels. In conclusion, chronic administration of aspartame caused marked hepatic GSH depletion, which should be ascribed to GCLc down-regulation and decreased cysteine levels. Aspartame triggered blockade of the trans-sulphuration pathway at two steps, cystathionine γ-lyase and methionine adenosyltransferases. NAC restored glutathione levels as well as the impairment of the trans-sulphuration pathway. Copyright © 2017. Published by Elsevier B.V.

The Triple Roles of Glutathione for a DNA-Cleaving DNAzyme and Development of a Fluorescent Glutathione/Cu2+-Dependent DNAzyme Sensor for Detection of Cu2+ in Drinking Water.

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Wang, Shijin; Liu, Chengcheng; Li, Guiying; Sheng, Yongjie; Sun, Yanhong; Rui, Hongyue; Zhang, Jin; Xu, Jiacui; Jiang, Dazhi

2017-03-24

Pistol-like DNAzyme (PLDz) is an oxidative DNA-cleaving catalytic DNA with ascorbic acid as cofactor. Herein, glutathione was induced into the reaction system to maintain reduced ascorbic acid levels for higher efficient cleavage. However, data indicated that glutathione played triple roles in PLDz-catalyzed reactions. Glutathione alone had no effect on PLDz, and showed inhibitory effect on ascorbic acid-induced PLDz catalysis, but exhibited stimulating effect on Cu 2+ -promoted self-cleavage of PLDz. Further analysis of the effect of glutathione/Cu 2+ on PLDz indicated that H 2 O 2 played a key role in PLDz catalysis. Finally, we developed a fluorescent Cu 2+ sensor (PL-Cu 1.0) based on the relationship between glutathione/Cu 2+ and catalytic activity of PLDz. The fluorescent intensity showed a linear response toward the logarithm concentration of Cu 2+ over the range from 80 nM to 30 μM, with a detection limit of 21.1 nM. PL-Cu 1.0 provided only detection of Cu 2+ over other divalent metal ions. Ca 2+ and Mg 2+ could not interfere with Cu 2+ detection even at a 1000-fold concentration. We further applied PL-Cu 1.0 for Cu 2+ detection in tap and bottled water. Water stored in copper taps overnight had relatively high Cu 2+ concentrations, with a maximum 22.3 μM. Trace Cu 2+ (52.2 nM) in deep spring was detected among the tested bottled water. Therefore, PL-Cu 1.0 is feasible to detect Cu 2+ in drinking water, with a practical application.

Comparative evaluation of serum superoxide dismutase and glutathione levels in periodontally diseased patients: An interventional study

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Thomas Biju

2014-01-01

Full Text Available Background: Periodontal disease is an immune-inflammatory disease characterized by connective tissue breakdown, loss of attachment, and alveolar bone resorption. Under normal physiological conditions, a dynamic equilibrium is maintained between the reactive oxygen species (ROS and antioxidant defense capacity. Oxidative stress occurs when this equilibrium shifts in favor of ROS. Oxidative stress is thought to play a causative role in the pathogenesis of periodontal diseases. Aim: The present study was designed to estimate and compare the superoxide dismutase (SOD and glutathione (GSH levels in the serum of periodontitis, gingivitis, and healthy individuals before and after nonsurgical periodontal therapy. Materials and Methods: The present study was conducted in the Department of Periodontics, A. B. Shetty Memorial Institute of Dental Sciences, Deralakatte, Mangalore. The study was designed as a single blinded interventional study comprising 75 subjects, inclusive of both sexes and divided into three groups of 25 patients each. Patients were categorized into chronic periodontitis, gingivitis, and healthy. The severity of inflammation was assessed using gingival index and pocket probing depth. Biochemical analysis was done to estimate the SOD and GSH levels before and after nonsurgical periodontal therapy. Results obtained were then statistically analyzed using ANOVA test and paired t-test. Results: The results showed a higher level of serum SOD and GSH in the healthy group compared to the other groups. The difference was found to be statistically significant (P < 0.0001. The post-treatment levels of SOD were statistically higher than the pre-treatment levels in periodontitis and gingivitis group.

Determination of glutaredoxin enzyme activity and protein S-glutathionylation using fluorescent eosin-glutathione.

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Coppo, Lucia; Montano, Sergio J; Padilla, Alicia C; Holmgren, Arne

2016-04-15

Glutaredoxins catalyze glutathione-dependent disulfide oxidoreductions, particularly reduction of glutathione (GSH)-protein mixed disulfides. Mammalian glutaredoxins are present in the cytosol/nucleus as Grx1 or in mitochondria as Grx2a. Here we describe di-eosin-glutathione disulfide (Di-E-GSSG) as a new tool to study glutaredoxin (Grx) activity. Di-E-GSSG has almost no fluorescence in its disulfide form due to self-quenching, whereas the reduced form (E-GSH) has a large fluorescence emission at 545 nm after excitation at 520 nm. Di-E-GSSG was a very poor substrate for glutathione reductase, but we discovered that the molecule was an excellent substrate for glutaredoxin in a coupled assay system with GSH, nicotinamide adenine dinucleotide phosphate (NADPH), and glutathione reductase or with lipoamide, NADH, and lipoamide dehydrogenase. In addition, Di-E-GSSG was used to glutathionylate the free SH group of bovine serum albumin (BSA), yielding eosin-glutathionylated BSA (E-GS-BSA) readily observed in ultraviolet (UV) light. E-GS-BSA also displayed a quenched fluorescence, and its Grx-catalyzed reduction could be followed by the formation of E-GSH by fluorescence emission using microtiter plates. This way of measuring Grx activity provided an ultrasensitive method that detected Grx1 and Grx2 at picomolar levels. Human Grx1 was readily quantified in 40 μl of plasma and determined to be 680 ± 208 pM in healthy controls. Copyright © 2016 Elsevier Inc. All rights reserved.

Intracellular cholesterol level regulates sensitivity of glioblastoma cells against temozolomide-induced cell death by modulation of caspase-8 activation via death receptor 5-accumulation and activation in the plasma membrane lipid raft.

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Yamamoto, Yutaro; Tomiyama, Arata; Sasaki, Nobuyoshi; Yamaguchi, Hideki; Shirakihara, Takuya; Nakashima, Katsuhiko; Kumagai, Kosuke; Takeuchi, Satoru; Toyooka, Terushige; Otani, Naoki; Wada, Kojiro; Narita, Yoshitaka; Ichimura, Koichi; Sakai, Ryuichi; Namba, Hiroki; Mori, Kentaro

2018-01-01

Development of resistance against temozolomide (TMZ) in glioblastoma (GBM) after continuous treatment with TMZ is one of the critical problems in clinical GBM therapy. Intracellular cholesterol regulates cancer cell biology, but whether intracellular cholesterol is involved in TMZ resistance of GBM cells remains unclear. The involvement of intracellular cholesterol in acquired resistance against TMZ in GBM cells was investigated. Intracellular cholesterol levels were measured in human U251 MG cells with acquired TMZ resistance (U251-R cells) and TMZ-sensitive control U251 MG cells (U251-Con cells), and found that the intracellular cholesterol level was significantly lower in U251-R cells than in U251-Con cells. In addition, treatment by intracellular cholesterol remover, methyl-beta cyclodextrin (MβCD), or intracellular cholesterol inducer, soluble cholesterol (Chol), regulated TMZ-induced U251-Con cell death in line with changes in intracellular cholesterol level. Involvement of death receptor 5 (DR5), a death receptor localized in the plasma membrane, was evaluated. TMZ without or with MβCD and/or Chol caused accumulation of DR5 into the plasma membrane lipid raft and formed a complex with caspase-8, an extrinsic caspase cascade inducer, reflected in the induction of cell death. In addition, treatment with caspase-8 inhibitor or knockdown of DR5 dramatically suppressed U251-Con cell death induced by combination treatment with TMZ, MβCD, and Chol. Combined treatment of Chol with TMZ reversed the TMZ resistance of U251-R cells and another GBM cell model with acquired TMZ resistance, whereas clinical antihypercholesterolemia agents at physiological concentrations suppressed TMZ-induced cell death of U251-Con cells. These findings suggest that intracellular cholesterol level affects TMZ treatment of GBM mediated via a DR5-caspase-8 mechanism. Copyright © 2017 Elsevier Inc. All rights reserved.

The Glutathione Derivative, GSH Monoethyl Ester, May Effectively Whiten Skin but GSH Does Not

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Bo Young Chung

2016-04-01

Full Text Available Glutathione in its reduced form (GSH is an antioxidant and also is involved in pheomelanin formation. Thus, it has been long believed that GSH has a skin whitening effect. However, its actual or direct effect is unproven. We evaluated the anti-melanogenic effects of GSH and its derivatives in vitro. We examined change of melanogenesis and its related proteins by GSH itself and its derivatives, including GSH monoethyl ester (GSH-MEE, GSH diethyl ester (GSH-DEE and GSH monoisopropyl ester (GSH-MIPE in Melan-A cells, Mel-Ab cells, and B16F10 cells. GSH and GSH-MEE did not display cytotoxic activity, but GSH-MIPE and GSH-DEE did. Intriguingly, GSH itself had no inhibitory effect on melanin production or intracellular tyrosinase activity. Rather, it was GSH-MEE and GSH-MIPE that profoundly reduced the amount of melanin and intracellular tyrosinase activity. Thus, GSH-MEE was selected as a suitable candidate skin-whitening agent and it did not alter melanogenesis-associated proteins such as microphthalmia-associated transcription factor (MITF, tyrosinase, tyrosinase-related protein (TRP-1, and TRP-2, but it did increase the amount of suggested pheomelanin and suggested pheomelanin/eumelanin ratio. GSH-MEE was effective for anti-melanogenesis, whereas GSH itself was not. GSH-MEE could be developed as a safe and efficient agent for the treatment of hyperpigmentation skin disorders.

Rutin improves spatial memory in Alzheimer's disease transgenic mice by reducing Aβ oligomer level and attenuating oxidative stress and neuroinflammation.

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Xu, Peng-Xin; Wang, Shao-Wei; Yu, Xiao-Lin; Su, Ya-Jing; Wang, Teng; Zhou, Wei-Wei; Zhang, He; Wang, Yu-Jiong; Liu, Rui-Tian

2014-05-01

Alzheimer's disease (AD) is a progressive, neurodegenerative disease characterized by extracellular β-amyloid (Aβ) plaques and intracellular neurofibrillary tangles in the brain. Aβ aggregation is closely associated with neurotoxicity, oxidative stress, and neuronal inflammation. The soluble Aβ oligomers are believed to be the most neurotoxic form among all forms of Aβ aggregates. We have previously reported a polyphenol compound rutin that could inhibit Aβ aggregation and cytotoxicity, attenuate oxidative stress, and decrease the production of nitric oxide and proinflammatory cytokines in vitro. In the current study, we investigated the effect of rutin on APPswe/PS1dE9 transgenic mice. Results demonstrated that orally administered rutin significantly attenuated memory deficits in AD transgenic mice, decreased oligomeric Aβ level, increased super oxide dismutase (SOD) activity and glutathione (GSH)/glutathione disulfide (GSSG) ratio, reduced GSSG and malondialdehyde (MDA) levels, downregulated microgliosis and astrocytosis, and decreased interleukin (IL)-1β and IL-6 levels in the brain. These results indicated that rutin is a promising agent for AD treatment because of its antioxidant, anti-inflammatory, and reducing Aβ oligomer activities. Copyright © 2014 Elsevier B.V. All rights reserved.

Effect of glutathione aerosol on oxidant-antioxidant imbalance in idiopathic pulmonary fibrosis.

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Borok, Z; Buhl, R; Grimes, G J; Bokser, A D; Hubbard, R C; Holroyd, K J; Roum, J H; Czerski, D B; Cantin, A M; Crystal, R G

1991-07-27

Idiopathic pulmonary fibrosis (IPF) is characterised by alveolar inflammation, exaggerated release of oxidants, and subnormal concentrations of the antioxidant glutathione in respiratory epithelial lining fluid (ELF). Glutathione (600 mg twice daily for 3 days) was given by aerosol to 10 patients with IPF. Total ELF glutathione rose transiently, ELF oxidised glutathione concentrations increased, and there was a decrease in spontaneous superoxide anion release by alveolar macrophages. Thus, glutathione by aerosol could be a means of reversing the oxidant-antioxidant imbalance in IPF.

Intracellular Drug Bioavailability: Effect of Neutral Lipids and Phospholipids.

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Treyer, Andrea; Mateus, André; Wiśniewski, Jacek R; Boriss, Hinnerk; Matsson, Pär; Artursson, Per

2018-06-04

Intracellular unbound drug concentrations are the pharmacologically relevant concentrations for targets inside cells. Intracellular drug concentrations are determined by multiple processes, including the extent of drug binding to intracellular structures. The aim of this study was to evaluate the effect of neutral lipid (NL) and phospholipid (PL) levels on intracellular drug disposition. The NL and/or PL content of 3T3-L1 cells were enhanced, resulting in phenotypes (in terms of morphology and proteome) reminiscent of adipocytes (high NL and PL) or mild phospholipidosis (only high PL). Intracellular bioavailability ( F ic ) was then determined for 23 drugs in these cellular models and in untreated wild-type cells. A higher PL content led to higher intracellular drug binding and a lower F ic . The induction of NL did not further increase drug binding but led to altered F ic due to increased lysosomal pH. Further, there was a good correlation between binding to beads coated with pure PL and intracellular drug binding. In conclusion, our results suggest that PL content is a major determinant of drug binding in cells and that PL beads may constitute a simple alternative to estimating this parameter. Further, the presence of massive amounts of intracellular NLs did not influence drug binding significantly.

A glutathione s-transferase confers herbicide tolerance in rice

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Tingzhang Hu

2014-07-01

Full Text Available Plant glutathione S-transferases (GSTs have been a focus of attention due to their role in herbicide detoxification. OsGSTL2 is a glutathione S-transferase, lambda class gene from rice (Oryza sativa L.. Transgenic rice plants over-expressing OsGSTL2 were generated from rice calli by the use of an Agrobacterium transformation system, and were screened by a combination of hygromycin resistance, PCR and Southern blot analysis. In the vegetative tissues of transgenic rice plants, the over-expression of OsGSTL2 not only increased levels of OsGSTL2 transcripts, but also GST and GPX expression, while reduced superoxide. Transgenic rice plants also showed higher tolerance to glyphosate and chlorsulfuron, which often contaminate agricultural fields. The findings demonstrate the detoxification role of OsGSTL2 in the growth and development of rice plants. It should be possible to apply the present results to crops for developing herbicide tolerance and for limiting herbicide contamination in the food chain.

Bioreducible Lipid-like Nanoparticles for Intracellular Protein Delivery

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Arellano, Carlos Luis

Protein-based therapy is one of the most direct ways to manipulate cell function and treat human disease. Although protein therapeutics has made its way to clinical practice, with five of the top fifteen global pharmaceuticals being peptide or protein-based drugs, one common limitation is that the effects of protein therapy are only achieved through the targeting of cell surface receptors and intracellular domains. Due to the impermeability of the cell membrane to most foreign materials, entire classes of potentially therapeutic proteins cannot thoroughly be studied without a safe and efficient method of transporting proteins into the cytosol. We report the use of a combinatorially-designed bioreducible lipid-like material (termed "lipidoid") - based protein delivery platform for the transfection of human cancer cell lines. Lipidoid nanoparticles are synthesized through a thin film dispersion method. The degradation of the bioreducible nanoparticles was observed when exposed to glutathione, a highly reductive compound present in the cytosol. We demonstrate that the nanoparticles are capable of transfecting a dose-dependent concentration of our model protein, beta-galactosidase into HeLa cells. Furthermore, formulations of the lipidoid containing the cytotoxic proteins saporin and RNase-A are both capable of inhibiting tumor cell proliferation as observed in in vitro treatment of different human cancer cell lines. There was no observed loss in protein activity after lyophilization and long--term storage, indicating the potential of pre-clinical applications. Overall, we demonstrate an effective approach to protein formulation and intracellular delivery. We believe that our formulations will lead to the study of a whole class of previously untapped therapeutics that may generate new solutions for previously untreatable diseases.

Changes in glutathione system and lipid peroxidation in rat blood during the first hour after chlorpyrifos exposure

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V. P. Rosalovsky

2015-10-01

Full Text Available Chlorpyrifos (CPF is a highly toxic organophosphate compound, widely used as an active substance of many insecticides. Along with the anticholinesterase action, CPF may affect other biochemical mechanisms, particularly through disrupting pro- and antioxidant balance and inducing free-radical oxidative stress. Origins and occurrence of these phenomena are still not fully understood. The aim of our work was to investigate the effects of chlorpyrifos on key parameters of glutathione system and on lipid peroxidation in rat blood in the time dynamics during one hour after exposure. We found that a single exposure to 50 mg/kg chlorpyrifos caused a linear decrease in butyryl cholinesterase activity, increased activity of glutathione peroxidase and glutathione reductase, alterations in the levels of glutathione, TBA-active products and lipid hydroperoxides during 1 hour after poisoning. The most significant changes in studied parameters were detected at the 15-30th minutes after chlorpyrifos exposure.

The Role Of Sevoflurane And Ionising Radiation On THE Level Of Primary DNA Damage Measured In Blood And Different Organs Of Swiss Albino Mice By THE Alkaline Comet Assay

International Nuclear Information System (INIS)

Benkovic, V.; Milic, M.; Horvat Knezevic, A.; Halovanovic, S.; Borojevic, N.; Orsolic, N.

2015-01-01

Sevoflurane is general anaesthetic suitable for short surgical procedures due to its quick induction of anaesthesia, maintaining spontaneous breathing frequency and hemodynamic stability of patients. However, it can directly trigger the formation of peroxynitrite, significantly increase intracellular levels of H2O2, peroxide, superoxide anion and nitric oxide in peripheral polymorphonuclear neutrophils 1h after the treatment; lowering the levels of intracellular glutathione, and increase radiosensitivity of cells also exposed to ionising radiation (IR). We wanted to evaluate the level of sinergistic effect and possible radiosensitivity DNA damage in blood, and different organs of Swiss albino mice after exposure to both sevoflurane (2.4 percent, 50:50) and the 1Gy gamma-ray radiation generally used in diagnostic purposes after 0, 2, 6 and 24 hours from the combined treatment with alkaline comet assay. Combined exposure to sevoflurane and IR has demonstrated synergistic effect. Due to metabolising of the sevoflurane, there was different sensitivity between blood, liver, kidney and brain cells. (author).

Avocado oil induces long-term alleviation of oxidative damage in kidney mitochondria from type 2 diabetic rats by improving glutathione status.

Science.gov (United States)

Ortiz-Avila, Omar; Figueroa-García, María Del Consuelo; García-Berumen, Claudia Isabel; Calderón-Cortés, Elizabeth; Mejía-Barajas, Jorge A; Rodriguez-Orozco, Alain R; Mejía-Zepeda, Ricardo; Saavedra-Molina, Alfredo; Cortés-Rojo, Christian

2017-04-01

Hyperglycemia and mitochondrial ROS overproduction have been identified as key factors involved in the development of diabetic nephropathy. This has encouraged the search for strategies decreasing glucose levels and long-term improvement of redox status of glutathione, the main antioxidant counteracting mitochondrial damage. Previously, we have shown that avocado oil improves redox status of glutathione in liver and brain mitochondria from streptozotocin-induced diabetic rats; however, the long-term effects of avocado oil and its hypoglycemic effect cannot be evaluated because this model displays low survival and insulin depletion. Therefore, we tested during 1 year the effects of avocado oil on glycemia, ROS levels, lipid peroxidation and glutathione status in kidney mitochondria from type 2 diabetic Goto-Kakizaki rats. Diabetic rats exhibited glycemia of 120-186 mg/dL the first 9 months with a further increase to 250-300 mg/dL. Avocado oil decreased hyperglycemia at intermediate levels between diabetic and control rats. Diabetic rats displayed augmented lipid peroxidation and depletion of reduced glutathione throughout the study, while increased ROS generation was observed at the 3rd and 12th months along with diminished content of total glutathione at the 6th and 12th months. Avocado oil ameliorated all these defects and augmented the mitochondrial content of oleic acid. The beneficial effects of avocado oil are discussed in terms of the hypoglycemic effect of oleic acid and the probable dependence of glutathione transport on lipid peroxidation and thiol oxidation of mitochondrial carriers.

Epithelial Cell Gene Expression Induced by Intracellular Staphylococcus aureus

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Xianglu Li

2009-01-01

Full Text Available HEp-2 cell monolayers were cocultured with intracellular Staphylococcus aureus, and changes in gene expression were profiled using DNA microarrays. Intracellular S. aureus affected genes involved in cellular stress responses, signal transduction, inflammation, apoptosis, fibrosis, and cholesterol biosynthesis. Transcription of stress response and signal transduction-related genes including atf3, sgk, map2k1, map2k3, arhb, and arhe was increased. In addition, elevated transcription of proinflammatory genes was observed for tnfa, il1b, il6, il8, cxcl1, ccl20, cox2, and pai1. Genes involved in proapoptosis and fibrosis were also affected at transcriptional level by intracellular S. aureus. Notably, intracellular S. aureus induced strong transcriptional down-regulation of several cholesterol biosynthesis genes. These results suggest that epithelial cells respond to intracellular S. aureus by inducing genes affecting immunity and in repairing damage caused by the organism, and are consistent with the possibility that the organism exploits an intracellular environment to subvert host immunity and promote colonization.

Glutathione-dependent responses of plants to drought: a review

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Mateusz Labudda

2014-02-01

Full Text Available Water is a renewable resource. However, with the human population growth, economic development and improved living standards, the world’s supply of fresh water is steadily decreasing and consequently water resources for agricultural production are limited and diminishing. Water deficiency is a significant problem in agriculture and increasing efforts are currently being made to understand plant tolerance mechanisms and to develop new tools (especially molecular that could underpin plant breeding and cultivation. However, the biochemical and molecular mechanisms of plant water deficit tolerance are not fully understood, and the data available is incomplete. Here, we review the significance of glutathione and its related enzymes in plant responses to drought. Firstly, the roles of reduced glutathione and reduced/oxidized glutathione ratio, are discussed, followed by an extensive discussion of glutathione related enzymes, which play an important role in plant responses to drought. Special attention is given to the S-glutathionylation of proteins, which is involved in cell metabolism regulation and redox signaling in photosynthetic organisms subjected to abiotic stress. The review concludes with a brief overview of future perspectives for the involvement of glutathione and related enzymes in drought stress responses.

Photoaffinity labelling of the active site of the rat glutathione transferases 3-3 and 1-1 and human glutathione transferase A1-1.

OpenAIRE

Cooke, R J; Björnestedt, R; Douglas, K T; McKie, J H; King, M D; Coles, B; Ketterer, B; Mannervik, B

1994-01-01

The glutathione transferases (GSTs) form a group of enzymes responsible for a wide range of molecular detoxications. The photoaffinity label S-(2-nitro-4-azidophenyl)glutathione was used to study the hydrophobic region of the active site of the rat liver GST 1-1 and 2-2 isoenzymes (class Alpha) as well as the rat class-Mu GST 3-3. Photoaffinity labelling was carried out using a version of S-(2-nitro-4-azidophenyl)glutathione tritiated in the arylazido ring. The labelling occurred with higher ...

Reexamination of the Physiological Role of PykA in Escherichia coli Revealed that It Negatively Regulates the Intracellular ATP Levels under Anaerobic Conditions.

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Zhao, Chunhua; Lin, Zhao; Dong, Hongjun; Zhang, Yanping; Li, Yin

2017-06-01

Pyruvate kinase is one of the three rate-limiting glycolytic enzymes that catalyze the last step of glycolysis, conversion of phosphoenolpyruvate (PEP) into pyruvate, which is associated with ATP generation. Two isozymes of pyruvate kinase, PykF and PykA, are identified in Escherichia coli PykF is considered important, whereas PykA has a less-defined role. Prior studies inactivated the pykA gene to increase the level of its substrate, PEP, and thereby increased the yield of end products derived from PEP. We were surprised when we found a pykA ::Tn 5 mutant in a screen for increased yield of an end product derived from pyruvate ( n -butanol), suggesting that the role of PykA needs to be reexamined. We show that the pykA mutant exhibited elevated intracellular ATP levels, biomass concentrations, glucose consumption, and n -butanol production. We also discovered that the pykA mutant expresses higher levels of a presumed pyruvate transporter, YhjX, permitting the mutant to recapture and metabolize excreted pyruvate. Furthermore, we demonstrated that the nucleotide diphosphate kinase activity of PykA leads to negative regulation of the intracellular ATP levels. Taking the data together, we propose that inactivation of pykA can be considered a general strategy to enhance the production of pyruvate-derived metabolites under anaerobic conditions. IMPORTANCE This study showed that knocking out pykA significantly increased the intracellular ATP level and thus significantly increased the levels of glucose consumption, biomass formation, and pyruvate-derived product formation under anaerobic conditions. pykA was considered to be encoding a dispensable pyruvate kinase; here we show that pykA negatively regulates the anaerobic glycolysis rate through regulating the energy distribution. Thus, knocking out pykA can be used as a general strategy to increase the level of pyruvate-derived fermentative products. Copyright © 2017 American Society for Microbiology.

Correlation between endogenous glutathione content and sensitivity of cultured human skin cells to radiation at defined wavelengths in the solar ultraviolet range

International Nuclear Information System (INIS)

Tyrrell, R.M.; Pidoux, M.

1988-01-01

Glutathione depletion of cultured human skin fibroblasts by treatment with buthionine-S.R.-sulfoximine (BSO) sensitises them to solar UV radiation. We now show that there is a close quantitative correlation between cellular glutathione content and sensitivity to radiation at 365 nm. A weaker correlation is observed when cells are depleted of glutathione using diethylmaleimide. Both fibroblasts and epidermal keratinocytes derived from the same foreskin biopsy are sensitised to radiation at 313 nm by glutathione depletion. At low to intermediate fluence levels, 10 mM cysteamine present during irradiation at 302 nm is able to almost completely reverse the sensitising effects of glutathione depletion suggesting that the endogenous thiol protects against radiation at this wavelength by a free radical scavenging mechanism. At 313 nm, the sensitisation is not reversed by cysteamine suggesting that glutathione plays a more specific role in protection against radiation at longer wavelengths. Xeroderma pigmentosum group A fibroblasts (excision deficient) are also sensitised to radiation at 313 and 365 nm by depletion of glutathione. The results provide further evidence that endogenous glutathione is involved in protecting human skin cells against a wide range of solar radiation damage. (author)

In Vivo EPR Assessment of pH, pO2, Redox Status, and Concentrations of Phosphate and Glutathione in the Tumor Microenvironment.

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Bobko, Andrey A; Eubank, Timothy D; Driesschaert, Benoit; Khramtsov, Valery V

2018-03-16

This protocol demonstrates the capability of low-field electron paramagnetic resonance (EPR)-based techniques in combination with functional paramagnetic probes to provide quantitative information on the chemical tumor microenvironment (TME), including pO2, pH, redox status, concentrations of interstitial inorganic phosphate (Pi), and intracellular glutathione (GSH). In particular, an application of a recently developed soluble multifunctional trityl probe provides unsurpassed opportunity for in vivo concurrent measurements of pH, pO2 and Pi in Extracellular space (HOPE probe). The measurements of three parameters using a single probe allow for their correlation analyses independent of probe distribution and time of the measurements.

Hemoglobin-catalyzed fluorometric method for the determination of glutathione

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Wang, Ruiqiang; Tang, Lin; Li, Hua; Wang, Yi; Gou, Rong; Guo, Yuanyuan; Fang, Yudong; Chen, Fengmei

2016-01-01

A new spectrofluorometric method for the determination of glutathione based on the reaction catalyzed by hemoglobin was reported. The reaction product gave a highly fluorescent intensity with the excitation and emission wavelengths of 320.0 nm and 413.0 nm, respectively. The optimum experimental conditions were investigated. Results showed that low concentration glutathione enhanced the fluorescence intensity significantly. The line ranges were 1.0 × 10-6-1.0 × 10-5 mol L-1 of glutathione and 6.0 × 10-10 mol L-1-1.0 × 10-8 mol L-1, respectively. The detection limit was calculated to be 1.1 × 10-11 mol L-1. The recovery test by the standard addition method gave values in the range of 90.78%-102.20%. This method was used for the determination of glutathione in synthetic and real samples with satisfactory results.

Extraction of glutathione from EFB fermentation waste using methanol with sonication process

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Muryanto, Muryanto; Alvin, Nurdin, Muhammad; Hanifah, Ummu; Sudiyani, Yanni

2017-11-01

Glutathione is important compound on the human body. Glutathione have a widely use at pharmacy and cosmetics as detoxification, skin whitening agent, antioxidant and many other. This study aims to obtain glutathione from Saccharomyces cerevisiae in fermentation waste of second generation bioethanol. The remaining yeast in the empty fruit bunch (EFB) fermentation was separated from the fermentation solution use centrifugation process and then extracted using a methanol-water solution. The extraction process was done by maceration which was assisted by sonication process. Solvent concentration and time of sonication were varied to see its effect on glutathione concentration. The concentration of glutathione from the extraction process was analyzed using alloxan method with UV-Vis spectrophotometer. The results show that the highest glutathione concentration was approximately 1.32 g/L obtained with methanol solvent at 90 minutes of maceration following with 15 minutes sonication.

[Induction of glutathione and activation of immune functions by low-dose, whole-body irradiation with gamma-rays].

Science.gov (United States)

Kojima, Shuji

2006-10-01

We first examined the relation between the induction of glutathione and immune functions in mice after low-dose gamma-ray irradiation. Thereafter, inhibition of tumor growth by radiation was confirmed in Ehrlich solid tumor (EST)-bearing mice. The total glutathione level of the splenocytes transiently increased soon after irradiation and reached a maximum at around 4 h postirradiation. Thereafter, the level reverted to the 0 h value by 24 h postirradiation. A significantly high splenocyte proliferative response was also recognized 4 h postirradiation. Natural killer (NK) activity was also increased significantly in a similar manner. The time at which the response reached the maximum coincided well with that of maximum total glutathione levels of the splenocytes in the gamma-ray-irradiated mice. Reduced glutathione exogenously added to splenocytes obtained from normal mice enhanced the proliferative response and NK activity in a dose-dependent manner. The inhibitory effects of radiation on tumor growth was then examined in EST-bearing mice. Repeated low-dose irradiation (0.5 Gy, four times, before and within an early time after inoculation) significantly delayed the tumor growth. Finally, the effect of single low-dose (0.5 Gy), whole-body gamma-ray irradiation on immune balance was examined to elucidate the mechanism underlying the antitumor immunity. The percentage of B cells in blood lymphocytes was selectively decreased after radiation, concomitant with an increase in that of the helper T cell population. The IFN-gamma level in splenocyte culture prepared from EST-bearing mice was significantly increased 48 h after radiation, although the level of IL-4 was unchanged. IL-12 secretion from macrophages was also enhanced by radiation. These results suggest that low-dose gamma-rays induce Th1 polarization and enhance the activities of tumoricidal effector cells, leading to an inhibition of tumor growth.

Albumin-gold-glutathione is a probable auranofin metabolite

International Nuclear Information System (INIS)

Shaw, C.F. III; Coffer, M.; Isab, A.A.

1989-01-01

The newly licensed gold drug, auranofin ((2,3,4,6-tetra-O-acetyl-β-1-D-gluco-pyranosato-S-)triethylphoshine-gold(I)) crosses cell membranes and enters cells which are inaccessible to parenteral gold drugs. In vivo, the triethylphosphine ligand and gold of auranofin, but not the thio-sugar moiety, accumulate in and subsequently efflux from red blood cells (RBCs). Extracellular albumin increases in the extent of gold efflux and acts as a gold binding site. The rate of efflux is first-order in RBC gold concentration. Studies using RBCs in which labelled [ 14 C]-glutathione is generated in situ incorporation of [ 14 C]- glycine demonstrate that glutathione also effluxes from the RBCs and forms a gold-glutathione-albumin complex. This may be the immunopharmacologically active complex

Manipulation of Glutathione and Amino Acid Biosynthesis in the Chloroplast1

Science.gov (United States)

Noctor, Graham; Arisi, Ana-Carolina M.; Jouanin, Lise; Foyer, Christine H.

1998-01-01

Poplars (Populus tremula × Populus alba) were transformed to overexpress Escherichia coli γ-glutamylcysteine synthetase (γ-ECS) or glutathione synthetase in the chloroplast. Five independent lines of each transformant strongly expressed the introduced gene and possessed markedly enhanced activity of the gene product. Glutathione (GSH) contents were unaffected by high chloroplastic glutathione synthetase activity. Enhanced chloroplastic γ-ECS activity markedly increased γ-glutamylcysteine and GSH levels. These effects are similar to those previously observed in poplars overexpressing these enzymes in the cytosol. Similar to cytosolic γ-ECS overexpression, chloroplastic overexpression did not deplete foliar cysteine or methionine pools and did not lead to morphological changes. Light was required for maximal accumulation of GSH in poplars overexpressing γ-ECS in the chloroplast. High chloroplastic, but not cytosolic, γ-ECS activities were accompanied by increases in amino acids synthesized in the chloroplast. We conclude that (a) GSH synthesis can occur in the chloroplast and the cytosol and may be up-regulated in both compartments by increased γ-ECS activity, (b) interactions between GSH synthesis and the pathways supplying the necessary substrates are similar in both compartments, and (c) chloroplastic up-regulation of GSH synthesis is associated with an activating effect on the synthesis of specific amino acids formed in the chloroplast. PMID:9765532

Protection of HepG2 cells against acrolein toxicity by 2-cyano-3,12-dioxooleana-1,9-dien-28-imidazolide via glutathione-mediated mechanism.

Science.gov (United States)

Shah, Halley; Speen, Adam M; Saunders, Christina; Brooke, Elizabeth A S; Nallasamy, Palanisamy; Zhu, Hong; Li, Y Robert; Jia, Zhenquan

2015-10-01

Acrolein is an environmental toxicant, mainly found in smoke released from incomplete combustion of organic matter. Several studies showed that exposure to acrolein can lead to liver damage. The mechanisms involved in acrolein-induced hepatocellular toxicity, however, are not completely understood. This study examined the cytotoxic mechanisms of acrolein on HepG2 cells. Acrolein at pathophysiological concentrations was shown to cause apoptotic cell death and an increase in levels of protein carbonyl and thiobarbituric acid reactive acid substances. Acrolein also rapidly depleted intracellular glutathione (GSH), GSH-linked glutathione-S-transferases, and aldose reductase, three critical cellular defenses that detoxify reactive aldehydes. Results further showed that depletion of cellular GSH by acrolein preceded the loss of cell viability. To further determine the role of cellular GSH in acrolein-mediated cytotoxicity, buthionine sulfoximine (BSO) was used to inhibit cellular GSH biosynthesis. It was observed that depletion of cellular GSH by BSO led to a marked potentiation of acrolein-mediated cytotoxicity in HepG2 cells. To further assess the contribution of these events to acrolein-induced cytotoxicity, triterpenoid compound 2-cyano-3,12-dioxooleana-1,9-dien-28-imidazolide (CDDO-Im) was used for induction of GSH. Induction of GSH by CDDO-Im afforded cytoprotection against acrolein toxicity in HepG2 cells. Furthermore, BSO significantly inhibited CDDO-Im-mediated induction in cellular GSH levels and also reversed cytoprotective effects of CDDO-Im in HepG2 cells. These results suggest that GSH is a predominant mechanism underlying acrolein-induced cytotoxicity as well as CDDO-Im-mediated cytoprotection. This study may provide understanding on the molecular action of acrolein which may be important to develop novel strategies for the prevention of acrolein-mediated toxicity. © 2014 by the Society for Experimental Biology and Medicine.

Glutathione transferase-mediated benzimidazole-resistance in Fusarium graminearum.

Science.gov (United States)

Sevastos, A; Labrou, N E; Flouri, F; Malandrakis, A

2017-09-01

Fusarium graminearum laboratory mutants moderately (MR) and highly (HR) benzimidazole-resistant, carrying or not target-site mutations at the β 2 -tubulin gene were utilized in an attempt to elucidate the biochemical mechanism(s) underlying the unique BZM-resistance paradigm of this fungal plant pathogen. Relative expression analysis in the presence or absence of carbendazim (methyl-2-benzimidazole carbamate) using a quantitative Real Time qPCR (RT-qPCR) revealed differences between resistant and the wild-type parental strain although no differences in expression levels of either β 1 - or β 2 -tubulin homologue genes were able to fully account for two of the highly resistant phenotypes. Glutathione transferase (GST)-mediated detoxification was shown to be -at least partly- responsible for the elevated resistance levels of a HR isolate bearing the β 2 -tubulin Phe200Tyr resistance mutation compared with another MR isolate carrying the same mutation. This benzimidazole-resistance mechanism is reported for the first time in F. graminearum. No indications of detoxification involved in benzimidazole resistance were found for the rest of the isolates as revealed by GST and glutathione peroxidase (GPx) activities and bioassays using monoxygenase and hydrolase detoxification enzyme inhibiting synergists. Interestingly, besides the Phe200Tyr mutation-carrying HR isolate, the remaining highly-carbendazim resistant phenotypes could not be associated with any of the target site modification/overproduction, detoxification or reduced uptake-increased efflux mechanisms. Copyright © 2016 Elsevier B.V. All rights reserved.

Role of glutathione in tolerance to arsenite in Salvinia molesta, an aquatic fern

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Adinan Alves da Silva

2017-09-01

Full Text Available ABSTRACT In many plant species, tolerance to toxic metals is highly dependent on glutathione, an essential metabolite for cellular detoxification. We evaluated the responses of glutathione metabolism to arsenite (AsIII in Salvinia molesta, an aquatic fern that has unexplored phytoremediation potential. Plants were exposed to different AsIII concentrations in nutrient solution for 24 h. AsIII caused cell membrane damage to submerged leaves, indicating oxidative stress. There was an increase in the glutathione content and ϒ-glutamylcysteine synthetase enzyme activity in the submerged and floating leaves. The glutathione peroxidase and glutathione sulfotransferase enzymes also showed increased activity in both plant parts, whereas glutathione reductase only showed increased activity in the submerged leaves. These findings suggest an important role for glutathione in the protection of S. molesta against the toxic effects of AsIII, with more effective tolerance responses in the floating leaves.

Glutathione Enhancer Protects Some Biochemical and Haematological Parameters from the Effect of Electromagnetic Field

International Nuclear Information System (INIS)

Marzook, E.A.; Marzook, F.A.

2016-01-01

The Present study was designed to study the effect of exposure to electromagnetic field (EMF), emitted from a cellular tower for mobile phone on some biochemical and haematological parameters in male albino rats and to evaluate of the possible protective role of the antioxidant glutathione enhancer on the studied parameters. Three groups of rats were studied, the control (unexposed), the exposed and the treated exposed groups. Exposed groups were subjected to EMF at frequency of 900 MHz, with a peak power of about 60 W, power density of 0.05 mW/ cm"2 at the site of exposure for 24 h/ day for 8 weeks, at the same time treated group was supplied with oral injection of glutathione enhancer three times weekly. At the end of experiment, serum levels of thyroid stimulating hormone (TSH), calcium (Ca), uric acid, malondialdehyde (MDA) beside, the activity of lactate dehydrogenase (LDH) were measured also, some haematological parameters were estimated. Impairment of TSH and serum calcium was expressed by a decrease in serum levels of the exposed group. Meanwhile, serum level of MDA, uric acid and the activity of LDH were increased by exposure. The haematological studies revealed that, exposure to electromagnetic spectrum induced significant reduction in red blood cell counts (RBC's), and haemoglobin concentration (Hb), meanwhile reticulocyte count (Ret) was elevated. The leucocyte counts (WBC's) and platelets count were not affected by exposure. The study demonstrated that glutathione enhancer can attenuate the side effects of exposure to electromagnetic field.

Uranyl complexes of glutathione

Energy Technology Data Exchange (ETDEWEB)

Marzotto, A [Consiglio Nazionale delle Ricerche, Padua (Italy). Lab. di Chimica e Tecnologia dei Radioelementi

1977-01-01

Dioxouranium(VI) complexes of the tripeptide glutathione having different molar ratios were prepared and studied by IR, PMR, electronic absorption and circular dichroism spectra. The results indicate that coordination occurs at the carboxylato groups, acting as monodentate ligands, whereas no significant interaction with the amino and sulfhydrylic groups takes place.

Thioredoxin-1 promotes survival in cells exposed to S-nitrosoglutathione: Correlation with reduction of intracellular levels of nitrosothiols and up-regulation of the ERK1/2 MAP Kinases

International Nuclear Information System (INIS)

Arai, Roberto J.; Ogata, Fernando T.; Batista, Wagner L.; Masutani, Hiroshi; Yodoi, Junji; Debbas, Victor; Augusto, Ohara; Stern, Arnold; Monteiro, Hugo P.

2008-01-01

Accumulating evidence indicates that post-translational protein modifications by nitric oxide and its derived species are critical effectors of redox signaling in cells. These protein modifications are most likely controlled by intracellular reductants. Among them, the importance of the 12 kDa dithiol protein thioredoxin-1 (TRX-1) has been increasingly recognized. However, the effects of TRX-1 in cells exposed to exogenous nitrosothiols remain little understood. We investigated the levels of intracellular nitrosothiols and survival signaling in HeLa cells over-expressing TRX-1 and exposed to S-nitrosoglutahione (GSNO). A role for TRX-1 expression on GSNO catabolism and cell viability was demonstrated by the concentration-dependent effects of GSNO on decreasing TRX-1 expression, activation of caspase-3, and increasing cell death. The over-expression of TRX-1 in HeLa cells partially attenuated caspase-3 activation and enhanced cell viability upon GSNO treatment. This was correlated with reduction of intracellular levels of nitrosothiols and increasing levels of nitrite and nitrotyrosine. The involvement of ERK, p38 and JNK pathways were investigated in parental cells treated with GSNO. Activation of ERK1/2 MAP kinases was shown to be critical for survival signaling. In cells over-expressing TRX-1, basal phosphorylation levels of ERK1/2 MAP kinases were higher and further increased after GSNO treatment. These results indicate that the enhanced cell viability promoted by TRX-1 correlates with its capacity to regulate the levels of intracellular nitrosothiols and to up-regulate the survival signaling pathway mediated by the ERK1/2 MAP kinases

Reduced glutathione as a persistence indicator of alien plants of the Amelancheir family

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L. G. Dolgova

2009-04-01

Full Text Available It was proved that glutathione is an important indicator of the vegetation condition and persistence. According to the amount of glutathione the studied mespilus species are adapted to the environmental conditions. Increase of the glutathione amount is caused by some abiotic factors, e.g. temperature. Some differences of the glutathione content may be explained by the plants species patterns.

Reduced glutathione concentration and glutathione reductase activity in various rat tissues after the administration of some radioprotective agents

International Nuclear Information System (INIS)

Pulpanova, J.; Kovarova, H.; Ledvina, M.

1982-01-01

The concentrations of reduced glutathione (GSH) and activity of glutathione reductase were investigated in rat liver, kidney and spleen after intraperitoneal administration of cystamine (50 mg/kg), mexamine (10 mg/kg), or a mixture of cystamine with mexamine (20 + 10 mg/kg). The GSH concentration increased after the administration of cystamine in the liver (maximum between the 20th and 30th min), in the kidney and spleen (maximum after 60 min). The cystamine + mexamine mixture also caused a significant increase of the GSH concentration in all the organs investigated; however, the values increased at earlier intervals as after the cystamine administration. No substantial effect was shown in the case of the mexamine administration, only 30 min after the administration the values were higher. The activity of glutathione reductase was significantly lower over the entire period examined. This was found in the liver and kidney as after the administration of cystamine, as after the radioprotective mixture. There was also a less pronounced inhibition of the enzyme activity in the spleen. Mexamine as a single radioprotector had practically no influence on the activity. (author)

Engineering of a Bacillus subtilis strain with adjustable levels of intracellular biotin for secretory production of functional streptavidin.

Science.gov (United States)

Wu, Sau-Ching; Wong, Sui-Lam

2002-03-01

Streptavidin is a biotin-binding protein which has been widely used in many in vitro and in vivo applications. Because of the ease of protein recovery and availability of protease-deficient strains, the Bacillus subtilis expression-secretion system is an attractive system for streptavidin production. However, attempts to produce streptavidin using B. subtilis face the problem that cells overproducing large amounts of streptavidin suffer poor growth, presumably because of biotin deficiency. This problem cannot be solved by supplementing biotin to the culture medium, as this will saturate the biotin binding sites in streptavidin. We addressed this dilemma by engineering a B. subtilis strain (WB800BIO) which overproduces intracellular biotin. The strategy involves replacing the natural regulatory region of the B. subtilis chromosomal biotin biosynthetic operon (bioWAFDBIorf2) with an engineered one consisting of the B. subtilis groE promoter and gluconate operator. Biotin production in WB800BIO is induced by gluconate, and the level of biotin produced can be adjusted by varying the gluconate dosage. A level of gluconate was selected to allow enhanced intracellular production of biotin without getting it released into the culture medium. WB800BIO, when used as a host for streptavidin production, grows healthily in a biotin-limited medium and produces large amounts (35 to 50 mg/liter) of streptavidin, with over 80% of its biotin binding sites available for future applications.

N-acetylcysteine stimulates protein synthesis in enterocytes independently of glutathione synthesis.

Science.gov (United States)

Yi, Dan; Hou, Yongqing; Wang, Lei; Long, Minhui; Hu, Shengdi; Mei, Huimin; Yan, Liqiong; Hu, Chien-An Andy; Wu, Guoyao

2016-02-01

Dietary supplementation with N-acetylcysteine (NAC) has been reported to improve intestinal health and treat gastrointestinal diseases. However, the underlying mechanisms are not fully understood. According to previous reports, NAC was thought to exert its effect through glutathione synthesis. This study tested the hypothesis that NAC enhances enterocyte growth and protein synthesis independently of cellular glutathione synthesis. Intestinal porcine epithelial cells were cultured for 3 days in Dulbecco's modified Eagle medium containing 0 or 100 μM NAC. To determine a possible role for GSH (the reduced form of glutathione) in mediating the effect of NAC on cell growth and protein synthesis, additional experiments were conducted using culture medium containing 100 μM GSH, 100 μM GSH ethyl ester (GSHee), diethylmaleate (a GSH-depletion agent; 10 μM), or a GSH-synthesis inhibitor (buthionine sulfoximine, BSO; 20 μM). NAC increased cell proliferation, GSH concentration, and protein synthesis, while inhibiting proteolysis. GSHee enhanced cell proliferation and GSH concentration without affecting protein synthesis but inhibited proteolysis. Conversely, BSO or diethylmaleate reduced cell proliferation and GSH concentration without affecting protein synthesis, while promoting protein degradation. At the signaling level, NAC augmented the protein abundance of total mTOR, phosphorylated mTOR, and phosphorylated 70S6 kinase as well as mRNA levels for mTOR and p70S6 kinase in IPEC-1 cells. Collectively, these results indicate that NAC upregulates expression of mTOR signaling proteins to stimulate protein synthesis in enterocytes independently of GSH generation. Our findings provide a hitherto unrecognized biochemical mechanism for beneficial effects of NAC in intestinal cells.

The Roles of Glutathione Peroxidases during Embryo Development.

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Ufer, Christoph; Wang, Chi Chiu

2011-01-01

Embryo development relies on the complex interplay of the basic cellular processes including proliferation, differentiation, and apoptotic cell death. Precise regulation of these events is the basis for the establishment of embryonic structures and the organ development. Beginning with fertilization of the oocyte until delivery the developing embryo encounters changing environmental conditions such as varying levels of oxygen, which can give rise to reactive oxygen species (ROS). These challenges are met by the embryo with metabolic adaptations and by an array of anti-oxidative mechanisms. ROS can be deleterious by modifying biological molecules including lipids, proteins, and nucleic acids and may induce abnormal development or even embryonic lethality. On the other hand ROS are vital players of various signaling cascades that affect the balance between cell growth, differentiation, and death. An imbalance or dysregulation of these biological processes may generate cells with abnormal growth and is therefore potentially teratogenic and tumorigenic. Thus, a precise balance between processes generating ROS and those decomposing ROS is critical for normal embryo development. One tier of the cellular protective system against ROS constitutes the family of selenium-dependent glutathione peroxidases (GPx). These enzymes reduce hydroperoxides to the corresponding alcohols at the expense of reduced glutathione. Of special interest within this protein family is the moonlighting enzyme glutathione peroxidase 4 (Gpx4). This enzyme is a scavenger of lipophilic hydroperoxides on one hand, but on the other hand can be transformed into an enzymatically inactive cellular structural component. GPx4 deficiency - in contrast to all other GPx family members - leads to abnormal embryo development and finally produces a lethal phenotype in mice. This review is aimed at summarizing the current knowledge on GPx isoforms during embryo development and tumor development with an emphasis on

Interactions of [alpha,beta]-unsaturated carbonyl compounds with the glutathione-related biotransformation system

NARCIS (Netherlands)

Iersel, van M.L.P.S.

1998-01-01

Introduction
Modulation of glutathione-related biotransformation steps may play a role in important phenomena as anticarcinogenicity and multidrug resistance. Glutathione-related biotransformation comprises three main aspects i.e. glutathione, the

Acrolein produces nitric oxide through the elevation of intracellular calcium levels to induce apoptosis in human umbilical vein endothelial cells: implications for smoke angiopathy.

Science.gov (United States)

Misonou, Yoshiko; Asahi, Michio; Yokoe, Shunichi; Miyoshi, Eiji; Taniguchi, Naoyuki

2006-03-01

Acrolein is a highly electrophilic alpha, beta-unsaturated aldehyde, the levels of which are increased in the blood of smokers. To determine if acrolein is involved in the pathology of smoke angiopathy, the effect of acrolein on human umbilical vein endothelial cells (HUVEC) was examined. Intracellular nitric oxide (NO) levels, determined using diaminofluorescein-2 diacetate (DAF-2 DA), an NO sensitive fluorescent dye, were found to be increased after treatment in HUVEC with 10 microM acrolein. The measurement of nitrite with 2,3-diaminonaphthalene and a Western blot analysis revealed that nitrite and S-nitroso-cysteine levels were increased in a dose-dependent manner, confirming that NO production is increased by acrolein. The increase was not reduced by treatment with 10mM N-acetyl-l-cysteine (NAC), an anti-oxidant, but was reduced with 10 microM of the intracellular calcium chelator, 1,2-bis (o-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid tetra (acetoxymethyl) ester. Acrolein-stimulated NO production was significantly reduced by pretreatment with 1mM N(G)-nitro-l-arginine-methyl ester (L-NAME), an NO synthase inhibitor. The cytotoxicity of acrolein was reduced by pretreatment with 10 microM 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (carboxy-PTIO), an intracellular NO scavenger, or 1mM L-NAME, whereas it was not reduced by 10mM NAC, 20 microM Curcumin, another peroxide scavenger, or 100 microM Mn(III)TMPyP, a superoxide dismutase mimic. Nuclear staining and a Western blot analysis using an anti-cleaved caspase 3 antibody revealed that the reduced viability of HUVEC by acrolein was due to apoptosis, which was reversed after pretreatment with 0.1mM carboxy-PTIO or 1mM L-NAME. Thus, acrolein increases intracellular calcium production to induce intracellular NO production by a calcium-dependent NO synthase, possibly eNOS, and the excess and rapid increase in NO might lead to the apoptosis of HUVEC. These data suggest that acrolein might be

Multiscale modelling approach combining a kinetic model of glutathione metabolism with PBPK models of paracetamol and the potential glutathione-depletion biomarkes ophthalmic acid and 5-oxoproline in humans and rats

NARCIS (Netherlands)

Geenen, S.; Yates, J.W.T.; Kenna, J.G.; Bois, F.Y.; Wilson, I.D.; Westerhoff, H.V.

2014-01-01

A key role of the antioxidant glutathione is detoxification of chemically reactive electrophilic drug metabolites within the liver. Therefore glutathione depletion can have severe toxic consequences. Ophthalmic acid and 5-oxoproline are metabolites involved in glutathione metabolism, which can be

Glutathione transferases are structural and functional outliers in the thioredoxin fold.

Science.gov (United States)

Atkinson, Holly J; Babbitt, Patricia C

2009-11-24

Glutathione transferases (GSTs) are ubiquitous scavengers of toxic compounds that fall, structurally and functionally, within the thioredoxin fold suprafamily. The fundamental catalytic capability of GSTs is catalysis of the nucleophilic addition or substitution of glutathione at electrophilic centers in a wide range of small electrophilic compounds. While specific GSTs have been studied in detail, little else is known about the structural and functional relationships between different groupings of GSTs. Through a global analysis of sequence and structural similarity, it was determined that variation in the binding of glutathione between the two major subgroups of cytosolic (soluble) GSTs results in a different mode of glutathione activation. Additionally, the convergent features of glutathione binding between cytosolic GSTs and mitochondrial GST kappa are described. The identification of these structural and functional themes helps to illuminate some of the fundamental contributions of the thioredoxin fold to catalysis in the GSTs and clarify how the thioredoxin fold can be modified to enable new functions.

Glutathione reductase: solvent equilibrium and kinetic isotope effects

International Nuclear Information System (INIS)

Wong, K.K.; Vanoni, M.A.; Blanchard, J.S.

1988-01-01

Glutathione reductase catalyzes the NADPH-dependent reduction of oxidized glutathione (GSSG). The kinetic mechanism is ping-pong, and we have investigated the rate-limiting nature of proton-transfer steps in the reactions catalyzed by the spinach, yeast, and human erythrocyte glutathione reductases using a combination of alternate substrate and solvent kinetic isotope effects. With NADPH or GSSG as the variable substrate, at a fixed, saturating concentration of the other substrate, solvent kinetic isotope effects were observed on V but not V/K. Plots of Vm vs mole fraction of D 2 O (proton inventories) were linear in both cases for the yeast, spinach, and human erythrocyte enzymes. When solvent kinetic isotope effect studies were performed with DTNB instead of GSSG as an alternate substrate, a solvent kinetic isotope effect of 1.0 was observed. Solvent kinetic isotope effect measurements were also performed on the asymmetric disulfides GSSNB and GSSNP by using human erythrocyte glutathione reductase. The Km values for GSSNB and GSSNP were 70 microM and 13 microM, respectively, and V values were 62 and 57% of the one calculated for GSSG, respectively. Both of these substrates yield solvent kinetic isotope effects greater than 1.0 on both V and V/K and linear proton inventories, indicating that a single proton-transfer step is still rate limiting. These data are discussed in relationship to the chemical mechanism of GSSG reduction and the identity of the proton-transfer step whose rate is sensitive to solvent isotopic composition. Finally, the solvent equilibrium isotope effect measured with yeast glutathione reductase is 4.98, which allows us to calculate a fractionation factor for the thiol moiety of GSH of 0.456

High-level intracellular expression of heterologous proteins in Brevibacillus choshinensis SP3 under the control of a xylose inducible promoter

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D’Urzo Nunzia

2013-02-01

Full Text Available Abstract Background In past years research has focused on the development of alternative Gram positive bacterial expression systems to produce industrially relevant proteins. Brevibacillus choshinensis is an easy to handle non-sporulating bacterium, lacking extracellular proteases, that has been already shown to provide a high level of recombinant protein expression. One major drawback, limiting the applicability of the Brevibacillus expression system, is the absence of expression vectors based on inducible promoters. Here we used the PxylA inducible promoter, commonly employed in other Bacillae expression systems, in Brevibacillus. Results Using GFP, α-amylase and TcdA-GT as model proteins, high level of intracellular protein expression (up to 250 mg/L for the GFP was achieved in Brevibacillus, using the pHis1522 vector carrying the B. megaterium xylose-inducible promoter (PxylA. The GFP expression yields were more than 25 fold higher than those reported for B. megaterium carrying the same vector. All the tested proteins show significant increment in their expression levels (2-10 folds than those obtained using the available plasmids based on the P2 constitutive promoter. Conclusion Combining the components of two different commercially available Gram positive expression systems, such as Brevibacillus (from Takara Bio and B. megaterium (from Mobitec, we demonstrate that vectors based on the B. megaterium PxylA xylose inducible promoter can be successfully used to induce high level of intracellular expression of heterologous proteins in Brevibacillus.

Glutathione mediation of papain inactivation by hydrogen peroxide and hydroxyl radicals

International Nuclear Information System (INIS)

Lin, W.S.; Armstrong, D.A.

1977-01-01

Glutathione reacts with papainCys 25 SOH, formed by the reaction of papain with hydrogen peroxide, to give papainCys 25 SSG. Subsequent reaction of this mixed disulfide with glutathione is slow (k -1 sec -1 ). However, at 30 0 C it is readily cleaved by cysteine to form active papain, i.e., papainCys 25 SH. Glutathione resembles cysteine in protecting papain by the scavenging of .OH radicals, but, unlike cysteine, glutathione gave no evidence for the repair of enzyme radical lesions or for the conversion of papainCys 25 S. radicals to repairable derivatives. Its overall effectiveness for reducing the radiation inactivation of papain in aqueous solution is much less than that of cysteine

Nuclear magnetic resonance studies of intracellular ions in perfused from heart

International Nuclear Information System (INIS)

Burnstein, D.; Fossel, E.T.

1987-01-01

Intracellular sodium, potassium, and lithium were observed in a perfused frog heart by nuclear magnetic resonance (NMR) spectroscopy. A perfusate buffer containing the shift reagent, dysprosium tripolyphosphate, was used in combination with mathematical filtering or presaturation of the extracellular resonance to separate the intra- and extracellular sodium NMR signals. Addition of 10 μM ouabain to the perfusate, perfusion with a zero potassium, low-calcium buffer, and replacement of 66% of the perfusate sodium with lithium resulted in changes in the intracellular sodium levels. An increase of 45% in the intracellular sodium was observed when changing the pacing rate from 0 to 60 beats/min (with proportional changes for intermediate pacing rates). The ratio of intracellular potassium to sodium concentration was determined to be 2.3 by NMR, indicating that a substantial amount of the intracellular potassium is undetectable with these NMR method. In addition, intracellular lithium was observed during perfusion with a lithium-containing perfusate

Glycine Increases Insulin Sensitivity and Glutathione Biosynthesis and Protects against Oxidative Stress in a Model of Sucrose-Induced Insulin Resistance

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Mohammed El-Hafidi

2018-01-01

Full Text Available Oxidative stress and redox status play a central role in the link between insulin resistance (IR and lipotoxicity in metabolic syndrome. This mechanistic link may involve alterations in the glutathione redox state. We examined the effect of glycine supplementation to diet on glutathione biosynthesis, oxidative stress, IR, and insulin cell signaling in liver from sucrose-fed (SF rats characterized by IR and oxidative stress. Our hypothesis is that the correction of glutathione levels by glycine treatment leads to reduced oxidative stress, a mechanism associated with improved insulin signaling and IR. Glycine treatment decreases the levels of oxidative stress markers in liver from SF rats and increases the concentrations of glutathione (GSH and γ-glutamylcysteine and the amount of γ-glutamylcysteine synthetase (γ-GCS, a key enzyme of GSH biosynthesis in liver from SF rats. In liver from SF rats, glycine also decreases the insulin-induced phosphorylation of insulin receptor substrate-1 (ISR-1 in serine residue and increases the phosphorylation of insulin receptor β-subunit (IR-β in tyrosine residue. Thus, supplementing diets with glycine to correct GSH deficiency and to reduce oxidative stress provides significant metabolic benefits to SF rats by improving insulin sensitivity.

Role of UBIAD1 in Intracellular Cholesterol Metabolism and Vascular Cell Calcification.

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Sha Liu

Full Text Available Vascular calcification is an important risk factor associated with mortality among patients with chronic kidney disease. Intracellular cholesterol metabolism is involved in the process of vascular cell calcification. In this study, we investigated the role of UbiA prenyltransferase domain containing 1 (UBIAD1 in intracellular cholesterol metabolism and vascular cell calcification, and identified its subcellular location. Primary human umbilical vein smooth muscle cells (HUVSMCs were incubated with either growth medium (1.4 mmol/L Pi or calcification medium (CM (3.0 mmol/L Pi. Under treatment with CM, HUVSMCs were further incubated with exogenous cholesterol, or menaquinone-4, a product of UBIAD1. The plasmid and small interfering RNA were transfected in HUVSMCs to alter the expression of UBIAD1. Matrix calcium quantitation, alkaline phosphatase activity, intracellular cholesterol level and menaquinone-4 level were measured. The expression of several genes involved in cholesterol metabolism were analyzed. Using an anti-UBIAD1 antibody, an endoplasmic reticulum marker and a Golgi marker, the subcellular location of UBIAD1 in HUVSMCs was analyzed. CM increased matrix calcium, alkaline phosphatase activity and intracellular cholesterol level, and reduced UBIAD1 expression and menaquinone-4 level. Addition of cholesterol contributed to increased matrix calcification and alkaline phosphatase activity in a dose-dependent manner. Elevated expression of UBIAD1 or menaquinone-4 in HUVSMCs treated with CM significantly reduced intracellular cholesterol level, matrix calcification and alkaline phosphatase activity, but increased menaquinone-4 level. Elevated expression of UBIAD1 or menaquinone-4 reduced the gene expression of sterol regulatory element-binding protein-2, and increased gene expression of ATP binding cassette transporters A1, which are in charge of cholesterol synthesis and efflux. UBIAD1 co-localized with the endoplasmic reticulum marker and

Subcellular distribution of glutathione and cysteine in cyanobacteria

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Zechmann, Bernd; Tomašić, Ana; Horvat, Lucija; Fulgosi, Hrvoje

2010-01-01

Glutathione plays numerous important functions in eukaryotic and prokaryotic cells. Whereas it can be found in virtually all eukaryotic cells, its production in prokaryotes is restricted to cyanobacteria and proteobacteria and a few strains of gram-positive bacteria. In bacteria, it is involved in the protection against reactive oxygen species (ROS), osmotic shock, acidic conditions, toxic chemicals, and heavy metals. Glutathione synthesis in bacteria takes place in two steps out of cysteine,...

Intracellular Cholesterol Trafficking and Impact in Neurodegeneration

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Fabian Arenas

2017-11-01

Full Text Available Cholesterol is a critical component of membrane bilayers where it plays key structural and functional roles by regulating the activity of diverse signaling platforms and pathways. Particularly enriched in brain, cholesterol homeostasis in this organ is singular with respect to other tissues and exhibits a heterogeneous regulation in distinct brain cell populations. Due to the key role of cholesterol in brain physiology and function, alterations in cholesterol homeostasis and levels have been linked to brain diseases and neurodegeneration. In the case of Alzheimer disease (AD, however, this association remains unclear with evidence indicating that either increased or decreased total brain cholesterol levels contribute to this major neurodegenerative disease. Here, rather than analyzing the role of total cholesterol levels in neurodegeneration, we focus on the contribution of intracellular cholesterol pools, particularly in endolysosomes and mitochondria through its trafficking via specialized membrane domains delineated by the contacts between endoplasmic reticulum and mitochondria, in the onset of prevalent neurodegenerative diseases such as AD, Parkinson disease, and Huntington disease as well as in lysosomal disorders like Niemann-Pick type C disease. We dissect molecular events associated with intracellular cholesterol accumulation, especially in mitochondria, an event that results in impaired mitochondrial antioxidant defense and function. A better understanding of the mechanisms involved in the distribution of cholesterol in intracellular compartments may shed light on the role of cholesterol homeostasis disruption in neurodegeneration and may pave the way for specific intervention opportunities.

Interaction of uranium(VI) towards glutathione. An example to study different functional groups in one molecule

International Nuclear Information System (INIS)

Frost, L.; Geipel, G.; Viehweger, K.; Bernhard, G.

2011-01-01

Glutathione, the most abundant thiol compound of the cell, has a great binding potential towards heavy metal ions. Hence it might influence the distribution of actinides on a cellular level. The unknown strength of the interaction of uranium(VI) with glutathione at physiologically relevant pH is subject of this paper and was studied with UV-vis spectroscopy and time-resolved laserinduced fluorescence spectroscopy (TRLFS). The complex stability constant of UO 2 H 2 GS + , logβ 121 0 , was calculated to be 39.09±0.15 and 39.04±0.02 in case of UV-vis spectroscopy and TRLFS respectively. Therefore the average formation constant for UO 2 2+ +H 2 GS - = UO 2 H 2 GS + can be assigned to be log K 0 11 = 19.83±0.15. Furthermore it was demonstrated that derivatization of the ligand associated with an enhancement of the ligand's spectroscopic properties can be used for the determination of complex stability constants and to assess the coordination chemistry in more detail. Using UV-vis spectroscopy, the stability constant of the complex between UO 2 2+ and glutathione pyruvate S-conjugate, a well absorbing ligand in contrast to glutathione, was calculated to be > 39.24±0.08. Furthermore the interaction of UO 2 2+ with glutathione derivatized with the fluorescent label monobromobimane was examined with femtosecond laser fluorescence spectroscopy. Thereby the stability constant of the 1: 1 complex was determined to be > 39.35±0.02. Although the thiol group of glutathione was blocked a strong coordination was found. Thus a significant involvement of the thiol group in the coordination of U(VI) can be excluded. (orig.)

Glutathione in Cellular Redox Homeostasis: Association with the Excitatory Amino Acid Carrier 1 (EAAC1

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Koji Aoyama

2015-05-01

Full Text Available Reactive oxygen species (ROS are by-products of the cellular metabolism of oxygen consumption, produced mainly in the mitochondria. ROS are known to be highly reactive ions or free radicals containing oxygen that impair redox homeostasis and cellular functions, leading to cell death. Under physiological conditions, a variety of antioxidant systems scavenge ROS to maintain the intracellular redox homeostasis and normal cellular functions. This review focuses on the antioxidant system’s roles in maintaining redox homeostasis. Especially, glutathione (GSH is the most important thiol-containing molecule, as it functions as a redox buffer, antioxidant, and enzyme cofactor against oxidative stress. In the brain, dysfunction of GSH synthesis leading to GSH depletion exacerbates oxidative stress, which is linked to a pathogenesis of aging-related neurodegenerative diseases. Excitatory amino acid carrier 1 (EAAC1 plays a pivotal role in neuronal GSH synthesis. The regulatory mechanism of EAAC1 is also discussed.

Glutathione Transferase from Trichoderma virens Enhances Cadmium Tolerance without Enhancing Its Accumulation in Transgenic Nicotiana tabacum

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Dixit, Prachy; Mukherjee, Prasun K.; Ramachandran, V.; Eapen, Susan

2011-01-01

Background Cadmium (Cd) is a major heavy metal pollutant which is highly toxic to plants and animals. Vast agricultural areas worldwide are contaminated with Cd. Plants take up Cd and through the food chain it reaches humans and causes toxicity. It is ideal to develop plants tolerant to Cd, without enhanced accumulation in the edible parts for human consumption. Glutathione transferases (GST) are a family of multifunctional enzymes known to have important roles in combating oxidative stresses induced by various heavy metals including Cd. Some GSTs are also known to function as glutathione peroxidases. Overexpression/heterologous expression of GSTs is expected to result in plants tolerant to heavy metals such as Cd. Results Here, we report cloning of a glutathione transferase gene from Trichoderma virens, a biocontrol fungus and introducing it into Nicotiana tabacum plants by Agrobacterium-mediated gene transfer. Transgenic nature of the plants was confirmed by Southern blot hybridization and expression by reverse transcription PCR. Transgene (TvGST) showed single gene Mendelian inheritance. When transgenic plants expressing TvGST gene were exposed to different concentrations of Cd, they were found to be more tolerant compared to wild type plants, with transgenic plants showing lower levels of lipid peroxidation. Levels of different antioxidant enzymes such as glutathione transferase, superoxide dismutase, ascorbate peroxidase, guiacol peroxidase and catalase showed enhanced levels in transgenic plants expressing TvGST compared to control plants, when exposed to Cd. Cadmium accumulation in the plant biomass in transgenic plants were similar or lower than wild-type plants. Conclusion The results of the present study suggest that transgenic tobacco plants expressing a Trichoderma virens GST are more tolerant to Cd, without enhancing its accumulation in the plant biomass. It should be possible to extend the present results to crop plants for developing Cd tolerance and

Glutathione transferase from Trichoderma virens enhances cadmium tolerance without enhancing its accumulation in transgenic Nicotiana tabacum.

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Prachy Dixit

Full Text Available BACKGROUND: Cadmium (Cd is a major heavy metal pollutant which is highly toxic to plants and animals. Vast agricultural areas worldwide are contaminated with Cd. Plants take up Cd and through the food chain it reaches humans and causes toxicity. It is ideal to develop plants tolerant to Cd, without enhanced accumulation in the edible parts for human consumption. Glutathione transferases (GST are a family of multifunctional enzymes known to have important roles in combating oxidative stresses induced by various heavy metals including Cd. Some GSTs are also known to function as glutathione peroxidases. Overexpression/heterologous expression of GSTs is expected to result in plants tolerant to heavy metals such as Cd. RESULTS: Here, we report cloning of a glutathione transferase gene from Trichoderma virens, a biocontrol fungus and introducing it into Nicotiana tabacum plants by Agrobacterium-mediated gene transfer. Transgenic nature of the plants was confirmed by Southern blot hybridization and expression by reverse transcription PCR. Transgene (TvGST showed single gene Mendelian inheritance. When transgenic plants expressing TvGST gene were exposed to different concentrations of Cd, they were found to be more tolerant compared to wild type plants, with transgenic plants showing lower levels of lipid peroxidation. Levels of different antioxidant enzymes such as glutathione transferase, superoxide dismutase, ascorbate peroxidase, guiacol peroxidase and catalase showed enhanced levels in transgenic plants expressing TvGST compared to control plants, when exposed to Cd. Cadmium accumulation in the plant biomass in transgenic plants were similar or lower than wild-type plants. CONCLUSION: The results of the present study suggest that transgenic tobacco plants expressing a Trichoderma virens GST are more tolerant to Cd, without enhancing its accumulation in the plant biomass. It should be possible to extend the present results to crop plants for

Atomic force microscopy for cellular level manipulation: imaging intracellular structures and DNA delivery through a membrane hole.

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Afrin, Rehana; Zohora, Umme Salma; Uehara, Hironori; Watanabe-Nakayama, Takahiro; Ikai, Atsushi

2009-01-01

The atomic force microscope (AFM) is a versatile tool for imaging, force measurement and manipulation of proteins, DNA, and living cells basically at the single molecular level. In the cellular level manipulation, extraction, and identification of mRNA's from defined loci of a cell, insertion of plasmid DNA and pulling of membrane proteins, for example, have been reported. In this study, AFM was used to create holes at defined loci on the cell membrane for the investigation of viability of the cells after hole creation, visualization of intracellular structure through the hole and for targeted gene delivery into living cells. To create large holes with an approximate diameter of 5-10 microm, a phospholipase A(2) coated bead was added to the AFM cantilever and the bead was allowed to touch the cell surface for approximately 5-10 min. The evidence of hole creation was obtained mainly from fluorescent image of Vybrant DiO labeled cell before and after the contact with the bead and the AFM imaging of the contact area. In parallel, cells with a hole were imaged by AFM to reveal intracellular structures such as filamentous structures presumably actin fibers and mitochondria which were identified with fluorescent labeling with rhodamine 123. Targeted gene delivery was also attempted by inserting an AFM probe that was coated with the Monster Green Fluorescent Protein phMGFP Vector for transfection of the cell. Following targeted transfection, the gene expression of green fluorescent protein (GFP) was observed and confirmed by the fluorescence microscope. Copyright (c) 2009 John Wiley & Sons, Ltd.

No net splanchnic release of glutathione in man during N-acetylcysteine infusion

DEFF Research Database (Denmark)

Poulsen, H E; Vilstrup, H; Almdal, T

1993-01-01

Glutathione and amino acid concentrations were measured in arterial and hepatic vein plasma in four healthy volunteers and two patients with cirrhosis. There was no significant splanchnic efflux of glutathione (95% confidence limits, -0.501 to 0.405 mumol/min). After infusion of N...... to 0.97 +/- 0.11 (mean +/- SEM; p amino acids corresponded to an increased load on hepatic metabolic N conversion and transamination among nonessential amino acids. Splanchnic uptake of serine, alanine, cystine, isoleucine, and phenylalanine increased...... after NAC compatible with stimulated hepatic glutathione synthesis. In contrast to the rat, plasma glutathione in man probably originates mainly from extrahepatic tissues....

The relationship between the violet pigment PP-V production and intracellular ammonium level in Penicillium purpurogenum

OpenAIRE

Kojima, Ryo; Arai, Teppei; Matsufuji, Hiroshi; Kasumi, Takafumi; Watanabe, Taisuke; Ogihara, Jun

2016-01-01

Penicillium purpurogenum is the fungus that produces an azaphilone pigment. However, details about the pigment biosynthesis pathway are unknown. The violet pigment PP-V is the one of the main pigments biosynthesized by this fungus. This pigment contains an amino group in a pyran ring as its core structure. We focused on this pigment and examined the relationship between intracellular ammonium concentration and pigment production using glutamine as a nitrogen source. The intracellular ammonium...

The Pseudomonas aeruginosa Chp Chemosensory System Regulates Intracellular cAMP Levels by Modulating Adenylate Cyclase Activity

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Fulcher, Nanette B.; Holliday, Phillip M.; Klem, Erich; Cann, Martin J.; Wolfgang, Matthew C.

2010-01-01

Summary Multiple virulence systems in the opportunistic pathogen Pseudomonas aeruginosa are regulated by the second messenger signaling molecule adenosine 3’, 5’-cyclic monophosphate (cAMP). Production of cAMP by the putative adenylate cyclase enzyme CyaB represents a critical control point for virulence gene regulation. To identify regulators of CyaB, we screened a transposon insertion library for mutants with reduced intracellular cAMP. The majority of insertions resulting in reduced cAMP mapped to the Chp gene cluster encoding a putative chemotaxis-like chemosensory system. Further genetic analysis of the Chp system revealed that it has both positive and negative effects on intracellular cAMP and that it regulates cAMP levels by modulating CyaB activity. The Chp system was previously implicated in the production and function of type IV pili (TFP). Given that cAMP and the cAMP-dependent transcriptional regulator Vfr control TFP biogenesis gene expression, we explored the relationship between cAMP, the Chp system and TFP regulation. We discovered that the Chp system controls TFP production through modulation of cAMP while control of TFP-dependent twitching motility is cAMP-independent. Overall, our data define a novel function for a chemotaxis-like system in controlling cAMP production and establish a regulatory link between the Chp system, TFP and other cAMP-dependent virulence systems. PMID:20345659

Determination of Glutathione and Its Redox Status in Isolated Vacuoles of Red Beetroot Cells

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E.V. Pradedova

2016-02-01

Full Text Available The glutathione of the red beetroot vacuoles (Beta vulgaris L. was measured using three well-known methods: the spectrofluorimetric method with orthophthalic aldehyde (OPT; the spectrophotometric method with 5.5'-dithiobis-2-nitrobenzoic acid (DTNB; the high-performance liquid chromatography (HPLC. The content of reduced (GSH and oxidized glutathione (GSSG differed depending on the research method. With OPT the concentration of glutathione was: GSH – 0.059 µmol /mg protein; GSSG – 0.019 µmol/mg protein and total glutathione (GSHtotal – 0.097 µmol/mg protein. In the case of determining with DTNB the concentration of glutathione was: GSH – 0.091 µmol/mg protein; GSSG – 0.031 µmol/mg protein; GSHtotal – 0.153 µmol/mg protein. HPLC-defined concentration of glutathione was lower: GSH – 0.039 µmol/mg protein; GSSG – 0.007 µmol/mg protein; GSHtotal – 0.053 µmol/mg protein. Redox ratio of GSH/GSSG was also dependent on the method of determination: with OPT – 3.11; with DTNB – 2.96 and HPLC – 5.57. Redox ratio of glutathione in vacuoles was much lower than the tissue extracts of red beetroot, which, depending on the method of determination, was: 7.23, 7.16 and 9.22. The results showed the vacuoles of red beetroot parenchyma cells contain glutathione. Despite the low value of the redox ratio GSH/GSSG, in vacuoles the pool of reduced glutathione prevailed over the pool of oxidized glutathione.

Inflammation and insulin resistance induced by trans-10, cis-12 conjugated linoleic acid depend on intracellular calcium levels in primary cultures of human adipocytes

DEFF Research Database (Denmark)

Kennedy, Arion; Martinez, Kristina; Chung, Soonkyu

2010-01-01

We previously demonstrated that trans-10, cis-12 (10,12) conjugated linoleic acid (CLA) induced inflammation and insulin resistance in primary human adipocytes by activating nuclear factor kappaB (NFkappaB) and extracellular signal-related kinase (ERK) signaling. In this study, we demonstrated...... that the initial increase in intracellular calcium ([Ca2+]i) mediated by 10,12 CLA was attenuated by TMB-8, an inhibitor of calcium release from the endoplasmic reticulum (ER), by BAPTA, an intracellular calcium chelator, and by D609, a phospholipase C (PLC) inhibitor. Moreover, BAPTA, TMB-8, and D609 attenuated......, and suppression of peroxisome proliferator activated receptor gamma protein levels and insulin-stimulated glucose uptake. These data suggest that 10,12 CLA increases inflammation and insulin resistance in human adipocytes, in part by increasing [Ca2+]i levels, particularly calcium from the ER....

Efficacy of glutathione mesotherapy in burns: an experimental study.

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Buz, A; Görgülü, T; Olgun, A; Kargi, E

2016-12-01

Thermal burns are the leading cause of trauma worldwide. Currently, no consensus on optimal treatment of deep partial-thickness (second-degree) burns has emerged, as reflected by the wide variability in available wound-care materials. The relative efficacies of products used for treatment of partial-thickness thermal burns remain unclear. Mesotherapy features intradermal administration of various agents, depending on burn location. In the present experimental study, we explored the efficacy of mesotherapy used to treat partial-thickness thermal burns in 50 male Wistar rats divided into five groups of equal number. No procedure was performed after infliction of thermal burns in control group (Group 1). Mesotherapy was applied with physiological saline in sham group (Group 2), glutathione, taurine, and L-carnitine were separately applied in Group 3, Group 4, and Group 5, respectively. Mesotherapeutic agents were injected intradermally into the reticular layer of the dermis using the point technique. The first course of mesotherapy was given within the first 2 h after infliction of thermal burns, and therapy was continued to day 10. On day 22, unhealed thermal burn areas were measured prior to sacrifice, and biopsies covering the total areas of burns were performed to allow of pathological evaluation. Group 3 (the glutathione group) showed the best extent of healing, followed by Group 4 (the taurine group) and Group 5 (the L-carnitine group). The healed thermal burn areas in these groups were significantly greater than those in the control and sham groups (P = 0.001). All of healing, acute and chronic inflammation, the amount of granulation tissue, the level of fibroblast maturation, the amount of collagen, the extent of re-epithelization and neovascularization, and ulcer depth were scored upon pathological examination of tissue cross-sections. The best outcomes were evident in the glutathione group, with statistical significance. Although wound healing in the L

Glutathione depletion in tissues after administration of buthionine sulphoximine

International Nuclear Information System (INIS)

Minchinton, A.I.; Rojas, A.; Smith, A.; Soranson, J.A.; Shrieve, D.C.; Jones, N.R.; Bremner, J.C.

1984-01-01

Buthionine sulphoximine (BSO) an inhibitor of glutathione (GSH) biosynthesis, was administered to mice in single and repeated doses. The resultant pattern of GSH depletion was studied in liver, kidney, skeletal muscle and three types of murine tumor. Liver and kidney exhibited a rapid depletion of GSH. Muscle was depleted to a similar level, but at a slower rate after a single dose. All three tumors required repeated administration of BSO over several days to obtain a similar degree of depletion to that shown in the other tissues

Lack of oxygen effect in glutathione-deficient human cells in culture

International Nuclear Information System (INIS)

Edgren, M.; Larsson, A.; Nilsson, K.; Revesz, L.; Scott, O.C.A.

1980-01-01

The frequency of X-ray-induced DNA breaks was determined in human cell lines which are deficient in glutathione synthetase and have a greatly reduced glutathione content. Hydroxyapatite chromatography was used for the estimation of the DNA breaks in cell cultures, which were derived either from lymphoblasts transformed by infection with EB virus or from fibroblasts. The dose-effect relationship for the induction of breaks when radiation exposure was made in argon, was similar to that found when exposure was made in air. In control cultures with normal glutathione content, the induction of breaks was enhanced when irradiation was made under aerobic, instead of anaerobic, conditions. Treatment of the glutathione-deficient cells with the hypoxic radiosensitizer misonidazole did not enhance the induction of breaks by radiation delivered either in air or in argon. In control cultures, radiation induction of breaks was enhanced by misonidazole under anaerobic but not under aerobic conditions. When the glutathione-deficient cells were pretreated with cysteamine however, irradiation in the absence of oxygen resulted in a decreased frequency of DNA breaks. (author)

Glutathione treatment of hepatocellular carcinoma

DEFF Research Database (Denmark)

Dalhoff, K; Ranek, L; Mantoni, M

1992-01-01

This prospective study was undertaken to substantiate observations that glutathione (GSH) inhibits or reverses tumor growth in humans with hepatocellular carcinoma (HCC), a neoplasm with an extremely poor prognosis. Eight patients with biopsy-proven HCC not amenable to surgery were given 5 g of GSH...

Glutathione in the human brain: Review of its roles and measurement by magnetic resonance spectroscopy.

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Rae, Caroline D; Williams, Stephen R

2017-07-15

We review the transport, synthesis and catabolism of glutathione in the brain as well as its compartmentation and biochemistry in different brain cells. The major reactions involving glutathione are reviewed and the factors limiting its availability in brain cells are discussed. We also describe and critique current methods for measuring glutathione in the human brain using magnetic resonance spectroscopy, and review the literature on glutathione measurements in healthy brains and in neurological, psychiatric, neurodegenerative and neurodevelopmental conditions In summary: Healthy human brain glutathione concentration is ∼1-2 mM, but it varies by brain region, with evidence of gender differences and age effects; in neurological disease glutathione appears reduced in multiple sclerosis, motor neurone disease and epilepsy, while being increased in meningiomas; in psychiatric disease the picture is complex and confounded by methodological differences, regional effects, length of disease and drug-treatment. Both increases and decreases in glutathione have been reported in depression and schizophrenia. In Alzheimer's disease and mild cognitive impairment there is evidence for a decrease in glutathione compared to age-matched healthy controls. Improved methods to measure glutathione in vivo will provide better precision in glutathione determination and help resolve the complex biochemistry of this molecule in health and disease. Copyright © 2017 Elsevier Inc. All rights reserved.

Thioredoxin and glutathione systems differ in parasitic and free-living platyhelminths

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Salinas Gustavo

2010-04-01

Full Text Available Abstract Background The thioredoxin and/or glutathione pathways occur in all organisms. They provide electrons for deoxyribonucleotide synthesis, function as antioxidant defenses, in detoxification, Fe/S biogenesis and participate in a variety of cellular processes. In contrast to their mammalian hosts, platyhelminth (flatworm parasites studied so far, lack conventional thioredoxin and glutathione systems. Instead, they possess a linked thioredoxin-glutathione system with the selenocysteine-containing enzyme thioredoxin glutathione reductase (TGR as the single redox hub that controls the overall redox homeostasis. TGR has been recently validated as a drug target for schistosomiasis and new drug leads targeting TGR have recently been identified for these platyhelminth infections that affect more than 200 million people and for which a single drug is currently available. Little is known regarding the genomic structure of flatworm TGRs, the expression of TGR variants and whether the absence of conventional thioredoxin and glutathione systems is a signature of the entire platyhelminth phylum. Results We examine platyhelminth genomes and transcriptomes and find that all platyhelminth parasites (from classes Cestoda and Trematoda conform to a biochemical scenario involving, exclusively, a selenium-dependent linked thioredoxin-glutathione system having TGR as a central redox hub. In contrast, the free-living platyhelminth Schmidtea mediterranea (Class Turbellaria possesses conventional and linked thioredoxin and glutathione systems. We identify TGR variants in Schistosoma spp. derived from a single gene, and demonstrate their expression. We also provide experimental evidence that alternative initiation of transcription and alternative transcript processing contribute to the generation of TGR variants in platyhelminth parasites. Conclusions Our results indicate that thioredoxin and glutathione pathways differ in parasitic and free-living flatworms and

Thioredoxin and glutathione systems differ in parasitic and free-living platyhelminths

Science.gov (United States)

2010-01-01

Background The thioredoxin and/or glutathione pathways occur in all organisms. They provide electrons for deoxyribonucleotide synthesis, function as antioxidant defenses, in detoxification, Fe/S biogenesis and participate in a variety of cellular processes. In contrast to their mammalian hosts, platyhelminth (flatworm) parasites studied so far, lack conventional thioredoxin and glutathione systems. Instead, they possess a linked thioredoxin-glutathione system with the selenocysteine-containing enzyme thioredoxin glutathione reductase (TGR) as the single redox hub that controls the overall redox homeostasis. TGR has been recently validated as a drug target for schistosomiasis and new drug leads targeting TGR have recently been identified for these platyhelminth infections that affect more than 200 million people and for which a single drug is currently available. Little is known regarding the genomic structure of flatworm TGRs, the expression of TGR variants and whether the absence of conventional thioredoxin and glutathione systems is a signature of the entire platyhelminth phylum. Results We examine platyhelminth genomes and transcriptomes and find that all platyhelminth parasites (from classes Cestoda and Trematoda) conform to a biochemical scenario involving, exclusively, a selenium-dependent linked thioredoxin-glutathione system having TGR as a central redox hub. In contrast, the free-living platyhelminth Schmidtea mediterranea (Class Turbellaria) possesses conventional and linked thioredoxin and glutathione systems. We identify TGR variants in Schistosoma spp. derived from a single gene, and demonstrate their expression. We also provide experimental evidence that alternative initiation of transcription and alternative transcript processing contribute to the generation of TGR variants in platyhelminth parasites. Conclusions Our results indicate that thioredoxin and glutathione pathways differ in parasitic and free-living flatworms and that canonical enzymes

Effect of Glutathione on the Taste and Texture of Type I Sourdough Bread.

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Tang, Kai Xing; Zhao, Cindy J; Gänzle, Michael G

2017-05-31

Type I sourdough fermentations with Lactobacillus sanfranciscensis as predominant organism accumulate reduced glutathione through glutathione reductase (GshR) activity of L. sanfranciscensis. Reduced glutathione acts as chain terminator for gluten polymerization but is also kokumi-active and may thus enhance bread taste. This study implemented a type I model sourdough fermentations to quantitate glutathione accumulation sourdough, bread dough, and bread and to assess the effect of L. sanfranciscensis GshR on bread volume by comparison of L. sanfranciscensis and an isogenic strain devoid of GshR. L. sanfranciscensis sourdough accumulated the highest amount of reduced glutathione during proofing. Bread produced with the wild type strain had a lower volume when compared to the gshR deficient mutant. The accumulation of γ-glutamyl-cysteine was also higher in L. sanfranciscensis sourdoughs when compared to doughs fermented with the gshR mutant strain. The accumulation of reduced glutathione in L. sanfranciscensis bread did not enhance the saltiness of bread.

Isolation of glutathione-deficient mutants of the yeast Saccharomyces cerevisiae

International Nuclear Information System (INIS)

Kistler, M.; Eckardt, F.; Summer, K.-H.

1986-01-01

Glutathione-deficient (gsh - ) mutants of the yeast Saccharomyces cerevisiae were isolated after UV treatment using MNNG as selective agent. For genetic and biochemical characterization 5 mutant strains were chosen which exhibited considerably decreased residual GSH contents varying from 2 to 6% of the wild-type levels. All 5 isolates showed a 2:2 segregation of the gsh - :GSH + phenotypes alluding to a monogenic recessive mutation. Complementation analysis indicates that all gsh - mutants belong to one complementation group. (Auth.)

Functional analysis and localisation of a delta-class glutathione S-transferase from Sarcoptes scabiei.

Science.gov (United States)

Pettersson, Eva U; Ljunggren, Erland L; Morrison, David A; Mattsson, Jens G

2005-01-01

The mite Sarcoptes scabiei causes sarcoptic mange, or scabies, a disease that affects both animals and humans worldwide. Our interest in S. scabiei led us to further characterise a glutathione S-transferase. This multifunctional enzyme is a target for vaccine and drug development in several parasitic diseases. The S. scabiei glutathione S-transferase open reading frame reported here is 684 nucleotides long and yields a protein with a predicted molecular mass of 26 kDa. Through phylogenetic analysis the enzyme was classified as a delta-class glutathione S-transferase, and our paper is the first to report that delta-class glutathione S-transferases occur in organisms other than insects. The recombinant S. scabiei glutathione S-transferase was expressed in Escherichia coli via three different constructs and purified for biochemical analysis. The S. scabiei glutathione S-transferase was active towards the substrate 1-chloro-2,4-dinitrobenzene, though the positioning of fusion partners influenced the kinetic activity of the enzyme. Polyclonal antibodies raised against S. scabiei glutathione S-transferase specifically localised the enzyme to the integument of the epidermis and cavities surrounding internal organs in adult parasites. However, some minor staining of parasite intestines was observed. No staining was seen in host tissues, nor could we detect any antibody response against S. scabiei glutathione S-transferase in sera from naturally S. scabiei infected dogs or pigs. Additionally, the polyclonal sera raised against recombinant S. scabiei glutathione S-transferase readily detected a protein from mites, corresponding to the predicted size of native glutathione S-transferase.

Functional Consequences of Intracellular Proline Levels Manipulation Affecting PRODH/POX-Dependent Pro-Apoptotic Pathways in a Novel in Vitro Cell Culture Model.

Science.gov (United States)

Zareba, Ilona; Surazynski, Arkadiusz; Chrusciel, Marcin; Miltyk, Wojciech; Doroszko, Milena; Rahman, Nafis; Palka, Jerzy

2017-01-01

The effect of impaired intracellular proline availability for proline dehydrogenase/proline oxidase (PRODH/POX)-dependent apoptosis was studied. We generated a constitutively knocked-down PRODH/POX MCF-7 breast cancer cell line (MCF-7shPRODH/POX) as a model to analyze the functional consequences of impaired intracellular proline levels. We have used inhibitor of proline utilization in collagen biosynthesis, 2-metoxyestradiol (MOE), inhibitor of prolidase that generate proline, rapamycin (Rap) and glycyl-proline (GlyPro), substrate for prolidase. Collagen and DNA biosynthesis were evaluated by radiometric assays. Cell viability was determined using Nucleo-Counter NC-3000. The activity of prolidase was determined by colorimetric assay. Expression of proteins was assessed by Western blot and immunofluorescence bioimaging. Concentration of proline was analyzed by liquid chromatography with mass spectrometry. PRODH/POX knockdown decreased DNA and collagen biosynthesis, whereas increased prolidase activity and intracellular proline level in MCF-7shPRODH/POX cells. All studied compounds decreased cell viability in MCF-7 and MCF-7shPRODH/POX cells. DNA biosynthesis was similarly inhibited by Rap and MOE in both cell lines, but GlyPro inhibited the process only in MCF-7shPRODH/POX and MOE+GlyPro only in MCF-7 cells. All the compounds inhibited collagen biosynthesis, increased prolidase activity and cytoplasmic proline level in MCF-7shPRODH/POX cells and contributed to the induction of pro-survival mode only in MCF-7shPRODH/POX cells. In contrast, all studied compounds upregulated expression of pro-apoptotic protein only in MCF-7 cells. PRODH/POX was confirmed as a driver of apoptosis and proved the eligibility of MCF-7shPRODH/POX cell line as a highly effective model to elucidate the different mechanisms underlying proline utilization or generation in PRODH/POX-dependent pro-apoptotic pathways. © 2017 The Author(s). Published by S. Karger AG, Basel.

Post-fermentative production of glutathione by baker's yeast (S. cerevisiae) in compressed and dried forms.

Science.gov (United States)

Musatti, Alida; Manzoni, Matilde; Rollini, Manuela

2013-01-25

The study was aimed at investigating the best biotransformation conditions to increase intracellular glutathione (GSH) levels in samples of baker's yeast (Saccharomyces cerevisiae) employing either the commercially available compressed and dried forms. Glucose, GSH precursors amino acids, as well as other cofactors, were dissolved in a biotransformation solution and yeast cells were added (5%dcw). Two response surface central composite designs (RSCCDs) were performed in sequence: in the first step the influence of amino acid composition (cysteine, glycine, glutamic acid and serine) on GSH accumulation was investigated; once their formulation was set up, the influence of other components was studied. Initial GSH content was found 0.53 and 0.47%dcw for compressed and dried forms. GSH accumulation ability of baker's yeast in compressed form was higher at the beginning of shelf life, that is, in the first week, and a maximum of 2.04%dcw was obtained. Performance of yeast in dried form was not found satisfactory, as the maximum GSH level was 1.18%dcw. When cysteine lacks from the reaction solution, yeast cells do not accumulate GSH. With dried yeast, the highest GSH yields occurred when cysteine was set at 3 g/L, glycine and glutamic acid at least at 4 g/L, without serine. Employing compressed yeast, the highest GSH yields occurred when cysteine and glutamic acid were set at 2-3 g/L, while glycine and serine higher than 2 g/L. Results allowed to set up an optimal and feasible procedure to obtain GSH-enriched yeast biomass, with up to threefold increase with respect to initial content. Copyright © 2012 Elsevier B.V. All rights reserved.

Glutathione and its antiaging and antimelanogenic effects

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Weschawalit S

2017-04-01

Full Text Available Sinee Weschawalit,1 Siriwan Thongthip,2 Phanupong Phutrakool,3 Pravit Asawanonda1 1Department of Medicine, Division of Dermatology, 2Chula Clinical Research Center, 3Chula Data Management Center, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand Background: Previous studies showed that supplementation of reduced form of glutathione (GSH, 500 mg/d has a skin-lightening efficacy in humans. This study was designed to evaluate the influences of both GSH and oxidized form (GSSG, at doses lower than 500 mg/d, on improving skin properties. Patients and methods: A randomized, double-blind, placebo-controlled, parallel, three-arm study was conducted. Healthy female subjects were equally randomized into three groups and took GSH (250 mg/d, GSSG (250 mg/d, or placebo orally for 12 weeks. At each visit at baseline and for 12 weeks, skin features including melanin index, wrinkles, and other relevant biophysical properties were measured. Blood samples were collected for safety monitoring. Results: In generalized estimating equation analyses, melanin index and ultraviolet spots of all sites including face and arm when given GSH and GSSG tended to be lower than placebo. At some sites evaluated, subjects who received GSH showed a significant reduction in wrinkles compared with those taking placebo. A tendency toward increased skin elasticity was observed in GSH and GSSG compared with placebo. There were no serious adverse effects throughout the study. Conclusion: We showed that oral glutathione, 250 mg/d, in both reduced and oxidized forms effectively influences skin properties. Overall, glutathione in both forms are well tolerated. Keywords: glutathione, melanin, pigment, aging, wrinkle, whitening

Enhanced depletion of glutathione and increased liver oxidative damage in aflatoxin-fed mice infected with Plasmodium berghei

DEFF Research Database (Denmark)

Ankrah, N A; Sittie, A; Addo, P G

1995-01-01

levels accompanied by a significant increase in serum cholinesterase and liver malonic dialdehyde levels in the mice fed aflatoxin compared with those in the control group. The results suggested that malaria parasites can enhance depletion of host glutathione and oxidative damage of the liver in mice fed...

Rational design of reversible fluorescent probes for live-cell imaging and quantification of fast glutathione dynamics

Science.gov (United States)

Umezawa, Keitaro; Yoshida, Masafumi; Kamiya, Mako; Yamasoba, Tatsuya; Urano, Yasuteru

2017-03-01

Alterations in glutathione (GSH) homeostasis are associated with a variety of diseases and cellular functions, and therefore, real-time live-cell imaging and quantification of GSH dynamics are important for understanding pathophysiological processes. However, existing fluorescent probes are unsuitable for these purposes due to their irreversible fluorogenic mechanisms or slow reaction rates. In this work, we have successfully overcome these problems by establishing a design strategy inspired by Mayr's work on nucleophilic reaction kinetics. The synthesized probes exhibit concentration-dependent, reversible and rapid absorption/fluorescence changes (t1/2 = 620 ms at [GSH] = 1 mM), as well as appropriate Kd values (1-10 mM: within the range of intracellular GSH concentrations). We also developed FRET-based ratiometric probes, and demonstrated that they are useful for quantifying GSH concentration in various cell types and also for real-time live-cell imaging of GSH dynamics with temporal resolution of seconds.

Radiosensitization of mouse skin by oxygen and depletion of glutathione

International Nuclear Information System (INIS)

Stevens, Graham; Joiner, Michael; Joiner, Barbara; Johns, Helen; Denekamp, Juliana

1995-01-01

Purpose: To determine the oxygen enhancement ratio (OER) and shape of the oxygen sensitization curve of mouse foot skin, the extent to which glutathione (GSH) depletion radiosensitized skin, and the dependence of such sensitization on the ambient oxygen tension. Methods and Materials: The feet of WHT mice were irradiated with single doses of 240 kVp x-rays while mice were exposed to carbogen or gases with oxygen/nitrogen mixtures containing 8-100% O 2 . The anoxic response was obtained by occluding the blood supply to the leg of anesthetized mice with a tourniquet, surrounding the foot with nitrogen, and allowing the mice to breathe 10% O 2 . Further experiments were performed to assess the efficacy of this method to obtain an anoxic response. Radiosensitivity of skin was assessed using the acute skin-reaction assay. Glutathione levels were modified using two schedules of dl-buthionine sulphoximine (BSO) and diethylmaleate (DEM), which were considered to produce extensive and intermediate levels of GSH depletion in the skin of the foot during irradiation. Results: Carbogen caused the greatest radiosensitization of skin, with a reproducible enhancement of 2.2 relative to the anoxic response. The OER of 2.2 is lower than other reports for mouse skin. This may indicate that the extremes of oxygenation were not produced, although there was no direct evidence for this. When skin radiosensitivity was plotted against the logarithm of the oxygen tension in the ambient gas, a sigmoid curve with a K value of 17-21% O 2 in the ambient gas was obtained. Depletion of GSH caused minimal radiosensitization when skin was irradiated under anoxic or well-oxygenated conditions. Radiosensitization by GSH depletion was maximal at intermediate oxygen tensions of 10-21% O 2 in the ambient gas. Increasing the extent of GSH depletion led to increasing radiosensitization, with sensitization enhancement ratios of 1.2 and 1.1, respectively, for extensive and intermediate levels of GSH

Single-bilayer graphene oxide sheet tolerance and glutathione redox system significance assessment in faba bean (Vicia faba L.)

International Nuclear Information System (INIS)

Anjum, Naser A.; Singh, Neetu; Singh, Manoj K.; Shah, Zahoor A.; Duarte, Armando C.; Pereira, Eduarda; Ahmad, Iqbal

2013-01-01

Adsorbents based on single-bilayer graphene oxide sheet (hereafter termed “graphene oxide”) are widely used in contaminated environments cleanup which may easily open the avenues for their entry to different environmental compartments, exposure to organisms and their subsequent transfer to human/animal food chain. Considering a common food crop—faba bean (Vicia faba L.) germinating seedlings as a model plant system, this study assesses the V. faba-tolerance to different concentrations (0, 100, 200, 400, 800, and 1600 mg L −1 ) of graphene oxide (0.5–5 μm) and evaluates glutathione (γ-glutamyl-cysteinyl-glycine) redox system significance in this context. The results showed significantly increased V. faba sensitivity under three graphene oxide concentrations (in order of impact: 1,600 > 200 > 100 mg graphene oxide L −1 ), which was accompanied by decreased glutathione redox (reduced glutathione-to-oxidized glutathione) ratio, reduced glutathione pool, as well as significant and equally elevated activities of glutathione-regenerating (glutathione reductase) and glutathione-metabolizing (glutathione peroxidase; glutathione sulfo-transferase) enzymes. Contrarily, the two graphene oxide concentrations (in order of impact: 800 > 400 graphene oxide mg L −1 ) yielded promising results; where, significant improvements in V. faba health status (measured as increased graphene oxide tolerance) were clearly perceptible with increased ratio of the reduced glutathione-to-oxidized glutathione, reduced glutathione pool and glutathione reductase activity but decreased activities of glutathione-metabolizing enzymes. It is inferred that V. faba seedlings-sensitivity and/or tolerance to graphene oxide concentrations depends on both the cellular redox state (reduced glutathione-to-oxidized glutathione ratio) and the reduced glutathione pool which in turn are controlled by a finely tuned modulation of the coordination between glutathione-regenerating and glutathione

Single-bilayer graphene oxide sheet tolerance and glutathione redox system significance assessment in faba bean (Vicia faba L.)

Energy Technology Data Exchange (ETDEWEB)

Anjum, Naser A. [University of Aveiro, Centre for Environmental and Marine Studies (CESAM) and Department of Chemistry (Portugal); Singh, Neetu; Singh, Manoj K. [University of Aveiro, Center for Mechanical Technology and Automation (TEMA) and Department of Mechanical Engineering (Portugal); Shah, Zahoor A. [University of Toledo, Department of Medicinal and Biological Chemistry, College of Pharmacy and Pharmaceutical Sciences (United States); Duarte, Armando C.; Pereira, Eduarda; Ahmad, Iqbal, E-mail: ahmadr@ua.pt [University of Aveiro, Centre for Environmental and Marine Studies (CESAM) and Department of Chemistry (Portugal)

2013-07-15

Adsorbents based on single-bilayer graphene oxide sheet (hereafter termed 'graphene oxide') are widely used in contaminated environments cleanup which may easily open the avenues for their entry to different environmental compartments, exposure to organisms and their subsequent transfer to human/animal food chain. Considering a common food crop-faba bean (Vicia faba L.) germinating seedlings as a model plant system, this study assesses the V. faba-tolerance to different concentrations (0, 100, 200, 400, 800, and 1600 mg L{sup -1}) of graphene oxide (0.5-5 {mu}m) and evaluates glutathione ({gamma}-glutamyl-cysteinyl-glycine) redox system significance in this context. The results showed significantly increased V. faba sensitivity under three graphene oxide concentrations (in order of impact: 1,600 > 200 > 100 mg graphene oxide L{sup -1}), which was accompanied by decreased glutathione redox (reduced glutathione-to-oxidized glutathione) ratio, reduced glutathione pool, as well as significant and equally elevated activities of glutathione-regenerating (glutathione reductase) and glutathione-metabolizing (glutathione peroxidase; glutathione sulfo-transferase) enzymes. Contrarily, the two graphene oxide concentrations (in order of impact: 800 > 400 graphene oxide mg L{sup -1}) yielded promising results; where, significant improvements in V. faba health status (measured as increased graphene oxide tolerance) were clearly perceptible with increased ratio of the reduced glutathione-to-oxidized glutathione, reduced glutathione pool and glutathione reductase activity but decreased activities of glutathione-metabolizing enzymes. It is inferred that V. faba seedlings-sensitivity and/or tolerance to graphene oxide concentrations depends on both the cellular redox state (reduced glutathione-to-oxidized glutathione ratio) and the reduced glutathione pool which in turn are controlled by a finely tuned modulation of the coordination between glutathione-regenerating and

Oxidation promotes insertion of the CLIC1 chloride intracellular channel into the membrane.

Science.gov (United States)

Goodchild, Sophia C; Howell, Michael W; Cordina, Nicole M; Littler, Dene R; Breit, Samuel N; Curmi, Paul M G; Brown, Louise Jennifer

2009-12-01

Members of the chloride intracellular channel (CLIC) family exist primarily as soluble proteins but can also auto-insert into cellular membranes to form ion channels. While little is known about the process of CLIC membrane insertion, a unique feature of mammalian CLIC1 is its ability to undergo a dramatic structural metamorphosis between a monomeric glutathione-S-transferase homolog and an all-helical dimer upon oxidation in solution. Whether this oxidation-induced metamorphosis facilitates CLIC1 membrane insertion is unclear. In this work, we have sought to characterise the role of oxidation in the process of CLIC1 membrane insertion. We examined how redox conditions modify the ability of CLIC1 to associate with and insert into the membrane using fluorescence quenching studies and a sucrose-loaded vesicle sedimentation assay to measure membrane binding. Our results suggest that oxidation of monomeric CLIC1, in the presence of membranes, promotes insertion into the bilayer more effectively than the oxidised CLIC1 dimer.

Glutathione metabolism modelling: a mechanism for liver drug-robustness and a new biomarker strategy

NARCIS (Netherlands)

Geenen, S.; du Preez, F.B.; Snoep, J.L.; Foster, A.J.; Sarda, S.; Kenna, J.G.; Wilson, I.D.; Westerhoff, H.V.

2013-01-01

Background Glutathione metabolism can determine an individual's ability to detoxify drugs. To increase understanding of the dynamics of cellular glutathione homeostasis, we have developed an experiment-based mathematical model of the kinetics of the glutathione network. This model was used to

Methionine sulfoximine supplementation enhances productivity in GS-CHOK1SV cell lines through glutathione biosynthesis.

Science.gov (United States)

Feary, Marc; Racher, Andrew J; Young, Robert J; Smales, C Mark

2017-01-01

In Lonza Biologics' GS Gene Expression System™, recombinant protein-producing GS-CHOK1SV cell lines are generated by transfection with an expression vector encoding both GS and the protein product genes followed by selection in MSX and glutamine-free medium. MSX is required to inhibit endogenous CHOK1SV GS, and in effect create a glutamine auxotrophy in the host that can be complemented by the expression vector encoded GS in selected cell lines. However, MSX is not a specific inhibitor of GS as it also inhibits the activity of GCL (a key enzyme in the glutathione biosynthesis pathway) to a similar extent. Glutathione species (GSH and GSSG) have been shown to provide both oxidizing and reducing equivalents to ER-resident oxidoreductases, raising the possibility that selection for transfectants with increased GCL expression could result in the isolation of GS-CHOKISV cell lines with improved capacity for recombinant protein production. In this study we have begun to address the relationship between MSX supplementation, the amount of intracellular GCL subunit and mAb production from a panel of GS-CHOK1SV cell lines. We then evaluated the influence of reduced GCL activity on batch culture of an industrially relevant mAb-producing GS-CHOK1SV cell line. To the best of our knowledge, this paper describes for the first time the change in expression of GCL subunits and recombinant mAb production in these cell lines with the degree of MSX supplementation in routine subculture. Our data also shows that partial inhibition of GCL activity in medium containing 75 µM MSX increases mAb productivity, and its more specific inhibitor BSO used at a concentration of 80 µM in medium increases the specific rate of mAb production eight-fold and the concentration in harvest medium by two-fold. These findings support a link between the inhibition of glutathione biosynthesis and recombinant protein production in industrially relevant systems and provide a process-driven method for

Determination of Intracellular Vitrification Temperatures for Unicellular Micro Organisms under Conditions Relevant for Cryopreservation.

Science.gov (United States)

Fonseca, Fernanda; Meneghel, Julie; Cenard, Stéphanie; Passot, Stéphanie; Morris, G John

2016-01-01

During cryopreservation ice nucleation and crystal growth may occur within cells or the intracellular compartment may vitrify. Whilst previous literature describes intracellular vitrification in a qualitative manner, here we measure the intracellular vitrification temperature of bacteria and yeasts under conditions relevant to cryopreservation, including the addition of high levels of permeating and nonpermeating additives and the application of rapid rates of cooling. The effects of growth conditions that are known to modify cellular freezing resistance on the intracellular vitrification temperature are also examined. For bacteria a plot of the activity on thawing against intracellular glass transition of the maximally freeze-concentrated matrix (Tg') shows that cells with the lowest value of intracellular Tg' survive the freezing process better than cells with a higher intracellular Tg'. This paper demonstrates the role of the physical state of the intracellular environment in determining the response of microbial cells to preservation and could be a powerful tool to be manipulated to allow the optimization of methods for the preservation of microorganisms.

Glutathione and Mitochondria

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Vicent eRibas

2014-07-01

Full Text Available Glutathione (GSH is the main nonprotein thiol in cells whose functions are dependent on the redox-active thiol of its cysteine moiety that serves as a cofactor for a number of antioxidant and detoxifying enzymes. While synthesized exclusively in the cytosol from its constituent amino acids, GSH is distributed in different compartments, including mitochondria where its concentration in the matrix equals that of the cytosol. This feature and its negative charge at physiological pH imply the existence of specific carriers to import GSH from the cytosol to the mitochondrial matrix, where it plays a key role in defense against respiration-induced reactive oxygen species and in the detoxification of lipid hydroperoxides and electrophiles. Moreover, as mitochondria play a central strategic role in the activation and mode of cell death, mitochondrial GSH has been shown to critically regulate the level of sensitization to secondary hits that induce mitochondrial membrane permeabilization and release of proteins confined in the intermembrane space that once in the cytosol engage the molecular machinery of cell death. In this review, we summarize recent data on the regulation of mitochondrial GSH and its role in cell death and prevalent human diseases, such as cancer, fatty liver disease and Alzheimer’s disease.

The role of glutathione in lymphocyte activation and proliferation

International Nuclear Information System (INIS)

Messina, J.P.

1990-01-01

The object of this work was to establish the requirement for GSH and cystine during the activation and proliferation of human peripheral blood mononuclear cells (PBMC). In the author's initial experiments the intracellular GSH content of PBMC was altered by continuous culture or pretreatment with BSO, a specific inhibitor of GSH synthesis. His results demonstrate that, continuous culture of mitogen stimulated PBMC in the presence of BSO inhibited entry into S-phase of the cell cycle and produced a simultaneous decrease in intracellular GSH. The influence of BSO on early activation events were determined by BSO pretreatment. Extensive depletion (>90%) of the intracellular GSH level prior to mitogenic stimulation did not impair the ability of these cells to produce IL-2 and express IL-2R, indicating that GSH may not be involved in the generation and response to early activation signals. Furthermore, the removal of BSO from these cultures rapidly reversed its inhibitory effects on DNA and GSH synthesis. Cystine transport activities and metabolism by PBMC were characterized in order to examine its contributions to intracellular GSH and early activation proteins. In spite of the ability of cystine to sustain the proliferative response of PBMC, differences in the kinetics of cystine and cysteine uptake indicated that separate transport systems may be operational. Treatment with 2ME enhanced cystine uptake, but lowered the proliferative responses of these cells. Metabolic studies with [ 35 S] cystine demonstrated that mitogen stimulation of PBMC enhance cystine uptake

Characterization of Leptin Intracellular Trafficking

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E Walum

2009-12-01

Full Text Available Leptin is produced by adipose tissue, and its concentration in plasma is related to the amount of fat in the body. The leptin receptor (OBR is a member of the class I cytokine receptor family and several different isoforms, produced by alternative mRNA splicing are found in many tissues, including the hypothalamus. The two predominant isoforms includes a long form (OBRl with an intracellular domain of 303 amino acids and a shorter form (OBRs with an intracellular domain of 34 amino acids. Since OBRl is mainly expressed in the hypotalamus, it has been suggested to be the main signalling form. The peripheral production of leptin by adipocyte tissue and its effects as a signal of satiety in the central nervous system imply that leptin gains access to regions of the brain regulating in energy balance by crossing the blood-brain barrier. In an attempt to characterize the intracellular transport of leptin, we have followed binding internalization and degradation of leptin in HEK293 cells. We have also monitored the intracellular transport pathway of fluorescent conjugated leptin in HEK293 cells. Phenylarsine oxide, a general inhibitor of endocytosis, as well as incubation at mild hypertonic conditions, prevented the uptake of leptin, confirming a receptor-mediated internalization process. When internalized, 125I-leptin was rapidly accumulated inside the cells and reached a maximum after 10 min. After 70 minutes about 40-50% of total counts in each time point were found in the medium as TCA-soluble material. Leptin sorting, at the level of early endosomes, did not seem to involve recycling endosomes, since FITC-leptin was sorted from Cy3- transferrin containing compartments at 37°C. At 45 minutes of continuos internalization, FITC-leptin appeared mainly accumulated in late endocytic structures colocalizing with internalized rhodamine coupled epidermial growth factor (EGF and the lysosomal marker protein lamp-1. The transport of leptin was also shown

Iodinated chlorin p6 copper complex induces anti-proliferative effect in oral cancer cells through elevation of intracellular reactive oxygen species.

Science.gov (United States)

Sarbadhikary, Paromita; Dube, Alok

2017-11-01

We investigated the anticancer chemotoxicity of previously reported iodinated chlorin p 6 copper complex (ICp 6 -Cu), a novel chlorophyll derivative in which copper is attached to the side chain carboxylate groups via coordination. Human oral carcinoma cells NT8e, 4451 and the non-cancerous keratinocyte HaCaT cells were treated with ICp 6 -Cu for 48 h in dark and cell viability, proliferation and morphological alterations were examined. ICp 6 -Cu showed pronounced cytotoxicity in cancer cells with IC 50 ∼40 μM, whereas, the viability of HaCaT cells was not affected. Cell proliferation assay revealed that ICp 6 -Cu at IC 50 concentration led to complete inhibition of cell proliferation in both the cell lines. Cell morphology studied by confocal microscopy showed absence of cell death via necrosis or apoptosis. Instead, the treated cells displayed distinct features of non-apoptotic death such as highly vacuolated cytoplasm, lysosomal membrane permeabilization and damage to cytoskeleton F-actin filaments. In addition, ICp 6 -Cu treatment led to time dependent increase in the intracellular level of reactive oxygen species (ROS) and the cytotoxicity of ICp 6 -Cu was significantly inhibited by pre-treatment of cells with antioxidants (glutathione and trolox). These findings revealed that ICp 6 -Cu is a potent chemotoxic agent which can induce cytotoxic effect in cancer cells through elevation of intracellular ROS. It is suggested that ICp 6 -Cu may provide tumor selective chemotoxicity by exploiting difference of redox environment in normal and cancer cells. Copyright © 2017 Elsevier B.V. All rights reserved.

Nanotoxicity of cobalt induced by oxidant generation and glutathione depletion in MCF-7 cells.

Science.gov (United States)

Akhtar, Mohd Javed; Ahamed, Maqusood; Alhadlaq, Hisham A; Alshamsan, Aws

2017-04-01

There are very few studies regarding the biological activity of cobalt-based nanoparticles (NPs) and, therefore, the possible mechanism behind the biological response of cobalt NPs has not been fully explored. The present study was designed to explore the potential mechanisms of the cytotoxicity of cobalt NPs in human breast cancer (MCF-7) cells. The shape and size of cobalt NPs were characterized by scanning and transmission electron microscopy (SEM and TEM). The crystallinity of NPs was determined by X-ray diffraction (XRD). The dissolution of NPs was measured in phosphate-buffered saline (PBS) and culture media by atomic absorption spectroscopy (AAS). Cytotoxicity parameters, such as [3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide] (MTT), neutral red uptake (NRU), and lactate dehydrogenase (LDH) release suggested that cobalt NPs were toxic to MCF-7 cells in a dose-dependent manner (50-200μg/ml). Cobalt NPs also significantly induced reactive oxygen species (ROS) generation, lipid peroxidation (LPO), mitochondrial outer membrane potential loss (MOMP), and activity of caspase-3 enzymes in MCF-7 cells. Moreover, cobalt NPs decreased intracellular antioxidant glutathione (GSH) molecules. The exogenous supply of antioxidant N-acetyl cysteine in cobalt NP-treated cells restored the cellular GSH level and prevented cytotoxicity that was also confirmed by microscopy. Similarly, the addition of buthionine-[S, R]-sulfoximine, which interferes with GSH biosynthesis, potentiated cobalt NP-mediated toxicity. Our data suggested that low solubility cobalt NPs could exert toxicity in MCF-7 cells mainly through cobalt NP dissolution to Co 2+ . Copyright © 2016 Elsevier Ltd. All rights reserved.

Glutathione system participation in thoracic aneurysms from patients with Marfan syndrome.

Science.gov (United States)

Zúñiga-Muñoz, Alejandra María; Pérez-Torres, Israel; Guarner-Lans, Verónica; Núñez-Garrido, Elías; Velázquez Espejel, Rodrigo; Huesca-Gómez, Claudia; Gamboa-Ávila, Ricardo; Soto, María Elena

2017-05-01

Aortic dilatation in Marfan syndrome (MFS) is progressive. It is associated with oxidative stress and endothelial dysfunction that contribute to the early acute dissection of the vessel and can result in rupture of the aorta and sudden death. We evaluated the participation of the glutathione (GSH) system, which could be involved in the mechanisms that promote the formation and progression of the aortic aneurysms in MFS patients. Aortic aneurysm tissue was obtained during chest surgery from eight control subjects and 14 MFS patients. Spectrophotometrical determination of activity of glutathione peroxidase (GPx), glutathione-S-transferase (GST), glutathione reductase (GR), lipid peroxidation (LPO) index, carbonylation, total antioxidant capacity (TAC), and concentration of reduced and oxidized glutathione (GSH and GSSG respectively), was performed in the homogenate from aortic aneurysm tissue. LPO index, carbonylation, TGF-β1, and GR activity were increased in MFS patients (p < 0.04), while TAC, GSH/GSSG ratio, GPx, and GST activity were significantly decreased (p < 0.04). The depletion of GSH, in spite of the elevated activity of GR, not only diminished the activity of GSH-depend GST and GPx, but increased LPO, carbonylation and decreased TAC. These changes could promote the structural and functional alterations in the thoracic aorta of MFS patients.

Role of glutathione transport processes in kidney function

International Nuclear Information System (INIS)

Lash, Lawrence H.

2005-01-01

The kidneys are highly dependent on an adequate supply of glutathione (GSH) to maintain normal function. This is due, in part, to high rates of aerobic metabolism, particularly in the proximal tubules. Additionally, the kidneys are potentially exposed to high concentrations of oxidants and reactive electrophiles. Renal cellular concentrations of GSH are maintained by both intracellular synthesis and transport from outside the cell. Although function of specific carriers has not been definitively demonstrated, it is likely that multiple carriers are responsible for plasma membrane transport of GSH. Data suggest that the organic anion transporters OAT1 and OAT3 and the sodium-dicarboxylate 2 exchanger (SDCT2 or NaDC3) mediate uptake across the basolateral plasma membrane (BLM) and that the organic anion transporting polypeptide OATP1 and at least one of the multidrug resistance proteins mediate efflux across the brush-border plasma membrane (BBM). BLM transport may be used pharmacologically to provide renal proximal tubular cells with exogenous GSH to protect against oxidative stress whereas BBM transport functions physiologically in turnover of cellular GSH. The mitochondrial GSH pool is derived from cytoplasmic GSH by transport into the mitochondrial matrix and is mediated by the dicarboxylate and 2-oxoglutarate exchangers. Maintenance of the mitochondrial GSH pool is critical for cellular and mitochondrial redox homeostasis and is important in determining susceptibility to chemically induced apoptosis. Hence, membrane transport processes are critical to regulation of renal cellular and subcellular GSH pools and are determinants of susceptibility to cytotoxicity induced by oxidants and electrophiles

[Effect of extremely low frequency magnetic field on glutathione in rat muscles].

Science.gov (United States)

Ciejka, Elzbieta; Jakubowska, Ewa; Zelechowska, Paulina; Huk-Kolega, Halina; Kowalczyk, Agata; Goraca, Anna

2014-01-01

Free radicals (FR) are atoms, molecules or their fragments. Their excess leads to the development of oxidizing stress, the cause of many neoplastic, neurodegenerative and inflammatory diseases, and aging of the organism. Industrial pollution, tobacco smoke, ionizing radiation, ultrasound and magnetic field are the major FR exogenous sources. The low frequency magnetic field is still more commonly applied in the physical therapy. The aim of the presented study was to evaluate the effect of extremely low frequency magnetic field used in the magnetotherapy on the level of total glutathione, oxidized and reduced, and the redox state of the skeletal muscle cells, depending on the duration of exposure to magnetic field. The male rats, weight of 280-300 g, were randomly devided into 3 experimental groups: controls (group I) and treatment groups exposed to extremely low frequency magnetic field (ELF-MF) (group II exposed to 40 Hz, 7 mT for 0.5 h/day for 14 days and group III exposed to 40 Hz, 7 mT for 1 h/day for 14 days). Control rats were kept in a separate room not exposed to extremely low frequency magnetic field. Immediately after the last exposure, part of muscles was taken under pentobarbital anesthesia. Total glutathione, oxidized and reduced, and the redox state in the muscle tissue of animals were determined after exposure to magnetic fields. Exposure to low magnetic field: 40 Hz, 7 mT for 30 min/day and 60 min/day for 2 weeks significantly increased the total glutathione levels in the skeletal muscle compared to the control group (p magnetic therapy plays an important role in the development of adaptive mechanisms responsible for maintaining the oxidation-reduction balance in the body and depends on exposure duration.

The effect of excimer laser keratectomy on corneal glutathione-related enzymes in rabbits.

Science.gov (United States)

Bilgihan, Ayşe; Bilgihan, Kamil; Yis, Ozgür; Yis, Nilgün Safak; Hasanreisoglu, Berati

2003-04-01

Glutathione related enzymes are involved in the metabolism and detoxification of cytotoxic and carcinogenic compounds as well as reactive oxygen species. Excimer laser is a very useful tool for the treatment of refractive errors and removing superficial corneal opacities. Previous studies have shown that excimer laser may initiate free radical formation in the cornea. In the present study, we evaluated the effect of excimer laser keratectomy on corneal glutathione-related enzyme activities in rabbits. Animals were divided into five groups, and all groups were compared with the controls (group 1), after epithelial scraping (group 2), transepithelial photorefractive keratectomy (PRK) (group 3), traditional PRK (group 4) and deep traditional PRK (group 5). Corneal glutathione peroxidase (GPx), glutathione S-transferase (GST) and glutathione reductase (GR) activities were measured after 24h. Corneal GPx and GR activities significantly decreased only in group 5 (p < 0.05) but GST activities significantly decreased in all groups when compared with the control group (p < 0.05). In conclusion, excimer laser inhibits the glutathione dependent defense system in the cornea, this effect becomes more prominent after high doses of excimer laser energy and antioxidants may be useful to reduce free radical mediated complications.

Kale Extract Increases Glutathione Levels in V79 Cells, but Does not Protect Them against Acute Toxicity Induced by Hydrogen Peroxide

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Paula B. Andrade

2012-05-01

Full Text Available This study aims to evaluate the antioxidant potential of extracts of Brassica oleracea L. var. acephala DC. (kale and several materials of Pieris brassicae L., a common pest of Brassica cultures using a cellular model with hamster lung fibroblast (V79 cells under quiescent conditions and subjected to H₂O₂-induced oxidative stress. Cytotoxicity was evaluated by 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyl tetrazolium bromide (MTT assay and glutathione was determined by the 5,5'-dithiobis(2-nitrobenzoic acid (DTNB-oxidized glutathione (GSSG reductase recycling assay. The phenolic composition of the extracts was also established by HPLC-DAD. They presented acylated and non acylated flavonoid glycosides, some of them sulfated, and hydroxycinnamic acyl gentiobiosides. All extracts were cytotoxic by themselves at high concentrations and failed to protect V79 cells against H₂O₂ acute toxicity. No relationship between phenolic composition and cytotoxicity of the extracts was found. Rather, a significant increase in glutathione was observed in cells exposed to kale extract, which contained the highest amount and variety of flavonoids. It can be concluded that although flavonoids-rich extracts have the ability to increase cellular antioxidant defenses, the use of extracts of kale and P. brassicae materials by pharmaceutical or food industries, may constitute an insult to health, especially to debilitated individuals, if high doses are consumed.

Acute effects of heavy metals on the expression of glutathione-related antioxidant genes in the marine ciliate Euplotes crassus

International Nuclear Information System (INIS)

Kim, Se-Hun; Kim, Se-Joo; Lee, Jae-Seong; Lee, Young-Mi

2014-01-01

Highlights: • Significant higher increases in the relative ROS and total GSH levels were observed after exposure to heavy metals. • Real-time PCR data showed expression levels of GPx and GR mRNA were sensitively modulated within 8 h of exposure to heavy metals. • E. crassus GPx and GR genes may be involved in cellular defense mechanisms against heavy metal-induced oxidative stress. • E. crassus GPx and GR genes will be useful as potential molecular markers for monitoring heavy metal contamination. - Abstract: Euplotes crassus, a single-celled eukaryote, is directly affected by environmental contaminants. Here, exponentially cultured E. crassus were exposed to cadmium, copper, lead, and zinc and then the reactive oxygen species (ROS) and total glutathione (GSH) levels were measured. Subsequently, the transcriptional modulation of glutathione peroxidase (GPx) and glutathione reductase (GR) were estimated by quantitative RT-PCR. After an 8-h exposure, significantly higher increases in the relative ROS and total GSH levels were observed in exposed group, compared to the controls. Real-time PCR data revealed that the expression levels of GPx and GR mRNA were sensitively modulated within 8 h of exposure to all heavy metals. These findings suggest that these genes may be involved in cellular defense mechanisms by modulating their gene expression against heavy metal-induced oxidative stress. Thus, they may be useful as potential molecular biomarkers to assess sediment environments for contaminants

Degree of glutathione deficiency and redox imbalance depend on subtype of mitochondrial disease and clinical status.

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Gregory M Enns

Full Text Available Mitochondrial disorders are associated with decreased energy production and redox imbalance. Glutathione plays a central role in redox signaling and protecting cells from oxidative damage. In order to understand the consequences of mitochondrial dysfunction on in vivo redox status, and to determine how this varies by mitochondrial disease subtype and clinical severity, we used a sensitive tandem mass spectrometry assay to precisely quantify whole blood reduced (GSH and oxidized (GSSG glutathione levels in a large cohort of mitochondrial disorder patients. Glutathione redox potential was calculated using the Nernst equation. Compared to healthy controls (n = 59, mitochondrial disease patients (n = 58 as a group showed significant redox imbalance (redox potential -251 mV ± 9.7, p<0.0001 with an increased level of oxidation by ∼ 9 mV compared to controls (-260 mV ± 6.4. Underlying this abnormality were significantly lower whole blood GSH levels (p = 0.0008 and GSH/GSSG ratio (p = 0.0002, and significantly higher GSSG levels (p<0.0001 in mitochondrial disease patients compared to controls. Redox potential was significantly more oxidized in all mitochondrial disease subgroups including Leigh syndrome (n = 15, electron transport chain abnormalities (n = 10, mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (n = 8, mtDNA deletion syndrome (n = 7, mtDNA depletion syndrome (n = 7, and miscellaneous other mitochondrial disorders (n = 11. Patients hospitalized in metabolic crisis (n = 7 showed the greatest degree of redox imbalance at -242 mV ± 7. Peripheral whole blood GSH and GSSG levels are promising biomarkers of mitochondrial dysfunction, and may give insights into the contribution of oxidative stress to the pathophysiology of the various mitochondrial disorders. In particular, evaluation of redox potential may be useful in monitoring of clinical status or response to redox-modulating therapies in clinical trials.

RED BLOOD CELL AND WHOLE BLOOD GLUTATHIONE REDOX STATUS IN ENDURANCE-TRAINED MEN FOLLOWING A SKI MARATHON

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Eve Unt

2008-09-01

Full Text Available The aim of the present study was to evaluate the changes in glutathione redox ratio (GSSG·GSH-1 in red blood cells (RBCs and whole blood in well-trained men following a ski marathon. 16 male subjects (27.0 ± 4.7 yrs, 1.81 ± 0.06 m, 77.6 ± 9.6 kg, VO2max 66.2 ± 5.7 ml·kg-1·min-1 were examined before the competition (pre- COMP, after the competition (post-COMP and during an 18-hour recovery period (RECOV. There was a slight decrease in reduced glutathione (GSH in blood and in RBCs in post-COMP. During RECOV, the GSH level in blood was reduced, the GSH level in RBCs was significantly elevated (a statistically significant difference as compared to the pre-COMP level. The post-COMP GSSG·GSH-1 in full blood did not increase significantly, but its increase was statistically significant during the 18-hour recovery period. During the post-COMP and RECOV, the GSSG·GSH-1 in RBCs slightly decreased in comparison with the pre-COMP. Vitamin C concentration in serum increased in post-COMP (49% vs. pre- COMP and decreased to the baseline level during RECOV. In conclusion, our data show that acute exercise slightly increases the GSSG·GSH-1 in whole blood, while GSSG·GSH-1 in RBCs significantly decreases. Thus, exercise-related changes in the non-enzymatic components of the glutathione system (GSSG and GSH in whole blood and RBCs are not identical

Silver ions increase plasma membrane permeability through modulation of intracellular calcium levels in tobacco BY-2 cells.

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Klíma, Petr; Laňková, Martina; Vandenbussche, Filip; Van Der Straeten, Dominique; Petrášek, Jan

2018-05-01

Silver ions increase plasma membrane permeability for water and small organic compounds through their stimulatory effect on plasma membrane calcium channels, with subsequent modulation of intracellular calcium levels and ion homeostasis. The action of silver ions at the plant plasma membrane is largely connected with the inhibition of ethylene signalling thanks to the ability of silver ion to replace the copper cofactor in the ethylene receptor. A link coupling the action of silver ions and cellular auxin efflux has been suggested earlier by their possible direct interaction with auxin efflux carriers or by influencing plasma membrane permeability. Using tobacco BY-2 cells, we demonstrate here that besides a dramatic increase of efflux of synthetic auxins 2,4-dichlorophenoxyacetic acid (2,4-D) and 1-naphthalene acetic acid (NAA), treatment with AgNO 3 resulted in enhanced efflux of the cytokinin trans-zeatin (tZ) as well as the auxin structural analogues tryptophan (Trp) and benzoic acid (BA). The application of AgNO 3 was accompanied by gradual water loss and plasmolysis. The observed effects were dependent on the availability of extracellular calcium ions (Ca 2+ ) as shown by comparison of transport assays in Ca 2+ -rich and Ca 2+ -free buffers and upon treatment with inhibitors of plasma membrane Ca 2+ -permeable channels Al 3+ and ruthenium red, both abolishing the effect of AgNO 3 . Confocal microscopy of Ca 2+ -sensitive fluorescence indicator Fluo-4FF, acetoxymethyl (AM) ester suggested that the extracellular Ca 2+ availability is necessary to trigger the response to silver ions and that the intracellular Ca 2+ pool alone is not sufficient for this effect. Altogether, our data suggest that in plant cells the effects of silver ions originate from the primal modification of the internal calcium levels, possibly by their interaction with Ca 2+ -permeable channels at the plasma membrane.

Quantifying intracellular hydrogen peroxide perturbations in terms of concentration

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Beijing K. Huang

2014-01-01

Full Text Available Molecular level, mechanistic understanding of the roles of reactive oxygen species (ROS in a variety of pathological conditions is hindered by the difficulties associated with determining the concentration of various ROS species. Here, we present an approach that converts fold-change in the signal from an intracellular sensor of hydrogen peroxide into changes in absolute concentration. The method uses extracellular additions of peroxide and an improved biochemical measurement of the gradient between extracellular and intracellular peroxide concentrations to calibrate the intracellular sensor. By measuring peroxiredoxin activity, we found that this gradient is 650-fold rather than the 7–10-fold that is widely cited. The resulting calibration is important for understanding the mass-action kinetics of complex networks of redox reactions, and it enables meaningful characterization and comparison of outputs from endogenous peroxide generating tools and therapeutics across studies.

A case of severe glutathione synthetase deficiency with novel GSS mutations

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Xia, H.; Ye, J.; Wang, L.; Zhu, J.; He, Z.

2018-01-01

Glutathione synthetase deficiency (GSSD) is a rare inborn error of glutathione metabolism with autosomal recessive inheritance. The severe form of the disease is characterized by acute metabolic acidosis, usually present in the neonatal period with hemolytic anemia and progressive encephalopathy. A case of a male newborn infant who had severe metabolic acidosis with high anion gap, hemolytic anemia, and hyperbilirubinemia is reported. A high level of 5-oxoproline was detected in his urine and a diagnosis of generalized GSSD was made. DNA sequence analysis revealed the infant to be compound heterozygous with two mutations, c.738dupG in exon 8 of GSS gene resulting in p.S247fs and a repetitive sequence in exon 3 of GSS gene. Treatment after diagnosis of GSSD included supplementation with antioxidants and oral sodium hydrogen bicarbonate. However, he maintained a variable degree of metabolic acidosis and succumbed shortly after his parents requested discontinuation of therapy because of dismal prognosis and medical futility when he was 18 days old. PMID:29340523

A case of severe glutathione synthetase deficiency with novel GSS mutations

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H. Xia

2018-01-01

Full Text Available Glutathione synthetase deficiency (GSSD is a rare inborn error of glutathione metabolism with autosomal recessive inheritance. The severe form of the disease is characterized by acute metabolic acidosis, usually present in the neonatal period with hemolytic anemia and progressive encephalopathy. A case of a male newborn infant who had severe metabolic acidosis with high anion gap, hemolytic anemia, and hyperbilirubinemia is reported. A high level of 5-oxoproline was detected in his urine and a diagnosis of generalized GSSD was made. DNA sequence analysis revealed the infant to be compound heterozygous with two mutations, c.738dupG in exon 8 of GSS gene resulting in p.S247fs and a repetitive sequence in exon 3 of GSS gene. Treatment after diagnosis of GSSD included supplementation with antioxidants and oral sodium hydrogen bicarbonate. However, he maintained a variable degree of metabolic acidosis and succumbed shortly after his parents requested discontinuation of therapy because of dismal prognosis and medical futility when he was 18 days old.

Isoniazid acetylating phenotype in patients with paracoccidioidomycosis and its relationship with serum sulfadoxin levels, glucose-6-phosphate dehydrogenase and glutathione reductase activities

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Benedito Barraviera

1991-06-01

Full Text Available The authors evaluated the isoniazid acetylating phenotype and measured hematocrit, hemoglobin, glucose-6-phosphate dehydrogenase and glutathione reductase activities plus serum sulfadoxin levels in 39 patients with paracoccidioidomycosis (33 males and 6 females aged 17 to 58 years. Twenty one (53.84% of the patients presented a slow acetylatingphenotype and 18(46.16% a fast acetylating phenotype. Glucose-6-phosphate- dehydrogenase (G6PD acti vity was decreased in 5(23.80% slow acetylators and in 4(22.22% fast acetylators. Glutathione reductase activity was decreased in 14 (66.66% slow acetylators and in 12 (66.66% fast acetylators. Serum levels of free and total sulfadoxin Were higher in slow acetylator (p Os autores avaliaram o fenótipo acetilador da isoniazida, hematócrito, hemoglobina, atividade da glicose-6- fosfato desidrogenase, glutationa redutase e os níveis séricos de sulfadoxina de 39 doentes com paracoccidíoidomicose, senão 33 do sexo masculino e 6 do feminino, com idades compreendidas entre 17 e 58 anos. Vinte e um (53,84% doentes apresentaram fenótipo acetilador lento e 18 (46,16% rápido. A atividade da glicose-6-fosfato desidrogenase (G6PD esteve diminuída em 5 (23,80% acetiladores lentos e 4 (22,22% rápidos. A atividade da glutationa redutase esteve diminuída em 14 (66,66% acetiladores lentos e 12 (66,66% rápidos. Os níveis séricos de sulfadoxina livre e total foram maiores nos acetiladores lentos (p < 0,02. A análise dos resultados permite concluir que os níveis séricos de sulfadoxina relaciona-se com o fenótipo acetilador. Além disso, os níveis estiveram sempre acima de 50 µg/ml, níveis estes considerados terapêuticos. Por outro lado, a deficiência de glutationa redutase pode estar relacionada com a má absorção intestinal de nutrientes, entre eles riboflavina, vitamina precursora de FAD.

Reduced glutathione and glutathione disulfide in the blood of glucose-6-phosphate dehydrogenase-deficient newborns.

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Gong, Zhen-Hua; Tian, Guo-Li; Huang, Qi-Wei; Wang, Yan-Min; Xu, Hong-Ping

2017-07-20

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is commonly detected during mass screening for neonatal disease. We developed a method to measure reduced glutathione (GSH) and glutathione disulfide (GSSG) using tandem mass spectrometry (MS/MS) for detecting G6PD deficiency. The concentration of GSH and the GSH/GSSG ratio in newborn dry-blood-spot (DBS) screening and in blood plus sodium citrate for test confirmation were examined by MS/MS using labeled glycine as an internal standard. G6PD-deficient newborns had a lower GSH content (242.9 ± 15.9 μmol/L)and GSH/GSSG ratio (14.9 ± 7.2) than neonatal controls (370.0 ± 53.2 μmol/L and 46.7 ± 19.6, respectively). Although the results showed a significance of P blood measured using MS/MS on the first day of sample preparation are consistent with G6PD activity and are helpful for diagnosing G6PD deficiency.

Comparison of the biological effects of {sup 18}F at different intracellular levels

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Kashino, Genro, E-mail: kashino@oita-u.ac.jp [Advanced Molecular Imaging Center, Faculty of Medicine, Oita University, 1-1 Idaigaoka, Hasama-machi, Yufu City, Oita 879-5593 (Japan); Hayashi, Kazutaka; Douhara, Kazumasa [Advanced Molecular Imaging Center, Faculty of Medicine, Oita University, 1-1 Idaigaoka, Hasama-machi, Yufu City, Oita 879-5593 (Japan); Kobashigawa, Shinko; Mori, Hiromu [Department of Radiology, Faculty of Medicine, Oita University, 1-1 Idaigaoka, Hasama-machi, Yufu City, Oita 879-5593 (Japan)

2014-11-07

Highlights: • We estimated the inductions of DNA DSB in cell treated with {sup 18}F-FDG. • We found that inductions of DNA DSB are dependent on accumulation of {sup 18}F in cell. • Accumulation of {sup 18}F in cell may be indispensable for risk estimation of PET. - Abstract: We herein examined the biological effects of cells treated with {sup 18}F labeled drugs for positron emission tomography (PET). The relationship between the intracellular distribution of {sup 18}F and levels of damaged DNA has yet to be clarified in detail. We used culture cells (Chinese Hamster Ovary cells) treated with two types of {sup 18}F labeled drugs, fluorodeoxyglucose (FDG) and fluorine ion (HF). FDG efficiently accumulated in cells, whereas HF did not. To examine the induction of DNA double strand breaks (DSB), we measured the number of foci for 53BP1 that formed at the site of DNA DSB. The results revealed that although radioactivity levels were the same, the induction of 53BP1 foci was stronger in cells treated with {sup 18}F-FDG than in those treated with {sup 18}F-HF. The clonogenic survival of cells was significantly lower with {sup 18}F-FDG than with {sup 18}F-HF. We concluded that the efficient accumulation of {sup 18}F in cells led to stronger biological effects due to more severe cellular lethality via the induction of DNA DSB.

Activation of dihaloalkanes by glutathione conjugation and formation of DNA adducts

International Nuclear Information System (INIS)

Guengerich, F.P.; Peterson, L.A.; Cmarik, J.L.; Koga, N.; Inskeep, P.B.

1987-01-01

Ethylene dibromide (1,2-dibromoethane, EDB) can be activated to electrophilic species by either oxidative metabolism or conjugation with glutathione. Although conjugation is generally a route of detoxication, in this case it leads to genetic damage. The major DNA adduct has been identified as S-[2-(N 7 -guanyl)ethyl]glutathione, which is believed to arise via half-mustard and episulfonium ion intermediates. The adduct has a half-life of about 70 to 100 hr and does not appear to migrate to other DNA sites. Glutathione-dependent DNA damage by EDB was also demonstrated in human hepatocyte preparations. The possible relevance of this DNA adduct to genetic damage is discussed

Evaluation of the precision-cut liver and lung slice systems for the study of induction of CYP1, epoxide hydrolase and glutathione S-transferase activities.

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Pushparajah, Daphnee S; Umachandran, Meera; Plant, Kathryn E; Plant, Nick; Ioannides, Costas

2007-02-28

The principal objective was to ascertain whether precision-cut tissue slices can be used to evaluate the potential of chemicals to induce CYP1, epoxide hydrolase and glutathione S-transferase activities, all being important enzymes involved in the metabolism of polycyclic aromatic hydrocarbons. Precision-cut rat liver and lung slices were incubated with a range of benzo[a]pyrene concentrations for various time periods. A rise in the O-deethylation of ethoxyresorufin was seen in both liver and lung slices exposed to benzo[a]pyrene, which was accompanied by increased CYP1A apoprotein levels. Pulmonary CYP1B1 apoprotein levels and hepatic mRNA levels were similarly enhanced. Elevated epoxide hydrolase and glutathione S-transferase activities were also observed in liver slices following incubation for 24h; similarly, a rise in apoprotein levels of both enzymes was evident, peak levels occurring at the same time point. When mRNA levels were monitored, a rise in the levels of both enzymes was seen as early as 4h after incubation, but maximum levels were attained at 24 h. In lung slices, induction of epoxide hydrolase by benzo[a]pyrene was observed after a 24-h incubation, and at a concentration of 1 microM; a rise in apoprotein levels was seen at this time point. Glutathione S-transferase activity was not inducible in lung slices by benzo[a]pyrene but a modest increase was observed in hepatic slices. Collectively, these studies confirmed CYP1A induction in rat liver slices and established that CYP1B1 expression, and epoxide hydrolase and glutathione S-transferase activities are inducible in precision-cut tissue slices.

Effect on the K/sub m/ for radiosensitization at 00C of thiol depletion by diethylmaleate pretreatment: quantitative differences found using the radiation sensitizing agent misonidazole or oxygen

International Nuclear Information System (INIS)

Koch, C.J.; Stobbe, C.C.; Bump, E.A.

1984-01-01

Pretreatment of V79-WNRE cells with 150 μM diethylmaleate for 1 hr at 37 0 C caused a decrease in intracellular glutathione levels to approximately 10-15% of control levels. The cells could be washed free of diethylmaleate and held at 0 0 C for several hours without toxicity and with no increase in glutathione concentration, although the glutathione concentration rapidly increased to normal levels at higher temperatures. Glutathione depletion itself caused a small but consistent radiosensitization of hypoxic cells (dose enhancement ratio of 1.2). However glutathione depletion caused a profound change in the radiosensitizing efficiency of misonidazole, with a decrease in K/sub m/ of about sevenfold from 0.6 to 0.09 mM. In contrast, only a 2.5-fold decrease was found in the K/sub m/ for radiosensitization by oxygen with diethylmaleate pretreatment. These results suggest a fundamental problem with the conventional theory of radiosensitivity whereby one considers a first-order competition for reaction with target radicals between radical-fixing versus radical-repairing species. It also suggests difficulties in the interpretation of glutathione as the only endogenous protective species

Antioxidant activity of melatonin and glutathione interacting with hydroxyl- and superoxide anion radicals

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T. Y. Kuznetsova

2017-12-01

Full Text Available Based on the analysis of the results obtained by quantum chemical modeling of interaction between reduced glutathione (GSH and melatonin (MLT molecules with oxygen radicals (•OH and • OOˉ it was found that this interaction occured following the acid-base mechanism, where MLT and GSH acted as a base in respect of •OH, and as acid in respect of •OOˉ. We have carried out the correlation of the results of quantum chemical calculations (density redistribution, energetic characteristics under the interaction of MLT and GSH molecules with •OH and •OOˉ in changing macroscopic properties of the process of electroreduction of free oxygen radicals in the presence of antioxidants (potential and maximal current wave reduction waves. This was a direct experimental macroscale evidence of the results of theoretical modeling at the nanoscale level that pointed to a marked antioxidant activity of glutathione compared with melatonin.

JS-K, a glutathione S-transferase-activated nitric oxide donor with antineoplastic activity in malignant gliomas.

Science.gov (United States)

Weyerbrock, Astrid; Osterberg, Nadja; Psarras, Nikolaos; Baumer, Brunhilde; Kogias, Evangelos; Werres, Anna; Bette, Stefanie; Saavedra, Joseph E; Keefer, Larry K; Papazoglou, Anna

2012-02-01

Glutathione S-transferases (GSTs) control multidrug resistance and are upregulated in many cancers, including malignant gliomas. The diazeniumdiolate JS-K generates nitric oxide (NO) on enzymatic activation by glutathione and GST, showing promising NO-based anticancer efficacy. To evaluate the role of NO-based antitumor therapy with JS-K in U87 gliomas in vitro and in vivo. U87 glioma cells and primary glioblastoma cell lines were exposed to JS-K and a variety of inhibitors to study cell death by necrosis, apoptosis, and other mechanisms. GST expression was evaluated by immunocytochemistry, polymerase chain reaction, and Western blot, and NO release from JS-K was studied with a NO assay. The growth-inhibitory effect of JS-K was studied in a U87 xenograft model in vivo. Dose-dependent inhibition of cell proliferation was observed in human U87 glioma cells and primary glioblastoma cells in vitro. Cell death was partially induced by caspase-dependent apoptosis, which could be blocked by Z-VAD-FMK and Q-VD-OPH. Inhibition of GST by sulfasalazine, cGMP inhibition by ODQ, and MEK1/2 inhibition by UO126 attenuated the antiproliferative effect of JS-K, suggesting the involvement of various intracellular death signaling pathways. Response to JS-K correlated with mRNA and protein expression of GST and the amount of NO released by the glioma cells. Growth of U87 xenografts was reduced significantly, with immunohistochemical evidence for increased necrosis and apoptosis and reduced proliferation. Our data show for the first time the potent antiproliferative effect of JS-K in gliomas in vitro and in vivo. These findings warrant further investigation of this novel NO-releasing prodrug in gliomas.

A glutathione conjugate of hepoxilin A3: Formation and action in the rat central nervous system

International Nuclear Information System (INIS)

Pace-Asciak, C.R.; Laneuville, O.; Su, W.G.; Corey, E.J.; Gurevich, N.; Wu, P.; Carlen, P.L.

1990-01-01

Incubation of (8R)- and (8S)-[1-14C]hepoxilin A3 [where hepoxilin A3 is 8-hydroxy-11,12-epoxyeicosa-(5Z,9E,14Z)-trienoic acid] and glutathione with homogenates of rat brain hippocampus resulted in a product that was identified as the (8R) and (8S) diastereomers of 11-glutathionyl hepoxilin A3 by reversed-phase high performance liquid chromatographic comparison with the authentic standard made by total synthesis. Identity was further confirmed by cleavage of the isolated product with gamma-glutamyltranspeptidase to yield the corresponding cysteinylglycinyl conjugate that was identical by reversed-phase high performance liquid chromatographic analysis with the enzymic cleavage product derived from the synthetic glutathionyl conjugate. The glutathionyl and cysteinylglycinyl conjugate are referred to as hepoxilin A3-C and hepoxilin A3-D, respectively, by analogy with the established leukotriene nomenclature. Formation of hepoxilin A3-C was greatly enhanced with a concomitant decrease in formation of the epoxide hydrolase product, trioxilin A3, when the epoxide hydrolase inhibitor trichloropropene oxide was added to the incubation mixture demonstrating the presence of a dual metabolic pathway in this tissue involving hepoxilin epoxide hydrolase and glutathione S-transferase processes. Hepoxilin A3-C was tested using intracellular electrophysiological techniques on hippocampal CA1 neurons and found to be active at concentrations as low as 16 nM in causing membrane hyperpolarization, enhanced amplitude and duration of the post-spike train afterhyperpolarization, a marked increase in the inhibitory postsynaptic potential, and a decrease in the spike threshold. These findings suggest that these products in the hepoxilin pathway of arachidonic acid metabolism formed by the rat brain may function as neuromodulators

Cadmium induced changes in subcellular glutathione contents within glandular trichomes of Cucurbita pepo L.

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Kolb, Dagmar; Müller, Maria; Zellnig, Günther; Zechmann, Bernd

2010-07-01

Plants cope with cadmium (Cd) stress by complexation with phytochelatins (Pc), metallothioneins and glutathione and sequestration within vacuoles. Especially glutathione was found to play a major role in Cd detoxification as Cd shows a high binding affinity towards thiols and as glutathione is a precursor for Pc synthesis. In the present study, we have used an immunohistochemical approach combined with computer-supported transmission electron microscopy in order to measure changes in the subcellular distribution of glutathione during Cd-stress in mesophyll cells and cells of different glandular trichomes (long and short stalked) of Cucurbita pepo L. subsp. pepo var. styriaca GREB: . Even though no ultrastructural alterations were observed in leaf and glandular trichome cells after the treatment of plants with 50 microM cadmium chloride (CdCl(2)) for 48 h, all cells showed a large decrease in glutathione contents. The strongest decrease was found in nuclei and the cytosol (up to 76%) in glandular trichomes which are considered as a major side of Cd accumulation in leaves. The ratio of glutathione between the cytosol and nuclei and the other cell compartments was strongly decreased only in glandular trichomes (more than 50%) indicating that glutathione in these two cell compartments is especially important for the detoxification of Cd in glandular trichomes. Additionally, these data indicate that large amounts of Cd are withdrawn from nuclei during Cd exposure. The present study gives a detailed insight into the compartment-specific importance of glutathione during Cd exposure in mesophyll cells and glandular trichomes of C. pepo L. plants.

Reduced intracellular c-di-GMP content increases expression of quorum sensing-regulated genes in Pseudomonas aeruginosa

DEFF Research Database (Denmark)

Chua, Song Lin; Liu, Yang; Li, Yingying

2017-01-01

Cyclic-di-GMP (c-di-GMP) is an intracellular secondary messenger which controls the biofilm life cycle in many bacterial species. High intracellular c-di-GMP content enhances biofilm formation via the reduction of motility and production of biofilm matrix, while low c-di-GMP content in biofilm...... cells leads to increased motility and biofilm dispersal. While the effect of high c-di-GMP levels on bacterial lifestyles is well studied, the physiology of cells at low c-di-GMP levels remains unclear. Here, we showed that Pseudomonas aeruginosa cells with high and low intracellular c-di-GMP contents...... possessed distinct transcriptome profiles. There were 535 genes being upregulated and 432 genes downregulated in cells with low c-di-GMP, as compared to cells with high c-di-GMP. Interestingly, both rhl and pqs quorum-sensing (QS) operons were expressed at higher levels in cells with low intracellular c-di-GMP...

PKC-η-MARCKS Signaling Promotes Intracellular Survival of Unopsonized Burkholderia thailandensis.

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Micheva-Viteva, Sofiya N; Shou, Yulin; Ganguly, Kumkum; Wu, Terry H; Hong-Geller, Elizabeth

2017-01-01

Pathogenic Burkholderia rely on host factors for efficient intracellular replication and are highly refractory to antibiotic treatment. To identify host genes that are required by Burkholderia spp. during infection, we performed a RNA interference (RNAi) screen of the human kinome and identified 35 host kinases that facilitated Burkholderia thailandensis intracellular survival in human monocytic THP-1 cells. We validated a selection of host kinases using imaging flow cytometry to assess efficiency of B. thailandensis survival in the host upon siRNA-mediated knockdown. We focused on the role of the novel protein kinase C isoform, PKC-η, in Burkholderia infection and characterized PKC-η/MARCKS signaling as a key event that promotes the survival of unopsonized B. thailandensis CDC2721121 within host cells. While infection of lung epithelial cells with unopsonized Gram-negative bacteria stimulated phosphorylation of Ser175/160 in the MARCKS effector domain, siRNA-mediated knockdown of PKC-η expression reduced the levels of phosphorylated MARCKS by >3-fold in response to infection with Bt CDC2721121. We compared the effect of the conventional PKC-α and novel PKC-η isoforms on the growth of B. thailandensis CDC2721121 within monocytic THP-1 cells and found that ≥75% knock-down of PRKCH transcript levels reduced intracellular bacterial load 100% more efficiently when compared to growth in cells siRNA-depleted of the classical PKC-α, suggesting that the PKC-η isoform can specifically mediate Burkholderia intracellular survival. Based on imaging studies of intracellular B. thailandensis , we found that PKC-η function stimulates phagocytic pathways that promote B. thailandensis escape into the cytoplasm leading to activation of autophagosome flux. Identification of host kinases that are targeted by Burkholderia during infection provides valuable molecular insights in understanding Burkholderia pathogenesis, and ultimately, in designing effective host

Intracellular mechanisms of solar water disinfection

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Castro-Alférez, María; Polo-López, María Inmaculada; Fernández-Ibáñez, Pilar

2016-12-01

Solar water disinfection (SODIS) is a zero-cost intervention measure to disinfect drinking water in areas of poor access to improved water sources, used by more than 6 million people in the world. The bactericidal action of solar radiation in water has been widely proven, nevertheless the causes for this remain still unclear. Scientific literature points out that generation of reactive oxygen species (ROS) inside microorganisms promoted by solar light absorption is the main reason. For the first time, this work reports on the experimental measurement of accumulated intracellular ROS in E. coli during solar irradiation. For this experimental achievement, a modified protocol based on the fluorescent probe dichlorodihydrofluorescein diacetate (DCFH-DA), widely used for oxidative stress in eukaryotic cells, has been tested and validated for E. coli. Our results demonstrate that ROS and their accumulated oxidative damages at intracellular level are key in solar water disinfection.

Glutathione Depletion Induced by c-Myc Downregulation Triggers Apoptosis on Treatment with Alkylating Agents1

Science.gov (United States)

Biroccio, Annamaria; Benassi, Barbara; Fiorentino, Francesco; Zupi, Gabriella

2004-01-01

Abstract Here we investigate the mechanism(s) involved in the c-Myc-dependent drug response of melanoma cells. By using three M14-derived c-Myc low-expressing clones, we demonstrate that alkylating agents, cisplatin and melphalan, trigger apoptosis in the c-Myc antisense transfectants, but not in the parental line. On the contrary, topoisomerase inhibitors, adriamycin and camptothecin, induce apoptosis to the same extent regardless of c-Myc expression. Because we previously demonstrated that c-Myc downregulation decreases glutathione (GSH) content, we evaluated the role of GSH in the apoptosis induced by the different drugs. In control cells treated with one of the alkylating agents or the others, GSH depletion achieved by l-buthionine-sulfoximine preincubation opens the apoptotic pathway. The apoptosis proceeded through early Bax relocalization, cytochrome c release, and concomitant caspase-9 activation, whereas reactive oxygen species production and alteration of mitochondria membrane potential were late events. That GSH was determining in the c-Myc-dependent drug-induced apoptosis was demonstrated by altering the intracellular GSH content of the c-Myc low-expressing cells up to the level of controls. Indeed, GSH ethyl ester-mediated increase of GSH abrogated apoptosis induced by cisplatin and melphalan by inhibition of Bax/cytochrome c redistribution. The relationship among c-Myc, GSH content, and the response to alkylating agent has been also evaluated in the M14 Myc overexpressing clones as well as in the melanoma JR8 c-Myc antisense transfectants. All together, these results demonstrate that GSH plays a key role in governing c-Myc-dependent drug-induced apoptosis. PMID:15153331

Structures of a putative ζ-class glutathione S-transferase from the pathogenic fungus Coccidioides immitis

International Nuclear Information System (INIS)

Edwards, Thomas E.; Bryan, Cassie M.; Leibly, David J.; Dieterich, Shellie H.; Abendroth, Jan; Sankaran, Banumathi; Sivam, Dhileep; Staker, Bart L.; Van Voorhis, Wesley C.; Myler, Peter J.; Stewart, Lance J.

2011-01-01

The pathogenic fungus C. immitis causes coccidioidomycosis, a potentially fatal disease. Here, apo and glutathione-bound crystal structures of a previously uncharacterized protein from C. immitis that appears to be a ζ-class glutathione S-transferase are presented. Coccidioides immitis is a pathogenic fungus populating the southwestern United States and is a causative agent of coccidioidomycosis, sometimes referred to as Valley Fever. Although the genome of this fungus has been sequenced, many operons are not properly annotated. Crystal structures are presented for a putative uncharacterized protein that shares sequence similarity with ζ-class glutathione S-transferases (GSTs) in both apo and glutathione-bound forms. The apo structure reveals a nonsymmetric homodimer with each protomer comprising two subdomains: a C-terminal helical domain and an N-terminal thioredoxin-like domain that is common to all GSTs. Half-site binding is observed in the glutathione-bound form. Considerable movement of some components of the active site relative to the glutathione-free form was observed, indicating an induced-fit mechanism for cofactor binding. The sequence homology, structure and half-site occupancy imply that the protein is a ζ-class glutathione S-transferase, a maleylacetoacetate isomerase (MAAI)

The effects of quercetin towards reactive oxygen species levels and glutathione in Toxoplasma gondii profilin-exposed adipocytes in vitro

Directory of Open Access Journals (Sweden)

Yulia D.S.

2018-04-01

Full Text Available Toxoplasma gondii (T. gondii has been found to potentially cause adipocyte dysfunction by activating the inflammatory pathways through its profilin. In response to inflammation, adipocytes produce Reactive Oxygen Species (ROS. To scavenge ROS, endogenous or exogenous antioxidants are required. Glutathione (GSH is one of enzimatic antioxidant that abundant in all of body cells. Quercetin, an exogenous antioxidant, can be widely found in natural products. This research aims to explore the effects of quercetin towards ROS and GSH stimulated from T. gondii profilin-exposed adipocytes. To achieve this, adipocytes were exposed to 20 µM T. gondii profilin and treated with four doses of quercetin; 31.25, 62.5, 125, and 250 µM. The results showed that quercetin significantly reduced the ROS levels (p <0,001 and significantly increased GSH (p <0,001 in T. gondii profilin-exposed adipocytes compared to untreated cells, with an effective dose of 62.5µM. This study implies that quercetin might be a promising candidate for development of antioxidant treatment interventions to prevent toxoplasmosis-mediated adipocytopathy.

Cosensitization Properties of Glutathione-Protected Au25 Cluster on Ruthenium Dye-Sensitized TiO2 Photoelectrode

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Kazuya Nakata

2013-01-01

Full Text Available Cosensitization by glutathione-protected Au25 clusters on Ru complex, N719-sensitized TiO2 photoelectrodes is demonstrated. Glutathione-protected Au25 clusters showed no significant changes in properties after adsorption onto TiO2 particles, as confirmed by optical absorption spectroscopy, transmission electron microscopy, and laser desorption/ionization mass spectrometry. Adsorption property of the glutathione-protected Au25 clusters depends on the pH, which affects the incident photon-to-current conversion efficiency (IPCE of the TiO2 photoelectrode containing Au25 clusters. When pH 7. The IPCE of a TiO2 photoelectrode sensitized by both glutathione-protected Au25 clusters and N719 was increased compared with photoelectrodes containing either glutathione-protected Au25 clusters or N719, which suggests that glutathione-protected Au25 clusters act as a coadsorbent for N719 on TiO2 photoelectrodes. This is also supported by the results that the IPCE of N719-sensitized TiO2 photoelectrodes increased upon addition of glutathione. Furthermore, cosensitization by glutathione-protected Au25 clusters on N719-sensitized TiO2 photoelectrodes allows that wavelength of photoelectric conversion was extended to the near infrared (NIR region. These results suggest that glutathione-protected Au25 clusters act not only as a coadsorbent to increase IPCE but also as an NIR-active sensitizer.

A novel method for screening the glutathione transferase inhibitors

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Węgrzyn Grzegorz

2009-03-01

Full Text Available Abstract Background Glutathione transferases (GSTs belong to the family of Phase II detoxification enzymes. GSTs catalyze the conjugation of glutathione to different endogenous and exogenous electrophilic compounds. Over-expression of GSTs was demonstrated in a number of different human cancer cells. It has been found that the resistance to many anticancer chemotherapeutics is directly correlated with the over-expression of GSTs. Therefore, it appears to be important to find new GST inhibitors to prevent the resistance of cells to anticancer drugs. In order to search for glutathione transferase (GST inhibitors, a novel method was designed. Results Our results showed that two fragments of GST, named F1 peptide (GYWKIKGLV and F2 peptide (KWRNKKFELGLEFPNL, can significantly inhibit the GST activity. When these two fragments were compared with several known potent GST inhibitors, the order of inhibition efficiency (measured in reactions with 2,4-dinitrochlorobenzene (CDNB and glutathione as substrates was determined as follows: tannic acid > cibacron blue > F2 peptide > hematin > F1 peptide > ethacrynic acid. Moreover, the F1 peptide appeared to be a noncompetitive inhibitor of the GST-catalyzed reaction, while the F2 peptide was determined as a competitive inhibitor of this reaction. Conclusion It appears that the F2 peptide can be used as a new potent specific GST inhibitor. It is proposed that the novel method, described in this report, might be useful for screening the inhibitors of not only GST but also other enzymes.

Lansoprazole induces apoptosis of breast cancer cells through inhibition of intracellular proton extrusion

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Zhang, Shangrong; Wang, Yifan; Li, Shu Jie, E-mail: shujieli@nankai.edu.cn

2014-06-13

Highlights: • Lansoprazole (LPZ) induces cell apoptosis in breast cancer cells. • LPZ markedly inhibits intracellular proton extrusion. • LPZ induces an increase in intracellular ATP level, lysosomal alkalinization and ROS accumulation. - Abstract: The increased glycolysis and proton secretion in tumors is proposed to contribute to the proliferation and invasion of cancer cells during the process of tumorigenesis and metastasis. Here, treatment of human breast cancer cells with proton pump inhibitor (PPI) lansoprazole (LPZ) induces cell apoptosis in a dose-dependent manner. In the implantation of the MDA-MB-231 xenografts in nude mice, administration of LPZ significantly inhibits tumorigenesis and induces large-scale apopotosis of tumor cells. LPZ markedly inhibits intracellular proton extrusion, induces an increase in intracellular ATP level, lysosomal alkalinization and accumulation of reactive oxygen species (ROS) in breast cancer cells. The ROS scavenger N-acetyl-L-cysteine (NAC) and diphenyleneiodonium (DPI), a specific pharmacological inhibitor of NADPH oxidases (NOX), significantly abolish LPZ-induced ROS accumulation in breast cancer cells. Our results suggested that LPZ may be used as a new therapeutic drug for breast tumor.

Lansoprazole induces apoptosis of breast cancer cells through inhibition of intracellular proton extrusion

International Nuclear Information System (INIS)

Zhang, Shangrong; Wang, Yifan; Li, Shu Jie

2014-01-01

Highlights: • Lansoprazole (LPZ) induces cell apoptosis in breast cancer cells. • LPZ markedly inhibits intracellular proton extrusion. • LPZ induces an increase in intracellular ATP level, lysosomal alkalinization and ROS accumulation. - Abstract: The increased glycolysis and proton secretion in tumors is proposed to contribute to the proliferation and invasion of cancer cells during the process of tumorigenesis and metastasis. Here, treatment of human breast cancer cells with proton pump inhibitor (PPI) lansoprazole (LPZ) induces cell apoptosis in a dose-dependent manner. In the implantation of the MDA-MB-231 xenografts in nude mice, administration of LPZ significantly inhibits tumorigenesis and induces large-scale apopotosis of tumor cells. LPZ markedly inhibits intracellular proton extrusion, induces an increase in intracellular ATP level, lysosomal alkalinization and accumulation of reactive oxygen species (ROS) in breast cancer cells. The ROS scavenger N-acetyl-L-cysteine (NAC) and diphenyleneiodonium (DPI), a specific pharmacological inhibitor of NADPH oxidases (NOX), significantly abolish LPZ-induced ROS accumulation in breast cancer cells. Our results suggested that LPZ may be used as a new therapeutic drug for breast tumor

Detection of Intracellular Factor VIII Protein in Peripheral Blood Mononuclear Cells by Flow Cytometry

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Gouri Shankar Pandey

2013-01-01

Full Text Available Flow cytometry is widely used in cancer research for diagnosis, detection of minimal residual disease, as well as immune monitoring and profiling following immunotherapy. Detection of specific host proteins for diagnosis predominantly uses quantitative PCR and western blotting assays. In this study, we optimized a flow cytometry-based detection assay for Factor VIII protein in peripheral blood mononuclear cells (PBMCs. An indirect intracellular staining (ICS method was standardized using monoclonal antibodies to different domains of human Factor VIII protein. The FVIII protein expression level was estimated by calculating the mean and median fluorescence intensities (MFI values for each monoclonal antibody. ICS staining of transiently transfected cell lines supported the method's specificity. Intracellular FVIII protein expression was also detected by the monoclonal antibodies used in the study in PBMCs of five blood donors. In summary, our data suggest that intracellular FVIII detection in PBMCs of hemophilia A patients can be a rapid and reliable method to detect intracellular FVIII levels.

Antisense Suppression of 2-Cysteine Peroxiredoxin in Arabidopsis Specifically Enhances the Activities and Expression of Enzymes Associated with Ascorbate Metabolism But Not Glutathione Metabolism1

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Baier, Margarete; Noctor, Graham; Foyer, Christine H.; Dietz, Karl-Josef

2000-01-01

The aim of this study was to characterize the effect of decreased 2-cysteine peroxiredoxin (2-CP) on the leaf anti-oxidative system in Arabidopsis. At three stages of leaf development, two lines of transgenic Arabidopsis mutants with decreased contents of chloroplast 2-CP were compared with wild type and a control line transformed with an empty vector. Glutathione contents and redox state were similar in all plants, and no changes in transcript levels for enzymes involved in glutathione metabolism were observed. Transcript levels for chloroplastic glutathione peroxidase were much lower than those for 2-CP, and both cytosolic and chloroplastic glutathione peroxidase were not increased in the mutants. In contrast, the foliar ascorbate pool was more oxidized in the mutants, although the difference decreased with plant age. The activities of thylakoid and stromal ascorbate peroxidase and particularly monodehydroascorbate reductase were increased as were transcripts for these enzymes. No change in dehydroascorbate reductase activity was observed, and effects on transcript abundance for glutathione reductase, catalase, and superoxide dismutase were slight or absent. The results demonstrate that 2-CP forms an integral part of the anti-oxidant network of chloroplasts and is functionally interconnected with other defense systems. Suppression of 2-CP leads to increased expression of other anti-oxidative genes possibly mediated by increased oxidation state of the leaf ascorbate pool. PMID:11027730

Delineation of the Pasteurellaceae-specific GbpA-family of glutathione-binding proteins

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Vergauwen Bjorn

2011-11-01

Full Text Available Abstract Background The Gram-negative bacterium Haemophilus influenzae is a glutathione auxotroph and acquires the redox-active tripeptide by import. The dedicated glutathione transporter belongs to the ATP-binding cassette (ABC-transporter superfamily and displays more than 60% overall sequence identity with the well-studied dipeptide (Dpp permease of Escherichia coli. The solute binding protein (SBP that mediates glutathione transport in H. influenzae is a lipoprotein termed GbpA and is 54% identical to E. coli DppA, a well-studied member of family 5 SBP's. The discovery linking GbpA to glutathione import came rather unexpectedly as this import-priming SBP was previously annotated as a heme-binding protein (HbpA, and was thought to mediate heme acquisition. Nonetheless, although many SBP's have been implicated in more than one function, a prominent physiological role for GbpA and its partner permease in heme acquisition appears to be very unlikely. Here, we sought to characterize five representative GbpA homologs in an effort to delineate the novel GbpA-family of glutathione-specific family 5 SBPs and to further clarify their functional role in terms of ligand preferences. Results Lipoprotein and non-lipoprotein GbpA homologs were expressed in soluble form and substrate specificity was evaluated via a number of ligand binding assays. A physiologically insignificant affinity for hemin was observed for all five GbpA homologous test proteins. Three out of five test proteins were found to bind glutathione and some of its physiologically relevant derivatives with low- or submicromolar affinity. None of the tested SBP family 5 allocrites interacted with the remaining two GbpA test proteins. Structure-based sequence alignments and phylogenetic analysis show that the two binding-inert GbpA homologs clearly form a separate phylogenetic cluster. To elucidate a structure-function rationale for this phylogenetic differentiation, we determined the crystal

CHARACTERIZATION OF DANSYLATED CYSTEINE, CYSTINE, GLUTATHIONE, AND GLUTATHIONE DISULFIDE BY NARROW BORE LIQUID CHROMATOGRAPHY - ELECTROSPRAY IONIZATION MASS SPECTROMETRY

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A method using reversed phase high performance liquid chromtography/electrospray ionization-mass spectrometry (RP-LC/ESI-MS) has been developed to confirm the dientity of dansylated derivatives of cysteine (C) and glutathione (GSH), and their respective dimers, cystine (CSSC) and...

Role of glutathione redox cycle and catalase in defense against oxidative stress induced by endosulfan in adrenocortical cells of rainbow trout (Oncorhynchus mykiss)

International Nuclear Information System (INIS)

Dorval, J.; Hontela, A.

2003-01-01

The role of antioxidants in maintaining the functional integrity of adrenocortical cells during in vitro exposure to endosulfan, an organochlorine pesticide, was investigated in rainbow trout (Oncorhynchus mykiss). Aminotriazole (ATA), an inhibitor of catalase (CAT), L-buthionine sulfoximine (L-BSO), an inhibitor of glutathione (GSH) synthesis, and N-acetyl cysteine (NAC), a glutathione precursor, were used to investigate the role of CAT and GSH redox cycle in protection against the adrenal toxicity of endosulfan, a pesticide that impairs cell viability (LC 50 366 μM) and cortisol secretion (EC 50 19 μM) in a concentration-related manner. Pretreatment with ATA and L-BSO enhanced the toxicity of endosulfan (LC 50 and EC 50 , respectively, 302 and 2.6 μM with ATA, 346 and 3.1 μM with L-BSO), while pretreatment with NAC had no significant effect on cell viability and increased the EC 50 of endosulfan to 51 μM. CAT activity was significantly reduced following exposure to endosulfan when cells were pretreated with ATA. Pretreatment with L-BSO significantly decreased glutathione peroxidase (GPx) activity and reduced glutathione (GSH) levels in a concentration-related manner following exposure to endosulfan, while GSH levels were significantly higher in NAC pretreated cells compared to untreated cells. Finally, pretreatment with ATA and L-BSO increased, while pretreatment with NAC decreased, lipid hydroperoxides (LOOH) levels. CAT, GPx, and GSH were identified as important antioxidants in maintaining the function and integrity of rainbow trout adrenocortical cells and ATA, L-BSO, and NAC were identified as effective modulators of CAT and GSH redox cycle. Moreover, this study suggests that the glutathione redox cycle may be more efficient than catalase in protecting adrenocortical cells against endosulfan-induced oxidative stress

Dietary docosahexaenoic acid-induced generation of liver lipid peroxides is not suppressed further by elevated levels of glutathione in ODS rats.

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Sekine, Seiji; Kubo, Kazuhiro; Tadokoro, Tadahiro; Saito, Morio

2006-04-01

We examined the effects of ascorbic acid (AsA) and glutathione (GSH; experiment 1) and of GSH in acetaminophen-fed rats (experiment 2) on dietary docosahexaenoic acid (DHA)-induced tissue lipid peroxidation. In experiment 1, AsA-requiring Osteogenic Disorder Shionogi/Shi-od/od (ODS) rats were fed soybean protein diets containing DHA (10.0% total energy) and AsA at 50 (low) or 300 (normal) mg/kg without (low) or with (normal) methionine at 2 g/kg for 32 d. In experiment 2, ODS rats were fed diets containing DHA (7.8% total energy) and acetaminophen (4 g/kg) with different levels of dietary methionine (low, moderate, high, and excessive at 0, 3, 6, and 9 g/kg, respectively) for 30 d. Tissue lipid peroxides and antioxidant levels were determined. In experiment 1, liver lipid peroxide levels in the low-AsA group were lower than those in the normal-AsA group, but kidney and testis lipid peroxide levels in the low-AsA group were higher than those in the normal-AsA group. Dietary methionine tended to decrease tissue lipid peroxide levels but did not decrease vitamin E (VE) consumption. In experiment 2, a high level of methionine (6 g/kg) decreased liver lipid peroxide levels and VE consumption. However, generation of tissue lipid peroxides and VE consumption were not decreased further by a higher dose of methionine (9 g/kg). Higher than normal levels of dietary methionine are not necessarily associated with decreased dietary DHA-induced generation of tissue lipid peroxides and VE consumption except that the GSH requirement is increased in a condition such as acetaminophen feeding.

Fluorescence detection of glutathione and oxidized glutathione in blood with a NIR-excitable cyanine probe

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Liu, Chang-hui; Qi, Feng-pei; Wen, Fu-bin; Long, Li-ping; Liu, Ai-juan; Yang, Rong-hua

2018-04-01

Cyanine has been widely utilized as a near infrared (NIR) fluorophore for detection of glutathione (GSH). However, the excitation of most of the reported cyanine-based probes was less than 800 nm, which inevitably induce biological background absorption and lower the sensitivity, limiting their use for detection of GSH in blood samples. To address this issue, here, a heptamethine cyanine probe (DNIR), with a NIR excitation wavelength at 804 nm and a NIR emission wavelength at 832 nm, is employed for the detection of GSH and its oxidized form (GSSG) in blood. The probe displays excellent selectivity for GSH over GSSG and other amino acids, and rapid response to GSH, in particular a good property for indirect detection of GSSG in the presence of enzyme glutathione reductase and the reducing agent nicotinamideadenine dinucleotide phosphate, without further separation prior to fluorescent measurement. To the best of our knowledge, this is the first attempt to explore NIR fluorescent approach for the simultaneous assay of GSH and GSSG in blood. As such, we expect that our fluorescence sensors with both NIR excitation and NIR emission make this strategy suitable for the application in complex physiological systems.

Toxicity of nickel and silver to Nostoc muscorum: interaction with ascorbic acid, glutathione, and sulfur-containing amino acids.

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Rai, L C; Raizada, M

1987-08-01

Exposure of Nostoc muscorum to different concentrations of Ni and Ag brought about reduction in growth, carbon fixation, heterocyst production, and nitrogenase activity and increase in the loss of ions (K+, Na+). In an attempt to ameliorate the toxicity of test metals by ascorbic acid, glutathione, and sulfur-containing amino acids (L-cysteine and L-methionine), it was found that the level of protection by ascorbic acid and glutathione was more for Ag than Ni. However, metal-induced inhibition of growth and carbon fixation was equally ameliorated by methionine. But the level of protection by cysteine was quite different, i.e., 27% for Ni and 22% for Ag. Protection of metal toxicity in N. muscorum by amino acids lends further support to self-detoxifying ability of cyanobacteria because they are known to synthesize all essential amino acids.

The role of nuclear factor E2-Related factor 2 and uncoupling protein 2 in glutathione metabolism: Evidence from an in vivo gene knockout study

International Nuclear Information System (INIS)

Chen, Yanyan; Xu, Yuanyuan; Zheng, Hongzhi; Fu, Jingqi; Hou, Yongyong; Wang, Huihui; Zhang, Qiang; Yamamoto, Masayuki; Pi, Jingbo

2016-01-01

Nuclear factor E2-related factor 2 (NRF2) and uncoupling protein 2 (UCP2) are indicated to protect from oxidative stress. They also play roles in the homeostasis of glutathione. However, the detailed mechanisms are not well understood. In the present study, we found Nrf2-knockout (Nrf2-KO) mice exhibited altered glutathione homeostasis and reduced expression of various genes involved in GSH biosynthesis, regeneration, utilization and transport in the liver. Ucp2-knockout (Ucp2-KO) mice exhibited altered glutathione homeostasis in the liver, spleen and blood, as well as increased transcript of cystic fibrosis transmembrane conductance regulator in the liver, a protein capable of mediating glutathione efflux. Nrf2-Ucp2-double knockout (DKO) mice showed characteristics of both Nrf2-KO and Ucp2-KO mice. But no significant difference was observed in DKO mice when compared with Nrf2-KO or Ucp2-KO mice, except in blood glutathione levels. These data suggest that ablation of Nrf2 and Ucp2 leads to disrupted GSH balance, which could result from altered expression of genes involved in GSH metabolism. DKO may not evoke more severe oxidative stress than the single gene knockout. - Highlights: • Nrf2/Ucp2 deficiency leads to alteration of glutathione homeostasis. • Nrf2 regulates expression of genes in glutathione generation and utilization. • Ucp2 affects glutathione metabolism by regulating hepatic efflux of glutathione. • Nrf2 deficiency may not aggravate oxidative stress in Ucp2-deficient mice.

The role of nuclear factor E2-Related factor 2 and uncoupling protein 2 in glutathione metabolism: Evidence from an in vivo gene knockout study

Energy Technology Data Exchange (ETDEWEB)

Chen, Yanyan [The First Affiliated Hospital, China Medical University, Shenyang, Liaoning (China); The Hamner Institutes for Health Sciences, Research Triangle Park, NC (United States); Xu, Yuanyuan, E-mail: yyxu@cmu.edu.cn [School of Public Health, China Medical University, Shenyang, Liaoning (China); Zheng, Hongzhi [The First Affiliated Hospital, China Medical University, Shenyang, Liaoning (China); The Hamner Institutes for Health Sciences, Research Triangle Park, NC (United States); Fu, Jingqi; Hou, Yongyong; Wang, Huihui [School of Public Health, China Medical University, Shenyang, Liaoning (China); Zhang, Qiang [Rollins School of Public Health, Emory University, Atlanta, GA (United States); Yamamoto, Masayuki [Graduate School of Medicine, Tohoku University, Sendai (Japan); Pi, Jingbo, E-mail: jbpi@cmu.edu.cn [School of Public Health, China Medical University, Shenyang, Liaoning (China); The Hamner Institutes for Health Sciences, Research Triangle Park, NC (United States)

2016-09-09

Nuclear factor E2-related factor 2 (NRF2) and uncoupling protein 2 (UCP2) are indicated to protect from oxidative stress. They also play roles in the homeostasis of glutathione. However, the detailed mechanisms are not well understood. In the present study, we found Nrf2-knockout (Nrf2-KO) mice exhibited altered glutathione homeostasis and reduced expression of various genes involved in GSH biosynthesis, regeneration, utilization and transport in the liver. Ucp2-knockout (Ucp2-KO) mice exhibited altered glutathione homeostasis in the liver, spleen and blood, as well as increased transcript of cystic fibrosis transmembrane conductance regulator in the liver, a protein capable of mediating glutathione efflux. Nrf2-Ucp2-double knockout (DKO) mice showed characteristics of both Nrf2-KO and Ucp2-KO mice. But no significant difference was observed in DKO mice when compared with Nrf2-KO or Ucp2-KO mice, except in blood glutathione levels. These data suggest that ablation of Nrf2 and Ucp2 leads to disrupted GSH balance, which could result from altered expression of genes involved in GSH metabolism. DKO may not evoke more severe oxidative stress than the single gene knockout. - Highlights: • Nrf2/Ucp2 deficiency leads to alteration of glutathione homeostasis. • Nrf2 regulates expression of genes in glutathione generation and utilization. • Ucp2 affects glutathione metabolism by regulating hepatic efflux of glutathione. • Nrf2 deficiency may not aggravate oxidative stress in Ucp2-deficient mice.

A multidomain fusion protein in Listeria monocytogenes catalyzes the two primary activities for glutathione biosynthesis.

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Gopal, Shubha; Borovok, Ilya; Ofer, Amos; Yanku, Michaela; Cohen, Gerald; Goebel, Werner; Kreft, Jürgen; Aharonowitz, Yair

2005-06-01

Glutathione is the predominant low-molecular-weight peptide thiol present in living organisms and plays a key role in protecting cells against oxygen toxicity. Until now, glutathione synthesis was thought to occur solely through the consecutive action of two physically separate enzymes, gamma-glutamylcysteine ligase and glutathione synthetase. In this report we demonstrate that Listeria monocytogenes contains a novel multidomain protein (termed GshF) that carries out complete synthesis of glutathione. Evidence for this comes from experiments which showed that in vitro recombinant GshF directs the formation of glutathione from its constituent amino acids and the in vivo effect of a mutation in GshF that abolishes glutathione synthesis, results in accumulation of the intermediate gamma-glutamylcysteine, and causes hypersensitivity to oxidative agents. We identified GshF orthologs, consisting of a gamma-glutamylcysteine ligase (GshA) domain fused to an ATP-grasp domain, in 20 gram-positive and gram-negative bacteria. Remarkably, 95% of these bacteria are mammalian pathogens. A plausible origin for GshF-dependent glutathione biosynthesis in these bacteria was the recruitment by a GshA ancestor gene of an ATP-grasp gene and the subsequent spread of the fusion gene between mammalian hosts, most likely by horizontal gene transfer.

Vitamin D is positively associated with sperm motility and increases intracellular calcium in human spermatozoa

DEFF Research Database (Denmark)

Blomberg Jensen, Martin; Bjerrum, Poul J; Jessen, Torben E

2011-01-01

BACKGROUND The vitamin D receptor (VDR) is expressed in human spermatozoa, and VDR-knockout mice and vitamin D (VD) deficiency in rodents results in impaired fertility, low sperm counts and a low number of motile spermatozoa. We investigated the role of activated VD (1,25(OH)(2)D(3)) in human...... spermatozoa and whether VD serum levels are associated with semen quality. METHODS Cross-sectional association study of semen quality and VD serum level in 300 men from the general population, and in vitro studies on spermatozoa from 40 men to investigate the effects of VD on intracellular calcium, sperm......M). 1,25(OH)(2)D(3) increased intracellular calcium concentration in human spermatozoa through VDR-mediated calcium release from an intracellular calcium storage, increased sperm motility and induced the acrosome reaction in vitro. CONCLUSIONS 1,25(OH)(2)D(3) increased intracellular calcium...

Insecticide resistance and intracellular proteases.

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Wilkins, Richard M

2017-12-01

Pesticide resistance is an example of evolution in action with mechanisms of resistance arising from mutations or increased expression of intrinsic genes. Intracellular proteases have a key role in maintaining healthy cells and in responding to stressors such as pesticides. Insecticide-resistant insects have constitutively elevated intracellular protease activity compared to corresponding susceptible strains. This increase was shown for some cases originally through biochemical enzyme studies and subsequently putatively by transcriptomics and proteomics methods. Upregulation and expression of proteases have been characterised in resistant strains of some insect species, including mosquitoes. This increase in proteolysis results in more degradation products (amino acids) of intracellular proteins. These may be utilised in the resistant strain to better protect the cell from stress. There are changes in insect intracellular proteases shortly after insecticide exposure, suggesting a role in stress response. The use of protease and proteasome inhibitors or peptide mimetics as synergists with improved application techniques and through protease gene knockdown using RNA interference (possibly expressed in crop plants) may be potential pest management strategies, in situations where elevated intracellular proteases are relevant. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

Possible role of glutathione in predicting radiotherapy response of cervix cancer

International Nuclear Information System (INIS)

Jadhav, Ganesh Krishna R.; Bhanumathi, Pullara; Devi, Pathirissery Uma; Seetharamaiah, Tattikonda; Vidyasagar, Mamudipudi S.; Rao, Kilari Koteshwer; Hospet, Chandrashekhar S.; Solomon, Johnson Gilbert R.

1998-01-01

Purpose: To see if changes in tumor/blood glutathione (GSH) levels after one fraction of radiotherapy can be correlated with the treatment response in patients with carcinoma of the uterine cervix. Methods and Materials: The study was done on 45 patients with squamous cell carcinoma of the uterine cervix, FIGO Stages IIB (17 patients) and IIIB (28 patients). Stage IIB patients received 35 Gy of cobalt-60 external radiotherapy (RT) in 16 fractions over 4 weeks with a concurrent high-dose-rate intracavitary dose of 8.5 Gy to point A once a week. Stage IIIB patients were given 45 Gy of RT in 20 fractions over 5 weeks, followed by two doses of intracavitary therapy once a week. Blood and tumor samples were collected before and after one dose of RT and GSH was estimated. Tumor response was assessed clinically at 1 month after treatment. Results: Glutathione levels in both blood and tumor showed a significant decrease after one fraction of RT, but the degree of decrease varied among patients. There was a good correlation between the extent of GSH decrease and the tumor response. All patients who had complete response (CR) (seven Stage IIB and eight Stage IIIB) showed ≥70% decrease in both tumor and blood GSH, while those who had <50% regression (NR) (five Stage IIB and 13 Stage IIIB) showed <50% decrease in GSH. The partial responders recorded an intermediate level (50-70%) of depletion in blood and tumor GSH. Conclusions: The results indicate that the changes in tumor/blood GSH levels after one fraction of RT could serve as an index of tumor response to therapy and may help in identifying radioresistant tumors, at least in the case of cervix carcinoma

Prolonged fasting increases glutathione biosynthesis in postweaned northern elephant seals

Science.gov (United States)

Vázquez-Medina, José Pablo; Zenteno-Savín, Tania; Forman, Henry Jay; Crocker, Daniel E.; Ortiz, Rudy M.

2011-01-01

SUMMARY Northern elephant seals experience prolonged periods of absolute food and water deprivation (fasting) while breeding, molting or weaning. The postweaning fast in elephant seals is characterized by increases in the renin–angiotensin system, expression of the oxidant-producing protein Nox4, and NADPH oxidase activity; however, these increases are not correlated with increased oxidative damage or inflammation. Glutathione (GSH) is a potent reductant and a cofactor for glutathione peroxidases (GPx), glutathione-S transferases (GST) and 1-cys peroxiredoxin (PrxVI) and thus contributes to the removal of hydroperoxides, preventing oxidative damage. The effects of prolonged food deprivation on the GSH system are not well described in mammals. To test our hypothesis that GSH biosynthesis increases with fasting in postweaned elephant seals, we measured circulating and muscle GSH content at the early and late phases of the postweaning fast in elephant seals along with the activity/protein content of glutamate-cysteine ligase [GCL; catalytic (GCLc) and modulatory (GCLm) subunits], γ-glutamyl transpeptidase (GGT), glutathione disulphide reductase (GR), glucose-6-phosphate dehydrogenase (G6PDH), GST and PrxVI, as well as plasma changes in γ-glutamyl amino acids, glutamate and glutamine. GSH increased two- to four-fold with fasting along with a 40–50% increase in the content of GCLm and GCLc, a 75% increase in GGT activity, a two- to 2.5-fold increase in GR, G6PDH and GST activities and a 30% increase in PrxVI content. Plasma γ-glutamyl glutamine, γ-glutamyl isoleucine and γ-glutamyl methionine also increased with fasting whereas glutamate and glutamine decreased. Results indicate that GSH biosynthesis increases with fasting and that GSH contributes to counteracting hydroperoxide production, preventing oxidative damage in fasting seals. PMID:21430206

Influence of dietary cholesterol on 26-hydroxycholesterol and the effect of 26-hydroxycholesterol on the intracellular free calcium level

International Nuclear Information System (INIS)

Kou, I.L.

1987-01-01

The purpose of this study was to investigate the factors influencing serum level of 26-hydroxycholesterol after long-term consumption of cholesterol by animals. It is also to examine the effect of this sterol on intracellular free calcium level. Purified 26-hydroxycholesterol was synthesized from kryptogenin by the Clemmemsen and Wolff-Kishner reduction method. 26-Hydroxycholesterol was also used for fatty acid esters syntheses, and to study its influence on membranes. Tritiated 26-hydroxycholesterol which was synthesized by an enzymatic method, was used to monitor the 26-hydroxycholesterol loss during the procedure. The ester form of 26-hydroxycholesterol was also synthesized, and used to investigate its effects on membranes. The HPLC method that was developed for the analysis of 26-hydroxycholesterol levels in animal tissues was accurate, efficient, and reproducible for the determination of 26-hydroxycholesterol in plasma. However, it was not suitable for the analysis of other tissues, due to the overlapping of peaks making quantitation difficult

Scanning electrochemical microscopy of menadione-glutathione conjugate export from yeast cells

Science.gov (United States)

Mauzeroll, Janine; Bard, Allen J.

2004-01-01

The uptake of menadione (2-methyl-1,4-naphthoquinone), which is toxic to yeast cells, and its expulsion as a glutathione complex were studied by scanning electrochemical microscopy. The progression of the in vitro reaction between menadione and glutathione was monitored electrochemically by cyclic voltammetry and correlated with the spectroscopic (UV–visible) behavior. By observing the scanning electrochemical microscope tip current of yeast cells suspended in a menadione-containing solution, the export of the conjugate from the cells with time could be measured. Similar experiments were performed on immobilized yeast cell aggregates stressed by a menadione solution. From the export of the menadione-glutathione conjugate detected at a 1-μm-diameter electrode situated 10 μm from the cells, a flux of about 30,000 thiodione molecules per second per cell was extracted. Numerical simulations based on an explicit finite difference method further revealed that the observation of a constant efflux of thiodione from the cells suggested the rate was limited by the uptake of menadione and that the efflux through the glutathione-conjugate pump was at least an order of magnitude faster. PMID:15148374

Formation of the accumulative human metabolite and human-specific glutathione conjugate of diclofenac in TK-NOG chimeric mice with humanized livers.

Science.gov (United States)

Kamimura, Hidetaka; Ito, Satoshi; Nozawa, Kohei; Nakamura, Shota; Chijiwa, Hiroyuki; Nagatsuka, Shin-ichiro; Kuronuma, Miyuki; Ohnishi, Yasuyuki; Suemizu, Hiroshi; Ninomiya, Shin-ichi

2015-03-01

3'-Hydroxy-4'-methoxydiclofenac (VI) is a human-specific metabolite known to accumulate in the plasma of patients after repeated administration of diclofenac sodium. Diclofenac also produces glutathione-conjugated metabolites, some of which are human-specific. In the present study, we investigated whether these metabolites could be generated in humanized chimeric mice produced from TK-NOG mice. After a single oral administration of diclofenac to humanized mice, the unchanged drug in plasma peaked at 0.25 hour and then declined with a half-life (t1/2) of 2.4 hours. 4'-Hydroxydiclofenac (II) and 3'-hydroxydiclofenac also peaked at 0.25 hour and were undetectable within 24 hours. However, VI peaked at 8 hours and declined with a t1/2 of 13 hours. When diclofenac was given once per day, peak and trough levels of VI reached plateau within 3 days. Studies with administration of II suggested VI was generated via II as an intermediate. Among six reported glutathione-conjugated metabolites of diclofenac, M1 (5-hydroxy-4-(glutathion-S-yl)diclofenac) to M6 (2'-(glutathion-S-yl)monoclofenac), we found three dichlorinated conjugates [M1, M2 (4'-hydroxy-3'-(glutathion-S-yl)diclofenac), and M3 (5-hydroxy-6-(glutathion-S-yl)diclofenac)], and a single monochlorinated conjugate [M4 (2'-hydroxy-3'-(glutathion-S-yl)monoclofenac) or M5 (4'-hydroxy-2'-(glutathion-S-yl)monoclofenac)], in the bile of humanized chimeric mice. M4 and M5 are positional isomers and have been previously reported as human-specific in vitro metabolites likely generated via arene oxide and quinone imine-type intermediates, respectively. The biliary monochlorinated metabolite exhibited the same mass spectrum as those of M4 and M5, and we discuss whether this conjugate corresponded to M4 or M5. Overall, humanized TK-NOG chimeric mice were considered to be a functional tool for the study of drug metabolism of diclofenac in humans. Copyright © 2015 by The American Society for Pharmacology and Experimental

Effect of an aqueous extract of Cucurbita ficifolia Bouché on the glutathione redox cycle in mice with STZ-induced diabetes.

Science.gov (United States)

Díaz-Flores, M; Angeles-Mejia, S; Baiza-Gutman, L A; Medina-Navarro, R; Hernández-Saavedra, D; Ortega-Camarillo, C; Roman-Ramos, R; Cruz, M; Alarcon-Aguilar, F J

2012-10-31

Cucurbita ficifolia is used in Mexican traditional medicine as an anti-diabetic and anti-inflammatory agent and its actions can be mediated by antioxidant mechanisms. Disturbance in the homeostasis of glutathione has been implicated in the etiology and progression of diabetes mellitus and its complications. It was evaluated, the effect of an aqueous extract of Cucurbita ficifolia on glycemia, plasma lipid peroxidation; as well as levels of reduced (GSH) and oxidized (GSSG) glutathione and activities of enzymes involved in glutathione redox cycle: glutathione peroxidase (GPx) and glutathione reductase (GR) in liver, pancreas, kidney and heart homogenates of streptozotocin-induced diabetic mice. Increased blood glucose and lipid peroxidation, together with decreased of GSH concentration, GSH/GSSG ratio and its redox potential (E(h)), and enhanced activity of GPx and GR in liver, pancreas and kidney were the salient features observed in diabetic mice. Administration of the aqueous extract of Cucurbita ficifolia to diabetic mice for 30 days, used at a dose of 200 mg/kg, resulted in a significant reduction in glycemia, polydipsia, hyperphagia and plasma lipid peroxidation. Moreover, GSH was increased in liver, pancreas and kidney, and GSSG was reduced in liver, pancreas and heart, therefore GSH/GSSG ratio and its E(h) were restored. Also, the activities involved in the glutathione cycle were decreased, reaching similar values to controls. An aqueous extract of Cucurbita ficifolia with hypoglycemic action, improve GSH redox state, increasing glutathione pool, GSH, GSH/GSSG ratio and its E(h), mechanism that can explain, at least in part, its antioxidant properties, supporting its use as an alternative treatment for the control of diabetes mellitus, and prevent the induction of complications by oxidative stress. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Induction of Apoptosis and Reduction of Endogenous Glutathione Level by the Ethyl-Acetate Soluble Fraction of the Methanol Extract of the Roots of Potentilla fulgens in Cancer Cells.

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Debabrata Tripathy

Full Text Available Potentilla fulgens root traditionally used as a folk remedy in Meghalaya, India. However, systematic evaluation of its anticancer efficacy was limited. We investigated the anticancer potentials of the various extracts prepared by partitioning of the methanol extract of the root with the aim to discover major contributing factors from the most effective fractions. Methanol extract of P. fulgens roots (PRE was prepared by maceration which was subsequently fractionated into hexane, ethyl-acetate (EA and n-butanol soluble fractions. Various assays (clonogenic assay, Flow cytometry analysis, western blot, semiquantitative RT-PCR and the level of endogenous glutathione were used to evaluate different parameters, such as Cell survivability, PARP-1 proteolysis, expression pattern of anti-apoptotic and γ-glutamyl-cysteine synthetase heavy subunit (GCSC genes in both MCF-7 and U87 cancer cell lines. Since the EA-fraction showed most efficient growth inhibitory effect, it was further purified and a total of nine compounds and some monomeric and dimeric flavan-3-ols were identified and characterized. Three compounds viz., epicatechin (EC, gallic acid (GA and ursolic acid (UA were taken on the basis of their higher yield and 10 μg/ml of each was mixed together. The concentration used in this study for PRE, EA- and Hex-fraction was 100 μg/ml, which was higher than the IC50 value. Apoptotic cell death in the PRE, EA-fraction and EC+GA+UA treated cancer cell cultures was significantly greater than in normal cells due to suppression of anti-apoptotic protein Bcl2 following treatment. Depletion of glutathione by downregulating GCSC was also observed. Induction of apoptosis and lowering the level of glutathione are considered to be positive activity for an anticancer agent. Therefore, modulation of GSH concentration in tumor cells by PRE and its EA-fraction opened up the possibility of a new therapeutic approach because these plant products are not harmful to

Depression of biliary glutathione excretion by chronic ethanol feeding in the rat

International Nuclear Information System (INIS)

Vendemiale, G.; Jayatilleke, E.; Shaw, S.; Lieber, C.S.

1984-01-01

The effects of chronic alcohol feeding on biliary glutathione excretion were studied in rats pair fed diets containing either ethanol (36% of total energy) or isocaloric carbohydrate for 4-6 weeks. An exteriorized biliary-duodenal fistula was established and total glutathione (GSH) and oxidized glutathione (GSSG) were measured. A significant decrease was observed in rats fed alcohol chronically compared to their pair fed controls in the biliary excretion of GSH (55.7 +/- 37.0 vs 243.1 +/- 29.0 μg/ml bile, p 35 -L-methionine incorporation into hepatic and biliary GSH was unchanged or even increased after chronic ethanol feeding. 22 references, 4 figures

Caveats and limitations of plate reader-based high-throughput kinetic measurements of intracellular calcium levels

International Nuclear Information System (INIS)

Heusinkveld, Harm J.; Westerink, Remco H.S.

2011-01-01

Calcium plays a crucial role in virtually all cellular processes, including neurotransmission. The intracellular Ca 2+ concentration ([Ca 2+ ] i ) is therefore an important readout in neurotoxicological and neuropharmacological studies. Consequently, there is an increasing demand for high-throughput measurements of [Ca 2+ ] i , e.g. using multi-well microplate readers, in hazard characterization, human risk assessment and drug development. However, changes in [Ca 2+ ] i are highly dynamic, thereby creating challenges for high-throughput measurements. Nonetheless, several protocols are now available for real-time kinetic measurement of [Ca 2+ ] i in plate reader systems, though the results of such plate reader-based measurements have been questioned. In view of the increasing use of plate reader systems for measurements of [Ca 2+ ] i a careful evaluation of current technologies is warranted. We therefore performed an extensive set of experiments, using two cell lines (PC12 and B35) and two fluorescent calcium-sensitive dyes (Fluo-4 and Fura-2), for comparison of a linear plate reader system with single cell fluorescence microscopy. Our data demonstrate that the use of plate reader systems for high-throughput real-time kinetic measurements of [Ca 2+ ] i is associated with many pitfalls and limitations, including erroneous sustained increases in fluorescence, limited sensitivity and lack of single cell resolution. Additionally, our data demonstrate that probenecid, which is often used to prevent dye leakage, effectively inhibits the depolarization-evoked increase in [Ca 2+ ] i . Overall, the data indicate that the use of current plate reader-based strategies for high-throughput real-time kinetic measurements of [Ca 2+ ] i is associated with caveats and limitations that require further investigation. - Research highlights: → The use of plate readers for high-throughput screening of intracellular Ca 2+ is associated with many pitfalls and limitations. → Single cell

Auranofin-induced oxidative stress causes redistribution of the glutathione pool in Taenia crassiceps cysticerci.

Science.gov (United States)

Martínez-González, J J; Guevara-Flores, A; Rendón, J L; Arenal, I P Del

2015-05-01

Previously, we have studied the effect of the gold-compound auranofin (AF) on both thioredoxin-glutathione reductasa (TGR) activity and viability of Taenia crassiceps cysticerci. It was demonstrated that micromolar concentrations of AF were high enough to fully inhibit TGR and kill the parasites. In this work, the dynamics of changes in the glutathione pool of T. crassiceps cysticerci following the addition of AF, was analyzed. A dose-dependent decrease in the internal glutathione concentration, concomitant with an increase in ROS production was observed. These changes were simultaneous with the formation of glutathione-protein complexes and the export of glutathione disulfide (GSSG) to the culture medium. Incubation of cysticerci in the presence of both AF and N-acetyl cysteine (NAC) prevents all the above changes, maintaining cysticerci viability. By contrast, the presence of both AF and buthionine sulfoximine (BSO) resulted in a potentiation of the effects of the gold compound, jeopardizing cysticerci viability. These results suggest the lethal effect of AF on T. crassiceps cysticerci, observed at micromolar concentrations, can be explained as a consequence of major changes in the glutathione status, which results in a significant increase in the oxidative stress of the parasites. Copyright © 2015 Elsevier B.V. All rights reserved.

Development and characterization of glutathione-conjugated albumin nanoparticles for improved brain delivery of hydrophilic fluorescent marker.

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Patel, Prerak J; Acharya, Niyati S; Acharya, Sanjeev R

2013-01-01

The glutathione-conjugated bovine serum albumin (BSA) nanoparticles were constructed in the present exploration as a novel biodegradable carrier for brain-specific drug delivery with evaluation of its in vitro and in vivo delivery properties. BSA nanocarriers were activated and conjugated to the distal amine functions of the glutathione via carbodiimide chemistry using EDAC as a mediator. These nanoparticles were characterized for particle shape, average size, SPAN value, drug entrapment and in vitro drug release. Further, presence of glutathione on the surface of BSA nanoparticles was confirmed by Ellman's assay, which has suggested that approximately 750 units of glutathione were conjugated per BSA nanoparticle. To evaluate the brain delivery properties of the glutathione-conjugated BSA nanoparticles fluorescein sodium was used as a model hydrophilic compound. Permeability and neuronal uptake properties of developed formulations were evaluated against the MDCK-MDR1 endothelial and neuro-glial cells, respectively. The permeability of glutathione-conjugated BSA nanoparticles across the monolayer of MDCK-MDR1 endothelial tight junction was shown significantly higher than that of unconjugated nanoparticles and fluorescein sodium solution. Similarly, glutathione-conjugated nanoparticles exhibited considerably higher uptake by neuro-glial cells which was inferred by high fluorescence intensity under microscope in comparison to unconjugated nanoparticles and fluorescein sodium solution. Following an intravenous administration, nearly three folds higher fluorescein sodium was carried to the rat brain by glutathione-conjugated nanoparticles as compared to unconjugated nanoparticles. The significant in vitro and in vivo results suggest that glutathione-conjugated BSA nanoparticles is a promising brain drug delivery system with low toxicity.

Toll-Like Receptor 4 Reduces Oxidative Injury via Glutathione Activity in Sheep

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Shoulong Deng

2016-01-01

Full Text Available Toll-like receptor 4 (TLR4 is an important sensor of Gram-negative bacteria and can trigger activation of the innate immune system. Increased activation of TLR4 can lead to the induction of oxidative stress. Herein, the pathway whereby TLR4 affects antioxidant activity was studied. In TLR4-overexpressing sheep, TLR4 expression was found to be related to the integration copy number when monocytes were challenged with lipopolysaccharide (LPS. Consequently, production of malondialdehyde (MDA was increased, which could increase the activation of prooxidative stress enzymes. Meanwhile, activation of an antioxidative enzyme, glutathione peroxidase (GSH-Px, was increased. Real-time PCR showed that expression of activating protein-1 (AP-1 and the antioxidative-related genes was increased. By contrast, the expression levels of superoxide dismutase 1 (SOD1 and catalase (CAT were reduced. In transgenic sheep, glutathione (GSH levels were dramatically reduced. Furthermore, transgenic sheep were intradermally injected with LPS in each ear. The amounts of inflammatory infiltrates were correlated with the number of TLR4 copies that were integrated in the genome. Additionally, the translation of γ-glutamylcysteine synthetase (γ-GCS was increased. Our findings indicated that overexpression of TLR4 in sheep could ameliorate oxidative injury through GSH secretion that was induced by LPS stimulation. Furthermore, TLR4 promoted γ-GCS translation through the AP-1 pathway, which was essential for GSH synthesis.

Glutathione, Glutaredoxins, and Iron.

Science.gov (United States)

Berndt, Carsten; Lillig, Christopher Horst

2017-11-20

Glutathione (GSH) is the most abundant cellular low-molecular-weight thiol in the majority of organisms in all kingdoms of life. Therefore, functions of GSH and disturbed regulation of its concentration are associated with numerous physiological and pathological situations. Recent Advances: The function of GSH as redox buffer or antioxidant is increasingly being questioned. New functions, especially functions connected to the cellular iron homeostasis, were elucidated. Via the formation of iron complexes, GSH is an important player in all aspects of iron metabolism: sensing and regulation of iron levels, iron trafficking, and biosynthesis of iron cofactors. The variety of GSH coordinated iron complexes and their functions with a special focus on FeS-glutaredoxins are summarized in this review. Interestingly, GSH analogues that function as major low-molecular-weight thiols in organisms lacking GSH resemble the functions in iron homeostasis. Since these iron-related functions are most likely also connected to thiol redox chemistry, it is difficult to distinguish between mechanisms related to either redox or iron metabolisms. The ability of GSH to coordinate iron in different complexes with or without proteins needs further investigation. The discovery of new Fe-GSH complexes and their physiological functions will significantly advance our understanding of cellular iron homeostasis. Antioxid. Redox Signal. 27, 1235-1251.

A mathematical modeling approach to assessing the reliabilty of biomarkers of glutathione metabolism.

NARCIS (Netherlands)

Geenen, S.; du Preez, F.B.; Reed, M.; Nijhout, H.F.; Kenna, J.G.; Wilson, I.D.; Westerhoff, H.V.; Snoep, J.L.

2012-01-01

One of the main pathways for the detoxification of reactive metabolites in the liver involves glutathione conjugation. Metabolic profiling studies have shown paradoxical responses in glutathione-related biochemical pathways. One of these is the increase in 5-oxoproline and ophthalmic acid

Cysteine- and glutathione-mediated uptake of lead and cadmium into Zea mays and Brassica napus roots

International Nuclear Information System (INIS)

Vadas, Timothy M.; Ahner, Beth A.

2009-01-01

This study examines a new mechanism for the uptake of Pb and Cd into Brassica napus and Zea mays roots. During hydroponic experiments, the uptake of Pb and Cd was enhanced in the presence of cysteine and glutathione, whereas no or very low uptake was observed in EDTA and penicillamine controls. Uptake rates were also enhanced after pre-exposure to cysteine or glutathione and inhibited in the presence of vanadate, suggesting a biological mechanism of uptake. Increasing concentrations of glutathione in solution resulted in decreasing Pb uptake rates, indicating competition for transport between free-glutathione and Pb-glutathione species. Pb uptake in the presence of increasing cysteine concentrations resulted in decreased uptake initially but linearly increasing uptake at higher concentrations. Experimentation showed concentration dependent Pb uptake rates. We speculate that there are specific transporters for these thiol ligands and describe what barriers remain for application of this novel transport mechanism in chelator-assisted phytoremediation. - Cysteine and glutathione mediate the transport of lead and cadmium into plant roots.

Cysteine- and glutathione-mediated uptake of lead and cadmium into Zea mays and Brassica napus roots

Energy Technology Data Exchange (ETDEWEB)

Vadas, Timothy M., E-mail: tvadas@umbc.ed [Department of Biological and Environmental Engineering, Cornell University, 320 Riley-Robb Hall, Ithaca, NY 14853 (United States); Ahner, Beth A., E-mail: baa7@cornell.ed [Department of Biological and Environmental Engineering, Cornell University, 320 Riley-Robb Hall, Ithaca, NY 14853 (United States)

2009-08-15

This study examines a new mechanism for the uptake of Pb and Cd into Brassica napus and Zea mays roots. During hydroponic experiments, the uptake of Pb and Cd was enhanced in the presence of cysteine and glutathione, whereas no or very low uptake was observed in EDTA and penicillamine controls. Uptake rates were also enhanced after pre-exposure to cysteine or glutathione and inhibited in the presence of vanadate, suggesting a biological mechanism of uptake. Increasing concentrations of glutathione in solution resulted in decreasing Pb uptake rates, indicating competition for transport between free-glutathione and Pb-glutathione species. Pb uptake in the presence of increasing cysteine concentrations resulted in decreased uptake initially but linearly increasing uptake at higher concentrations. Experimentation showed concentration dependent Pb uptake rates. We speculate that there are specific transporters for these thiol ligands and describe what barriers remain for application of this novel transport mechanism in chelator-assisted phytoremediation. - Cysteine and glutathione mediate the transport of lead and cadmium into plant roots.

Effect of insulin resistance on intracellular signal transduction of vessels in diabetic

International Nuclear Information System (INIS)

Cen Rongguang; Wei Shaoying; Mo Xingju

2003-01-01

To investigate the relationship between the insulin resistance (IR) and the intracellular signal transduction of vessels, changes in fasting blood glucose (FBG), fasting insulin (FINS), triglyceride (TG), total cholesterol (TC), inositol triphosphate (IP 3 ), protein kinase C(PKC) and intracellular total calcium concentration in 31 diabetic patients were compared with those of 39 normal controls. The levels of FBG, FINS, TG and TC in diabetic patients were significantly higher than those of normal controls (P 3 and PKC in diabetic patients were significantly lower than those of normal controls (P<0.01). The results suggest that there is a causal relation between insulin resistance and abnormalities of cellular calcium metabolism and intracellular signal transduction of vessels

Novel agmatine analogue, γ-guanidinooxypropylamine (GAPA) efficiently inhibits proliferation of Leishmania donovani by depletion of intracellular polyamine levels

International Nuclear Information System (INIS)

Singh, Sushma; Jhingran, Anupam; Sharma, Ankur; Simonian, Alina R.; Soininen, Pasi; Vepsalainen, Jouko; Khomutov, Alex R.; Madhubala, Rentala

2008-01-01

The efficacy of γ-guanidinooxypropylamine (GAPA), a novel agmatine analogue against protozoan parasite, Leishmaniadonovani was evaluated. Wild-type and ornithine decarboxylase-overexpressors of L. donovani were used to study the effect and mode of action of this inhibitor. GAPA inhibited the growth of both promastigotes and amastigotes. Ornithine decarboxylase (ODC) activity and polyamine levels were markedly lower in cells treated with GAPA and proliferation was rescued by addition of putrescine or spermidine. GAPA inhibited L. donovani recombinant ODC with K i value of ∼60 μM. The ODC-overexpressors showed significant resistance to GAPA. GAPA has pK a 6.71 and at physiological pH the analogue can mimic protonated state of putrescine and can probably use putrescine transport system. Transport of putrescine in wild-type L. donovani promastigotes was inhibited by GAPA. We for the first time report that GAPA is a potential antileishmanial lead compound and it possibly inhibits L. donovani growth by depletion of intracellular polyamine levels

Protective effect of Pycnogenol in human neuroblastoma SH-SY5Y cells following acrolein-induced cytotoxicity.

Science.gov (United States)

Ansari, Mubeen A; Keller, Jeffrey N; Scheff, Stephen W

2008-12-01

Oxidative stress is one of the hypotheses involved in the etiology of Alzheimer's disease (AD). Considerable attention has been focused on increasing the intracellular glutathione (GSH) levels in many neurodegenerative diseases, including AD. Pycnogenol (PYC) has antioxidant properties and stabilizes intracellular antioxidant defense systems including glutathione levels. The present study investigated the protective effects of PYC on acrolein-induced oxidative cell toxicity in cultured SH-SY5Y neuroblastoma cells. Decreased cell survival in SH-SY5Y cultures treated with acrolein correlated with oxidative stress, increased NADPH oxidase activity, free radical production, protein oxidation/nitration (protein carbonyl, 3-nitrotyrosine), and lipid peroxidation (4-hydroxy-2-nonenal). Pretreatment with PYC significantly attenuated acrolein-induced cytotoxicity, protein damage, lipid peroxidation, and cell death. A dose-response study suggested that PYC showed protective effects against acrolein toxicity by modulating oxidative stress and increasing GSH. These findings provide support that PYC may provide a promising approach for the treatment of oxidative stress-related neurodegenerative diseases such as AD.

Interleukin-6, intracellular adhesion molecule-1, and glycodelin A levels in serum and peritoneal fluid as biomarkers for endometriosis.

Science.gov (United States)

Mosbah, Alaa; Nabiel, Yasmin; Khashaba, Eman

2016-09-01

To compare levels of interleukin-6 (IL-6), intracellular adhesion molecule-1 (ICAM-1), and glycodelin A in serum and peritoneal fluid of patients with and without endometriosis, and to correlate levels with disease stage. An observational study was undertaken at Mansoura University Hospital, Egypt, between March 2014 and June 2015. Patients aged 21-48 years laparoscopically diagnosed with endometriosis and those without endometriosis who underwent laparoscopy for tubal ligation were included. Levels of IL-6, ICAM-1, and glycodelin A were measured in samples of serum and peritoneal fluid. Receiver operating characteristic curves were used to evaluate diagnostic accuracy. Forty-eight women with endometriosis and 20 without the disorder were included. IL-6 and glycodelin A levels in serum and peritoneal fluid were higher in the endometriosis group than in the control group (Pperitoneal fluid markers were 85.4% and 89.0%, 60.4% and 50.0%, and 89.6% and 90.0%, respectively. IL-6 and glycodelin A levels in serum and peritoneal fluid increased with disease stage (Pendometriosis and are positively correlated with the disease stage. Copyright © 2016 International Federation of Gynecology and Obstetrics. Published by Elsevier Ireland Ltd. All rights reserved.

Characterization of Affinity-Purified Isoforms of Acinetobacter calcoaceticus Y1 Glutathione Transferases

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Chin-Soon Chee

2014-01-01

Full Text Available Glutathione transferases (GST were purified from locally isolated bacteria, Acinetobacter calcoaceticus Y1, by glutathione-affinity chromatography and anion exchange, and their substrate specificities were investigated. SDS-polyacrylamide gel electrophoresis revealed that the purified GST resolved into a single band with a molecular weight (MW of 23 kDa. 2-dimensional (2-D gel electrophoresis showed the presence of two isoforms, GST1 (pI 4.5 and GST2 (pI 6.2 with identical MW. GST1 was reactive towards ethacrynic acid, hydrogen peroxide, 1-chloro-2,4-dinitrobenzene, and trans,trans-hepta-2,4-dienal while GST2 was active towards all substrates except hydrogen peroxide. This demonstrated that GST1 possessed peroxidase activity which was absent in GST2. This study also showed that only GST2 was able to conjugate GSH to isoproturon, a herbicide. GST1 and GST2 were suggested to be similar to F0KLY9 (putative glutathione S-transferase and F0KKB0 (glutathione S-transferase III of Acinetobacter calcoaceticus strain PHEA-2, respectively.

Characterization of Affinity-Purified Isoforms of Acinetobacter calcoaceticus Y1 Glutathione Transferases

Science.gov (United States)

Chee, Chin-Soon; Tan, Irene Kit-Ping; Alias, Zazali

2014-01-01

Glutathione transferases (GST) were purified from locally isolated bacteria, Acinetobacter calcoaceticus Y1, by glutathione-affinity chromatography and anion exchange, and their substrate specificities were investigated. SDS-polyacrylamide gel electrophoresis revealed that the purified GST resolved into a single band with a molecular weight (MW) of 23 kDa. 2-dimensional (2-D) gel electrophoresis showed the presence of two isoforms, GST1 (pI 4.5) and GST2 (pI 6.2) with identical MW. GST1 was reactive towards ethacrynic acid, hydrogen peroxide, 1-chloro-2,4-dinitrobenzene, and trans,trans-hepta-2,4-dienal while GST2 was active towards all substrates except hydrogen peroxide. This demonstrated that GST1 possessed peroxidase activity which was absent in GST2. This study also showed that only GST2 was able to conjugate GSH to isoproturon, a herbicide. GST1 and GST2 were suggested to be similar to F0KLY9 (putative glutathione S-transferase) and F0KKB0 (glutathione S-transferase III) of Acinetobacter calcoaceticus strain PHEA-2, respectively. PMID:24892084

Targeting Glutathione-S Transferase Enzymes in Musculoskeletal Sarcomas: A Promising Therapeutic Strategy

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Michela Pasello

2011-01-01

Full Text Available Recent studies have indicated that targeting glutathione-S-transferase (GST isoenzymes may be a promising novel strategy to improve the efficacy of conventional chemotherapy in the three most common musculoskeletal tumours: osteosarcoma, Ewing's sarcoma, and rhabdomyosarcoma. By using a panel of 15 drug-sensitive and drug-resistant human osteosarcoma, Ewing's sarcoma, and rhabdomyosarcoma cell lines, the efficay of the GST-targeting agent 6-(7-nitro-2,1,3-benzoxadiazol-4-ylthiohexanol (NBDHEX has been assessed and related to GST isoenzymes expression (namely GSTP1, GSTA1, GSTM1, and MGST. NBDHEX showed a relevant in vitro activity on all cell lines, including the drug-resistant ones and those with higher GSTs levels. The in vitro activity of NBDHEX was mostly related to cytostatic effects, with a less evident apoptotic induction. NBDHEX positively interacted with doxorubicin, vincristine, cisplatin but showed antagonistic effects with methotrexate. In vivo studies confirmed the cytostatic efficay of NBDHEX and its positive interaction with vincristine in Ewing's sarcoma cells, and also indicated a positive effect against the metastatisation of osteosarcoma cells. The whole body of evidence found in this study indicated that targeting GSTs in osteosarcoma, Ewing's sarcoma and rhabdomyosarcoma may be an interesting new therapeutic option, which can be considered for patients who are scarcely responsive to conventional regimens.

Promotion and Rescue of Intracellular Brucella neotomae Replication during Coinfection with Legionella pneumophila.

Science.gov (United States)

Kang, Yoon-Suk; Kirby, James E

2017-05-01

We established a new Brucella neotomae in vitro model system for study of type IV secretion system-dependent (T4SS) pathogenesis in the Brucella genus. Importantly, B. neotomae is a rodent pathogen, and unlike B. abortus , B. melitensis , and B. suis , B. neotomae has not been observed to infect humans. It therefore can be handled more facilely using biosafety level 2 practices. More particularly, using a series of novel fluorescent protein and lux operon reporter systems to differentially label pathogens and track intracellular replication, we confirmed T4SS-dependent intracellular growth of B. neotomae in macrophage cell lines. Furthermore, B. neotomae exhibited early endosomal (LAMP-1) and late endoplasmic reticulum (calreticulin)-associated phagosome maturation. These findings recapitulate prior observations for human-pathogenic Brucella spp. In addition, during coinfection experiments with Legionella pneumophila , we found that defective intracellular replication of a B. neotomae T4SS virB4 mutant was rescued and baseline levels of intracellular replication of wild-type B. neotomae were significantly stimulated by coinfection with wild-type but not T4SS mutant L. pneumophila Using confocal microscopy, it was determined that intracellular colocalization of B. neotomae and L. pneumophila was required for rescue and that colocalization came at a cost to L. pneumophila fitness. These findings were not completely expected based on known temporal and qualitative differences in the intracellular life cycles of these two pathogens. Taken together, we have developed a new system for studying in vitro Brucella pathogenesis and found a remarkable T4SS-dependent interplay between Brucella and Legionella during macrophage coinfection. Copyright © 2017 American Society for Microbiology.

Glutathione S-transferase gene polymorphisms in presbycusis.

Science.gov (United States)

Ateş, Nurcan Aras; Unal, Murat; Tamer, Lülüfer; Derici, Ebru; Karakaş, Sevim; Ercan, Bahadir; Pata, Yavuz Selim; Akbaş, Yücel; Vayisoğlu, Yusuf; Camdeviren, Handan

2005-05-01

Glutathione and glutathione-related antioxidant enzymes are involved in the metabolism and detoxification of cytotoxic and carcinogenic compounds as well as reactive oxygen species. Reactive oxygen species generation occurs in prolonged relative hypoperfusion conditions such as in aging. The etiology of presbycusis is much less certain; however, a complex genetic cause is most likely. The effect of aging shows a wide interindividual range; we aimed to investigate whether profiles of (glutathione S-transferase (GST) M1, T1 and P1 genotypes may be associated with the risk of age-related hearing loss. We examined 68 adults with presbycusis and 69 healthy controls. DNA was extracted from whole blood, and the GSTM1, GSTT1 and GSTP1 polymorphisms were determined using a real-time polymerase chain reaction and fluorescence resonance energy transfer with a Light-Cycler Instrument. Associations between specific genotypes and the development of presbycusis were examined by use of logistic regression analyses to calculate odds ratios and 95% confidence intervals. Gene polymorphisms at GSTM1, GSTT1, and GSTP1 in subjects with presbycusis were not significantly different than in the controls (p > 0.05). Also, the combinations of different GSTM1, GSTT1, and GSTP1 genotypes were not an increased risk of presbycusis (p > 0.05). We could not demonstrate any significant association between the GSTM1, GSTT1, and GSTP1 polymorphism and age-related hearing loss in this population. This may be because of our sample size, and further studies need to investigate the exact role of GST gene polymorphisms in the etiopathogenesis of the presbycusis.

Selenium concentrations and enzyme activities of glutathione metabolism in wild long-tailed ducks and common eiders

Science.gov (United States)

Franson, J. Christian; Hoffman, David J.; Flint, Paul L.

2011-01-01

The relationships of selenium (Se) concentrations in whole blood with plasma activities of total glutathione peroxidase, Se-dependent glutathione peroxidase, and glutathione reductase were studied in long-tailed ducks (Clangula hyemalis) and common eiders (Somateria mollissima) sampled along the Beaufort Sea coast of Alaska, USA. Blood Se concentrations were >8 μg/g wet weight in both species. Linear regression revealed that the activities of total and Se-dependent glutathione peroxidase were significantly related to Se concentrations only in long-tailed ducks, raising the possibility that these birds were experiencing early oxidative stress.

Prenatal methylmercury exposure hampers glutathione antioxidant system ontogenesis and causes long-lasting oxidative stress in the mouse brain

International Nuclear Information System (INIS)

Stringari, James; Nunes, Adriana K.C.; Franco, Jeferson L.; Bohrer, Denise; Garcia, Solange C.; Dafre, Alcir L.; Milatovic, Dejan; Souza, Diogo O.; Rocha, Joao B.T.; Aschner, Michael; Farina, Marcelo

2008-01-01

During the perinatal period, the central nervous system (CNS) is extremely sensitive to metals, including methylmercury (MeHg). Although the mechanism(s) associated with MeHg-induced developmental neurotoxicity remains obscure, several studies point to the glutathione (GSH) antioxidant system as an important molecular target for this toxicant. To extend our recent findings of MeHg-induced GSH dyshomeostasis, the present study was designed to assess the developmental profile of the GSH antioxidant system in the mouse brain during the early postnatal period after in utero exposure to MeHg. Pregnant mice were exposed to different doses of MeHg (1, 3 and 10 mg/l, diluted in drinking water, ad libitum) during the gestational period. After delivery, pups were killed at different time points - postnatal days (PND) 1, 11 and 21 - and the whole brain was used for determining biochemical parameters related to the antioxidant GSH system, as well as mercury content and the levels of F 2 -isoprostane. In control animals, cerebral GSH levels significantly increased over time during the early postnatal period; gestational exposure to MeHg caused a dose-dependent inhibition of this developmental event. Cerebral glutathione peroxidase (GPx) and glutathione reductase (GR) activities significantly increased over time during the early postnatal period in control animals; gestational MeHg exposure induced a dose-dependent inhibitory effect on both developmental phenomena. These adverse effects of prenatal MeHg exposure were corroborated by marked increases in cerebral F 2 -isoprostanes levels at all time points. Significant negative correlations were found between F 2 -isoprostanes and GSH, as well as between F 2 -isoprostanes and GPx activity, suggesting that MeHg-induced disruption of the GSH system maturation is related to MeHg-induced increased lipid peroxidation in the pup brain. In utero MeHg exposure also caused a dose-dependent increase in the cerebral levels of mercury at

Current status and emerging role of glutathione in food grade lactic acid bacteria

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Pophaly Sarang

2012-08-01

Full Text Available Abstract Lactic acid bacteria (LAB have taken centre stage in perspectives of modern fermented food industry and probiotic based therapeutics. These bacteria encounter various stress conditions during industrial processing or in the gastrointestinal environment. Such conditions are overcome by complex molecular assemblies capable of synthesizing and/or metabolizing molecules that play a specific role in stress adaptation. Thiols are important class of molecules which contribute towards stress management in cell. Glutathione, a low molecular weight thiol antioxidant distributed widely in eukaryotes and Gram negative organisms, is present sporadically in Gram positive bacteria. However, new insights on its occurrence and role in the latter group are coming to light. Some LAB and closely related Gram positive organisms are proposed to possess glutathione synthesis and/or utilization machinery. Also, supplementation of glutathione in food grade LAB is gaining attention for its role in stress protection and as a nutrient and sulfur source. Owing to the immense benefits of glutathione, its release by probiotic bacteria could also find important applications in health improvement. This review presents our current understanding about the status of glutathione and its role as an exogenously added molecule in food grade LAB and closely related organisms.

Non-linear impact of glutathione depletion on C. elegans life span and stress resistance

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Nadine Urban

2017-04-01

Full Text Available The redox environment in cells and organisms is set by low-molecular mass and protein-bound thiols, with glutathione (GSH representing a major intracellular redox buffer. Subtle thiol oxidation elicits signal transduction processes and adaptive responses to cope with stressors, whereas highly oxidizing conditions may provoke cell death. We here tested how thiol depletion affects life span, stress resistance and stress signaling in the model organism Caenorhabditis elegans. Diethyl maleate (DEM, an α,β-unsaturated carbonyl compound that conjugates to GSH and other thiols, decreased C. elegans life span at a concentration of 1 mM. In contrast, low and moderate doses of DEM (10–100 µM increased mean and maximum life span and improved resistance against oxidative stress. DEM-induced life span extension was not detectable in worms deficient in either the FoxO orthologue, DAF-16, or the Nrf2 orthologue, SKN-1, pointing to a collaborative role of the two transcription factors in life span extension induced by thiol depletion. Cytoprotective target genes of DAF-16 and SKN-1 were upregulated after at least 3 days of exposure to 100 µM DEM, but not 1 mM DEM, whereas only 1 mM DEM caused upregulation of egl-1, a gene controlled by a p53-orthologue, CEP-1. In order to test whether depletion of GSH may elicit effects similar to DEM, we suppressed GSH biosynthesis in worms by attenuating γ-glutamylcysteine synthetase (gcs-1 expression through RNAi. The decline in GSH levels elicited by gcs-1 knockdown starting at young adult stage did not impair viability, but increased both stress resistance and life expectancy of the worms. In contrast, gcs-1 knockdown commencing right after hatching impaired nematode stress resistance and rendered young adult worms prone to vulval ruptures during egg-laying. Thus, modest decrease in GSH levels in young adult worms may promote stress resistance and life span, whereas depletion of GSH is detrimental to freshly

Non-linear impact of glutathione depletion on C. elegans life span and stress resistance.

Science.gov (United States)

Urban, Nadine; Tsitsipatis, Dimitrios; Hausig, Franziska; Kreuzer, Katrin; Erler, Katrin; Stein, Vanessa; Ristow, Michael; Steinbrenner, Holger; Klotz, Lars-Oliver

2017-04-01

The redox environment in cells and organisms is set by low-molecular mass and protein-bound thiols, with glutathione (GSH) representing a major intracellular redox buffer. Subtle thiol oxidation elicits signal transduction processes and adaptive responses to cope with stressors, whereas highly oxidizing conditions may provoke cell death. We here tested how thiol depletion affects life span, stress resistance and stress signaling in the model organism Caenorhabditis elegans. Diethyl maleate (DEM), an α,β-unsaturated carbonyl compound that conjugates to GSH and other thiols, decreased C. elegans life span at a concentration of 1mM. In contrast, low and moderate doses of DEM (10-100µM) increased mean and maximum life span and improved resistance against oxidative stress. DEM-induced life span extension was not detectable in worms deficient in either the FoxO orthologue, DAF-16, or the Nrf2 orthologue, SKN-1, pointing to a collaborative role of the two transcription factors in life span extension induced by thiol depletion. Cytoprotective target genes of DAF-16 and SKN-1 were upregulated after at least 3 days of exposure to 100µM DEM, but not 1mM DEM, whereas only 1mM DEM caused upregulation of egl-1, a gene controlled by a p53-orthologue, CEP-1. In order to test whether depletion of GSH may elicit effects similar to DEM, we suppressed GSH biosynthesis in worms by attenuating γ-glutamylcysteine synthetase (gcs-1) expression through RNAi. The decline in GSH levels elicited by gcs-1 knockdown starting at young adult stage did not impair viability, but increased both stress resistance and life expectancy of the worms. In contrast, gcs-1 knockdown commencing right after hatching impaired nematode stress resistance and rendered young adult worms prone to vulval ruptures during egg-laying. Thus, modest decrease in GSH levels in young adult worms may promote stress resistance and life span, whereas depletion of GSH is detrimental to freshly hatched and developing worms

A Lipid Transfer Protein Increases the Glutathione Content and Enhances Arabidopsis Resistance to a Trichothecene Mycotoxin.

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John E McLaughlin

Full Text Available Fusarium head blight (FHB or scab is one of the most important plant diseases worldwide, affecting wheat, barley and other small grains. Trichothecene mycotoxins such as deoxynivalenol (DON accumulate in the grain, presenting a food safety risk and health hazard to humans and animals. Despite considerable breeding efforts, highly resistant wheat or barley cultivars are not available. We screened an activation tagged Arabidopsis thaliana population for resistance to trichothecin (Tcin, a type B trichothecene in the same class as DON. Here we show that one of the resistant lines identified, trichothecene resistant 1 (trr1 contains a T-DNA insertion upstream of two nonspecific lipid transfer protein (nsLTP genes, AtLTP4.4 and AtLTP4.5. Expression of both nsLTP genes was induced in trr1 over 10-fold relative to wild type. Overexpression of AtLTP4.4 provided greater resistance to Tcin than AtLTP4.5 in Arabidopsis thaliana and in Saccharomyces cerevisiae relative to wild type or vector transformed lines, suggesting a conserved protection mechanism. Tcin treatment increased reactive oxygen species (ROS production in Arabidopsis and ROS stain was associated with the chloroplast, the cell wall and the apoplast. ROS levels were attenuated in Arabidopsis and in yeast overexpressing AtLTP4.4 relative to the controls. Exogenous addition of glutathione and other antioxidants enhanced resistance of Arabidopsis to Tcin while the addition of buthionine sulfoximine, an inhibitor of glutathione synthesis, increased sensitivity, suggesting that resistance was mediated by glutathione. Total glutathione content was significantly higher in Arabidopsis and in yeast overexpressing AtLTP4.4 relative to the controls, highlighting the importance of AtLTP4.4 in maintaining the redox state. These results demonstrate that trichothecenes cause ROS accumulation and overexpression of AtLTP4.4 protects against trichothecene-induced oxidative stress by increasing the glutathione

Glutathione protects liver and kidney tissue from cadmium- and lead-provoked lipid peroxidation

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Jovanović Jasmina M.

2013-01-01

Full Text Available Cd and Pb represent a serious ecological problem due to their soluble nature, their mobility and ability to accumulate in the soil. The exposure to these heavy metals can originate from different sources (drinking water, food, air, and they can make their way into the human body through the respiratory and digestive system. We investigated the effects of glutathione on Cd and Pb accumulation and lipid peroxidation effects in the liver and kidneys of heavy metal intoxicated rats. The content of the marker of lipid peroxidation - malondialdehyde was increased several fold the in tissues of exposed animals, the effects being more pronounced in liver. The treatment of intoxicated animals with glutathione drastically suppressed lipid peroxidation. Our results imply that the application of glutathione may have protective role in heavy metal intoxication by inhibiting lipid peroxidation. However, precaution should be made when it comes to Cd, since it seems that glutathione promoted Cd accumulation in the liver.

Targeting brain cells with glutathione-modulated nanoliposomes: in vitro and in vivo study

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Salem HF

2015-07-01

Full Text Available Heba F Salem,1 Sayed M Ahmed,2 Ashraf E Hassaballah,3 Mahmoud M Omar1,4 1Department of Pharmaceutics and Industrial Pharmacy, Beni-suef University, 2Department of Industrial Pharmacy, Assiut University, 3Department of Clinical Pathology, Faculty of Medicine, Assiut University, Assuit, 4Department of Pharmaceutics and Industrial Pharmacy, Deraya University, Egypt Background: The blood–brain barrier prevents many drug moieties from reaching the central nervous system. Therefore, glutathione-modulated nanoliposomes have been engineered to enhance the targeting of flucytosine to the brain. Methods: Glutathione-modulated nanoliposomes were prepared by thin-film hydration technique and evaluated in the primary brain cells of rats. Lecithin, cholesterol, and span 65 were mixed at 1:1:1 molar ratio. The molar percentage of PEGylated glutathione varied from 0 mol% to 0.75 mol%. The cellular binding and the uptake of the targeted liposomes were both monitored by epifluorescent microscope and flow cytometry techniques. A biodistribution and a pharmacokinetic study of flucytosine and flucytosine-loaded glutathione–modulated liposomes was carried out to evaluate the in vivo brain-targeting efficiency. Results: The size of glutathione-modulated nanoliposomes was <100 nm and the zeta potential was more than -65 mV. The cumulative release reached 70% for certain formulations. The cellular uptake increased as molar percent of glutathione increased to reach the maximum at 0.75 mol%. The uptake of the targeted liposomes by brain cells of the rats was three times greater than that of the nontargeted liposomes. An in vivo study showed that the relative efficiency was 2.632±0.089 and the concentration efficiency was 1.590±0.049, and also, the drug-targeting index was 3.670±0.824. Conclusion: Overall, these results revealed that glutathione-PEGylated nanoliposomes enhance the effective delivery of flucytosine to brain and could become a promising new

Cytosolic NADP(+)-dependent isocitrate dehydrogenase status modulates oxidative damage to cells.

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Lee, Su Min; Koh, Ho-Jin; Park, Dong-Chan; Song, Byoung J; Huh, Tae-Lin; Park, Jeen-Woo

2002-06-01

NADPH is an important cofactor in many biosynthesis pathways and the regeneration of reduced glutathione, critically important in cellular defense against oxidative damage. It is mainly produced by glucose 6-phosphate dehydrogenase (G6PD), malic enzyme, and the cytosolic form of NADP(+)-dependent isocitrate dehydrogenase (IDPc). Little information is available about the role of IDPc in antioxidant defense. In this study we investigated the role of IDPc against cytotoxicity induced by oxidative stress by comparing the relative degree of cellular responses in three different NIH3T3 cells with stable transfection with the cDNA for mouse IDPc in sense and antisense orientations, where IDPc activities were 3-4-fold higher and 35% lower, respectively, than that in the parental cells carrying the vector alone. Although the activities of other antioxidant enzymes, such as superoxide dismutase, catalase, glutathione reductase, glutathione peroxidase, and G6PD, were comparable in all transformed cells, the ratio of GSSG to total glutathione was significantly higher in the cells expressing the lower level of IDPc. This finding indicates that IDPc is essential for the efficient glutathione recycling. Upon transient exposure to increasing concentrations of H(2)O(2) or menadione, an intracellular source of free radicals and reactive oxygen species, the cells with low levels of IDPc became more sensitive to oxidative damage by H(2)O(2) or menadione. Lipid peroxidation, oxidative DNA damage, and intracellular peroxide generation were higher in the cell-line expressing the lower level of IDPc. However, the cells with the highly over-expressed IDPc exhibited enhanced resistance against oxidative stress, compared to the control cells. This study provides direct evidence correlating the activities of IDPc and the maintenance of the cellular redox state, suggesting that IDPc plays an important role in cellular defense against oxidative stress.

Activation of the microsomal glutathione-S-transferase and reduction of the glutathione dependent protection against lipid peroxidation by acrolein

NARCIS (Netherlands)

Haenen, G R; Vermeulen, N P; Tai Tin Tsoi, J N; Ragetli, H M; Timmerman, H; Blast, A

1988-01-01

Allyl alcohol is hepatotoxic. It is generally believed that acrolein, generated out of allyl alcohol by cytosolic alcohol dehydrogenase, is responsible for this toxicity. The effect of acrolein in vitro and in vivo on the glutathione (GSH) dependent protection of liver microsomes against lipid

Oxidative Stress Markers and Genetic Polymorphisms of Glutathione ...

African Journals Online (AJOL)

2017-10-26

Oct 26, 2017 ... stress such as asthma, lung cancer, and type 2 diabetes mellitus.[11-13]. Although ... epigenetic, and environmental factors. Little is known ..... glutathione Stransferase genes increase risk of prostate cancer biochemical ...

Glutathione-dependent extracellular ferric reductase activities in dimorphic zoopathogenic fungi

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Zarnowski, Robert; Woods, Jon P.

2009-01-01

In this study, extracellular glutathione-dependent ferric reductase (GSH-FeR) activities in different dimorphic zoopathogenic fungal species were characterized. Supernatants from Blastomyces dermatitidis, Histoplasma capsulatum, Paracoccidioides brasiliensis and Sporothrix schenckii strains grown in their yeast form were able to reduce iron enzymically with glutathione as a cofactor. Some variations in the level of reduction were noted amongst the strains. This activity was stable in acidic, neutral and slightly alkaline environments and was inhibited when trivalent aluminium and gallium ions were present. Using zymography, single bands of GSH-FeRs with apparent molecular masses varying from 430 to 460 kDa were identified in all strains. The same molecular mass range was determined by size exclusion chromatography. These data demonstrate that dimorphic zoopathogenic fungi produce and secrete a family of similar GSH-FeRs that may be involved in the acquisition and utilization of iron. Siderophore production by these and other fungi has sometimes been considered to provide a full explanation of iron acquisition in these organisms. Our work reveals an additional common mechanism that may be biologically and pathogenically important. Furthermore, while some characteristics of these enzymes such as extracellular location, cofactor utilization and large size are not individually unique, when considered together and shared across a range of fungi, they represent an important novel physiological feature. PMID:16000713

Glutathione-mediated detoxification of halobenzoquinone drinking water disinfection byproducts in T24 cells.