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A study of glutathione status in the blood and tissues of patients with breast cancer.

Glutathione plays an important role in the antioxidant system that is required for the maintenance of the redox status of the cell, defence against free radicals and detoxification of toxic compounds. Reduced glutathione (redGSH) can be converted to oxidized glutathione (GSSG) during oxidative stress. The ratio of redGSH/total glutathione can be regarded as an index of the redox status and a useful indicator of disease risks. We conducted experiments by the capillary zone electrophoresis method to investigate the alterations of the glutathione status in the blood and tissue samples from patients with breast cancer. The results showed that the levels of redGSH, GSSG, total glutathione and the ratio of redGSH/total glutathione were significantly decreased in the blood of the patients with breast cancer compared to those of the control subjects. The levels of various forms of glutathione were lower and more pronounced in stage III. In contrast, the levels of redGSH, GSSG, total glutathione and the redGSH/total glutathione ratio in breast cancer tissues were significantly increased relative to those of the adjacent cancer-free tissues, especially in stage II. We suggest that the high redGSH levels are associated with the enhancement of cell proliferation and resistance to apoptosis in the cancer cells, and the loss of the large amount of erythrocyte redGSH may be due to increased detoxification capacities and defence against oxidative stress. We propose that redGSH should be regarded as an important biochemical parameter for detecting breast malignancy.

Adult↗

PubMed1994

Decreased expression of liver glutathione peroxidase in Long-Evans cinnamon mutant rats predisposed to hepatitis and hepatoma.

The Long-Evans Cinnamon rat is a mutant strain that contracts hereditary hepatitis and, eventually, spontaneous hepatoma. Recently, abnormal copper accumulations in Long-Evans Cinnamon rat livers were shown to be genetically linked to the development of hepatitis. Because reduced glutathione and glutathione-related enzymes are known to play important roles in cellular resistance to transition metal toxicity, we determined the levels of reduced glutathione and glutathione-related enzymes in seven different tissues of Long-Evans Cinnamon and control Long-Evans Agouti rats. Of the enzymes examined, only hepatic glutathione peroxidase was markedly decreased in Long-Evans Cinnamon rats. Glutathione peroxidase content in the liver of Long-Evans Cinnamon rats was 39%, 53% and 58% of the control values at 9 (normal stage), 19 (acute hepatitis stage) and 27 (chronic hepatitis stage) wk of age, respectively. Northern-blot analysis revealed that messenger RNA levels of glutathione peroxidase in the livers of Long-Evans Cinnamon rats were about 40% of the control levels. The activity of glutathione S-transferase was slightly decreased in the livers of Long-Evans Cinnamon rats. These data suggest that the liver of the Long-Evans Cinnamon rat is poorly protected against active oxygen species, the production of which is enhanced in the presence of excess copper. Glutathione-reductase activity in the livers of Long-Evans Cinnamon rats increased to 166% and 148% of the control levels at 19 and 27 wk of age, respectively. No significant changes were observed in the activity of gamma-glutamylcysteine synthetase or in the content of total reduced glutathione in the liver of the Long-Evans Cinnamon rat.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1995

Heterologous expression of selenium-dependent glutathione peroxidase affords cellular resistance to paraquat.

Transfection of murine NIH3T3 fibroblasts and human MCF7 breast carcinoma cells with a pSV2-derived eukaryotic expression vector for human cytosolic glutathione peroxidase resulted in clones with increased glutathione peroxidase activity. This heterologous expression indicates that murine cells recognize the human "selenocysteine insertion sequence" in the 3' untranslated region of the mRNA which facilitates insertion of selenocysteine directed by the opal codon. Though most clones from both cell lines eventually lost their enhanced glutathione peroxidase activity despite continuous selection on G418, some NIH3T3 clones retained enhanced enzyme activity without continuous G418 exposure. Transfection of MCF7 cells with an Epstein-Barr virus (EBV)-derived episomally replicating expression vector carrying the glutathione peroxidase gene also revealed increased glutathione peroxidase activity. These MCF7 cells, however, all required exposure to G418 to maintain enhanced glutathione peroxidase activity. Detailed biochemical analysis of a stably expressing NIH3T3 clone and MCF7 expressing cells revealed no alterations in activities of copper-zinc superoxide dismutase, manganese superoxide dismutase, catalase, phospholipid-glutathione peroxidase, glutathione reductase, glutathione transferase, or NADPH-P450 reductase. Both pSV2- and EBV-derived glutathione peroxidase-expressing clones exhibited enhanced resistance to paraquat as well as to peroxides.

3T3 Cells↗

PubMed1998

Loss of glutathione, ascorbate recycling, and free radical scavenging in human erythrocytes exposed to filtered cigarette smoke.

Exposure of human erythrocytes to filtered cigarette smoke in vitro inhibited their capacity to reduce dehydroascorbic acid (ascorbate recycling activity). Glucose uptake was not affected, implying that dehydroascorbic acid transport was not inhibited by the smoke treatment. The intracellular reduction of cationic nitroxide free radicals, which provides a measure of ascorbate recycling, was also inhibited by cigarette smoke. A major factor in the inhibition of free radical reduction was glutathione depletion. However, glutathione depletion alone could not account for the inhibition of free radical reduction because a restoration of the glutathione pool in hemolyzed cells only partially restored free radical reduction activity. Another factor inhibiting free radical reduction was a lowering of pH, which was attributed mainly to the uptake of CO2 and was reversible by restoring the physiological pH. Exogenous glutathione spared both intracellular glutathione and free radical reduction activity. The rate of depletion of intracellular glutathione was similar to that of extracellular glutathione, indicating that the erythrocyte membrane did not significantly attenuate thiol-reactive species in smoke. Protein thiols were also depleted by cigarette smoke, but to a much lesser extent than was glutathione. Ascorbate was relatively unaffected by cigarette smoke; significant intracellular ascorbate levels remained after glutathione was barely detectable. Autooxidizable reducing agents, capable of reducing both reduced piperidinyl (Tempo) and pyrrolidinyl (Proxyl) nitroxides partitioned from filtered cigarette smoke into aqueous solutions. Attempts to detect cigarette smoke-derived oxidants in buffer solutions or in cell suspensions with a prereduced Tempo nitroxide, whose oxidation properties resemble those of ascorbate, were unsuccessful. The results of this study suggest that chemical modification of glutathione is a major damage mechanism of filtered cigarette smoke, whereas free radical oxidations are relatively insignificant.

Ascorbic Acid↗

PubMed1995

Effects of glutathione depletion on the cytotoxic actions of cadmium in LLC-PK1 cells.

Recent studies have shown that exposure of LLC-PK1 cells to micromolar concentrations of Cd2+ for 1-4 hr causes the disruption of the junctions between the cells, whereas exposure to higher concentrations of Cd2+ for longer periods of time causes more severe toxic effects and cell death. Studies suggesting that glutathione may serve a protective role against Cd2+ toxicity in other tissues and cells led us to examine the effects of glutathione depletion on the cytotoxic actions of Cd2+ in the LLC-PK1 cell line. Confluent cells on Falcon cell culture inserts were depleted of glutathione by exposing them to 250 microM buthionine sulfoximine for 18 hr and then exposed to various concentrations of Cd2+ for up to 24 hr. The integrity of cell-cell junctions was assessed by morphologic observation of the cells and by monitoring the transepithelial electrical resistance. Cell viability was evaluated by monitoring the release of lactate dehydrogenase into the medium. The results showed that depleting the cells of glutathione did not alter the early junction-perturbing effects of Cd2+, but greatly enhanced the lethal effects. In both the glutathione-depleted and the normal cells, junctional changes were evident after as little as 1 hr of Cd2+ exposure. While the normal cells did not begin to die until they had been exposed to Cd2+ for 12-24 hr, the glutathione-depleted cells began to die after only 8 hr of Cd2+ exposure. Additional results showed that Cd2+ exposure had no effect on the total levels of glutathione at the time in which the junctional effects were occurring, but caused a marked decrease in glutathione levels at the time the cells were dying. These results indicate that the early junctional effects of Cd2+ do not result from alterations in intracellular glutathione or sulfhydryl metabolism, whereas the more severe cytotoxic effects and cell death may involve glutathione-sensitive mechanisms.

Animals↗

PubMed1997

Alterations in the distribution of glutathione in the substantia nigra in Parkinson's disease.

Depletion of reduced glutathione occurs in the substantia nigra in Parkinson's disease and in incidental Lewy body disease (presymptomatic Parkinson's disease) which may implicate oxidative stress in the neurodegenerative process. In this study mercury orange fluorescent staining and immunostaining with an antibody to reduced glutathione have been used to determine the distribution of reduced glutathione in the substantia nigra in Parkinson's disease compared with normal individuals. Mercury orange staining showed moderate background levels of fluorescence in the neuropil in both control and Parkinson's disease substantia nigra and localised reduced glutathione to the somata of melanized nigral neurons and glial elements of the neuropil. Neuronal nuclei revealed a relative lack of fluorescence after mercury orange staining. There was a significant depletion of reduced glutathione in surviving neurons in Parkinson's disease compared to nerve cell populations in control tissue. Mercury orange fluorescence indicated a high concentration of reduced glutathione in a subpopulation of non-neuronal cells, most likely astrocytes or microglia. Immunohistochemical examination of nigral tissue from the same Parkinson's disease and control patients with an antibody to glutathione showed staining in neuronal perikarya and axonodendritic processes of melanized nigral neurons which was generally most intense in control neurons. Moderately intense staining of the background neuropil, most prominent in control nigras, and staining of capillary walls was also detected. Intense staining was seen in cells with the morphological features of glial cells in both control and PD nigra. These data show a significant presence of reduced glutathione in the cell bodies and axons of nigral neurons. They are in agreement with biochemical studies showing depletion of reduced glutathione in substantia nigra in Parkinson's disease, and indicate a significant loss of neuronal reduced glutathione in surviving nigral neurons in Parkinson's disease.

Aged↗

PubMed1997

Glutathione content but not gamma glutamyl cysteine synthetase mRNA expression predicts cisplatin resistance in head and neck cancer cell lines.

PURPOSE: To correlate cellular glutathione content and gamma-glutamyl cysteine synthetase (gamma GCS) mRNA expression with cisplatin sensitivity in a panel of seven head and neck squamous cancer cell lines. METHODS: Cisplatin IC50 was determined for each cell line using a sodium tetreazolium (XTT) assay. Cellular glutathione content was measured by using a previously reported enzymic method. gamma GCS mRNA expression was measured using an RNase protection assay. RESULTS: Total cellular glutathione was an excellent predictor of cisplatin sensitivity in this series of cell lines. The IC50 for cisplatin in the cell line with the highest glutathione concentration was approximately 90 times higher than in the cell line with the lowest glutathione concentration. Regression analysis showed a highly statistically significant positive correlation between cisplatin IC50 and cellular glutathione (coefficient of determination R2 = 0.81, P = 0.0012). Some-what surprisingly, in contrast to previous studies in ovarian cancer, gamma GCS mRNA expression in these cell lines was not significantly predictive of either total cellular glutathione or cisplatin sensitivity (R2 = 0.005, P = 0.84). As expected, treatment of resistant cell lines with buthionine sulfoximine resulted in decreased cellular glutathione and enhanced cisplatin sensitivity. CONCLUSIONS: Our results suggest that glutathione may be an important determinant of cisplatin sensitivity in clinical head and neck cancer. Since cisplatin is the most active chemotherapy drug for the treatment of this disease, this correlation may have important clinical relevance. The lack of correlation between glutathione level and gamma GCS expression suggests that salvage or alternate synthetic pathways may be critical in these cells.

Buthionine Sulfoximine↗

PubMed2006

Artificial elevation of glutathione affects symptom development in ZYMV-infected Cucurbita pepo L. plants.

Styrian oil pumpkin seedlings (Cucurbita pepo L. subsp. pepo var. styriaca GREB: .) were treated for 48 h with 1 mM OTC (L-2-oxothiazolidine-4-carboxylic acid) in order to artificially increase cellular glutathione content. They were inoculated with zucchini yellow mosaic virus (ZYMV) 10 days later. The effects of OTC treatment and ZYMV infection on glutathione levels were examined at the subcellular level by immunogold labeling of glutathione using a transmission electron microscope (TEM). These effects were further tested at the whole-tissue level by high performance liquid chromatography (HPLC). Such tests were carried out a) on roots, cotyledons and the first true leaves immediately after OTC treatment in order to analyze to which extent OTC increases glutathione levels in different cell compartments as well as in the whole organ; and b) in older and younger leaves and in roots three weeks after ZYMV inoculation in order to study how possible effects of OTC on symptom development would correlate with glutathione levels at the subcellular level and in the whole organ. Immunocytological and biochemical investigations revealed that, 48 h after OTC treatment, glutathione content had increased in all investigated organs, up to 144% in peroxisomes of cotyledons. Three weeks after ZYMV inoculation, glutathione labeling density had significantly increased within intact cells of infected leaves, up to 124% in the cytosol of younger leaves. Roots showed decreased amounts of glutathione in the TEM. Biochemical studies revealed that OTC treatment resulted in 41 and 51% higher glutathione content in older and younger ZYMV-infected leaves, respectively, in comparison to untreated and ZYMV-infected plants. Evaluation of symptom development at this point revealed that all untreated ZYMV-infected plants had symptoms, whereas only 42% of OTC-treated ZYMV-infected plants showed signs of symptoms. Quantification of ZYMV particles revealed that all organs of OTC-treated and ZYMV-infected plants contained significantly decreased amounts of ZYMV particles over a period of five weeks when compared to the same organs of untreated ZYMV-infected plants. We can conclude that OTC treatment and subsequently elevated glutathione contents within Styrian oil pumpkin plants led to a strong decrease in virus content, which was accompanied by a suppression of ZYMV-induced symptoms as well as reduced and delayed symptom development within plants exhibiting symptoms.

Antiviral Agents↗

PubMed1982

Purification from pig liver of a protein which protects liposomes and biomembranes from peroxidative degradation and exhibits glutathione peroxidase activity on phosphatidylcholine hydroperoxides.

The cell sap from pig liver contains a protein which protects phosphatidylcholine liposomes and biomembranes from peroxidative degradation in the presence of glutathione. The activity of this protein has been assayed by measuring the inhibition of aged phosphatidylcholine liposome peroxidation induced by the Fe3+-triethylenetetramine complex. The peroxidation-inhibiting protein from pig liver has been purified 585-fold to homogeneity with overall recovery of activity of 12%. (NH4)2SO4 precipitation, ion-exchange chromatography on DEAE-Sepharose CL-6B and CM23-cellulose, affinity chromatography on glutathione-bromosulfophthalein-Sepharose and gel filtration on Sephadex G-50 were used. Gel filtration and SDS- polyacrylamide gel electrophoresis indicated a molecular weight of approximately 20 000. The protein inhibited peroxidation by Fe3+-triethylenetetramine following a 15 min preincubation of phosphatidylcholine liposomes in the presence of 5mM glutathione or 2-mercapthoethanol. The pure protein exhibited glutathione peroxidase activity on hydroperoxide groups of phosphatidylcholine and on cumene and t-butyl hydroperoxides, with specific activities of 2.2, 3.8 and 0.9 mumol/min per mg protein, respectively. The protein appears to be distinct from the selenoenzyme glutathione peroxidase and from any known glutathione S-transferase. The peroxidation was studied also with fresh phosphatidylcholine liposomes and was induced in this case by Fe-ascorbate. To obtain protection by the peroxidation-inhibiting protein and glutathione, preincubation was not necessary, but alpha-tocopherol, incorporated in the liposomes in the molar ratio 1:250 to phosphatidylcholine, was required. Lipid peroxidation of rat liver mitoplasts and microsomes was blocked when these preparations were incubated in the peroxidizing mixture in the presence of peroxidation-inhibiting protein and glutathione. The protection from Fe3+-triethylenetetramine-induced peroxidation is related apparently to reduction of hydroperoxide groups in polyunsaturated fatty acid residues of phospholipids and to inhibition of free radicals formation by chain branching. Protection from the Fe-ascorbate-induced peroxidation is apparently attributable to the same mechanism. However, the requirement of alpha-tocopherol for protection in the Fe-ascorbate-induced peroxidation suggests that the cooperation of a free-radical scavenger is necessary. It is probable that the glutathione peroxidase activity is involved also in the glutathione-dependent protection exhibited by the protein on lipid peroxidation of biomembranes.

Animals↗

PubMed1984

Inhibition of soluble glutathione S-transferase by diuretic drugs.

Glutathione transferases are believed to play an important protective role in the various tissues of animals and man by catalysing the glutathione conjugation of electrophilic drugs and electrophilic drug metabolites. Many of these compounds have the potential to react with vital cellular macromolecules in the absence of this enzyme system. We have investigated the interaction of a number of high ceiling diuretics with the glutathione transferases contained in the cytosolic fraction of the rat liver. Of bumetanide, ethacrynic acid, furosemide, indacrynic acid and tienilic acid, only ethacrynic acid was conjugated with glutathione. Further experiments revealed that ethacrynic, indacrynic and tienilic acids are all potent inhibitors of glutathione S- aryltransferase . Glutathione S- alkyltransferase and glutathione S-epoxide transferase were also inhibited by the diuretics, but to a lesser extent than glutathione S- aryltransferase . The diuretics giving the greatest inhibition of these reactions were chemically related to ethacrynic acid. The concept where inhibition of glutathione-S-transferase by a drug may enhance its own toxicity is considered. This mechanism has also the potential of enhancing the toxicity of other concurrently administered drugs which normally require glutathione S-transferase for detoxication.

Animals↗

PubMed1985

Diurnal variation of methotrexate transport and accumulation in hepatocytes--a consequence of variations in cellular glutathione.

Diurnal variation of the methotrexate (MTX) initial influx and net uptake in isolated rat liver cells was studied in dependence on diurnal variation of cellular glutathione. It was found that the most significant differences concerning the MTX initial transport and accumulation were observed between hepatocytes prepared at 1200 hr when cellular glutathione reached its maximum, and those isolated at 0000 hr when liver glutathione had its minimal concentration. The initial influx of MTX was the biggest in cells isolated at 0000 hr and the smallest in cells prepared at 1200 hr. The Km values in cells with low cellular glutathione (at 1800 and 0000 hr) were about three times smaller than in cells with high glutathione (at 0700 and at 1200 hr), whereas the Vmax value remained unchanged. Titration of external membrane SH groups by [203Hg]p-CMBS revealed a much larger amount of free SH groups on cells having low glutathione level than on those with high glutathione. Despite big initial influx of MTX found in cells with low cellular glutathione, net accumulation of MTX was significantly smaller in these cells as compared with hepatocytes having high glutathione level. In conclusion, the present studies confirmed a statement of the preceding paper on the important role of cellular glutathione played in methotrexate transport and accumulation in rat liver cells.

Animals↗

PubMed1989

Inhibition of stress-induced gastric injury in the rat by glutathione.

Glutathione metabolism occurs via interorgan cycles in which hepatic synthesis of reduced glutathione and its transfer to extrahepatic tissues play an important role. To elucidate the physiologic significance of the cycles and tissue thiol status during stress-induced gastric mucosal injury, dynamic aspects of glutathione metabolism were analyzed in rats that were treated with water-immersion restraint. This treatment induced gastric mucosal lesion with concomitant decrease in the levels of perchloric acid-soluble thiols in various tissues, particularly in the liver and stomach. During the treatment, glutathione levels markedly decreased in the liver but not in other tissues. Depletion of hepatic glutathione by buthionine sulfoximine, a specific inhibitor for gamma-glutamyl cysteine synthetase, markedly decreased hepatic glutathione levels and increased the gastric injury. Intraperitoneal injection of reduced glutathione significantly increased plasma levels of glutathione and inhibited the occurrence of gastric injury without affecting intracellular glutathione levels. These results indicate that extracellular glutathione and its interorgan metabolism might play a critical role in the protection of gastric mucosa particularly when animals were challenged with various stress.

Acetylcysteine↗

PubMed1988

Effect of glutathione depletion on the irreversible association of acrylonitrile with tissue macromolecules after oral administration to rats.

Binding of acrylonitrile and its reactive metabolites to tissue macromolecules, especially nucleic acids, may be responsible for its carcinogenicity in rats. Both acrylonitrile and its primary metabolite, 2-cyanoethylene oxide, also react with glutathione. To better understand the role of glutathione in the manifestation of acrylonitrile toxicity, the irreversible binding to tissue macromolecules was assessed in control and glutathione-depleted F-344 rats treated with a 4 mg/kg dose (po) of [2,3-14C]acrylonitrile. Glutathione was depleted in rat tissues by the administration of a combined intraperitoneal phorone/buthionine sulfoximine treatment (300 mg/kg and 2 mmol/kg, respectively) given 30 min prior to acrylonitrile administration. The amount of total radioactivity recovered from brain, stomach (target organs), liver, kidney, lung, and blood (nontarget organs) was similar between control and glutathione-depleted rats. However, stomach, lung, blood, and liver showed an increase in total radioactivity content after glutathione depletion by phorone/buthionine sulfoximine treatment. Glutathione depletion also caused an increase in acrylonitrile-derived non-dialysable radioactivity (MW greater than 3500 Da) in liver, lung, kidney, stomach, blood, and brain macromolecules between 6 and 24 hr after the dose. There was no organ-specific accumulation of radiolabel in RNA in control rats. However, an increase in the radiolabel associated with nucleic acids in the target organs but not in the nontarget organs was measured in glutathione-depleted rats. Urinary excretion of thiocyanate, a metabolite derived from the epoxide pathway, was also increased by 300% in glutathione-depleted rats. These results suggest that glutathione might play a role in the extent of 2-cyanoethylene oxide formation and in the distribution of the radiolabel among tissues.

Acrylonitrile↗

PubMed1990

Hydrogen peroxide production by monoamine oxidase in isolated rat-brain mitochondria: its effect on glutathione levels and Ca2+ efflux.

H2O2 production and accumulation during incubation of isolated rat-brain mitochondria with substrates of monoamine oxidase A and B were investigated. All substrates gave rise to an accumulation of H2O2 which was inhibited by malate + pyruvate or isocitrate, consistent with a need for mitochondrial NADPH to maintain glutathione in the reduced state. However, in the absence of these additions the level of reduced glutathione decreased only by about 30%, indicating that only a fraction of the mitochondrial glutathione pool was accessible to the glutathione peroxidase and glutathione reductase activities responsible for the continuous removal of H2O2 generated by monoamine oxidase. The H2O2 accumulation was also inhibited by externally added reduced glutathione or NADPH but not NADH. External NADPH was oxidized by added oxidized glutathione but not alpha-ketoglutarate + NH4+. These results suggest that the removal of H2O2 generated by monoamine oxidase proceeds by way of special fractions of glutathione peroxidase and glutathione reductase that are located in the intermembrane space of mitochondria in such a way that they can react with both intra- and extra-mitochondrial glutathione and NADPH, possibly at the contact sites between the inner and outer mitochondrial membranes. Evidence is also presented that H2O2 generated by monoamine oxidase enhances Ca2+ release from mitochondria and may thus function as a regulator of mitochondrial Ca2+ efflux.

Animals↗

PubMed1995

The purification and properties of glutathione reductase from the cestode Moniezia expansa.

Glutathione reductase has a central role in glutathione metabolism and as such is a potential target for chemotherapy. The aim of the work was to purify and characterise glutathione reductase from the cestode Moniezia expansa and to compare the properties of the helminth enzyme with its mammalian counterpart. The enzyme was purified by a combination of anion exchange and affinity chromatography and further characterized by chromatofocusing and gel electrophoresis. Analysis revealed a single isoenzyme of glutathione reductase in Moniezia expansa, with a pI of 5.8. The enzyme was a homodimer of native molecular weight 114 kDa, subunit weight 63 kDa. Enzyme activity was affected by buffer concentration and the presence of monovalent sodium salts. The pH optimum was 7.4 with NADPH as cofactor and 5 with NADH. The Kma for oxidized glutathione was 76 microM and for NADPH and NADH, 21 and 350 microM respectively. In addition to oxidized glutathione only the mixed disulphide between CoA and glutathione (CoASSG) showed any significant activity as substrate. The cestode enzyme was inhibited by a variety of compounds including arsonic derivatives, 2,4,6 trinitrobenzene sulfonate 1,3-bis (2-chlorethyl)-1-nitrosourea and oxidized glutathione. In conclusion the glutathione reductase of M. expansa resembles the mammalian enzyme in its general physical properties and its substrate and inhibitor profile. However, the parasite enzyme shows an unusually high activity with the mixed disulphide of coenzyme A and glutathione (CoASSG) and appears to be more sensitive to inhibition by sodium ions.

Animals↗

PubMed2005

Alterations in glutathione levels of brain structures caused by acute restraint stress and by nitric oxide synthase inhibition but not by intraspecific agonistic interaction.

Glutathione is the major non-protein thiol to which many different roles in the central nervous system (CNS) are attributed. To further investigate the glutathione response in the CNS, we tested the effect of three stress models on glutathione levels in the brain. We tested the effect of two models of repeated intraspecific agonistic interaction in mice. No influence was observed over the glutathione levels in the mice cerebral cortex, cerebellum, liver, and blood. Acute restraint stress in rats was found to induce an increase in glutathione levels in the cerebellum after 2 and 4 h of immobilization, an effect not observed in the cerebral cortex, striatum, and hippocampus. To investigate the interference of an inhibitor of nitric oxide synthase (NOS), N(omega)Nitro-L-arginine-methyl-ester (L-NAME, 50 mg/kg) was applied i.p. at the beginning of restraint stress. L-NAME alone did not lead to a change in glutathione levels although, in combination with restraint stress, it induced an increase in such levels. This effect was observed in all four structures studied, i.e. cortex, hippocampus, striatum, and cerebellum. The values returned to basal levels after 6h of immobilization. In conclusion, the pattern of dominance, after repeated intraspecific agonistic interaction, was ineffective in producing alterations in brain glutathione, whereas acute restraint stress led to an increase in glutathione levels within a window of 2-4 h, and the inhibition of NOS increased glutathione levels in all studied rat brain structures, suggesting a specificity interference of acute restraint stress with the glutathione system.

Agonistic Behavior↗

PubMed2004

Expression of selenocysteine-containing glutathione S-transferase in Escherichia coli.

Evolution of a probable 'glutathione-binding ancestor' resulting in a common thioredoxin-fold for glutathione S-transferases and glutathione peroxidases may possibly suggest that a glutathione S-transferase could be engineered into a selenium-containing glutathione S-transferase (seleno-GST), having glutathione peroxidase (GPX) activity. Here, we addressed this question by production of such protein. In order to obtain a recombinant seleno-GST produced in Escherichia coli, we introduced a variant bacterial-type selenocysteine insertion sequence (SECIS) element which afforded substitution with selenocysteine for the catalytic Tyr residue in the active site of GST from Schistosoma japonica. Utilizing coexpression with the bacterial selA, selB, and selC genes (encoding selenocysteine synthase, SelB, and tRNA(Sec), respectively) the yield of recombinant seleno-GST was about 2.9 mg/L bacterial culture, concomitant with formation of approximately 85% truncation product as a result of termination of translation at the selenocysteine-encoding UGA codon. The mutations inferred as a result of the introduction of a SECIS element did not affect the glutathione-binding capacity (Km = 53 microM for glutathione as compared to 63 microM for the wild-type enzyme) nor the GST activity (kcat = 14.3 s(-1) vs. 16.6 s(-1)), provided that the catalytic Tyr residue was intact. When this residue was changed to selenocysteine, however, the resulting seleno-GST lost the GST activity. It also failed to display any novel GPX activity towards three standard peroxide substrates (hydrogen peroxide, butyl hydroperoxide or cumene hydroperoxide). These results show that recombinant selenoproteins with internal selenocysteine residues may be heterologously produced in E. coli at sufficient amounts for purification. We also conclude that introduction of a selenocysteine residue into the catalytic site of a glutathione S-transferase is not sufficient to induce GPX activity in spite of a maintained glutathione-binding capacity.

Amino Acid Sequence↗

PubMed2004

High glutathione turnover in human cell lines revealed by acivicin inhibition of gamma-glutamyltranspeptidase and the effects of thiol-reactive metals during acivicin inhibition.

BACKGROUND: Glutathione is the most abundant nonprotein sulfhydryl-containing compound and constitutes the largest component of the endogenous thiol buffer. Glutathione is known to have multifaceted physiological functions and is a critical factor in protecting organisms against toxicity and disease. Intracellular cysteine concentration is a limiting factor for glutathione synthesis. METHODS: In the present study, the metabolism of intra- and extracellular glutathione in HeLa and hepatoma cell cultures is investigated by using different transport inhibitors for cellular uptake of cystine/cysteine. RESULTS: There exist several ways of cystine/cysteine transport into HeLa and hepatoma cells, and inhibition of them decreased intracellular concentration of cystine/cysteine and in some cases also of glutathione. It was also shown that a large pool of total cell culture glutathione was located extracellularly in both HeLa and hepatoma cell cultures when gamma-glutamyltranspeptidase (GT) activity was inhibited by acivicin (ACI). Furthermore, the addition of thiol-reactive metal ions significantly increased the total amount of glutathione in hepatoma cell cultures during acivicin inhibition. Thus, occasional determinations of extracellular concentrations of glutathione without GT inhibition strongly underestimate the total turnover of glutathione in a cell culture. CONCLUSION: This finding has important implications for future research in glutathione metabolism and the understanding of its role in human health and disease.

Carcinoma, Hepatocellular↗