The role of glutathione in rat adipocyte pentose phosphate cycle activity

An assay for reduced and oxidized glutathione was adapted to isolated rat epididymal adipocytes in order to correlate pentose phosphate cycle activity and glutathione metabolism. In collagenase-digested adipocytes the [GSH/GSSG] molar ratio was in excess of 100. Cells incubated for 1 hr with low glucose concentrations (0.28–0.55 mm) had higher GSH contents (3.2 μg/10⁶ cells) than in the absence of glucose (2.3 μg/10⁶ cells). The glutathione oxidant diamide caused a dose-related decrease in intracellular GSH, an increase in GSSG released into the medium, but no detectable change in the low intracellular GSSG content. The intracellular content of GSH and amount of GSSG released into the medium were therefore taken to reflect the glutathione status of the adipocytes most closely. Addition of H₂O₂ to a concentration of 60 μm to adipocytes caused to decline within 5 min in GSH content, which was less severe and more rapid to recover in the presence of 1.1 mm glucose, suggesting that the concomitant stimulation of glucose C-1 oxidation induced by the peroxide in the presence of glucose provided NADPH for regeneration of GSH. Further evidence for tight coupling between adipocyte [GSH/GSSG] ratios and pentose phosphate cycle activity was that (i) lowering intracellular GSH to 35–60% of control values by agents as diverse in action as t-butyl hydroperoxide, diamide, or the sulfhydryl blocker N-ethylmaleimide resulted in optimal stimulation of glucose C-1 oxidation and fractional pentose phosphate cycle activity, and (ii) incubating adipocytes directly with 2.5 mm GSSG resulted in a slight increase in glucose C-1 oxidation and when 0.5 mm NADP⁺ was also added a synergistic effect on pentose phosphate cycle activity was found. On the other hand, electron acceptors such as methylene blue did not lower cellular GSH content, but did stimulate the pentose phosphate cycle, confirming a site of action independent of glutathione metabolism. The results show that (i) glucose metabolism by the pentose phosphate cycle contributes to regeneration of GSH and that (ii) glutathione metabolism either directly or via coupled changes in [NADPH/NADP⁺] ratios may play a significant role in short-term control of the pentose phosphate cycle.     

Oxidized glutathione fermentation using Saccharomyces cerevisiae engineered for glutathione metabolism

Glutathione is a valuable tripeptide that is widely used in the pharmaceutical, food, and cosmetic industries. Intracellular glutathione exists in two forms, reduced glutathione (GSH) and oxidized glutathione (GSSG). Most of the glutathione produced by fermentation using yeast is in the GSH form because intracellular GSH concentration is higher than GSSG concentration. However, the stability of GSSG is higher than GSH, which makes GSSG more advantageous for industrial production and storage after extraction. In this study, an oxidized glutathione fermentation method using Saccharomyces cerevisiae was developed by following three metabolic engineering steps. First, over-expression of the glutathione peroxidase 3 (GPX3) gene increased the GSSG content better than over-expression of other identified peroxidase (GPX1 or GPX2) genes. Second, the increase in GSSG brought about by GPX3 over-expression was enhanced by the over-expression of the GSH1/GSH2 genes because of an increase in the total glutathione (GSH + GSSG) content. Finally, after deleting the glutathione reductase (GLR1) gene, the resulting GPX3/GSH1/GSH2 over-expressing ΔGLR1 strain yielded 7.3-fold more GSSG compared with the parental strain without a decrease in cell growth. Furthermore, use of this strain also resulted in an enhancement of up to 1.6-fold of the total glutathione content compared with the GSH1/GSH2 over-expressing strain. These results indicate that the increase in the oxidized glutathione content helps to improve the stability and total productivity of glutathione.     

Hyperglycemia in diabetic rats reduces the glutathione content in the aortic tissue

The glutathione redox cycle plays a major role in scavenging hydrogen peroxide (H2O2) under physiological conditions. Recently, we demonstrated that a high glucose concentration in the culture medium reduced the level of H2O2 scavenging activity of human vascular smooth muscle cells (hVSMCs). We also showed that a high glucose concentration reduced the intracellular glutathione (GSH) content and the rate of uptake of cystine, which itself is a rate-limiting factor that maintains the GSH level (FEBS Lett.421: 19-22,1998). In the present study, we investigated whether the hyperglycemic condition in diabetic rats impairs the glutathione content in the aortic tissue in vivo. Wistar rats were divided into the following three groups: streptozotocin-induced diabetic rats (STZ-D, n=7), insulin-treated STZ-D rats (I-STZ-D, n=8), and non-diabetic controls (C, n=7). Fourteen days after streptozotocin injection, the aortic tissue was extracted and the GSH content in the aortic tissue was measured. Furthermore, the relationship between the GSH content in the aortic tissue and blood glucose level in Otsuka Long-Evans Tokushima Fatty (OLETF) rats aged 30 weeks, which developed diabetes spontaneously, was investigated. The GSH content in the aortic tissue of the STZ-D group (0.99+/-0.14 nmol/mg protein) was significantly lower than that of the control group (1.68+/-0.15 nmol/mg protein). Insulin treatment to the diabetic rats restored the GSH content in the aortic tissue (I-STZ-D group; 1.45+/-0.11 nmol/mg protein). Among the 22 Wistar rats, the GSH content in the aortic tissue was negatively correlated with the blood glucose level (r=-0.69, p<0.01, n=22). Among the OLETF rats, a similar negative correlation between the GSH content in the aortic tissue and blood glucose level was seen (r=-0.64, p<0.05, n=10). We demonstrated in vivo that the hyperglycemic condition in STZ-induced diabetic Wistar rats and OLETF rats reduced the GSH content in aortic tissue. This suggested reduced glutathione redox cycle function of aorta.     

Glutathione metabolizing enzymes and oxidative stress in ferric nitrilotriacetate mediated hepatic injury

Summary

Glutathione (GSH) plays several important roles in the protection of cells against oxidative damage, particularly following exposure to xenobiotics. Ferric nitrilotriacetate (Fe-NTA) is a potent depletor of GSH and also enhances tissue lipid peroxidation. In this study, we show the effect of Fe-NTA treatment on hepatic GSH and some of the glutathione metabolizing enzymes, oxidant generation and liver damage. The level of hepatic GSH and the activities of glutathione reductase, glutathione S-transferase, glutathione peroxidase, and glucose 6-phosphate dehydrogenase all decrease following Fe-NTA administration. In these parameters the maximum decrease occurred at 12 h following Fe-NTA treatment. In contrast, γ-glutamyl transpeptidase was increased at this time. Not surprisingly, the increase in the activity of γ-glutamyl transpeptidase and decreases in GSH, glutathione peroxidase, glutathione reductase, glucose 6-phosphate dehydrogenase and glutathione S-transferase were found to be dependent on the dose of Fe-NTA administered. Fe-NTA administration also enhances the production of H₂O₂ and increases hepatic lipid peroxidation. Parallel to these changes, Fe-NTA enhances liver damage as evidenced by increases in serum transaminases. Once again, the liver damage is dependent on the dose of Fe-NTA and is maximal at 12 h. Pretreatment of animals with antioxidant, butylated hydroxy anisole (BHA), protects against Fe-NTA-mediated hepatotoxicity further supporting the involvement of oxidative stress in Fe-NTA-mediated hepatic damage. In aggregate, our results indicate that Fe-NTA administration eventuates in decreased hepatic GSH, a fall in the activities of glutathione metabolizing enzymes and excessive production of oxidants, all of which are involved in the cascade of events leading to iron-mediated hepatic injury.     

The transport of oxidized glutathione from the erythrocytes of various species in the prescence of chromate

1. Erythrocytes from normal and glucose 6-phosphate dehydrogenase-deficient humans were subjected to hydrogen peroxide diffusion to oxidize the GSH. Studies were carried out in the presence and absence of chromate to inhibit glutathione reductase and with or without the addition of glucose. 2. The GSH content of erythrocytes from other species was oxidized by subjecting them to hydrogen peroxide diffusion in the presence of chromate and glucose. 3. Chromate (1.3mm) inhibited glutathione reductase by about 80%, whereas glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, hexokinase, phosphofructokinase and pyruvate kinase were not inhibited. 4. The GSSG formed was transported from the erythrocytes to the medium. 5. The transport rate of GSSG from glucose 6-phosphate dehydrogenase-deficient erythrocytes subjected to hydrogen peroxide diffusion in the presence of chromate was comparable with that from normal and glucose 6-phosphate dehydrogenase-deficient erythrocytes. 6. The rate of transport of GSSG from erythrocytes of various species studied could be ranked: pigeon>rabbit>rat>donkey>man>dog>horse>sheep>chicken>fish.     

Glutathione metabolism under the influence of hydroperoxides in the lactating mammary gland of the rat. Effect of glucose and extracellular ATP

Tert-butyl hydroperoxide decreases GSH and total free glutathione (GSH+2GSSG) contents of acini from lactating mammary glands. The decrease in total free glutathione can be explained by an increase in mixed disulfide formation and by excretion of GSS G to the extracellular medium, and subsequent degradation catalyzed by gamma-glutamyl transpeptidase. Low concentrations of glucose prevented the changes in glutathione levels induced by the peroxide. In the presence of extracellular ATP, glucose did not prevent these changes. However, incubations with the peroxide, did not alter the rate of other metabolic pathways by acini.Abbreviations used GSH Reduced glutathione - GSSG Glutathione disulfide - GSSR Glutathione mixed disulfide - GGT Gamma-glutamyl transpaptidase - tbOOH Tert-butyl hydroperoxide     

Signal-on fluorescent sensor based on N-CQDs for the detection of glutathione in human serum and pharmaceutic preparation

Nitrogen-doped carbon quantum dots (N-CQDs) with citric acid and ethylenediamine as raw materials were synthesized by an efficient one-step strategy. The N-CQDs showed a special property that the fluorescence was quenched by Fe³⁺. The quenched fluorescence of N-CQDs could be recovered by glutathione (GSH). Therefore, a “signal-on” fluorescent sensor was developed to detect GSH. The fluorescent sensor could favorably avoid the interference of ascorbic acid, dopamine, glucose, oxidized glutathione, and other amino acids in the detecting process of GSH. The proposed sensor showed a great feature that GSH can be accurately detected in the range from 0.001 to 0.1?mol/L and can be applied to detect GSH in the human serum. Therefore, the proposed method has a promising application for monitoring the blood drug concentration of GSH in clinical studies.     

Ameliorative potential of S-allyl cysteine on oxidative stress in STZ induced diabetic rats

Increased oxidative stress and impaired antioxidant defense mechanism are important factors in the pathogenesis and progression of diabetes mellitus and other oxidant-related diseases. The present study was undertaken to evaluate the possible protective effects of S-allyl cysteine (SAC) against oxidative stress in streptozotocin (STZ) induced diabetic rats. SAC was administered orally for 45 days to control and STZ induced diabetic rats. The effects of SAC on glucose, plasma insulin, thiobarbituric acid reactive substances (TBARS), hydroperoxide, superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), reduced glutathione (GSH), oxidized glutathione (GSSG) and GSH/GSSG ratio were studied. The levels of glucose, TBARS, hydroperoxide, and GSSG were increased significantly whereas the levels of plasma insulin, reduced glutathione, GSH/GSSG ratio, superoxide dismutase, catalase and GPx were decreased in STZ induced diabetic rats. Administration of SAC to diabetic rats showed a decrease in plasma glucose, TBARS, hydroperoxide and GSSG. In addition, the levels of plasma insulin, superoxide dismutase, catalase, GPx and reduced glutathione (GSH) were increased in SAC treated diabetic rats. The above findings were supported by histological observations of the liver and kidney. The antioxidant effect of SAC was compared with glyclazide, a well-known antioxidant and antihyperglycemic drug. The present study indicates that the SAC possesses a significant favorable effect on antioxidant defense system in addition to its antidiabetic effect.     

The Detoxification of Cumene Hydroperoxide by the Glutathione System of Cultured Astroglial Cells Hinges on Hexose Availability for the Regeneration of NADPH

The ability of astroglia-rich primary cultures derived from the brains of newborn rats to detoxify exogenously applied cumene hydroperoxide (CHP) was analyzed as a model to study glutathione-mediated peroxide detoxification by astrocytes. Under the conditions used, 200 microM CHP disappeared from the incubation buffer with a half-time of approximately 10 min. The half-time of CHP in the incubation buffer was found strongly elevated (a) in cultures depleted of glutathione by a preincubation with buthionine sulfoximine, an inhibitor of glutathione synthesis, (b) in the presence of mercaptosuccinate, an inhibitor of glutathione peroxidase, and (c) in the absence of glucose, a precursor for the regeneration of NADPH. The involvement of glutathione peroxidase in the clearance of CHP was confirmed by the rapid increase in the level of GSSG after application of CHP. The restoration of the initial high ratio of GSH to GSSG depended on the presence of glucose during the incubation. The high capacity of astroglial cells to clear CHP and to restore the initial ratio of GSH to GSSG was fully maintained when glucose was replaced by mannose. In addition, fructose and galactose at least partially substituted for glucose, whereas exogenous isocitrate and malate were at best marginally able to replace glucose during peroxide detoxification and regeneration of GSH. These results demonstrate that CHP is detoxified rapidly by astroglial cells via the glutathione system. This metabolic process strongly depends on the availability of glucose or mannose as hydride donors for the regeneration of the NADPH that is required for the reduction of GSSG by glutathione reductase.     

Oxidant effect of acetylphenylhydrazine: a comparative study with erythrocytes of several animal species.

Red cells from several animal species were treated with acetylphenylhydrazine and a comparative study of the oxidation of hemoglobin (Hb) and glutathione (GSH) made. Wide interspecies differences were observed but the oxidation of GSH paralleled that of Hb. Added glucose protected both Hb and GSH from oxidation; GSH by itself exercises a protective effect on Hb. The characteristic rates of oxidation of GSH in the different species can be observed only in the presence of oxyhemoglobin but not carboxyhemoblobin or methemoglobin. The oxidation of Hb appears to be the primary event, the oxidation of GSH being a consequence thereof.     

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

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, glucose metabolism through the pentose phosphate pathway is a major source of NADPH, the cofactor necessary for the regeneration of reduced glutathione. This study aims at investigating glucose metabolism in cultured astrocytes from GCLM knockout mice, which show decreased GSH levels. No difference in the basal metabolism of glucose was observed between wild-type and knockout cells. In contrast, glycogen levels were lower and its turnover was higher in knockout astrocytes. These changes were accompanied by a decrease in the expression of the genes involved in its synthesis and degradation, including the protein targeting to glycogen. During an oxidative challenge induced by tert-Butylhydroperoxide, wild-type cells increased their glycogen mobilization and glucose uptake. However, knockout astrocytes were unable to mobilize glycogen following the same stress and they could increase their glucose utilization only following a major oxidative insult. Altogether, these results show that glucose metabolism and glycogen utilization are dysregulated in astrocytes showing a chronic deficit in GSH, suggesting that alterations of a fundamental aspect of brain energy metabolism is caused by GSH deficit and may therefore be relevant to metabolic dysfunctions observed in schizophrenia.     

Glutathione production in copper-deficient isolated rat hepatocytes.

Dietary copper deficiency has been shown to reduce copper-dependent superoxide dismutase (SOD) activity and to increase lipid peroxidation in rats. Circulating reduced glutathione (GSH) concentrations are elevated in copper-deficient (CuD) rats, which suggests an increased GSH synthesis or decreased degradation, perhaps as an adaptation to the oxidative stress of copper deficiency. GSH synthesis was examined in isolated hepatocytes from CuD rats. Isolated hepatocytes were prepared by collagenase perfusion and incubated in Krebs-Henseleit bicarbonate buffer, pH 7.4, 10 mM glucose, 2.5 mM Ca2+ in the presence and absence of 1.0 mM buthionine sulfoximine (BSO), a specific inhibitor of GSH synthesis. Cell viability was assessed by trypan blue exclusion. GSH and oxidized glutathione (GSSG) were measured by the glutathione reductase recycling assay. Copper deficiency depressed hepatocyte Cu by greater than 90% and increased intracellular GSH by 41-117% over the 3-h incubation, with a two- to threefold increase in the rate of intracellular GSH synthesis. Intracellular GSSG values were minimally influenced by CuD, with a constant mol% GSSG. Extracellular total glutathione (GSH + 2GSSG) synthesis was increased by approximately 33%. Both intracellular GSH and extracellular total glutathione synthesis were inhibited by BSO. The pattern of food consumption in CuD rats, meal fed versus ad libitum fed, had no effect on glutathione synthesis. The results indicate an increased hepatic GSH synthesis as a response to dietary copper deficiency and suggest an interrelationship between the essential nutrients involved in oxyradical metabolism.     

2-Deoxyribose Deprives Cultured Astrocytes of their Glutathione

High concentrations of 2-deoxy-d-ribose (2dRib) have been reported to cause oxidative stress and to disturb the glutathione (GSH) metabolism of various cell types. Exposure of astrocyte-rich primary cultures to millimolar concentrations of 2dRib or its stereoisomer 2-deoxy-l-ribose, but not the incubation with ribose, 2-deoxyglucose, glucose, fructose or saccharose, lowered the cellular GSH content in a time and concentration dependent manner. After exposure for 4 h to 30 mM 2dRib the cells contained 2dRib in a concentration of about 24 mM. Under these conditions 2dRib did not compromise cell viability and the ability of the cells to synthesise GSH, nor were the cellular ratio of glutathione disulfide (GSSG) to GSH and the extracellular concentrations of GSH or GSSG increased. These data demonstrate that 2dRib deprives viable cultured astrocytes of GSH and suggest that a cellular reaction of GSH with 2dRib or its metabolites is involved in the deprivation of astrocytic GSH.     

Acute hypoglycemia induces retinal cell death in mouse

Background

Glucose is the most important metabolic substrate of the retina and maintenance of normoglycemia is an essential challenge for diabetic patients. Glycemic excursions could lead to cardiovascular disease, nephropathy, neuropathy and retinopathy. A vast body of literature exists on hyperglycemia namely in the field of diabetic retinopathy, but very little is known about the deleterious effect of hypoglycemia. Therefore, we decided to study the role of acute hypoglycemia in mouse retina.

Methodology/Principal Findings

To test effects of hypoglycemia, we performed a 5-hour hyperinsulinemic/hypoglycemic clamp; to exclude an effect of insulin, we made a hyperinsulinemic/euglycemic clamp as control. We then isolated retinas from each group at different time-points after the clamp to analyze cells apoptosis and genes regulation. In parallel, we used 661W photoreceptor cells to confirm in vivo results. We showed herein that hypoglycemia induced retinal cell death in mouse via caspase 3 activation. We then tested the mRNA expression of glutathione transferase omega 1 (Gsto1) and glutathione peroxidase 3 (Gpx3), two genes involved in glutathione (GSH) homeostasis. The expression of both genes was up-regulated by low glucose, leading to a decrease of reduced glutathione (GSH). In vitro experiments confirmed the low-glucose induction of 661W cell death via superoxide production and activation of caspase 3, which was concomitant with a decrease of GSH content. Moreover, decrease of GSH content by inhibition with buthionine sulphoximine (BSO) at high glucose induced apoptosis, while complementation with extracellular glutathione ethyl ester (GSHee) at low glucose restored GSH level and reduced apoptosis.

Conclusions/Significance

We showed, for the first time, that acute insulin-induced hypoglycemia leads to caspase 3-dependant retinal cell death with a predominant role of GSH content.     

Differential action of methylselenocysteine in control and alloxan-diabetic rabbits

Antidiabetic action of inorganic selenium compounds is commonly accepted. Since in diet selenium mainly exists as selenoamino acids, potential hypoglycemic properties of methylselenocysteine (MSC) were investigated in four groups of rabbits: untreated and MSC-treated control animals as well as alloxan-diabetic and MSC-treated diabetic rabbits. MSC (at a dose of 1 mg/kg body weight) was administered daily for 3 weeks via intraperitoneal injection. The data show, that in MSC-treated control animals plasma glucose concentration was diminished, while plasma urea and creatinine levels as well as urine albumin content were elevated and necrotic changes occurred in kidney-cortex. Decreased GSH/GSSG ratios in blood, liver and kidney-cortex were accompanied by increased glutathione peroxidase and glutathione reductase activities and a diminished renal γ-glutamylcysteine synthetase activity. Death of 50% of control animals was preceded by a dramatic decline in blood glucose concentration. Surprisingly, in MSC-treated diabetic rabbits, plasma glucose levels were either normalized or significantly decreased. Blood and liver GSH/GSSG ratios were increased and renal functions were markedly improved, as indicated by a diminished albuminuria and attenuated histological changes characteristic of diabetes. However, after administration of MSC to diabetic rabbits plasma urea and creatinine levels as well as renal GSH/GSSG ratios were not altered. In view of MSC-induced marked accumulation of selenium in kidneys and liver of control rabbits, accompanied by a decline in blood glucose level, disturbance of glutathione homeostasis and kidney-injury, application of MSC in chemotherapy needs a careful evaluation. On the contrary, MSC supplementation might be beneficial for diabetes therapy due to an improvement of both glycemia and renal function.     

Blood glutathione oxidation during human exercise

To examine the effects of increased O2 utilization on the glutathione antioxidant system in blood, eight moderately trained male volunteers were exercised to peak O2 consumption (VO2peak) and for 90 min at 65% of VO2peak on a cycle ergometer. Blood samples were taken during exercise, and for up to 4 days of recovery from submaximal exercise. During exercise to VO2peak, blood reduced glutathione (GSH) and total glutathione [GSH + oxidized glutathione (GSSG)] did not change significantly. Lactate (L), pyruvate (P), and L/P increased significantly from rest values (P less than 0.01). During prolonged submaximal exercise, GSH decreased 60% from control, and GSSG increased 100%. Total glutathione, glucose, pyruvate, and lactate concentrations and L/P did not change significantly during sustained exercise. During recovery, GSH and GSH/GSSG increased from exercise levels and significantly overshot preexercise levels, reaching maximum values after 3 days. Oxidation of GSH during submaximal exercise and its reduction in recovery suggest increased formation of active O2-. species in blood during physical exercise in moderately trained males.     

External GSSG enhances intracellular glutathione level in isolated cardiac myocytes

The addition of external GSSG at concentrations in the range 50-500 microM produces in isolated adult rat heart myocytes an increase of GSH level and only a slight increase of GSSG level. On the contrary, external GSH at the above same indicated concentrations did not change the cell glutathione pool. The pretreatment of the cells with diethylamaleate depleted the myocytes of glutathione and enhanced the GSSG-induced replenishment effect on GSH level. On the contrary, the addition of GSH did not increase the concentration of cell glutathione. The level of cell GSH in diethylmaleate-treated myocytes was not increased after 30 min of incubation with cysteine, or acetylcysteine. The GSSG induced-stimulation on GSH level was not inhibited by buthionine sulfoximine, an inhibitor of glutathione synthesis. On the contrary, this stimulatory effect was inhibited by N, N-bis(2-chloroethyl)-N-nitrosourea, an inhibitor of glutathione reductase, or partially, by the remotion of glucose from the incubation medium. These results support the idea that the isolated adult rat heart myocytes are able to utilize external GSSG in order to increase the intracellular glutathione pool, probably through the reduction of the imported GSSG to GSH.     

Effects of t-butyl hydroperoxide on NADPH, glutathione, and the respiratory burst of rat alveolar macrophages

The effects of t-butyl hydroperoxide on glutathione and NADPH and the respiratory burst (an NADPH-dependent function) in rat alveolar macrophages was investigated. Alveolar macrophages were exposed for 15 min to t-butyl hydroperoxide in the presence or absence of added glucose. Cells were then assayed for concanavalin A-stimulated O2 production or for NADPH, NADP, reduced glutathione, glutathione disulfide, glutathione released into the medium and glutathione mixed disulfides. Exposure of rat alveolar macrophages to 1 X 10(-5) M t-butyl hydroperoxide causes a loss of concanavalin A-stimulated superoxide production (the respiratory burst) that can be prevented or reversed by added glucose. Cells incubated without glucose had a higher oxidation state of the NADPH/NADP couple than cells incubated with glucose. With t-butyl hydroperoxide, NADP rose to almost 100% of the NADP + NADPH pool; however, addition of glucose prevented this alteration of the NADPH oxidation state. Cells exposed to 1 X 10(-5) M t-butyl hydroperoxide in the absence of glucose showed a significant increase in the percentage GSSG in the GSH + GSSG pool and increased glutathione mixed disulfides. These changes in glutathione distribution could also be prevented or reversed by glucose. With 1 X 10(-4) M t-butyl hydroperoxide, changes in glutathione oxidation were not prevented by glucose and cells were irreversibly damaged. We conclude that drastic alteration of the NADPH/NADP ratio does not itself reflect toxicity and that significant alteration of glutathione distribution can also be tolerated; however, when oxidative stress exceeds the ability of glucose to prevent alterations in oxidation state, irreversible damage to cell function and structure may occur.