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.     

Glutathione oxidation and activation of pentose phosphate cycle during hydroperoxide metabolism. A comparison of livers from fed and fasted rats

Perfusion of livers from fed and fasted rats with 0.07--0.1 mM t-butyl hydroperoxide for 15 min decreased the levels of reduced glutathione (GSH) by 1.5 mumol/g liver in both nutritional states. Glutathione disulfide (GSSG) was increased by 70 and 140 nmol/g liver and glutathione mixed disulfides enhanced by 45 and 150 nmol/g liver in livers from fed and fasted animals, respectively. The ratio of GSH/GSSG was decreased from 243 to 58 in fed animals, and from 122 to 8 in fasted animals. The increase of GSSG and the mixed disulfides was nearly parallel until an apparently critical low GSH content of 1.5 mumol/g was reached. Only in livers from fasted rats 14CO2-production from [1-14C]glucose was stimulated upon t-butyl hydroperoxide infusion at the employed rates. Flux of glucose through pentose phosphate cycle rose from 8 to 12% of glucose utilization via glycolysis, whereas in livers from fed animals this portion remained unchanged at 8% Dithio-erythritol reversed pentose phosphate cycle activity as well as GSSG and protein-bound glutathione contents to the original levels. In livers from fasted rats the activity of glucose-6-phosphate dehydrogenase was increased by 34% by t-butyl hydroperoxide infusion.     

Changes in the levels of glutathione after cellular and cutaneous damage induced by squalene monohydroperoxide.

Squalene monohydroperoxide (Sq-OOH), the initial product of ultraviolet-peroxidated squalene, was used to investigate the effect of peroxidative challenge upon the glutathione contents in rabbit ear skin and primary-cultured fibroblasts derived from rabbit ear skin. The cellular reduced glutathione (GSH) contents decreased during 30-minute incubations in vitro with Sq-OOH, and oxidized glutathione (GSSG) was formed concomitantly, indicating that Sq-OOH had a potential for GSH-depleting activity in vitro. When Sq-OOH was applied topically to the skin in vivo, only GSSG contents increased significantly within 30 minutes. Moreover, pretreatment with the GSH depletors, DL-buthionine sulfoximine (BSO) and diethyl maleate (DEM), could potentiate the cytotoxicity and comedogenicity induced by Sq-OOH. These findings suggest that the endogenous antioxidant, glutathione, is quite sensitive to Sq-OOH and may be an important material for protecting cells and/or tissues against the oxidative stress induced by Sq-OOH treatment.     

Oxidative stress stimulates the synthesis of the eosinophil chemoattractant 5-oxo-6,8,11,14-eicosatetraenoic acid by inflammatory cells

5-Oxo-ETE (5-oxo-6,8,11,14-eicosatetraenoic acid) is a highly potent granulocyte chemoattractant that acts through a selective G-protein coupled receptor. It is formed by oxidation of the 5-lipoxygenase product 5-HETE (5S-hydroxy-6,8,11,14-eicosatetraenoic acid) by 5-hydroxyeicosanoid dehydrogenase (5-HEDH). Although leukocytes and platelets display high microsomal 5-HEDH activity, unstimulated intact cells do not convert 5-HETE to appreciable amounts of 5-oxo-ETE. To attempt to resolve this dilemma we explored the possibility that 5-oxo-ETE synthesis could be enhanced by oxidative stress. We found that hydrogen peroxide and t-butyl hydroperoxide strongly stimulate 5-oxo-ETE formation by U937 monocytic cells. This was dependent on the GSH redox cycle, as it was blocked by depletion of GSH or inhibition of glutathione reductase and mimicked by oxidation of GSH to GSSG by diamide. Glucose inhibited the response to H2O2 through its metabolism by the pentose phosphate pathway, as its effect was reversed by the glucose-6-phosphate dehydrogenase inhibitor dehydroepiandrosterone. 5-Oxo-ETE synthesis was also strongly stimulated by hydroperoxides in blood monocytes, lymphocytes, and platelets, but not neutrophils. Unlike monocytic cells, lymphocytes and platelets were resistant to the inhibitory effects of glucose. 5-Oxo-ETE synthesis following incubation of peripheral blood mononuclear cells with arachidonic acid and calcium ionophore was also strongly enhanced by t-butyl hydroperoxide. Oxidative stress could act by depleting NADPH, resulting in the formation NADP+, the cofactor for 5-HEDH. This is opposed by the pentose phosphate pathway, which converts NADP+ back to NADPH. Oxidative stress could be an important mechanism for stimulating 5-oxo-ETE production in inflammation, promoting further infiltration of granulocytes into inflammatory sites.     

Glutathione dependent metabolism and detoxification of 4-hydroxy-2-nonenal.

The involvement of glutathione (GSH) dependent processes in the detoxification of 4-hydroxy-2-nonenal (4HNE) was investigated using Chinese hamster fibroblasts and clonogenic cell survival. GSH reacted, in a dose-dependent fashion, with 4HNE in phosphate buffer at pH 6.5, leading to the disappearance of 4HNE. The addition of glutathione transferase activity (GST) facilitated a more rapid disappearance of 4HNE but the reaction was still dependent on the concentration of GSH. When cell cultures were exposed to the reaction mixtures, 4HNE cytotoxicity was also reduced in a manner which was dependent on the concentration of GSH. When 2.16- or 1.08-mM GSH were incubated in phosphate buffer with 1.08-mM 4HNE in the presence or absence of GST, then mixed with media and placed on cells for 1 h, the cytotoxicity associated with exogenous exposure to free 4HNE was abolished. GSH depletion (greater than 90%) using buthionine sulfoximine (BSO) was accomplished in control (HA1) and H2O2-resistant variants derived from HA1. GSH depletion resulted in enhanced cytotoxicity of 4HNE in all cell lines. This BSO-induced sensitization to 4HNE cytotoxicity was accompanied by a significant reduction in the ability of cells to metabolize 4HNE. The magnitude of the sensitization to 4HNE toxicity caused by GSH depletion was similar to the magnitude of the reduction in the ability of cells to metabolize 4HNE. These results support the hypothesis that GSH and GST provide a biologically significant pathway for protection against aldehydic by-products of lipid peroxidation.     

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     

Functional significance of the pentose phosphate pathway and glutathione reductase in the antioxidant defenses of human sperm

Glutathione peroxidase is one of the principal antioxidant defense enzymes in human spermatozoa, but it requires oxidized glutathione to be reduced by glutathione reductase using NADPH generated in the pentose phosphate pathway. We investigated whether flux through the pentose phosphate pathway would increase in response to oxidative stress and whether glutathione reductase was required to protect sperm from oxidative damage. Isotopic measurements of the pentose phosphate pathway and glycolytic flux, thiobarbituric acid assay of malondialdehyde for lipid peroxidation, and computer-assisted sperm analysis for sperm motility were assessed in a group of normal, healthy semen donors. Applying moderate oxidative stress to human spermatozoa by adding cumene hydroperoxide, H(2)O(2), or xanthine plus xanthine oxidase or by promoting lipid peroxidation with ascorbate increased flux through the pentose phosphate pathway without changing the glycolytic rate. However, adding higher concentrations of oxidants inhibited both the pentose phosphate pathway and glycolytic flux. At concentrations of 50 microg/ml or greater, the glutathione reductase-inhibitor 1,3-bis-(2-chloroethyl) 1-nitrosourea decreased flux through the pentose phosphate pathway and blocked the response to cumene hydroperoxide. It also increased lipid peroxidation and impaired the survival of motility in sperm incubated under 95% O(2). These data show that the pentose phosphate pathway in human spermatozoa can respond dynamically to oxidative stress and that inhibiting glutathione reductase impairs the ability of sperm to resist lipid peroxidation. We conclude that the glutathione peroxidase-glutathione reductase-pentose phosphate pathway system is functional and provides an effective antioxidant defense in normal human spermatozoa.     

Variation in glutathione status associated with induction and initiation of diapause in eggs of the bivoltine strain of the silkworm Bombyx mori

For the bivoltine (Dazao) strain of the silkworm Bombyx mori L., diapause expression in progeny is induced by exposure to conditions of 25 °C and continuous illumination (LL) during the maternal generation, whereas an environment of 15 °C and constant darkness (DD) results in nondiapause progeny. Initiation of diapause in progeny can be prevented by treatment of diapause‐programmed eggs with hydrochloric acid (HCl) at approximately 24 h post‐oviposition. To investigate whether glutathione is involved in the regulation of diapause induction and initiation in this species, measurements of total glutathione, reduced glutathione (GSH), oxidised glutathione (GSSG), GSH/GSSG ratio, glutathione S‐transferase (GST) and peroxiredoxins (Prdx) are compared in eggs incubated under LL and DD conditions, and between diapause eggs and those treated with HCl. Compared with DD, eggs incubated under LL have higher total glutathione (GSH + 2GSSG), lower GSH, higher GSSG, a lower GSH/GSSG ratio, lower GST activity and higher Prdx activity at stages 20–25 of maternal embryogenesis. The lower ratio of GSH/GSSG is indicative of pro‐oxidative conditions during diapause induction, which may result from the stronger oxidation of GSH. Compared with HCl‐treated eggs, diapause eggs have lower total glutathione, no difference in GSH, lower GSSG, a higher GSH/GSSG ratio, no difference in GST activity and lower Prdx between 36 and 72 h post‐oviposition. The higher ratio GSH/GSSG is indicative of reducing conditions during diapause initiation, which may a result of the weaker oxidation of GSH. Moreover, variations of Prdx and GST suggest that Prdx rather than GST plays an important role in the oxidation of GSH during the induction and initiation of diapause.     

Glutathione system adaptation to acute stress in the heart of rats during different regimes of hypoxia training

The intensity of lipid peroxidation (LPO), reduced and oxidized glutathione (GSH and GSSG) contents, glutathione reductase, glutathione peroxidase, glutathione-S-transferase, glucose-6-phosphate dehydrogenase (G-6-PDH), and NADP-isocitrate dehydrogenase (NADP-IDH) activities were studied in the heart of male rats exposed to two modes of intermittent hypoxic training (IHT): I-breathing in normobaric chamber with 7% O2 gas mixture for 5 min with 15 min normoxic intervals 4 times daily during 3 weeks; II-breathing by 12% O2 gas mixture in the same manner). After adaptation to hypoxia, the rats were subjected to 6h-immobilization stress. It has been shown that stress action after IHT (regime I) caused the increase in LPO and the shift of GSH/GSSG to disulfides. A disbalance in antioxidative defense system was determined by the decrease in glutatione peroxidase, G-6-PDH activities, and GSH content. The support of glutathione reductase activity under stress in this group with simultaneous decrease of enzyme activity in the pentose phosphate pathway was realized through the participation of NADP-IDH. Hypoxic training in regime II induced LPO decrease in the heart tissue after stress. The increase in the heart GSH content, optimal balance of glutathione-related enzymes in this group evidences for the dependence of adaptation effects on the vigor of hypoxic exposition. Our results suggest the active participation of glutathione system in the formation of adaptation reactions under the extreme factor influences through the action on intracellular red/ox potential as well as effectiveness of antioxidant defense.     

Hydroperoxide-metabolizing systems in rat liver.

1. Metabolism of added hydroperoxides was studied in hemoglobin-free perfused rat liver and in isolated rat hepatocytes as well as microsomal and mitochondrial fractions. 2. Perfused liver is capable of removing organic hydroperoxides [cumene and tert-butyl hydroperoxide] at rates up to 3--4 mumol X min-1 X gram liver-1. Concomitantly, there is a release of glutathione disulfide (GSSG) into the extracellular space in a relationship approx. linear with hydroperoxide infusion rates. About 30 nmol GSSG are released per mumol hydroperoxide added per min per gram liver. GSSG release is interpreted to indicate GSH peroxidase activity. 3. GSSG release is observed also with added H2O2. At rates of H2O2 infusion of about 1.5 mumol X min-1 X gram liver-1 a maximum of GSSG release is attained which, however, can be increased by inhibition of catalase with 3-amino-1,2,4-aminotriazole. 4. A contribution of the endoplasmic reticulum in addition to glutathione peroxidase in organic hydroperoxide removal is demonstrated (a) by comparison of perfused livers from untreated and phenobarbital-pretreated rats and (b) in isolated microsomal fractions, and a possible involvement of reactive iron species (e.g. cytochrome P-450-linked peroxidase activity) is discussed. 5. Hydroperoxide addition to microsomes leads to rapid and substantial lipid peroxidation as evidenced by formation of thiobarbituric-acid-reactive material (presumably malondialdehyde) and by O2 uptake. Like in other types of induction of lipid peroxidation, malondialdehyde/O2 ratios of 1/20 are observed. Cumene hydroperoxide (0.6 mM) gives rise to 4-fold higher rates of malondialdehyde formation than tert-butyl hydroperoxide (1 mM). Ethylenediamine tetraacetate does not inhibit this type of lipid peroxidation. 6. Lipid peroxidation in isolated hepatocytes upon hydroperoxide addition is much lower than in isolated microsomes or mitochondria, consistent with the presence of effective hydroperoxide-reducing systems. However, when NADPH is oxidized to the maximal extent as evidenced by dual-wavelength spectrophotometry, lipid peroxidation occurs at large amounts. 7. A dependence of hydroperoxide removal rates upon flux through the pentose phosphate pathway is suggested by a stimulatory effect of glucose in hepatocytes from fasted rats and by an increased rate of 14CO2 release from [1-14C]glucose during hydroperoxide metabolism in perfused liver.     

Mitochondrial poisons cause depletion of reduced glutathione in isolated hepatocytes.

Antimycin A, KCN, and 1-methyl-4-phenylpyridinium ion (MPP+) all produced a marked depletion of cellular GSH levels in freshly isolated hepatocytes. This effect was consistently observed before the onset of cytotoxicity and seemed to be correlated with the loss of cellular ATP induced by these mitochondrial poisons. Concentrations of GSSG remained unchanged both intracellularly and extracellularly, indicating that oxidation was not involved in the events leading to GSH depletion. Approximately 40% of the decrease of intracellular GSH was accounted for by efflux of this tripeptide, assessed by increased formation of cysteinyl-glutathione when hepatocytes were incubated in the presence of 0.2 mM cystine. Therefore, an overall loss of glutathione was observed during incubations with all three inhibitors of mitochondrial function. Addition of 10 mM fructose to the incubation media substantially protected against GSH depletion caused by antimycin A, KCN, and MPP+. These results indicate that energy-dependent mechanisms are involved in the maintenance of intracellular GSH levels, and suggest that GSH depletion may be a general phenomenon associated with impairment of mitochondrial function.     

Differences in glutathione oxidation and transpeptidylation between normal liver and hepatomas (author's transl)

Total homogenates from liver tissues, as well from Morris 3924 A and Yoshida AH-I30 hepatomas display a different degree of thiobarbituric acid reacting substances (TBArs) when incubated "in vitro". It is well known that carbonyl compounds arising from lipoperoxidative decomposition of unsaturated fatty acids can easily react with reduced glutathione (GSH). So, the decay in GSH we have shown in previous experiments could be accounted for GSH trapping by the formed aldehydes. Some discrepancies were, however, seen when the decay in GSH and the increase in GSSG were compared, both in normal and in tumour tissues. It is known that GSH can be destroyed not only through oxidative process, but also through the action of gamma-glutamyl-transpeptidase. In the present paper the decrease of total (TG) and reduced (GSH) glutathione was followed and compared with both the increase in GSSG and the increase in the production of TBArs, during "in vitro" incubation. In normal liver, increase in TBArs production parallels the decay in GSH concentration; GSSG, on the contrary, increases. In AH-I30 Yoshida hepatoma cells, TBArs production is lower and GSSG is also decreased. In 3924 A Morris hepatoma GSH decrease is similar to that observed in the liver, while TBArs production is lower and GSSG is also decreased. Analysis of TG content during the incubation-time suggests that GSH decay in both hepatoma types is essentially due to gamma-glutamyl-transpeptidase action, whilst GSH oxidation to GSSG is decreased.     

Age-associated analysis of oxidative stress parameters in human plasma and erythrocytes

Oxidative damage accumulation in macromolecules has been considered as a cause of cellular damage and pathology. Rarely, the oxidative stress parameters in healthy humans related to the individual age have been reported. The purpose of this study was to examine the redox status in plasma and erythrocytes of healthy individuals and determine correlations between these parameters and the aging process. The following parameters were used: malondialdehyde (MDA), protein carbonyls (PCO), 4-hydroxy-2,3-trans-nonenal (HNE), reduced glutathione (GSH), glutathione disulfide (GSSG) and uric acid (UA) in blood and plasma samples of 194 healthy women and men of ages ranging from 18 to 84 years. The results indicate that the balance of oxidant and antioxidant systems in plasma shifts in favor of accelerated oxidation during ageing. That is demonstrated by increases of MDA, HNE, GSSG and by the slight decrease of erythrocytic GSH with age. As the content of UA is more determined by metabolic and nutritional influences than by the balance between prooxidants and antioxidants there was no significant age-related change observed. For plasma concentrations of HNE the first time age-dependent reference values for healthy humans are presented.     

Hepatic response to the oxidative stress induced by E. coli endotoxin: Glutathione as an index of the acute phase during the endotoxic shock

Reactive oxygen species are important mediators of cellular damage during endotoxic shock. In order to investigate the hepatic response to the oxidative stress induced by endotoxin, hepatic and plasma glutathione (total, GSH and GSSG), GSSG/GSH ratio as well as Mn-superoxide dismutase and catalase activities were determined during the acute and recovery phases of reversible endotoxic shock in the rat. A significant increase in liver and plasma total glutathione content was observed 5 h after endotoxin treatment (acute phase), followed by a diminution of these parameters below control values at 48 h (recovery phase). The significant increases of GSSG levels and GSSG/GSH ratio are indicative of oxidative stress occurring during the acute phase. Liver Mn-SOD activity showed a similar time dependency as the GSSG/GSH ratio; however, a marked decrease in the liver catalase activity was observed during the process. These results indicate the participation of liver glutathione in the response to endotoxin and the possible use of plasma glutathione levels and GSSG/GSH ratio as indicators of the acute phase during the endotoxic process. (Mol Cell Biochem 159: 115-121, 1996)     

Lipid peroxide-induced redox imbalance differentially mediates CaCo-2 cell proliferation and growth arrest

Dietary oxidants like lipid hydroperoxides (LOOH) can perturb cellular glutathione/glutathione disulphide (GSH/GSSG) status and disrupt mucosal turnover. This study examines the effect of LOOH on GSH/GSSG balance and phase transitions in the human colon cancer CaCo-2 cell. LOOH at 1 or 5 micro m were noncytotoxic, but disrupted cellular GSH/GSSG and stimulated proliferative activity at 6 h that paralleled increases in ornithine decarboxylase activity, thymidine incorporation, expression of cyclin D1/cyclin-dependent kinase 4, phosphorylation of retinoblastoma protein, and cell progression from G0/G1 to S. At 24 h, LOOH-induced sustained GSH/GSSG imbalance mediated growth arrest at G0/G1 that correlated with suppression of proliferative activity and enhanced oxidative DNA damage. LOOH-induced cell transitions were effectively blocked by N-acetylcysteine. Collectively, the study shows that subtoxic LOOH levels induce CaCo-2 GSH/GSSG imbalance that elicits time-dependent cell proliferation followed by growth arrest. These results provide insights into the mechanism of hydroperoxide-induced disruption of mucosal turnover with implications for understanding oxidant-mediated genesis of gut pathology.     

Glutathiolation of the proteasome is enhanced by proteolytic inhibitors

The proteasome inhibitors lactacystin, clastro lactacystin beta-lactone, or tri-leucine vinyl sulfone (NLVS), in the presence of [(35)S]cysteine/methionine, caused increased incorporation of (35)S into cellular proteins, even when protein synthesis was inhibited by cycloheximide. This effect was blocked by incubation with the glutathione synthesis inhibitor buthionine sulfoximine. Proteasome inhibitors also enhanced total glutathione levels, increased reduced/oxidized glutathione ratio (GSH/GSSG) and upregulated gamma-glutamylcysteine synthetase (rate-limiting in glutathione synthesis). Micromolar concentrations of GSH, GSSG, or cysteine stimulated the chymotrypsin-like activity of purified 20S proteasome, but millimolar GSH or GSSG was inhibitory. Interestingly, GSH did not affect 20S proteasome's trypsin-like activity. Enhanced proteasome glutathiolation was verified when purified preparations of the 20S core enzyme complex were incubated with [(35)S]GSH after pre-incubation with any of the inhibitors. NLVS, lactacystin or clastro lactacystin beta-lactone may promote structural modification of the 20S core proteasome, with increased exposure of cysteine residues, which are prone to S-thiolation. Three main conclusions can be drawn from the present work. First, proteasome inhibitors alter cellular glutathione metabolism. Second, proteasome glutathiolation is enhanced by inhibitors but still occurs in their absence, at physiological GSH and GSSG levels. Third, proteasome glutathiolation seems to be a previously unknown mechanism of proteasome regulation in vivo.     

Regulation of the human-erythrocyte hexose-monophosphate shunt under conditions of oxidative stress. A study using NMR spectroscopy, a kinetic isotope effect, a reconstituted system and computer simulation

The regulation of the hexose monophosphate shunt of human erythrocytes under conditions of oxidative stress has been investigated by monitoring the reduction of oxidised glutathione (GSSG) to reduced glutathione (GSH) in erythrocytes containing high levels of GSSG; 1H NMR and a biochemical assay were used to measure the changes. A reconstituted metabolic system prepared with the purified erythrocyte enzymes was used in conjunction with studies of intact cells and haemolysates to determine the dependence of the rate of GSH production on the activities of hexokinase and glucose-6-phosphate dehydrogenase. Both of these enzymes have previously been claimed to be the rate-limiting step of oxidatively stimulated flux through the hexose monophosphate shunt. The absence of a kinetic isotope effect on the rate of GSH production in these systems, when [1-2H]glucose replaced glucose as the source of reducing equivalents, showed that glucose-6-phosphate dehydrogenase activity was not a strong determinant of the flux. The dependence of the rate of GSH production on the concentration of the hexokinase inhibitors glucose 1,6-bisphosphate and glycerate 2,3-bisphosphate showed that, under conditions of oxidative stress, hexokinase was the principal determinant of flux through the shunt. Glucose 1,6-bisphosphate at the concentration present in vivo appears to be more important in limiting hexokinase activity, and thus the rate of glucose utilisation, than was previously assumed. A detailed computer model of the system was developed based on the reported kinetic parameters of the enzymes involved. A sensitivity analysis of this model predicted that the hexokinase reaction would have a sensitivity coefficient of 0.995 with respect to the maximal rate of GSH production.     

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.     

"In vitro" effect of CCl4, PG and EDTA on GSH levels at different time of incubation of rat liver homogenates

During CCl4-induced lipid peroxidation GSH content in total homogenate from rat liver falls very rapidly in the first 30 min. of incubation "in vitro". CCl4 does not enhance the decrease in total glutathione (TG) during the incubation time, so GSH loss is mainly due to its oxidation to GSSG. On the contrary PG and EDTA, two substances decreasing lipid peroxidation rate, are able to decrease GSH oxidation, without affecting TG content. At 25 degrees C EDTA and PG completely prevent GSH decrease at pH 7.4, while at pH 6 PG affords only a partial prevention. At 37 degrees C both compounds are able to limit GSH decrease at a large extent. Lipid peroxidation seems to have a great importance in the kinetics of GSH decrease and GSSG formation, at least "in vitro". It is noteworthy that PG which inhibits lipid peroxidation stimulated by CCl4 is also able to limit the high GSH loss observed in the homogenates incubated in the presence of halogeno-alkane.