Altered GSH Research Articles

Altered metabolism of glutathione in cells transformed by oncogenes which cause resistance to ionizing radiations

We measured glutathione (GSH) metabolism in normal NIH/3T3 fibroblasts, and in cells transformed by the oncogenes sis, erbB, src, ras, dbl, and raf.erbB, src,ras and raf, but not sis and dbltransformants, showed increased level of total and reduced GSH as compared with normal NIH/3T3 fibroblasts; oxidized GSH was elevated only in src- and ras-transformed cells. Increased total GSH content was associated with decreased activity of the synthetic enzyme γ-glutamylcysteine synthetase, and oxidized GSH level with increased activity of GSH reductase. These data suggest that GSH synthesis was selectively enhanced in cells transformed by specific oncogenes, with resulting down-regulation of its synthetic enzyme; alterations of GSH metabolism appeared to be peculiar of transformation by specific oncogenes, and not trivial epiphenomena of neoplastic transformation. Oncogenic transformants that presented elevated level of GSH were also those reported to be resistant to antineoplastic drugs and ionizing radiations, thus confirming a possible link between altered GSH metabolism and resistance to antineoplastic treatment.

Effects of dietary selenium and vitamin E on glutathione concentrations and glutathione S-transferase activities in chick liver and plasma

Studies were conducted to determine the effects of the antioxidant nutrients selenium (Se) and vitamin E (VE) on the concentrations of glutathione (GSH) and activities of two GSH-metabolizing enzymes, Se-dependent GSH-peroxidase (SeGSHpx) and GSH S-transferase (GSH-T), in the chick. The hepatic activity of GSH-T, assayed using 1-chloro-2,4-dinitrobenzene as substrate (GSH-T CDNB), was greater in chicks depleted of both Se and VE than in chicks fed adequate levels of either nutrient. This effect was only observed when the Se deficiency was severe enough to result in a very substantial loss of SeGSHpx activity (e.g., 8% of control levels); it was not observed unless chicks were also deficient in VE. Chicks fed the Se-deficient diet supplemented with a low level of VE (2 IU/kg) failed to show hepatic GSH-T CDNB activities greater than controls. Chicks fed the Se-deficient diet had plasma GSH concentrations greater than those of Se-fed chicks, which difference was intensified by concomittant VE deficiency. These results show that GSH metabolism in the chick can be affected by nutritional status with respect to VE as well as Se. It is suggested that the increased hepatic activity of GSH-T CDNB that occurs in nutritional Se- and VE-deficiency may be a response to altered GSH metabolism rather than a compensation for loss of SeGSHpx.

Increase in rat brain glutathione following intracerebroventricular administration of γ-glutamylcysteine

The effects of intracereboventricularly (i.c.v.) administered γ-glutamylcysteine (γ-GC) and glutathione (GSH) monoethyl ester, subcutaneously (s.c.) injected l-2-oxo-4-thiazolidinecarboxylic acid (OTC) and intraperitoneally (i.p.) administered cysteine on the concentration of GSH in rat brain were investigated. The brain content of GSH, cysteine and γ-GC was determined by HPLC with electrochemical detection (gold/mercury electrode) using N-acetylcysteine as internal standard. A dosedependent increase in the GSH concentration (145–170% of controls) was found in the substantia nigra (SN) and in the rest of the brain stem after injection of γ-GC, whereas no significant alterations in GSH were observed in the striatum and in the cerebral cortex. High levels of γ-GC could be detected in the brain tissue after the administration, and the concentration of cysteine did also increase markedly after γ-GC injection in all brain regions assessed. I.c.v. administration of l-buthionine sulfoximine (L-BSO) reduced the brain concentration of GSH by 50–70% within 24 hr. Injection of γ-GC 24 hr after L-BSO resulted in an increase in GSH up to control values within 1–3 hr in the SN and the rest of the brain stem, whereas only a slight increase in GSH was observed in the striatum and the cerebral cortex. The concentration of GSH in the striatum and SN did not change after i.p. injection of cysteine, but a slight increase in the GSH concentration in the limbic region was observed. GSH monoethyl ester (i.c.v.) and OTC (s.c.) did not produce any significant increase in the GSH concentration in the brain. When the GSH concentration had been reduced by administration of L-BSO (i.c.v.; 24 hr) subsequent injection of GSH monoethyl ester led to a slight increase in the striatal and limbic GSH levels. These data show that, of the drugs studied, γ-GC was the most effective in increasing brain GSH. It could thus serve as a valuable tool in future studies regarding metabolism and function of GSH in the brain. The observed difference in the effects of γ-GC in different brain regions indicate that the brain tissue is not homogeneous with regard to GSH synthesizing capacity.

NADPH, Not Glutathione, Status Modulates Oxidant Sensitivity in Normal and Glucose-6-Phosphate Dehydrogenase-Deficient Erythrocytes

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is characterized by the loss of NADPH and enhanced erythrocyte oxidant sensitivity. Historically, it has been theorized that the elevated oxidant sensitivity of G6PD-deficient erythrocytes arises as the direct consequence of decreased intracellular glutathione (GSH) concentrations. To directly investigate the basis of G6PD deficiency oxidant sensitivity, the effects of altered GSH and NADPH concentrations were examined in normal and G6PD-deficient erythrocytes. The results of this study demonstrated that GSH depletion, by 1-chloro-2,4-dinitrobenzene (CDNB), had no effect on hemoglobin oxidation in response to hydrogen peroxide (H2O2) generating systems (phenazine methosulfate and menadione bisulfite) in either normal or G6PD-deficient cells. Furthermore, a fourfold to sixfold increase in intracellular GSH concentration also did not protect against H2O2-generating systems in the normal or G6PD-deficient erythrocytes. Conversely, introduction of an NADPH-generating system (purified G6PD) into G6PD-deficient cells resulted in a significant decrease in oxidant sensitivity and an ability to cycle GSH. Further experiments demonstrated that the reduced oxidant sensitivity of the G6PD-reconstituted erythrocytes was not due to the maintenance of GSH levels because CDNB-mediated depletion of GSH did not alter this protective effect. Analysis of these results demonstrated a direct correlation between NADPH, but not GSH, concentration and hemoglobin oxidant sensitivity.

NADPH, not glutathione, status modulates oxidant sensitivity in normal and glucose-6-phosphate dehydrogenase-deficient erythrocytes

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is characterized by the loss of NADPH and enhanced erythrocyte oxidant sensitivity. Historically, it has been theorized that the elevated oxidant sensitivity of G6PD-deficient erythrocytes arises as the direct consequence of decreased intracellular glutathione (GSH) concentrations. To directly investigate the basis of G6PD deficiency oxidant sensitivity, the effects of altered GSH and NADPH concentrations were examined in normal and G6PD-deficient erythrocytes. The results of this study demonstrated that GSH depletion, by 1-chloro- 2,4-dinitrobenzene (CDNB), had no effect on hemoglobin oxidation in response to hydrogen peroxide (H2O2) generating systems (phenazine methosulfate and menadione bisulfite) in either normal or G6PD- deficient cells. Furthermore, a fourfold to sixfold increase in intracellular GSH concentration also did not protect against H2O2- generating systems in the normal or G6PD-deficient erythrocytes. Conversely, introduction of an NADPH-generating system (purified G6PD) into G6PD-deficient cells resulted in a significant decrease in oxidant sensitivity and an ability to cycle GSH. Further experiments demonstrated that the reduced oxidant sensitivity of the G6PD- reconstituted erythrocytes was not due to the maintenance of GSH levels because CDNB-mediated depletion of GSH did not alter this protective effect. Analysis of these results demonstrated a direct correlation between NADPH, but not GSH, concentration and hemoglobin oxidant sensitivity.

Cytotoxicity of alkylating agents in isolated rat kidney proximal tubular and distal tubular cells

Patterns of chemical-induced cytotoxicity in different regions of the nephron were studied with freshly isolated proximal tubular and distal tubular cells from rat kidney. Three model alkylating agents, methyl vinyl ketone, allyl alcohol, and N-dimethylnitrosamine, were used as test chemicals. Methyl vinyl ketone and a metabolite of allyl alcohol, acrolein, are Michael acceptors that bind to cellular protein sulfhydryl groups and GSH. N-Dimethylnitrosamine binds to cellular protein and DNA. Lactate dehydrogenase leakage was used to assess irreversible cellular injury. Distal tubular cells were more susceptible than proximal tubular cells to injury produced by methyl vinyl ketone or allyl alcohol while the two cell populations were equally susceptible to injury produced by N-dimethylnitrosamine. Preincubation of both proximal tubular and distal tubular cells with GSH protected them from methyl vinyl ketone- and allyl alcohol-induced cytotoxicity but had no effect on N-dimethylnitrosamine-induced cytotoxicity. Similarly, incubation of cells with methyl vinyl ketone or allyl alcohol, but not N-dimethylnitrosamine, altered cellular GSH status. As with GSH status, incubation of cells with methyl vinyl ketone or allyl alcohol, but not N-dimethylnitrosamine, caused pronounced inhibitory effects on mitochondrial function, as evidenced by ATP depletion and inhibition of cellular oxygen consumption. These results demonstrate that alkylating agents are cytotoxic to both proximal tubular and distal tubular cells, and that interaction with cellular GSH is a factor determining nephron cell type specificity of injury.

Myocardial sulfhydryl pool alterations occur during reperfusion after brief and prolonged myocardial ischemia in vivo.

Myocardial sulfhydryl (SH)-containing compounds, including reduced glutathione (GSH), are both defenses against and potential markers of reactive oxygen metabolite injury during ischemia and reperfusion. We examined the alterations in GSH and other myocardial SH pools during reperfusion in anesthetized dogs exposed to brief (15 minutes, n = 7) or prolonged (90 minutes, n = 6) regional ischemia caused by occlusion of the left anterior descending artery. Ninety minutes of ischemia followed by 5 hours of reperfusion, which resulted in myocardial necrosis of 43.9 +/- 4.0% of the area at risk, caused a 22% reduction in total myocardial SH groups (p less than 0.01), a 57% decrease in nonprotein myocardial SH groups (p less than 0.01), a 56% decrease in GSH (p less than 0.01), and a 62% decrease in non-GSH, nonprotein SH groups (p less than 0.02). However, protein SH groups were not significantly reduced (12% decrease, p = NS). Also, myocardial release of GSH and oxidized glutathione (GSSG) into the coronary venous effluent occurred during early reperfusion. In contrast, 15 minutes of ischemia, followed by 30 minutes of reperfusion, did not alter myocardial total SH groups, protein SH groups, or GSH (9% decrease, p = NS); nor was there reperfusion release of GSH or GSSG. However, even with brief ischemia, nonprotein SH groups decreased 23% (p less than 0.05), due mainly to a 59% decrease in the non-GSH, nonprotein SH pool (p less than 0.05). These changes after brief ischemia occurred without alterations in myocardial GSSG or the GSH/GSSG ratio.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of glutathione depletion on the biodistribution of Cu(PTSM) in rats

The tissue retention of radiocopper afforded by intravenous injection of the proposed blood flow imaging agent, 62Cu-labeled copper(II) pyruvaldehyde bis( N 4-methylthiosemicarbazone) [Cu(PTSM)], is thought to result from reductive decomposition of the copper(II) complex by intracellular sulfhydryls (e.g. glutathione, GSH). To determine if the tissue uptake and retention of this tracer adequately measures perfusion in tissues containing altered GSH concentrations, the biodistribution of copper-67 labeled Cu(PTSM) was determined in GSH-depleted rats. Despite treatment to induce relatively large reductions in tissue GSH levels, it was found that only very small changes in the biodistribution of copper-labeled Cu(PTSM) occurred in the treated rats compared to untreated controls.

Influence of ethanol on glutathione- S-transferase activity and glutathione content in the isolated perfused rabbit lung

The induction of pulmonary glutathione- S-transferase (GST) by ethanol was investigated using the isolated perfused rabbit lung (IPRL) preparation with particular attention paid to the duration and route of ethanol administration. For perfusion with buffer containing 0.2% ethanol or acute ethanol treatment (4 g/kg by gastric intubation) 4 h before the IPRL preparation, there were no differences in the rate of glutathione (GSH) conjugation with 1-chloro-2,4-dinitrobenzene (CDNB) at low substrate concentrations (100–400 μM) but a decrease was observed in the rate at high substrate concentrations (500–1000 μM). Lungs from rabbits treated acutely showed the lowest maximal rate of GSH conjugation in the IPRL. Prolonged treatment with ethanol (10% in drinking water for 3 weeks) increased GSH conjugation (CDNB concentration of 300–750 μM). None of these ethanol treatments altered GSH conjugation with 1,2-epoxy( p-nitrophenoxy)propane (ENP). Upon termination of perfusion, there were no differences in pulmonary GSH concentration between control and ethanol-treated groups. Therefore, the effect of altered GSH level as a co-substrate on GST activity in lung might be excluded as an explanation for the effects of ethanol. These data suggest that ethanol has differential effects on GST activity depending upon the substrate and duration of ethanol administration.

Influences of glutathione status on different cytocidal responses of monolayer rat hepatocytes exposed to aflatoxin B 1 or acetaminophen

In short-term primary monolayer cultures of rat hepatocytes, aflatoxin B 1 (AFB 1) causes a characteristic prelethal cytomorphological response in which peripheral attached cytoplasm contracts segmentally to form finger-like blebs. This response precedes lethal injury as detected by release of lactate dehydrogenase (LDH) into culture medium. We compared the influences of various modifiers of cellular glutathione (GSH) status on cytocidal responses of Fischer 344 rats hepatocytes exposed to AFB 1 or acetaminophen (AAP), a hepatotoxin which does not produce segmental cytoplasmic contraction. N-Acetylcysteine (4 m m) reduced the degree of LDH release by AAP (4 to 16 m m) but was not protective against cell killing by AFB 1, although it slightly reduced the percentage of hepatocytes with segmental cytoplasmic contraction at 6 hr. BCNU (1,3-bis(2-chloroethyl)-1-nitrosourea) at 40 μ m markedly inhibited glutathione reductase and also strongly potentiated cell killing by AAP but did not significantly influence segmental cytoplasmic contraction or LDH release in response to AFB 1. Diethylmaleate (40 to 160 μ m), a depletor of hepatocellular GSH, and buthionine- d,l-sulfoximine (4 m m), an inhibitor of GSH synthesis, each did not alter hepatocyte killing by AFB 1 but were strong potentiators of toxicity of AAP. AAP inhibited glutathione reductase but AFB 1 did not. Total GSH concentrations at 6 and 18 hr were reduced by AAP and to a lesser extent by AFB 1 in comparison with control cultures. These findings demonstrate that, in contrast to AAP toxicity, the characteristic mode of hepatocyte killing by AFB 1 in monolayer cultures is substantially independent of induced alterations in GSH. These results indicate that GSH-dependent detoxification mechanisms do not play a major role in removing necrogenic metabolites of AFB 1 in Fischer 344 rat hepatocytes. They further suggest that prelethal responses of AFB 1-injured hepatocytes are not affected by GSH-dependent cytoprotective mechanisms.

Effect of fasting, diethyl maleate, and alcohols on carbon tetrachloride-induced hepatotoxicity

The effect of fasting, diethyl maleate treatment, and methanol, ethanol, iso-propanol, or tert-butanol treatment on carbon tetrachloride (CCl 4)-induced hepatotoxicity in male Sprague-Dawley rats was studied. A 24-hr fast and the administration of diethyl maleate were found to be equipotent and not additive in potentiating CCl 4-induced hepatotoxicity; this potentiation appeared to be due to a depletion of hepatic glutathione (GSH) levels. Concomitant diurnal variations in hepatic GSH content and in CCl 4-induced hepatotoxicity also suggested hepatic GSH involvement in CCl 4-induced hepatic damage. Whereas ethanol has been reported to potentiate CCl 4-induced hepatotoxicity and to lower hepatic GSH levels, the present study suggested that these effects are due to an ethanol-induced loss of body weight. Methanol, iso-propanol, and tert-butanol, on the other hand, show the same maximal potentiation of CCl 4-induced hepatotoxicity and do so without inducing a depletion of hepatic GSH content or producing a loss of body weight. In contrast to a previous report, however, methanol was found to be markedly less effective on an equimolar basis than either iso-propanol or tert-butanol in potentiating CCl 4-induced hepatotoxicity. The study suggests that GSH is an important modulator of CCl 4-induced hepatotoxicity and also suggests that methanol, iso-propanol, and tert-butanol, but not ethanol, potentiate CCl 4-induced hepatotoxicity by a mechanism that does not involve altered hepatic GSH levels.