Basal Glutathione Levels Research Articles

Exercise, depletion of antioxidants and antioxidant manipulation.

Strenuous physical activity is known to increase the production of reactive oxygen species (ROS), associated with depletion of antioxidant defence. In the present work we evaluated the level of lipid peroxidation and antioxidant components in blood of sportsmen under resting conditions and compared the data obtained with those in age- and sex-matched sedentary controls. A significant increase was noted in the levels of thiobarbituric acid reactive substances (TBARS) and conjugated dienes while a decrease was observed in ascorbic acid and glutathione levels in sportsmen. alpha-Tocopherol was unaltered in plasma of sportsmen as compared to controls. The activity of superoxide dismutase was increased (52 per cent) and glutathione peroxidase was decreased (43 per cent) in the erythrocytes of sportsmen compared to controls. Basal glutathione levels were negatively correlated with conjugated dienes and maximal oxygen uptake (VO2max) of the subjects. Dietary supplementation with antioxidant vitamins has been shown to be beneficial in combating oxidative stress without enhancing performance while exogenous glutathione was found to influence the endurance capacity of athletes. Such studies demonstrate the critical role played by glutathione and suggest that intervention trials should include a mixture of antioxidants rather than a single antioxidant.

Exercise, depletion of antioxidants and antioxidant manipulation

Strenuous physical activity is known to increase the production of reactive oxygen species (ROS), associated with depletion of antioxidant defence. In the present work we evaluated the level of lipid peroxidation and antioxidant components in blood of sportsmen under resting conditions and compared the data obtained with those in age- and sex-matched sedentary controls. A significant increase was noted in the levels of thiobarbituric acid reactive substances (TBARS) and conjugated dienes while a decrease was observed in ascorbic acid and glutathione levels in sportsmen. α-Tocopherol was unaltered in plasma of sportsmen as compared to controls. The activity of superoxide dismutase was increased (52 per cent) and glutathione peroxidase was decreased (43 per cent) in the erythrocytes of sportsmen compared to controls. Basal glutathione levels were negatively correlated with conjugated dienes and maximal oxygen uptake (VO2max) of the subjects. Dietary supplementation with antioxidant vitamins has been shown to be beneficial in combating oxidative stress without enhancing performance while exogenous glutathione was found to influence the endurance capacity of athletes. Such studies demonstrate the critical role played by glutathione and suggest that intervention trials should include a mixture of antioxidants rather than a single antioxidant. Copyright © 1998 John Wiley & Sons, Ltd.

Potentiation of Treosulfan Toxicity by the Glutathione-Depleting Agent Buthionine Sulfoximine in Human Malignant Glioma Cells: The Role of Bcl-2

Median survival of human malignant glioma patients is less than one year even with cytoreductive surgery and postoperative radiotherapy. Adjuvant chemotherapy has been rather ineffective. Here, we studied the potentiation by l-buthionine-[S,R]-sulfoximine (BSO), a glutathione-depleting agent, of anticancer drug actions on two human malignant glioma cell lines, LN-229 and T98G. LN-229 has wild-type p53 status, T98G is mutant for p53. Glutathione levels were depleted by BSO with similar kinetics in both cell lines. Only LN-229 cells were growth-inhibited by BSO. BSO had minor effects on the toxicity of doxorubicin, ACNU (1-[(4-amino-2-methyl-5-pyrimidinyl)methyl]-3-(2-chloroethyl)-3-nitrosourea, nimustine) and vincristine. BSO failed to alter teniposide or cytarabine toxicity. BSO induced prominent sensitization to the alkylating agent, treosulfan, in both cell lines, as assessed by viability assays, in situ DNA end labeling and quantitative DNA fragmentation. Treosulfan is thought to mediate toxicity via formation of reactive epoxides. In the absence of BSO, treosulfan had little acute cytotoxic and moderate antiproliferative effects. Synergistic glioma cell cytotoxicity induced by treosulfan and BSO was not associated with reactive oxygen species formation. Ectopic expression of bcl-2 did not alter basal glutathione levels but attenuated glutathione depletion induced by BSO. Bcl-2 provided only moderate protection from synergistic induction of glioma cell death by treosulfan and BSO. Glutathione depletion may play a role in BSO-mediated chemosensitization, but other mechanisms are probably involved as well. BSO may be a useful agent for glioma cell sensitization to specific chemotherapeutic drugs such as treosulfan.

Induction of nitric oxide synthesis in J774 cells lowers intracellular glutathione: effect of modulated glutathione redox status on nitric oxide synthase induction.

Under pathological conditions, the induction of nitric oxide synthase (NOS) in macrophages is responsible for NO production to a cytotoxic concentration. We have investigated changes to, and the role of, intracellular glutathione in NO production by the activated murine macrophage cell line J774. Total glutathione concentrations (reduced, GSH, plus the disulphide, GSSG) were decreased to 45% of the control 48 h after cells were activated with bacterial lipopolysaccharide plus interferon gamma. This was accompanied by a decrease in the GSH/GSSG ratio from 12:1 to 2:1. The intracellular decrease was not accounted for by either GSH or GSSG efflux; on the contrary, rapid export of glutathione in control cells was abrogated during activation. The loss of intra- and extracellular glutathione indicates either a decrease in synthesis de novo, or an increase in utilization, rather than competition for available NADPH. All changes in activated cells were prevented by pretreatment with the NOS inhibitor L-N-(1-iminoethyl)ornithine. Basal glutathione levels in J774 cells were manipulated by pretreatment with (1) buthionine sulphoximine (glutathione synthase inhibitor), (2) acivicin (gamma-glutamyltranspeptidase inhibitor), (3) bromo-octane (glutathione S-transferase substrate) and (4) diamide/zinc (thiol oxidant and glutathione reductase inhibitor). All treatments significantly decreased the output of NO following activation. The degree of inhibition was dependent on (i) duration of treatment prior to activation, (ii) rate of depletion or subsequent recovery and (iii) thiol end product. The level of GSH did not significantly affect the production of NO, after induction of NOS. Thus, glutathione redox status appears to plays an important role in NOS induction during macrophage activation.

Liver transplantation: does prolonged storage promote non-anastomatic biliary strictures [correction of structures] ?

The occurrence of biliary stricutures in allografts following liver transplantation correlates with the duration of preservation time. The correlation between preservation time and biliary strictures suggests that anoxic or reperfusion injury of the bile duct epithelium causes stricture formation. However, the relative susceptibility of bile duct cells to anoxic or reoxygenation injury is unknown. Our aims were to determine the vulnerability of rat liver bile duct cells to anoxic and reoxygenation injury and to compare the results with hepatocytes. During anoxia, bile duct epithelial cells were significantly more resistant to cell killing than hepatocytes. Rates of cellular proteolysis were also 2.5-fold lower in bile duct cells than in hepatocytes during anoxia. In contrast to anoxia, reoxygenation of anoxic cells increased cell killing of bile duct cells but improved viability of hepatocytes. The rate of toxic oxygen species formation by bile duct cells was 5-fold greater than in hepatocytes during reoxygenation. In addition, basal levels of glutathione are lower in bile duct cells than in hepatocytes. These data suggests that bile duct cells are more susceptible to reoxygenation injury than to anoxia. These studies support the hypothesis that reoxygenation injury during liver preservation leads to bile duct injury during liver transplantation.

THE GREATER VULNERABILITY OF BILE DUCT CELLS TO REOXYGENATION INJURY THAN TO ANOXIA

The occurrence of biliary strictures in allografts following liver transplantation correlates with the duration of preservation time. The correlation between preservation time and biliary strictures suggests that anoxic or reperfusion injury of the bile duct epithelium causes stricture formation. However, the relative susceptibility of bile duct cells to anoxic or reoxygenation injury is unknown. Our aims were to determine the vulnerability of rat liver bile duct cells to anoxic and reoxygenation injury and to compare the results with hepatocytes. During anoxia, bile duct epithelial cells were significantly more resistant to cell killing than hepatocytes. Rates of cellular proteolysis were also 2.5-fold lower in bile duct cells than in hepatocytes during anoxia. In contrast to anoxia, reoxygenation of anoxic cells increased cell killing of bile duct cells but improved viability of hepatocytes. The rate of toxic oxygen species formation by bile duct cells was 5-fold greater than in hepatocytes during reoxygenation. In addition, basal levels of glutathione are lower in bile duct cells than in hepatocytes. These data suggest that bile duct cells are more susceptible to reoxygenation injury than to anoxia. These studies support the hypothesis that reoxygenation injury during liver preservation leads to bile duct injury during liver transplantation.

A Simple Technique to Determine Glutathione (GSH) Levels and Synthesis in Ocular Tissues as GSH-bimane Adduct: Application to Normal and Galactosemic Guinea-pigs

A fluorimetric technique previously described for other tissues has been applied to determine levels of glutathione and its synthetic rates in ocular tissues of Hartley guinea-pigs. Monochlorobimane forms a stable, fluorescent adduct with glutathione in a reaction catalyzed by glutathione-S-transferase. The fluorescent signal recorded over time is directly proportional to the synthetic rate of glutathione. Lens, cornea and retina were homogenized and cytosolic fractions dialyzed overnight to deplete endogeneous glutathione. Glutathione synthetic rates were determined from a mixture of glutathione precursors and co-factors, viz. cysteine+dithiothreitol, glutamate+glycine, ATP and Mg++ in the presence of monochlorobimane. The mixture was supplemented with glutathione-S-transferase to catalyze the formation of the fluorescent adduct. Glutathione synthetic rates were determined in the absence and presence of buthionine sulfoximine, an inhibitor of γ-glutamyl cysteine synthetase. The difference in fluorescence change over time in the presence and absence of buthionine sulfoximine was used to estimate glutathione synthesis. Basal levels of glutathione in pre-dialyzed cytosolic fractions of the lens, cornea, and retina were 21·8±2·2, 36·5±4·1 and 38·6±2·8 nmol mg-1 protein, respectively. The maximal glutathione synthetic rates in these tissues were 0·52±0·04, 2·25±0·67 and 3·35±0·65 nmol min-1 mg-1 protein, respectively. When γ-glutamyl cysteine is used as a precursor instead of cysteine, the glutathione synthetase activities from lenses and retinas were 0·19 ±0·08 and 1·54± 0·76 nmol-1 min mg-1 protein. The present technique when applied to galactosemic guinea-pigs, showed that galactose feeding for 14 days caused a significant depletion (65 %) of lens glutathione levels, but the rates of glutathione synthesis in those lenses were not altered. The present technique permits measurements of glutathione levels and synthesis in different ocular tissues. It is suggested that the monochlorobimane method may be of value in delineating the role of glutathione in various ocular disorders such as in the galactose-induced cataract.

Species-related variations in tissue antioxidant status—I. Differences in antioxidant enzyme profiles

1. 1. Antioxidants enzyme activity profiles in red cells of man, rabbit, quail, pig, and rat have been investigated and found to exhibit striking differences. 2. 2. No direct correlations between activities of “functionally coupled” enzymes (superoxide dismutase/ catalase and glutathione peroxidase/glutathione reductase) were apparent, suggesting their independent regulation. 3. 3. However, activities of red cell catalase and glutathione peroxidase in the various species studied were inversely correlated. 4. 4. This was most evident in quail red cells, which showed negligible catalase activity but the highest levels of glutathione peroxidase of all the species examined. 5. 5. A significant correlation between catalase and glutathione reductase activities was also demonstrated. 6. 6. This may be relevant to the suggestion that the binding of NADPH to catalase may serve to decrease the intracellular inactivation of this reducing cofactor which may be limiting in the glutathione reductase reaction. 7. 7. Basal levels of glutathione, which have been claimed to be limiting for the glutathione peroxidase reaction, were found to correlate positively with the activity of this enzyme in red cells. 8. 8. Myocardial tissues also exhibited species-related differences in antioxidant enzyme profiles but these did not bear any obvious relationship to patterns observed in the corresponding red cells.

The effect of in vivo glutathione depletion with buthionine sulfoximine on rat embryo development

Glutathione is an abundant endogenous nucleophile whose depletion may have adverse effects on a number of cellular processes, including protein and DNA synthesis, amino acid transport, and detoxification of reactive electrophiles. Previous studies have indicated that a certain basal level of glutathione is essential for normal development in vitro in the whole rat embryo culture system. The objective of this study was to determine the effect of glutathione depletion with buthionine sulfoximine on the development of rat embryos in vivo. Timed pregnant Sprague-Dawley rats were treated by gavage on days 10 and 11 of gestation with saline (control) or with L-buthionine-(S,R)-sulfoximine (4 or 8 mmol/kg). Pregnancy outcome was assessed on day 12 or day 20 of gestation. The glutathione content of embryos and of maternal organs was measured on day 12. Glutathione concentrations were significantly decreased in the embryos and in maternal muscle and ovary but not in maternal liver and kidney. Glutathione depletion, when assessed on day 12 of gestation, was found to result in an increase in the numbers of dead and malformed embryos. Treatment with the higher dose of buthionine sulfoximine resulted in the death of 13.2% of the total implanted embryos; of the surviving embryos, 21.7% were malformed. There was also a significant decrease on day 12 after treatment with the higher dose of buthionine sulfoximine in the embryonic total morphological score, a measure of the developmental stage of the embryos (control, 57.7 +/- 1.0; 8 mmol/kg buthionine sulfoximine, 52.8 +/- 1.8).(ABSTRACT TRUNCATED AT 250 WORDS)

Inverse correlation between resistance towards copper and towards the redox-cycling compound paraquat: A study in copper-tolerant hepatocytes in tissue culture

The essential mediatory role of copper or iron in the manifestation of paraquat toxicity has been demonstrated (Kohen and Chevion (1985) Free Rad. Res. Commun. 1, 79–88; Korbashi, P. et al. (1986) J. Biol. Chem. 261, 12472–12476). Several liver cell lines, characterized by their resistance to copper, were challenged with paraquat and their cross-resistance to paraquat and copper was studied. Cell growth and survival data showed that copper-resistant cells, containing elevated copper, are more sensitive towards paraquat than wild type cells. Copper-deprived resistant cells did not have this sensitivity. Paraquat was also shown to cause a marked degradation of cellular glutathione in all cell lines. Albeit the fact that the basal glutathione levels are higher in copper-resistant than in wild type cells, there is more paraquat-induced degradation of cellular glutathione (GSH + GSSG) in resistant cells. It is suggested that in copper-resistant cells which contain elevated levels of copper, paraquat-induced cellular injury is potentiated even where glutathione levels are elevated. Additionally, in vitro experiments are presented that support the in vivo findings demonstrating a role for copper in glutathione degradation.

Glutathione in neutrophils from human infants.

The concentration of total glutathione (GSH + GSSG) and its depletion during oxidant stress were studied in neutrophils obtained from human neonates, their mothers and adult controls to determine if the previously reported increased susceptibility of infant neutrophils to oxidant damage could be related to glutathione deficiency. We found the basal level of glutathione to be lower and the oxidant-induced depletion of glutathione to be greater in infant neutrophils than in cells obtained from the blood of the infants' mothers and adult controls. In studies reported earlier, neutrophils from infants were found to be deficient in the oxidant protective enzymes glutathione peroxidase and catalase. These abnormalities, in a fashion similar to that observed for patients with congenital defects of glutathione and catalase metabolism, may render infant neutrophils prone to oxidant damage and consequent dysfunction.

Levels of glutathione in Escherichia coli

Log phase cells of Escherichia coli growing in minimal medium contain a basal level of glutathione (5 pmol/mL per Klett unit) which can increase more than sixfold when the cells reach stationary phase. Since the addition of cysteine alone to log phase cells illicits the same response, the increase in the intracellular pool of glutathione appears to be influenced by the amount of cysteine available for glutathione synthesis. Glucose depletion at low cell densities resulted in a decrease in the glutathione pool while the addition of amino acids other than cysteine did not affect the glutathione pool. Depletion of ammonia or proline as the nitrogen source also resulted in a decrease in the glutathione pool to one-third of the original basal levels as did a shift to anaerobic growth. The large glutathione pool in stationary phase cells dropped from 31.5 to 4.5 pmol/mL per Klett unit within 30 min of transfer to fresh medium. There was no apparent correlation between changes in the glutathione and coenzyme A--glutathione disulfide (CoASSG) pools after a variety of metabolic disruptions.