Low-dose γ-ray irradiation reduces oxidative damage induced by CCl4 in mouse liver

We examined the effects of irradiation (50 cGy of γ-ray) reducing the oxidative damage in carbon tetrachloride (CCl₄)-hepatopathy mice. We made pathological examinations and analyzed transaminase activity (glutamic oxaloacetic transaminase and glutamic pyruvic transaminase), lipid peroxide level and the activities of endogenous antioxidants in the mouse. The irradiation was found to accelerate the recovery. Based on pathological examination as well as changes in each transaminase activity and lipid peroxide levels, it was shown that hepatopathy improved 3 d after the irradiation. The activities of glutathione reductase and glutathione peroxidase rapidly elevated after irradiation, and the total glutathione content gradually increased in the irradiation group. Both activities of γ-glutamylcysteine synthetase and catalase were higher than normal at all times after the irradiation and gradually increased. In addition, the γ-glutamylcysteine synthetase activity changed in a similar fashion to the total glutathione content. However, superoxide dismutase activity in both groups decreased and that of the irradiation group was significantly lower than that of the sham-irradiation group. These findings suggest that low-dose radiation relieved functional disorder at least in the liver of mice with active oxygen diseases.     

Inhibitory effects of post low dose gamma-ray irradiation on ferric-nitrilotriacetate-induced mice liver damage

We studied the effects of a single post whole-body low-dose irradiation (50 cGy of γ-ray) on mice with ferric nitrilotriacetate (Fe³⁺-NTA)-induced transient hepatopathy. As a result, low-dose irradiation accelerated the rate of recovery. Based on the changes in glutamic oxaloacetic transaminase (GOT) activities, glutamic pyruvic transaminase (GPT) activities and lipid peroxide levels, it was shown that hepatopathy was improved by low-dose irradiation 3 h after Fe³⁺-NTA administration. This may be because of the enhancement of antioxidant agents such as total glutathione (GSH + GSSG), glutathione peroxidase (GPX), glutathione reductase (GR) and γ-glutamylcysteine synthetase (γ-GCS) by low-dose irradiation. These findings suggest that low-dose irradiation relieved functional disorders at least in the livers of mice with active oxygen species related diseases.     

Inhibitory effects of post low dose γ-ray irradiation on ferric-nitrilotriacetate-induced mice liver damage

We studied the effects of a single post whole-body low-dose irradiation (50 cGy of γ-ray) on mice with ferric nitrilotriacetate (Fe³⁺-NTA)-induced transient hepatopathy. As a result, low-dose irradiation accelerated the rate of recovery. Based on the changes in glutamic oxaloacetic transaminase (GOT) activities, glutamic pyruvic transaminase (GPT) activities and lipid peroxide levels, it was shown that hepatopathy was improved by low-dose irradiation 3 h after Fe³⁺-NTA administration. This may be because of the enhancement of antioxidant agents such as total glutathione (GSH + GSSG), glutathione peroxidase (GPX), glutathione reductase (GR) and γ-glutamylcysteine synthetase (γ-GCS) by low-dose irradiation. These findings suggest that low-dose irradiation relieved functional disorders at least in the livers of mice with active oxygen species related diseases.     

Glutathione metabolism in normal and cystinotic fibroblasts

Intracellular concentrations of glutathione and activities of the enzymes gamma-glutamylcysteine synthetase, glutathione synthetase, and gamma-glutamyl transpeptidase were measured in confluent cultured human fibroblasts cell lines from 14 normal cell lines and four cystinotic cell lines. gamma-Glutamyl transpeptidase had a wide range of variability while the glutathione synthetic enzymes, gamma-glutamylcysteine synthetase and glutathione synthetase, had narrower variations and also exhibited no apparent relationship to glutathione content. No differences in the activities of these enzymes were found between normal and cystinotic cells in confluent cell cultures. The activities of the above enzymes and the cell number and content of glutathione, cystine, DNA, and total protein in two normal and two cystinotic fibroblast cell lines were measured during growth. The following growth-dependency patterns were observed: (1) gamma-glutamylcysteine synthetase activity increased markedly in lag and early log phases in both normal and cystinotic cells and decreased rapidly to low confluent levels thereafter. (2) gamma-Glutamyl transpeptidase showed the same wide range of activity noted at confluency but activities decreased in the log phase of growth, a pattern also seen in cystinotic cells. (3) Glutathione synthetase activity remained relatively constant during growth of normal cells but exhibited a peak of activity during lag and early growth of cystinotic cells. (4) Comparative glutathione levels of normal and cystinotic cells were not significantly different and exhibited similar fluctuations with time. (5) The cystine content of normal and cystinotic cells unexpectedly rose to high levels in the lag phase, then decreased to 0.1 nmol 1/2 cystine/mg protein in normal cells and to 0.3 to 1.2 nmol 1/2 cystine/mg protein in cystinotic cells during the log phase. As confluency was approached, normal cell cystine remained at low levels while cystinotic cell cystine rose to characteristically high levels of 50- to 100-fold greater than normal cells at late confluency. These studies extend our understanding of the regulation of glutathione and cystine content in cultured fibroblasts and suggest that glutathione content is closely controlled throughout the cell cycle in the face of varying activities of its anabolic and catabolic enzymes.     

Effects of post low-dose X-ray irradiation on carbon tetrachloride-induced acatalasemic mice liver damage

The catalase activities in the blood and organs of the acatalasemic (C3H/AnLCsb-Csb) mouse of the C3H strain are lower than those of the normal (C3H/AnLCSa-Csa) mouse. We examined the effects of post low-dose (0.5 Gy) X-ray irradiation which reduced the oxidative damage under carbon tetrachloride-induced hepatopathy in acatalasemic or normal mice. As a result, the 0.5 Gy irradiation after carbon tetrachloride administration decreased the glutamic oxaloacetic and glutamic pyruvic transaminase activity in the acatalasemic mouse blood to a level similar to that of the acatalasemic mouse blood not treated with carbon tetrachloride; this is in contrast to a high-dose (15 Gy) irradiation. In the same manner, pathological disorder was improved by 0.5 Gy irradiation. The fat degeneration in normal mice was quickly reduced, in contrast to acatalasemic mice. These findings suggest that low-dose irradiation after carbon tetrachloride administration accelerates the rate of recovery and that catalase plays an important role in the recovery from hepatopathy induced by carbon tetrachloride, in contrast to high-dose irradiation.     

Ocimum sanctum aqueous leaf extract provides protection against mercury induced toxicity in Swiss albino mice

HgCl2 (5.0 mg/kg body weight) induced toxicity led to significant elevation of lipid peroxidation (LPO) level but decline in the glutathione content in liver of Swiss albino mice. In serum of HgCl2 treated mice there was significant elevation in serum glutamate oxaloacetate transaminase (SGOT) and serum glutamate pyruvate transaminase (SGPT) activities but significant decline in the alkaline phosphatase activity. Animals treated with O. sanctum extract (10 mg/kg body weight, po) before and after mercury intoxication showed a significant decrease in LPO level, SGOT and SGPT activities and increase in serum alkaline phosphatase activity and glutathione (GSH) content. Ocimum treatment alone did not alter SGOT, SGPT and alkaline phosphatase activities but significantly enhanced reduced glutathione. The results suggest that oral administration of Ocimum extract provides protection against HgCl2 induced toxicity in Swiss albino mice.     

Significance of glutathione S-conjugate for glutathione metabolism in human erythrocytes

The significance of glutathione S-conjugate in the regulation of glutathione synthesis was studied using human erythrocyte gamma-glutamylcysteine synthetase. Feedback inhibition of the enzyme by reduced glutathione was released by the addition of the glutathione S-conjugate (S-2,4-dinitrophenyl glutathione). A half-maximal effect of glutathione S-conjugate on gamma-glutamylcysteine synthetase activity was obtained at approximately 1 microM; 50 microM glutathione S-conjugate in the presence of 10 mM glutathione actually increased the enzyme activity twofold above uninhibited levels. Glutathione S-conjugate had no effect on the enzyme activity in the absence of glutathione. When erythrocytes were exposed to the electrophile 1-chloro-2,4-dinitrobenzene, which forms a glutathione S-conjugate by the catalytic reaction of glutathione S-transferase, the level of glutathione synthesis increased. These data suggest that glutathione S-conjugate plays a role in stimulating the synthesis of glutathione.     

Stimulation of glutathione synthesis in iron-loaded mice

We have previously reported that the iron-loading of mice, by feeding them carbonyl iron, caused an elevation of hepatic glutathione concentration and an increase in glutathione excretion from the liver (Kawabata, T., Ogino, T. and Awai, M. (1989) Biochim. Biophys. Acta 1004, 89-94). To elucidate the mechanism of glutathione elevation, hepatic cysteine concentration and gamma-glutamylcysteine synthetase (L-glutamate: L-cysteine gamma-ligase (ADP-forming), EC 6.3.2.2) activity were measured and possible changes in cysteine metabolism were also compared between iron-loaded and control mice. Hepatic cysteine concentration was higher in iron-loaded mice (185 +/- 12 nmol/g wet wt.) than in the controls (164 +/- 8 nmol/g wet wt.), and gamma-glutamylcysteine synthetase activity was also elevated in iron-loaded mice (34.3 +/- 3.2 nmol/mg protein per min) compared with the controls (28.6 +/- 3.8 nmol/mg protein per min). A comparison of the metabolic pathways with intravenously injected [35S]cysteine showed that organ distribution of the isotope was not significantly different, and also the rate of [35S]cysteine uptake into the hepatic glutathione fraction exhibited no difference between the two groups of mice. This shows that hepatic cysteine turnover may not be different between the two groups of mice. Since hepatic cysteine concentration was higher in iron-loaded mice, the apparently equal turnover of hepatic cysteine suggests that GSH synthesis may be elevated in iron-loaded mice. The high gamma-glutamylcysteine synthetase activity is suggested to stimulate GSH synthesis in iron-loaded mice.     

Some properties of glutathione biosynthesis-deficient mutants of Escherichia coli B

Mutants of Escherichia coli B that contain essentially no detectable glutathione were isolated. These mutants had a very low activity of gamma-glutamylcysteine synthetase or glutathione synthetase. No significant differences in growth in minimal medium were observed between the mutants and the parental strain. The mutants lacking gamma-glutamylcysteine synthetase activity were more susceptible to toxic compounds than either the parental strain or a glutathione synthetase-deficient strain. The mutants lacking gamma-glutamylcysteine synthetase activity were also susceptible to oxygen.     

Intermediates of the gamma-glutamyl cycle in mouse tissues. Influence of administration of amino acids on pyrrolidone carboxylate and gamma-glutamyl amino acids.

GAMMA-Glutamyl transpeptidase, gamma-glutamyl cyclotransferase, L-pyrrolidone carboxylate hydrolase, gamma-glutamylcysteine synthetase and glutathione synthetase, the enzymes of the gamma-glutamyl cycle, were found in mouse brain, liver and kidney. The activity of L-pyrrolidone carboxylate hydrolase was many times lower than the activities of the other enzymes, and thus the conversion of L-pyrrolidone carboxylate to L-glutamate is likely to be the rate-limiting step of the cycle. The specificity of gamma-glutamyl cyclotransferase from mouse tissues was similar to that from rat tissues. The concentration of pyrrolidone carboxylate and gamma-glutamyl amino acids, intermediates of the gamma-glutamyl cycle, was determined by a gas chromatographic procedure coupled with electron capture detection. Administration of L-2-aminobutyrate, an amino acid that is utilized as substrate in the reaction catalyzed by gamma-glutamylcysteine synthetase, led to a large accumulation of gamma-glutamyl-2-aminobutyrate and pyrrolidone carboxylate in mouse tissues. L-Methionine-RS-sulfoximine, an inhibitor of gamma-glutamylcysteine synthetase, abolished the increase in concentration of pyrrolidone carboxylate. No accumulation of pyrrolidone carboxylate was observed after L-cysteine. The separate administration of several protein amino acids had little effect on the concentration of pyrrolidone carboxylate; however formation of small amounts of the corresponding gamma-glutamyl derivatives (e.g. gamma-glutamylmethionine and gamma-glutamylphenylalanine) was detected. These intermediates are probably formed by transpeptidation between glutathione and the corresponding amino acid, catalyzed by gamma-glutamyl transpeptidase. The concentration of pyrrolidone carboxylate increased significantly after administration of a mixture containing all protein amino acids, the highest increase occurring in the kidney. The results suggest that two separate pathways for the formation of gamma-glutamyl amino acids and pyrrolidone carboxylate exist in vivo. One of these results from the function of gamma-glutamylcysteine synthetase in glutathione synthesis. The other pathway involves the amino-acid-dependent degradation of glutathione, mediatedby gamma-glutamyl transpeptidase. Only very small amounts of free intermediates are apparently derived from the latter pathway, suggesting that the gamma-glutamyl amino acids formed in this pathway are either enzyme-bound or are directly hydrolyzed to glutamate and free amino acid.     

Role of glutathione in apoptosis induced by radiation as determined by 1H MR spectra of cultured tumor cells

The relationship between apoptosis induced by gamma radiation and glutathione in cells of two human cancer cell lines, HeLa from cervix carcinoma and MCF-7 from mammary carcinoma, was examined. MCF-7 cells appeared to be more radioresistant than HeLa cells, and radiation-induced apoptosis, which was monitored by assessing phosphatidylserine externalization, was observed in HeLa cells but not in MCF-7 cells. Glutathione levels monitored by (1)H MRS were higher in MCF-7 cells than in HeLa cells, while the opposite was true for the free glu signals. MCF-7 cells became more radiosensitive when treated with 0.1 mM buthionine sulfoximine, which inhibits GSH synthesis through inactivation of gamma-glutamylcysteine synthetase, with the concomitant appearance of radiation-induced apoptosis. We can thus reasonably associate, at least in part, the resistance of MCF-7 cells to apoptosis with a high level of glutathione and probably with a high activity of gamma-glutamylcysteine synthetase. A late decrease in glutathione concentration after irradiation was observed in MCF-7 cells, but not in HeLa cells and to a lesser degree in buthionine sulfoximine-treated MCF-7 cells. This would indicate that the radiation-induced decrease in glutathione concentration is not related to the onset of apoptosis, but it is more likely related to glutathione consumption as a result of detoxification reactions.     

Coordinate induction of glutathione biosynthesis and glutathione-metabolizing enzymes is correlated with salt tolerance in tomato

The acclimation of reduced glutathione (GSH) biosynthesis and GSH-utilizing enzymes to salt stress was studied in two tomato species that differ in stress tolerance. Salt increased GSH content and GSH:GSSG (oxidized glutathione) ratio in oxidative stress-tolerant Lycopersicon pennellii (Lpa) but not in Lycopersicon esculentum (Lem). These changes were associated with salt-induced upregulation of gamma-glutamylcysteine synthetase protein, an effect which was prevented by preincubation with buthionine sulfoximine. Salt treatment induced glutathione peroxidase and glutathione-S-transferase but not glutathione reductase activities in Lpa. These results suggest a mechanism of coordinate upregulation of synthesis and metabolism of GSH in Lpa, that is absent from Lem.     

Regulation of the gene encoding glutathione synthetase from the fission yeast

The fission yeast cells that contained the cloned glutathione synthetase (GS) gene showed 1.4-fold higher glutathione (GSH) content and 1.9-fold higher GS activity than the cells without the cloned GS gene. Interestingly, gamma-glutamylcysteine synthetase activity increased 2.1-fold in the S. pombe cells that contained the cloned GS gene. The S. pombe cells that harbored the multicopy-number plasmid pRGS49 (containing the cloned GS gene) showed a higher level of survival on solid media with cadmium chloride (1 mM) or mercuric chloride (10 microM) than the cells that harbored the YEp357R vector. The 506 bp upstream sequence from the translational initiation point and N-terminal 8 amino acid-coding region were fused into the promoterless beta-galactosidase gene of the shuttle vector YEp367R to generate the fusion plasmid pUGS39. Synthesis of beta-galactosidase from the fusion plasmid pUGS39 was significantly enhanced by cadmium chloride and NO-generating S-nitroso-N-acetylpenicillamine (SNAP) and sodium nitroprusside (SN). It was also induced by L-buthionine-(S,R)-sulfoximine, a specific inhibitor of gamma-glutamylcysteine synthetase (GCS). We also found that the expression of the S. pombe GS gene is regulated by the Atf1-Spc1-Wis1 signal pathway.     

Role of glutathione in the adaptive tolerance to H2O2

Endogenous antioxidant defense systems are enhanced by various physiological stimuli including sublethal oxidative challenges, which induce tolerance to subsequent lethal oxidative injuries. We sought to evaluate the contributions of catalase and the glutathione system to the adaptive tolerance to H2O2. For this purpose, H9c2 cells were stimulated with 100 microM H2O2, which was the maximal dose at which no significant acute cell damage was observed. Twenty-four hours after stimulation, control and pretreated cells were challenged with a lethal concentration of H2O2 (300 microM). Compared with the control cells, pretreated cells were significantly tolerant of H2O2, with reduced cell lysis and improved survival rate. In pretreated cells, glutathione content increased to 48.20 +/- 6.38 nmol/mg protein versus 27.59 +/- 2.55 nmol/mg protein in control cells, and catalase activity also increased to 30.82 +/- 2.64 versus 15.46 +/- 1.29 units/mg protein in control cells, whereas glutathione peroxidase activity was not affected. Increased glutathione content was attributed to increased gamma-glutamylcysteine synthetase activity, which is known as the rate-limiting enzyme of glutathione synthesis. To elucidate the relative contribution of the glutathione system and catalase to tolerance of H2O2, control and pretreated cells were incubated with specific inhibitors of gamma-glutamyl cysteine synthetase (L-buthionine sulfoximine) or catalase (3-amino-1,2,4-triazole), and challenged with H2O2. Cytoprotection by the low-dose H2O2 pretreatment was almost completely abolished by L-buthionine sulfoximine, while it was preserved after 3-amino-1,2,4-triazole treatment. From these results, it is concluded that both the glutathione system and catalase can be enhanced by H2O2 stimulation, but increased glutathione content rather than catalase activity was operative in the tolerance of lethal oxidative stress.     

Induction of gamma-glutamylcysteine synthetase by prostaglandin A2 in L-1210 cells

Effects of prostaglandin A2 (PGA2) on glutathione (GSH) status in L-1210 cells were examined. When the cells were cultured in the presence of PGA2, a persistent rise of cellular GSH concentration was observed 6 h after the addition of PGA2. This stimulatory effect of PGA2 was abolished if the cells were pretreated with an enzyme inhibitor of GSH synthesis, buthionine sulfoximine. Subsequent study with cell free extract of cultured L-1210 has revealed that PGA2 stimulated the biosynthesis of gamma-glutamylcysteine synthetase (EC 6.3.2.2). Actinomycin D inhibited this stimulatory effect of PGA2 on cultured cells. The optimal pH, Km value for glutamic acid and sensitivity to inhibitors of gamma-glutamylcysteine synthetase from PGA2 treated and nontreated cells were virtually the same. Thus, our findings suggest that PGA2 induced gamma-glutamylcysteine synthetase in cultured L-1210 cells which is responsible for the elevated level of GSH in these cells.     

Peroxisome proliferator perfluorodecanoic acid alters glutathione and related enzymes

Previously we have shown that treatment with the peroxisome proliferator perfluorodecanoic acid (PFDA) significantly increased hepatic reduced glutathione (GSH) content without altering the activity of selenium-glutathione peroxidase. In this study we examined some potential mechanisms by which PFDA treatment increases GSH levels. Male Sprague-Dawley rats were given a single injection of 0, 8.8, 17.5, and 35 mg PFDA in corn oil per kg body weight. Twelve days later the effects of PFDA on the activities of enzymes associated with GSH synthesis, utilization, and regeneration were assessed. The results showed that in a dose-dependent manner, PFDA treatment significantly decreased the activity of gamma-glutamylcysteine synthetase, while the activities of NADPH-generating enzymes, malic enzyme, glucose-6-phosphate dehydrogenase, and 6-phosphogluconate dehydrogenase were increased. PFDA treatment also dose dependently decreased cytosolic, but not microsomal, glutathione S-transferase activity, and the activity of glutathione reductase was decreased by the highest dose of PFDA. The data obtained suggest that increased hepatic GSH levels following PFDA treatment may result from increased regeneration and/or decreased utilization.     

Effects of redox cycling compounds on glutathione content and activity of glutathione-related enzymes in rainbow trout liver

In fish, as in other aerobic organisms, glutathione and glutathione-related enzymes are important components in the defences against oxidative stress. To study if hepatic glutathione levels and/or activities of glutathione-related enzymes can act as indicators of oxidative stress in fish, we injected rainbow trout (Oncorhynchus mykiss) intraperitoneally with paraquat (PQ), menadione (MD), naphthazarin (DHNQ), or beta-naphthoflavone (beta-NF), all known to cause a rise in reactive oxygen species (ROS). After 2 and 5 days of exposure, we measured the activities of hepatic glutathione peroxidase (GPox), glutathione S-transferase (GST), gamma-glutamylcysteine synthetase (GCS), and glutathione reductase (GR). We also measured total glutathione (tGSH) and oxidised glutathione (GSSG) in the liver of fish treated with PQ and MD. All chemicals caused an increase in GR activity after 5 days, which ranged from 160% in fish treated with beta-NF to 1,500% in fish treated with PQ. All chemicals except beta-NF caused moderate elevation in GST activity; GPox activity was lower in fish treated with DHNQ and MD, while GCS activity increased twofold in the fish treated with DHNQ, without being affected by beta-NF, PQ or MD. After 5 days of treatment with PQ or MD, tGSH content was elevated. Our findings demonstrated that of the parameters included in the study, GR activity was the most responsive to treatment with redox cycling compounds, indicating that GR activity is a promising biomarker of such compounds and possibly indicating oxidative stress in rainbow trout.     

Assay of the glutathione-synthesizing enzymes by high-performance liquid chromatography

We report a new convenient assay of the activity of gamma-glutamylcysteine synthetase (EC 6.3.2.2) and glutathione synthetase (EC 6.3.2.3) in crude microbial extracts as well as in purified enzyme preparations. The assay is based on the quantitative analysis of the reaction products by high-performance liquid chromatography after derivatization of the thiol group with 5,5'-dithiobis-(2-nitrobenzoic acid) as described by J. Reeve, J. Kuhlenkamp, and N. Kaplowitz [(1980) J. Chromatogr. 194, 424-428]. In addition, the procedure yields information on basal levels of gamma-glutamylcysteine and glutathione in crude microbial extracts. The two enzymes responsible for glutathione biosynthesis can be determined in parallel under the same chromatographic conditions. No prior separation from substrates and by-products is necessary. Product formation is linear with time for at least 30 min between 0.03 and 12 mU for both enzymes. Even in crude extracts 0.2-0.5 nmol of products formed can be detected with certainty. The method was found to be sensitive and highly reproducible.