Gamma-glutamylcysteine Synthetase Activity Research Articles

Increased lipoperoxide levels and antioxidant system in colorectal cancer.

In this study, lipid peroxide and glutathione (GSH) levels, GSH peroxidase, GSH S-transferase, superoxide dismutase, gamma-glutamylcysteine synthetase and gamma-glutamyl transpeptidase activities were investigated in tumorous and nontumorous colorectal tissues obtained from ten patients diagnosed with colon and rectum cancer. Histopathological evaluations, including type, stage, necrosis and lymphocyte infiltration, were also performed for each patient. According to the results, lipid peroxide and GSH levels and the activities of GSH peroxidase, superoxide dismutase, gamma-glutamylcysteine synthetase were found to be increased, while GSH S-transferase and gamma-glutamyl transpeptidase activities remained unchanged in tumors compared to adjacent normal tissues of subjects with colorectal cancer. However, the considerable interindividual variations were found in these parameters. A definite interrelation between histopathological results with lipid peroxidation and antioxidant system was not observed.

The glutathione-deficient, cadmium-sensitive mutant, cad2-1, of Arabidopsis thaliana is deficient in gamma-glutamylcysteine synthetase.

This paper reports that the glutathione (GSH)-deficient mutant, cad2-1, of Arabidopsis is deficient in the first enzyme in the pathway of GSH biosynthesis, gamma-glutamylcysteine synthetase (GCS). The mutant accumulates a substrate of GCS, cysteine, and is deficient in the product, gamma-glutamylcysteine. In vitro enzyme assays showed that the cad2-1 mutant has 40% of wild-type levels of GCS activity but is unchanged in the activity of the second enzyme in the pathway, GSH synthetase. The CAD2 locus maps to chromosome 4 and is tightly linked to a gene, GSHA, identified by a previously isolated cDNA. A genomic clone of GSHA complements both the phenotypic and biochemical deficiencies of the cad2-1 mutant. The nucleotide sequence of the gene has been determined and, in the mutant, this gene contains a 6 bp deletion within an exon. These data demonstrate that the CAD2 gene encodes GCS. The cad2-1 mutation is close to the conserved cysteine which is believed to bind the substrate glutamate and the specific inhibitor L-buthionine-[S,R] sulfoximine (BSO). Both root growth and GCS activity of the cad2-1 mutant was less sensitive than the wild-type to inhibition by BSO, indicating that the mutation may alter the affinity of the inhibitor binding site.

Glutathione reverses the growth abnormalities of skin fibroblasts from insulin-dependent diabetic patients with nephropathy.

Oxidative stress has been proposed as a possible pathogenic factor for diabetic complications. It is relevant in determining cell replicative capacity and life span, and in vitro antioxidant treatment is able to reverse the impaired proliferative activity of different cell types. It was recently demonstrated that cultured skin fibroblasts from insulin-dependent diabetic patients with nephropathy age prematurely and have a shorter life cell cycle. To test whether the growth phenotype of cells from patients with diabetic nephropathy was related to a lack of protection from oxidative stress, the effect of reduced glutathione (GSH) on cultured skin fibroblasts from 13 insulin-dependent diabetes mellitus (IDDM) patients with nephropathy (DN), 10 IDDM patients without kidney disease (D), and 10 nondiabetic control subjects (C), in normal (5 mM) glucose (NG) and high (22 mM) glucose (HG) medium was studied. After 6 to 8 passages, fibroblasts from DN showed impaired growth both in NG (mean +/- SD fold increase over baseline counts in DN 1.17 +/- 0.6 versus D 1.7 +/- 0.5 versus C 1.95 +/- 0.8; P = 0.04 by ANOVA) and in HG (mean +/- SD fold increase over baseline counts DN 1.16 +/- 0.41 versus D 1.89 +/- 0.66 versus C 2.24 +/- 0.9; P = 0.003 by ANOVA). GSH prevented the growth abnormalities of cells from DN restoring it to values similar to that of the other two groups (mean +/- SD fold increase over baseline counts NG +/- GSH: DN 1.68 +/- 0.9 versus D 1.78 +/- 0.49 versus C 1.99 +/- 0.7, P = 0.6; and in HG + GSH: DN 1.66 +/- 0.69 versus D 1.87 +/- 0.75 versus C 2.2 +/- 0.9, P = 0.3). Growth rates were not affected by the addition of GSH in fibroblasts from D and C. The treatment of fibroblasts from D and C with the inhibitor of the gamma-glutamylcysteine synthetase activity, L-buthionine-S,R-sulfoximine, resulted in growth impairment, and the addition to the culture medium of another antioxidant, superoxide dismutase, corrected the growth abnormalities in fibroblasts from DN. The impaired growth of cultured fibroblasts from IDDM patients with nephropathy is prevented by GSH and superoxide dismutase and is independent of prevailing glucose concentrations. This suggests that oxidative stress is an important mechanism of intrinsic cell dysfunction in these patients.

Changes in glutathione homeostasis during liver regeneration in the rat.

We have shown previously that plating primary cultures of rat hepatocytes under low density, which stimulates hepatocytes to shift from the G0 to the G1 phase of the cell cycle, resulted in increased levels of glutathione (GSH) and cysteine, and increased activity of gamma-glutamylcysteine synthetase (GCS), the rate-limiting enzyme in GSH synthesis (Lu et al., Am. J. Physiol. 1992;263:C1181-C1189). In the current work we examined changes in GSH homeostasis after two-thirds partial hepatectomy (PH). Male Sprague-Dawley rats underwent two-thirds PH or sham operation. GSH, oxidized glutathione (GSSG), cysteine, GSH efflux, DNA synthesis, changes in GCS subunit messenger RNA (mRNA), and protein levels were measured 12 and 24 hours after PH. Both liver GSH and cysteine levels were doubled at 12 hours and remained elevated at 24 hours after PH. GSSG levels also increased, but the ratio of GSH to GSSG levels remained unchanged. The increase in GSH and cysteine levels preceded the increase in DNA synthesis. Sinusoidal GSH efflux was unchanged after two-thirds PH, but biliary GSH efflux decreased. However, total GSH efflux was minimally altered after two-thirds PH. The increase in GSH can be largely accounted for by the increase in both cysteine availability and the activity of GCS. The steady-state mRNA and protein levels of the GCS heavy subunit were increased at 12 hours after PH. The mRNA level of the GCS light subunit was unchanged. In summary, early in the course of liver regeneration the steady-state hepatic GSH levels double because of an increase in the biosynthesis of GSH.

Nitric oxide synthase inhibition reduces gamma-glutamylcysteine synthetase activity in rat kidneys: [formula omitted], Juha Laakso, Timo Vaskonen, Risto Lapatto, Hospital for Children and Adolescents and Institute of Biomedicine, University of Helsinki, Finland

Nitric oxide synthase inhibition reduces gamma-glutamylcysteine synthetase activity in rat kidneys: [formula omitted], Juha Laakso, Timo Vaskonen, Risto Lapatto, Hospital for Children and Adolescents and Institute of Biomedicine, University of Helsinki, Finland

Efflux of reduced glutathione after exposure of human lung epithelial cells to crocidolite asbestos.

This study investigated glutathione (GSH) homeostasis in human lung epithelial cells (A549) exposed to crocidolite. Exposure of A549 cells to 3 micrograms/cm2 crocidolite resulted in a decrease in intracellular reduced glutathione by 36% without a corresponding increase in GSH disulfide. After a 24-hr exposure to crocidolite, 75% of the intracellular GSH lost was recovered in the extracellular medium, of which 50% was in reduced form. Since the half-life of reduced GSH in culture medium was less than 1 hr, this suggests that reduced GSH was released continuously from the cells after treatment. The release of GSH did not appear to result from nonspecific membrane damage, as there was no concomitant release of lactate dehydrogenase or 14C-adenine from loaded cells after crocidolite treatment for 24 hr. Crocidolite exposure resulted in the formation of S-nitrosothiols but no increase in the level of GSH-protein mixed disulfides or GSH conjugates. Exposure of A549 cells to crocidolite for 24 hr decreased gamma glutamylcysteine synthetase (gamma-GCS) activity by 47% without changes in the activities of GSH reductase, GSH peroxidase, GSH S-transferase, or glucose-6-phosphate dehydrogenase. Treatment of cells with crocidolite pretreated with the iron chelator desferrioxamine B resulted in the same level of intracellular GSH depletion and efflux and the same decrease in gamma-GCS activity as treatment with unmodified crocidolite, which suggests that iron-catalyzed reactions were not responsible for the GSH depletion.

Elevation of glutathione and related enzyme activities in high-grade and metastatic extremity soft tissue sarcoma

Glutathione is a free radical scavenger implicated in the chemoresistance of certain tumors. As treatment with chemotherapy has added little to improved survival in adult soft tissue sarcoma and little is known concerning the mechanisms of chemoresistance in sarcoma, we studied concentrations of glutathione (nmol/mg protein) and activities of gamma-glutamylcysteine synthetase (GCS; nmol/mg protein/h) and gamma-glutamyl transpeptidase (GGTP; U/mg protein) in extremity soft tissue sarcoma. Tumor specimens (n = 65) were frozen in liquid nitrogen at the time of resection. Fourteen low-grade tumors, 40 high-grade tumors, and 11 pulmonary metastases were analyzed. Glutathione concentrations and GGTP activity were significantly lower in low-grade (3.97 +/- 0.7 nmol/mg protein and 1.07 +/- 0.2 U/mg protein) than in high-grade (8.98 +/- 1.2 nmol/mg protein, p < 0.001; 2.10 +/- 0.4 U/mg protein, p < 0.002) tumors and pulmonary metastases (10.05 +/- 1.8 nmol/mg protein, p < 0.008; 3.14 +/- 2.8 U/mg protein, p < 0.04). While GCS activity was lower in low-grade (0.81 +/- 0.3 nmol/mg protein/h) than high-grade (1.49 +/- 0.5 nmol/mg protein/h) tumors and pulmonary metastases (1.03 +/- 0.2 nmol/mg protein/h), these differences were not significant. In those patients with a high-grade tumor presenting with a local recurrence, glutathione levels were higher in those patients who had received preoperative doxorubicin-based chemotherapy (9.25 +/- 1.7 nmol/mg protein; n = 7) than in those who had no preoperative chemotherapy (4.71 +/- 3.1 nmol/mg protein; n = 4, p = 0.08). In extremity soft tissue sarcoma, glutathione concentration and GGTP activity are significantly elevated in patients with high-grade and metastatic sarcomas. In addition, there is a trend for increased glutathione levels in tumors previously exposed to doxorubicin-based chemotherapy. Glutathione may play a role in soft tissue sarcoma chemoresistance.

Dietary sulphur amino acid adequacy influences glutathione synthesis and glutathione-dependent enzymes during the inflammatory response to endotoxin and tumour necrosis factor-alpha in rats.

1. Glutathione concentrations in liver and lung fall when food intake or sulphur amino acid intake is inadequate. However, concentrations may be restored during inflammation, despite anorexia, provided that prior sulphur amino acid intake is adequate. 2. We studied the mechanisms of these changes by measuring the effect of sulphur amino acid and protein intake on hepatic glutathione synthesis and gamma-glutamylcysteine synthetase activity, hepatic and lung glutathione concentrations, glutathione reductase and glutathione peroxidase activities in young rats given an inflammatory challenge by intraperitoneal injection of tumour necrosis factor-alpha or endotoxin (lipopolysaccharide). 3. Diets containing 200 g of casein and 8 g of L-cysteine/kg (normal-protein diet), or 80 g of casein and 8 g of L-cysteine, or isonitrogenous amounts of L-methionine or L-alanine (low-protein diets) were fed ad libitum to young Wistar rats for 8 days. Dietary groups were subdivided into three: one subgroup continued feeding ad libitum, a second was given tumour necrosis factor or lipopolysaccharide and killed 24 h thereafter, while the third was pair-fed to the intakes of the second subgroup for 24 h before being killed. 4. Glutathione concentrations in liver and lung were reduced in rats fed the low-protein diet containing alanine, and in all dietary groups when food intake was restricted. The inflammatory challenges restored hepatic glutathione concentrations in all groups but the diet supplemented with alanine, which had an inadequate sulphur amino acid content. In lung, restoration occurred only in animals fed the normal-protein diet. 5. The activity of gamma-glutamylcysteine synthetase, which is rate limiting for glutathione synthesis, was unaffected by dietary or sulphur amino acid intake or by the inflammatory response. Substrate supply may therefore be a major determinant in glutathione synthesis in vivo. 6. Total hepatic glutathione synthesis was affected by food intake, the type and amount of sulphur amino acids in the diet and by inflammation. Total synthesis was 207, 137, 421 and 90 mumol/day for animals fed ad libitum the normal-protein diet, or low-protein diets supplemented with cysteine, methionine or alanine respectively, ad libitum. Pair-feeding resulted in values of 76, 31, 71, and 0 mumol/day respectively. After lipopolysaccharide injection, rates increased to 200, 117, 151 and 56 mumol/day respectively. 8. Reductase and peroxidase activities increased in liver and lung, when low-protein diets which contained supplemental methionine or alanine were consumed ad libitum. A reduction in food intake resulted in enzyme activity changes, which suggested that recycling of glutathione increased in lung and decreased in liver. Injection of tumour necrosis factor reversed this effect. 9. The restoration of glutathione concentrations in liver after an inflammatory challenge is closely associated with an enhanced rate of synthesis and increased recycling. The former is impaired when inadequate sulphur amino acid is consumed before the challenge. In lung, increased recycling of glutathione may help maintain concentrations when food intake is restricted, but not during inflammation.

Adaptations of glutathione antioxidant system to endurance training are tissue and muscle fiber specific.

The effect of endurance training on glutathione (GSH) status and antioxidant enzyme system was investigated in skeletal muscle, heart, and liver of female Sprague-Dawley rats pair fed an isocaloric diet. Ten weeks of treadmill training (25 m/min, 10% grade for 2 h/day, 5 days/wk) increased citrate synthase activity in the deep vastus lateralis (DVL) and soleus muscles by 79 and 39%, respectively (P < 0.01), but not in the heart or liver. In DVL, GSH content was increased 33% (P < 0.05) with training, accompanied by a 64% (P < 0.05) increase in glutamate content but no change in cysteine. Trained rats showed a 62 and 27% higher GSH peroxidase (GPX) and superoxide dismutase (SOD) activity, respectively (P < 0.05), in DVL compared with control rats. In contrast, GSH content and glutathione reductase (GR) activity in soleus declined with training (P < 0.05), whereas activities of GPX and SOD remained unchanged. Training did not alter GSH status in the liver or plasma but significantly decreased the GSH-to glutathione disulfide ratio in the heart. In addition, GR activity in the liver and GSH sulfur-transferase activity in the heart and DVL were significantly lower in the trained vs control rats DVL muscle had threefold higher gamma-glutamyl transpeptidase activity compared with other tissues; however no significant alteration was observed in the activity of gamma-glutamyltranspeptidase or gamma-glutamylcysteine synthetase in the liver, heart, or skeletal muscle. These data indicate that endurance training can cause tissue- and muscle fiber-specific adaptation of antioxidant systems and that GSH homeostasis in extrahepatic tissues may be determined by utilization and uptake of GSH via the gamma-glutamyl cycle.

Elevated hepatic gamma-glutamylcysteine synthetase activity and abnormal sulfate levels in liver and muscle tissue may explain abnormal cysteine and glutathione levels in SIV-infected rhesus macaques.

To establish whether the low cysteine and glutathione levels in HIV-infected patients and SIV-infected rhesus macaques may be consequences of an abnormal cysteine catabolism, we analyzed sulfate and glutathione levels in macaques. Muscle tissue (m. vastus lateralis and m. gastrocnemius) of SIV-infected macaques (n = 25) had higher sulfate and lower glutathione and glutamate levels than that of uninfected controls (n =9). Hepatic tissue, in contrast, showed decreased sulfate and glutathione disulfide (GSSG) levels, and increased gamma-glutamylcysteine synthetase (gamma-GCS) activity. These findings suggest drainage of the cysteine pool by increased cysteine catabolism in skeletal muscle tissue, and by increased hepatic glutathione biosynthesis. Cachectic macaques also showed increased urea levels and decreased glutamine/urea ratios in the liver, which are obviously related to the abnormal urea excretion and negative nitrogen balance commonly observed in cachexia. As urea production and net glutamine synthesis in the liver are strongly influenced by proton-generating processes, the abnormal hepatic urea production may be the direct consequence of the cysteine deficiency and the decreased catabolic conversion of cysteine into sulfate and protons in the liver.

Regulation of gamma-glutamylcysteine synthetase by protein phosphorylation.

We previously reported that the activity of gamma-glutamylcysteine synthetase (GCS; EC 6.3.2.2), the rate-limiting enzyme in GSH synthesis, can be acutely inhibited approximately 20-40% by agonists of various signal transduction pathways in rat hepatocytes [Lu, Kuhlenkamp, Garcia-Ruiz and Kaplowitz (1991) J. Clin. Invest. 88, 260-269]. We have now examined the possibility that GCS is phosphorylated directly by activation of protein kinase A (PKA), protein kinase C (PKC) and Ca2+/calmodulin-dependent kinase II (CMK). Phosphorylation of GCS was studied using both purified rat kidney GCS and cultured rat hepatocytes by immunoprecipitating the reaction product with specific rabbit anti-(rat GCS heavy subunit) (anti-GCS-HS) antibodies. All three kinases, PKA, PKC and CMK, phosphorylated rat kidney GCS-HS in a Mg(2+)-concentration-dependent manner, with the highest degree of phosphorylation occurring at 20 mM Mg2+. The maximum incorporation of phosphate in mol/mol of GCS was 1.17 for PKA, 0.70 for PKC and 0.62 for CMK. The degree of phosphorylation was correlated with the degree of loss of GCS activity, and no additional inhibition occurred when GCS was phosphorylated by all three kinases, suggesting that the kinases phosphorylated the same site(s). Phosphoamino analysis showed that all three kinases phosphorylated serine and threonine residues. Two-dimensional phosphopeptide mapping demonstrated that all three kinases phosphorylated the same five peptides, both PKA and PKC phosphorylated two other peptides, and only PKA phosphorylated one additional peptide. Phosphorylation of GCS decreased its Vmax for cysteine and glutamate without changing its K(m). Finally, treatment of cultured rat hepatocytes with dibutyryl cAMP and phenylephrine significantly increased the phosphorylation of GCS, suggesting a potentially important physiological role. In summary, we have demonstrated that GCS is phosphorylated and suggest that phosphorylation/dephosphorylation may regulate GCS activity.

Alterations in tissue glutathione antioxidant system in streptozotocin-induced diabetic rats.

Changes in tissue glutathione antioxidant system in streptozotocin-induced diabetic rats for a period of 15 weeks were examined. Total glutathione level was significantly increased in kidney tissue, but were slightly decreased and increased in liver and heart tissues, respectively. The small changes in total glutathione level in the liver and heart, though not statistically significant, were associated with reciprocal alterations in the activity of gamma-glutamylcysteine synthetase (GCS). While the GCS activity was not changed in kidney tissue, the activity of gamma-glutathione peroxidase was significantly increased in kidney tissue. Insulin treatment could completely or partly normalize almost all of these changes induced by diabetes. However, the decrease in hepatic glutathione S-transferases activity in diabetic rats was not reversed by the insulin treatment. The ensemble of results suggests that the diabetes-induced alterations in tissue glutathione antioxidant system may possibly reflect an inter-organ antioxidant response to a generalized increase in tissue oxidative stress associated with diabetes.

Alterations in glutathione homeostasis in mutant Eisai hyperbilirubinemic rats.

Eisai hyperbilirubinemic rats (EHBR) are mutant Sprague-Dawley rats that exhibit impaired biliary organic anion and reduced glutathione (GSH) secretion. In addition, liver GSH levels are twice that of age- matched controls. The mechanisms for the defect in biliary GSH secretion and the increase in cell GSH are not fully understood. We previously showed that canalicular membrane-enriched vesicles isolated from EHBR livers exhibited normal GSH transport. In the present study, we examined the steady-state rat canalicular reduced glutathione transporter (RcGshT) messenger RNA (mRNA) and protein levels, as well as the mechanisms for the increase in cell GSH. Both Northern and Western blot analyses of EHBR livers showed nearly identical RcGshT mRNA and polypeptide levels, respectively, as compared with controls. Treatment with phenobarbital, which increased steady-state RcGshT mRNA by five- to sixfold, RcGshT polypeptide, and biliary GSH secretion by onefold in controls, had a smaller effect on steady-state RcGshT-mRNA level in EHBR (by 1.5-fold) and did not increase RcGshT polypeptide or biliary GSH secretion. In examining possible mechanisms for increased liver GSH, both cysteine level and gamma-glutamylcysteine synthetase (GCS) activity were significantly higher than controls, while the activity of GSH synthetase was unchanged. Northern and Western blot analyses also showed increased steady-state GCS heavy subunit (GCS-HS) mRNA and polypeptide levels, respectively. In addition to liver, GSH levels in kidney, duodenal, jejunal, and ileal mucosa of EHBR were 200% to 300% of age-matched control rats. GCS activity was also increased in kidney cytosol of EHBR. Thus, the defect in biliary GSH secretion in EHBR most likely is either at the posttranslational level of RcGshT or in the inhibition exerted by retained endogenous organic anions. In addition, there is a widespread up-regulation of GSH synthesis capacity in the tissues of EHBR. (Hepatology 1996 Jul;24(1):253-8)

Sulphur dioxide: A potent glutathione depleting agent

Sulphur dioxide (SO 2) is an air pollutant implicated in the initiation of asthmatic symptoms. Glutathione (gsh) has been proposed to play a role in detoxification of SO 2 through the sulfitolysis of glutathione disulphide (gssg) to S-sulphoglutathione (gsso 3 −). Rats were exposed to concentrations of so 2 between 5 and 100 ppm for 5 hr a day between 7 and 28 days. Lung injury as assessed by bronchoalveolar lavage and tissue GSH status were evaluated. SO 2 5 ppm failed to elicit any lung injury or inflammatory response but did deplete GSH pools in lung, liver, heart and kidney. Activities of gamma-glutamylcysteine synthetase (GCS), glutathione peroxidase (GPx), glutathione S-transferase (GST) and glutathione reductase (GRed) in lung were lowered relative to those in control animals. In liver, GRed activity was decreased. SO 2 50 ppm exposure also failed to elicit injury or inflammation but did lower inflammatory cell numbers in the circulation. Rats exposed to 50 ppm SO 2 maintained tissue GSH status, but activities of GCS, GPx, GRed and gamma-glutamyltranspeptidase in lung and hepatic GRed and GPx were significantly lower than in control rats. Unaltered GST activity in lung and liver was suggestive of an impairment of the sulfitolysis reaction in these animals, perhaps through lower substrate flux through the GPx reaction, as gsso 3 − is a known inhibitor of GST in the rat. Rats exposed to 100 ppm SO 2 exhibited evidence of inflammation (120-fold increase in neutrophil numbers recovered in lavage fluid) and like the 5 ppm exposed rats had lower tissue GSH concentrations and GSH-related enzyme activities in lung. We conclude that sulfitolysis of GSSG does occur in vivo during SO 2 exposure and that SO 2, even in the absence of pulmonary injury, is a potent glutathione depleting agent.

Increased gamma-glutamylcysteine synthetase and gamma-glutamyl transpeptidase activities enhance resistance of rat lung epithelial L2 cells to quinone toxicity.

Tert-butylhydroquinone (TBHQ) is a monofunctional Phase II enzyme inducer, which produces reactive oxygen species. Incubation with a sublethal concentration of TBHQ increased the activities of both gamma-glutamyl transpeptidase (GGT) and gamma-glutamylcysteine synthetase (GCS), although the mechanisms are different (Liu and colleagues, accompanying manuscript). In this study, we found that TBHQ increased intracellular glutathione (GSH) content in rat lung epithelial L2 cells. L2 cells pretreated with a nontoxic concentration of TBHQ (50 microM) acquired resistance to a subsequent challenge with a normally lethal concentration of TBHQ (200 microM). Pretreatment with L-buthionine S,R-sulfoximine (BSO), an inhibitor of GCS, prevented the TBHQ-induced increase in GSH and markedly diminished resistance to 200 microM TBHQ. Similarly, pretreatment with acivicin, an inhibitor of GGT, also prevented the TBHQ-induced increase in GSH and markedly diminished resistance to 200 microM TBHQ. Nevertheless, blockage of GGT by acivicin could be bypassed using 2-oxothiazolidine-4-carboxylate (procysteine) to provide the cell with a source of cysteine. This allowed an increase in GSH and restored resistance in the TBHQ-pretreated cells. The results suggest that elevation of GCS and GGT activities participated in acquired resistance to quinone toxicity.

Differential enhancement of gamma-glutamyl transpeptidase and gamma-glutamylcysteine synthetase by tert-butylhydroquinone in rat lung epithelial L2 cells.

Sublethal quinone-mediated oxidative stress stimulates increases in the activities and mRNA levels of gamma-glutamyl transpeptidase (GGT) and gamma-glutamylcysteine synthetase (GCS) in rat lung epithelial L2 cells [Kugelman, A. et al. 1994. Am. J. Respir. Cell Mol. Biol. 11:586-592; Shi, M. M. et al. 1994. J. Biol. Chem. 269:26512-26517]. The present study demonstrated that the quinone-induced increases in these two enzymes were differentially regulated. L2 cells were exposed to various concentrations of tertiary-butylhydroquinone (TBHQ) for different periods of times. TBHQ increased the activities and the mRNAs for GGT and the catalytic subunit of GCS; however, the time- and concentration-dependencies differed. With 50 microM TBHQ, GCS activity increased significantly by 6 h whereas the activity of GGT was not increased until later. Under the same conditions, the highest GCS-mRNA level observed was at 6 h whereas the mRNA level of GGT increased after 6 h, reached a higher level at 12 h, and then returned to the control level by 24 h. Differences were also observed in the concentration-dependence of mRNA increases between the GGT and GCS. Actinomycin D (an inhibitor of RNA synthesis) abolished the increase of GCS-mRNA but not the increase in GGT-mRNA, suggesting a difference in regulation by TBHQ between these two genes. Nuclear run-on experiments confirmed that the increase of GCS-mRNA, but not GGT-mRNA was due to increased transcription. The increase in GGT-mRNA probably results from a decreased degradation rate. The differences between these two enzymes demonstrate how cells can use multiple mechanisms for regulating gene expression in response to oxidative stress.

Influence of selenium on glutathione and some associated enzymes in rats with mammary tumor induced by 7,12-dimethylbenz(a)anthracene.

A recent finding in epidemiological and laboratory studies suggests that the ratio of selenium to glutathione is lower in breast cancer subjects than its control counterparts. Selenium, an antioxidant and anticarcinogen, can modify the status of glutathione and some associated enzymes by blocking peroxidation of lipids in membranes of cancer subjects. Studies were conducted using female albino rats of Wistar strain bearing mammary tumor induced by 7,12-dimethylbenz(a) anthracene to assess the biological role of selenium on some antioxidant enzymes associated with the maintenance of glutathione status. For induction of mammary tumor, 25 mg DMBA in a 1 ml emulsion of sunflower oil and physiological saline was injected subcutaneously to each rat. One group in each of control and tumor bearing rats, were fed 5 mg sodium selenite/kg diet from the day of tumor induction for 24 weeks. Increase in the reduced glutathione concentration was preceded by significant increase in the oxidized glutathione as well as in the activities of gamma-glutamylcysteine synthetase, glutathione peroxidase, glutathione reductase, glutathione S-transferase, and glucose-6-phosphate dehydrogenase by selenium administration in rats bearing tumor. However, selenium administration to rats bearing tumor decreased the activity of gamma-glutamyl transpeptidase. These observations clearly demonstrate the influence of dietary selenium supplementation in correcting abnormal changes in glutathione turnover and some associated enzymes in tumor induced rats.

Augmentation of human and rat lenticular glutathione in vitro by prodrugs of gamma-L-glutamyl-L-cysteine.

A marked age-related decrease in glutathione (GSH) levels as well as depression of gamma-glutamylcysteine synthetase activity are factors that are believed to render the aged lens more susceptible to oxidative stress and, therefore, to cataractogenesis. Providing gamma-L-glutamyl-L-cysteine, the dipeptide precursor of GSH, would effectively bypass the compromised first step in its biosynthesis and should protect the lens from GSH depletion. Accordingly, some bioreversible sulfhydryl-, amino-, and C-terminal carboxyl-protected prodrug forms of this dipeptide were prepared. Sulfhydryl protection was in the form of an acetyl thioester, while the carboxyl group was protected as the ethyl ester. These prodrugs were evaluated for their GSH-enhancing activity in cultured human and rat lenses in vitro using an assay that measured the incorporation of [14C]glycine into lens GSH. Ethyl S-acetyl-gamma-L-glutamyl-L-cysteinate (2) raised GSH levels in human lenses by 25% and in rat lenses by >150%. These data suggest that 2 may have potential as an anticataract agent since ethyl gamma-L-glutamyl-L-cysteinate (1a), the des-S-acetyl analog of 2, had been shown (by others) to protect against experimental rodent cataracts. GSH augmentation by 1a was 2% in human lenses and 25% in rat lenses, considerably less than that shown by 2.

Alterations in renal cellular glutathione metabolism after in vivo administration of a subtoxic dose of mercuric chloride.

Renal cellular concentration of glutathione (GSH) increases after exposure to a subtoxic dose of inorganic mercury (Hg2+). In the present study, we tested the hypothesis that the increase in renal cellular concentration of GSH after exposure to a subtoxic dose of Hg2+ (0.5 mumol HgCl2/kg body wt) is due to induction of GSH synthesis. Rats were treated in vivo with HgCl2, and renal proximal tubular (PT) and distal tubular (DT) cells were isolated 24 hours later. PT cells were studied as the presumed target site for Hg2+, and DT cells were investigated as a nontarget cell population. gamma-Glutamylcysteine synthetase activity increased after exposure to Hg2+ in PT cells when expressed on a per cell basis. Increases in activities of glutathione disulfide (GSSG) reductase, GSH peroxidase, and several enzymes involved in cellular energetics occurred after exposure to Hg2+. Many of these increases were observed in both PT and DT cells, indicating that the responses to Hg2+ were not restricted to the PT cells. These results are consistent with the hypothesis that in vivo exposure to a subtoxic dose of Hg2+ is also associated with induction of GSH synthesis and other key cellular enzymes. Early changes in GSH metabolism associated with exposure to Hg2+ appear to occur both in the primary target cell population and in more distal nephron sites.

Hydrogen peroxide increases the activity of gamma-glutamylcysteine synthetase in cultured Chinese hamster V79 cells.

Hydrogen peroxide (H2O2) caused a rapid and a concentration-dependent increase in the activity of gamma-glutamylcysteine synthetase (gamma-GCS) in cultured Chinese hamster V79 cells. The increase in the activity was transient and declined rapidly during post-treatment incubation. Inhibition of protein synthesis by cycloheximide, chelation of divalent iron by o-phenanthroline, and scavenging of free radicals by butyl-4-hydroxyanisole failed to suppress the increase in activity of gamma-GCS caused by H2O2. However, catalase completely inhibited the increase in the activity of the enzyme. H2O2 did not change the level of total glutathione (GSH + GSSG) but it oxidized GSH. The increase in levels of GSSG caused by H2O2 was enhanced by o-phenanthroline. These results suggest that the increase in activity of gamma-GCS caused by H2O2 is not an inducible phenomenon, nor it is attributable to the action of free radicals generated by an iron-catalyzed Fenton reaction. Furthermore, the changes in levels of GSH and GSSG caused by H2O2 appear not to be responsible for the increase in activity of gamma-GCS caused by the hydroperoxide. However, chemical reduction of the enzyme, the activity of which had been increased by H2O2, resulted in a decrease in tha activity, suggesting the involvement of oxidation of the enzyme in the increased activity of gamma-GCS caused by H2O2. The results also suggest that the activity of gamma-GCS in cultured V79 cells can be regulated by the cellular oxidation-reduction state.