Pyrrolidine dithiocarbamate up-regulates the expression of the genes encoding the catalytic and regulatory subunits of gamma-glutamylcysteine synthetase and increases intracellular glutathione levels.
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Time- and dose-dependent increases in the steady-state mRNA levels of the genes encoding the catalytic and regulatory subunits of the enzyme gamma-glutamylcysteine synthetase (GCS) were observed in HepG2 human hepatocarcinoma cells after exposure to pyrrolidine dithiocarbamate (PDTC). PDTC was demonstrated to manifest both antioxidant and pro-oxidant properties in HepG2 cells, as assessed by the decreased fluorescence of the redox-sensitive dye Dihydrorhodamine 123 and by the oxidation of glutathione respectively. Attempts to characterize the signalling pathway from PDTC exposure to increases in the expression of the GCS catalytic and regulatory subunit genes demonstrated that induction by PDTC could be partially blocked by treatment with the thiol agent N-acetylcysteine and by the copper chelator bathocuproine disulphonic acid. These findings suggested that the up-regulation of the two genes resulted from a PDTC-induced pro-oxidant signal, which was partially copper-dependent. In summary, these studies demonstrate that PDTC exposure elicits a cellular response in HepG2 cells, characterized by the induction of the genes encoding the two subunits of the enzyme GCS and increased de novo synthesis of the cellular protectant GSH. (+info)
Depletion of colonic detoxication enzyme activity in mice with dextran sulphate sodium-induced colitis.
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BACKGROUND: The increased risk of colonic malignancies in individuals with ulcerative colitis has prompted a search for early biomarkers of disease progression. AIM: To characterize Phase II detoxication enzyme expression during acute and chronic colitis. The mouse model of dextran sulphate sodium (DSS)-induced colitis represents a relevant system with which to sequentially evaluate the spectrum of biochemical changes associated with colorectal cancer risk. METHODS: Acute and chronic colitis were induced in Swiss Webster mice by administering DSS in the drinking water (5%) for 1-4 cycles. Each cycle consisted of 7 days DSS and 14 days of water. The glutathione S-transferase (GST) activity, gamma-glutamylcysteine synthetase (gamma-GCS) activity and glutathione content of the colonic tissues were determined at various time points throughout the experiment. Alterations in GST isozyme expression were confirmed by Western and Northern blot. RESULTS: GST activity was reduced significantly in the colon by the end of Cycle 1 (84% of control values). Specific activities continued to decrease with subsequent cycles of DSS exposure. By the end of Cycle 4, glutathione levels and gamma-GCS activity had reached 29% and 56% of control, respectively. CONCLUSIONS: These data suggest that detoxication enzyme depletion is associated with both acute and chronic colitis and may be an important event in the progression of ulcerative colitis to colon cancer. (+info)
The induction of GSH synthesis by nanomolar concentrations of NO in endothelial cells: a role for gamma-glutamylcysteine synthetase and gamma-glutamyl transpeptidase.
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Nitric oxide protects cells from oxidative stress through a number of direct scavenging reactions with free radicals but the effects of nitric oxide on the regulation of antioxidant enzymes are only now emerging. Using bovine aortic endothelial cells as a model, we show that nitric oxide, at physiological rates of production (1-3 nM/s), is capable of inducing the synthesis of glutathione through a mechanism involving gamma-glutamylcysteine synthetase and gamma-glutamyl transpeptidase. This novel nitric oxide signalling pathway is cGMP-independent and we hypothesize that it makes an important contribution to the anti-atherosclerotic and antioxidant properties of nitric oxide. (+info)
Significance of glutathione-dependent antioxidant system in diabetes-induced embryonic malformations.
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Hyperglycemia-induced embryonic malformations may be due to an increase in radical formation and depletion of intracellular glutathione (GSH) in embryonic tissues. In the past, we have investigated the role of the glutathione-dependent antioxidant system and GSH on diabetes-related embryonic malformations. Embryos from streptozotocin-induced diabetic rats on gestational day 11 showed a significantly higher frequency of embryonic malformations (neural lesions 21.5 vs. 2.8%, P<0.001; nonneural lesions 47.4 vs. 6.4%, P<0.001) and growth retardation than those of normal mothers. The formation of intracellular reactive oxygen species (ROS), estimated by flow cytometry, was increased in isolated embryonic cells of diabetic rats on gestational day 11. The concentration of intracellular GSH in embryonic tissues of diabetic pregnant rats on day 11 was significantly lower than that of normal rats. The activity of y-glutamylcysteine synthetase (gamma-GCS), the rate-limiting GSH synthesizing enzyme, in embryos of diabetic rats was significantly low, associated with reduced expression of gamma-GCS mRNA. Administration of buthionine sulfoxamine (BSO), a specific inhibitor of gamma-GCS, to diabetic rats during the period of maximal teratogenic susceptibility (days 6-11 of gestation) reduced GSH by 46.7% and increased the frequency of neural lesions (62.1 vs. 21.5%, P<0.01) and nonneural lesions (79.3 vs. 47.4%, P<0.01). Administration of GSH ester to diabetic rats restored GSH concentration in the embryos and reduced the formation of ROS, leading to normalization of neural lesions (1.9 vs. 21.5%) and improvement in nonneural lesions (26.7 vs. 47.4%) and growth retardation. Administration of insulin in another group of pregnant rats during the same period resulted in complete normalization of neural lesions (4.3 vs. 21.5%), nonneural lesions (4.3 vs. 47.4%), and growth retardation with the restoration of GSH contents. Our results indicate that GSH depletion and impaired responsiveness of GSH-synthesizing enzyme to oxidative stress during organogenesis may have important roles in the development of embryonic malformations in diabetes. (+info)
Cellular balance of glutathione levels through the expression of gamma-glutamylcysteine synthetase and glutathione thiol transferase genes in human hepatic cells resistant to a glutathione poison.
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Buthionine sulfoximine (BSO) is a synthetic amino acid that irreversibly inhibits glutathione biosynthesis and deranges reduced glutathione (GSH) metabolism in liver cells. We isolated two BSO-resistant lines, HLE/BSO2-1 and HLE/BSO2-2, from human hepatic HLE/WT cells. Cellular levels of the Pi class glutathione thiol transferase (GSTP1) were 3-fold lower in BSO-resistant lines than in HLE/WT cells. By contrast, gamma-glutamylcysteine synthetase (GCS) heavy subunit (GCSh) mRNA levels were markedly decreased in HLE/BSO2-1 and HLE/BSO2-2 as compared with HLE/WT. The expression of a dominant-negative mutant of c-Jun inhibited the GCSh promoter activity in HLE/WT, but not in HLE/BSO2-1. Cellular levels of AP-1, however, were not decreased in either BSO-resistant cell line. Transfection of GCSh promoter of various lengths driven reporter constructs showed no sequence-specific increase in the promoter activities in HLE/BSO2-1. However, transfection of GSTP1 cDNA into HLE/BSO2-1 and HLE/BSO2-2 restored the levels of GCSh mRNA and the GCSh promoter activity to those of HLE/WT. Sequences between -315 and -241 bp of the 5' region contained an AP-1 site responsible for the enhanced GCSh promoter activity in GSTP1 transfectants of HLE/BSO2-1. In vivo footprint analysis showed a specific protection of the AP-1 site on GCSh promoter in GSTP1 transfected HLE/BSO2-1. GSH homeostasis thus appears to be maintained by an interaction between GSTP1 and GCS in human hepatic cells resistant to the GSH poison. (+info)
Induction of gamma-glutamylcysteine synthetase gene expression by platinum drugs in peripheral mononuclear cells of lung cancer patients.
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BACKGROUND: To investigate in vivo the roles of gamma-glutamylcysteine synthetase (gamma-GCS), multidrug resistance-associated protein (MRP), human canalicular multispecific organic anion transporter (cMOAT) and DNA topoisomerase I (topo I) in relation to platinum drug resistance, we monitored the changes of the steady-state levels of the mRNAs for these factors in peripheral mononuclear cells (PMN) after completing platinum drug administration. PATIENTS AND METHODS: PMN from 46 subjects were studied. We obtained PMN from 14 previously untreated lung cancer patients and 14 normal volunteers to measure the baseline gene expression levels. We then obtained PMN from 18 patients with previously untreated advanced lung cancer before and after they received platinum drug treatment. We analyzed the gene expression levels by using the quantitative reverse transcription polymerase chain reaction (RT-PCR). RESULTS: There were no differences in the baseline expression levels between normal volunteers and lung cancer patients in any of the genes. After platinum drug administration, the heavy subunit of gamma-GCS (gamma-GCSh) expression level increased 2.5-fold within 24 hours and the increase persisted for a month, whereas the light subunit of gamma-GCS (gamma-GCSl) expression level did not show an early response but had increased after a month. By contrast, the MRP, cMOAT and topo I expression levels were similar before, during and after chemotherapy. CONCLUSIONS: These results suggest that the gene expression levels of both subunits of gamma-GCS play an important in vivo role in platinum drug resistance. (+info)
Induction of the multispecific organic anion transporter (cMoat/mrp2) gene and biliary glutathione secretion by the herbicide 2,4,5-trichlorophenoxyacetic acid in the mouse liver.
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The canalicular multispecific organic anion transporter, cMoat, is an ATP-binding-cassette protein expressed in the canalicular domain of hepatocytes. In addition to the transport of endo- and xenobiotics, cMoat has also been proposed to transport GSH into bile, the major driving force of bile-acid-independent bile flow. We have shown previously that the herbicide 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), a peroxisome-proliferator agent, significantly increases bile-acid-independent bile flow in mice. On this basis, the effect of the herbicide on cMoat gene expression was studied. A 3.6-fold increase in cMoat mRNA levels and a 2.5-fold increase in cMoat protein content were observed in the liver of mice fed on a diet supplemented with 0.125% 2,4,5-T. These effects were due to an increased rate of gene transcription (3.9-fold) and were not associated with peroxisome proliferation. Significant increases in bile flow (2.23+/-0.39 versus 1.13+/-0.15 microl/min per g of liver; P<0.05) and biliary GSH output (7.40+/-3.30 versus 2.65+/-0.34 nmol/min per g of liver; P<0.05) were observed in treated animals. The hepatocellular concentration of total glutathione also increased in hepatocytes of treated mice (10.95+/-0.84 versus 5.12+/-0.47 mM; P<0.05), because of the induction (2.4-fold) of the heavy subunit of the gamma-glutamylcysteine synthetase (GCS-HS) gene. This is the first model of co-induction of cMoat and GCS-HS genes in vivo in the mouse liver, associated with increased glutathione synthesis and biliary glutathione output. Our observations are consistent with the hypothesis that the cMoat transporter plays a crucial role in the secretion of biliary GSH. (+info)
Regulation of gamma-glutamylcysteine synthetase subunit gene expression in retinal Muller cells by oxidative stress.
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PURPOSE: To study regulation of gamma-glutamylcysteine synthetase (GCS) heavy and light subunit gene expression in Muller cells under conditions of oxidative stress. METHODS: Experiments were carried out with an SV40 transformed cell line (rMC-1) that exhibits the phenotype of rat retinal Muller cells. Endogenous glutathione levels were modified by treating cells with diethyl maleate (DEM), D,L-buthionine sulfoximine (BSO), or tert-butylhydroquinone (TBH). In other experiments, cells were grown in either high (28 mM) or normal (5.5 mM) glucose medium for 1 week to examine the effects of hyperglycemia. Cells were processed for reduced glutathione (GSH) measurement, RNA extraction, cell count, and, in some cases, lactate dehydrogenase activity. The steady state mRNA levels of GCS heavy and light subunits were measured by northern blot analysis using specific cDNA probes. Changes in mRNA levels were normalized to beta-actin or 18S rRNA. RESULTS: Treatment with DEM for 30 minutes depleted cell GSH to 20% to 30% of the normal value. GSH content recovered completely 6 hours after returning to normal medium. BSO treatment for 12 hours followed by a medium change for 6 hours resulted in a cell GSH level that was 26% that of untreated cells. If cells were left in BSO for 18 hours, however, GSH levels were reduced to < 1%. Treatment with TBH for 12 hours led to a 77% increase in cellular GSH level. Treatment with DEM, TBH, or BSO for 18 hours led to a significant induction of the mRNA level of the GCS subunits, regardless of glucose concentration in the medium. Shorter BSO treatment exerted no effect. Prolonged hyperglycemia resulted in 30% lower GSH level, 55% lower GCS heavy subunit, and 30% lower GCS light subunit mRNA levels. CONCLUSIONS: Oxidative stress induced the gene expression of GCS heavy and light subunits in Muller cells. The effect of BSO on mRNA levels correlated with the degree of GSH depletion. Prolonged hyperglycemia lowered GCS subunit mRNA and GSH levels. (+info)