Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.5.1.18 (glutathione S-transferase)
 22,582 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In this study, the role of glutathione S-transferase (GST) P1-1, the cellular reduced glutathione (GSH) status, and ATP-dependent efflux pumps in the cellular glutathione-dependent biotransformation of thiotepa and transport of the main metabolite monoglutathionylthiotepa in relation to cytotoxicity was studied in control and GST-P1-1-transfected MCF-7 cell lines. It was demonstrated that an enhanced cellular level of GST-P1-1 leads to an enhanced formation of monoglutathionylthiotepa, which is transported out of the cell into the medium. Monoglutathionylthiotepa was able to reversibly inhibit the activity of purified GST-P1-1, but only at nonphysiological concentrations, indicating that feedback inhibition of GST by its metabolites is not a relevant process in vivo. The GST activity, cellular GSH level, and/or ATP-dependent efflux of monoglutathionylthiotepa were modulated using ethacrynic acid, D,L-buthionine-S,R-sulfoximine, probenecid, and verapamil to understand the interplay between GSTs, glutathione conjugation, and efflux of glutathione conjugates in more detail. Inhibition of the GSH biosynthesis by D,L-buthionine-R,S-sulfoximine, a specific inhibitor of gamma-glutamylcysteine synthetase, significantly reduced the glutathione conjugation of thiotepa and potentiated the cytotoxicity of thiotepa. Pretreatment of cells with ethacrynic acid resulted in decreased formation of monoglutathionylthiotepa as a result of inhibition of GST in the GST-P1-1 transfectant. In addition, the intracellular amount of monoglutathionylthiotepa increased in both of the cell lines on exposure to ethacrynic acid, indicating that transport of the glutathione conjugate was partially inhibited by the glutathione conjugate of ethacrynic acid. Transport activity of monoglutathionylthiotepa could also be inhibited by probenecid and verapamil, inhibitors of organic anion transport, without influencing the biotransformation capacity of the cells. It was demonstrated that inhibition of glutathione conjugate efflux by probenecid and verapamil leads to enhanced cytotoxicity, which indicates that besides thiotepa, monoglutathionylthiotepa is also cytotoxic for the cells. Only enhanced biotransformation and subsequent transport of the glutathione conjugate into the medium (which occurs with the GST-P1-1 transfectant) results in enhanced viability. Therefore, it was concluded that only enhanced biotransformation of thiotepa represents a real detoxification pathway when the resulting conjugate is transported out of the cells. Altogether, the results indicate that it is not the overexpression of GST per se but the interplay between GSH/GST and glutathione conjugate efflux pumps that results in increased resistance to alkylating anticancer drugs such as thiotepa.
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PMID:Glutathione-dependent biotransformation of the alkylating drug thiotepa and transport of its metabolite monoglutathionylthiotepa in human MCF-7 breast cancer cells. 978 13

Pentachlorobenzene (PeCB) is an important environmental contaminant derived primarily from the by-product contamination of the popular fungicides hexachlorobenzene and pentachloronitrobenzene. Its tumor-promoting activity was studied in a medium-term initiation/promotion assay in male F344 rats. Animals were given a single i.p. injection of diethylnitrosamine (200 mg/kg body weight) and 2 weeks later were administered 0.1 or 0.4 mmol/kg per day PeCB by gavage in a corn oil vehicle, 7 days/week. At the end of week 3, rats were subjected to a partial hepatectomy. Results showed that PeCB, at both doses, significantly increased both the number and area of glutathione S-transferase pi (GST-P) foci (>0.2 mm diameter) (P < 0.05). This trend was dose-dependent. In addition to increases in preneoplastic foci, liver glutathione concentrations and glutathione-associated enzymes showed significant changes in animals treated with PeCB. Glutathione reductase (GR) and gamma-glutamylcysteine synthetase (gamma-GCS) were both significantly induced in the centrilobular region. Changes in oxidized glutathione concentrations corresponded with the increase in GR activity with decreases of 40 and 30% in the low and high dose groups, respectively. No significant changes were detected in reduced glutathione concentrations. Together with changes in GR and gamma-GCS expression, a decrease in GST-P foci around the central veins was significant (P = 0.004) at the high dose. In these animals, 26% of the foci were classified as centrilobular whereas 37 and 39% of the foci were centrilobular in the low dose and control groups, respectively. Because of the co-localized nature of the changes in glutathione-associated enzymes and the decreased incidence of centrilobular foci, our results suggest that the reduced cellular environment may ultimately play a role in negatively selecting for foci growth.
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PMID:Evidence for hepatocarcinogenic activity of pentachlorobenzene with intralobular variation in foci incidence. 980 69

Because acute infection and inflammation affect drug metabolism and drug-metabolizing enzymes, the effect of the acute-phase response on the expression of glutathione S-transferase (GST) isoenzymes, glutathione synthesis, and several antioxidant enzymes was investigated. Hepatic expression of GST isozymes, positive and negative acute-phase reactants, and antioxidant enzymes were determined by Northern blotting and hybridization with gene-specific oligonucleotide probes after lipopolysaccharide treatment of rats. Lipopolysaccharide caused the expected acute-phase response as judged by the increased expression of positive and decreased expression of negative acute-phase proteins. The messenger RNA (mRNA) expression of the major hepatic rat GST isozymes A1, A2, A3, M1, and M2 was decreased 50% to 90%. Total hepatic GST activity toward 1-chloro-2,4-dinitrobenzene was also significantly decreased. mRNA expression of gamma-glutamylcysteine synthetase (GCS) large subunit and catalase was reduced by approximately 60%. GCS enzyme activity was also decreased, resulting in a 35% decrease in the hepatic content of reduced glutathione 4 days after lipopolysaccharide challenge. Mn-Superoxide dismutase expression was increased 13-fold, and thioredoxin level was elevated 3-fold after lipopolysaccharide challenge. The expression of all parameters determined returned to near control levels 7 days after treatment. Together, these data show that GSTs and GCS are negative acute-phase proteins and that decreased GCS activity results in a decrease in hepatic glutathione content. Thus, in addition to the phase I drug-metabolizing enzymes known to be decreased during the acute-phase response, some phase II enzymes involved in the elimination of xenobiotics and carcinogens are also decreased.
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PMID:Identification of glutathione S-transferase isozymes and gamma-glutamylcysteine synthetase as negative acute-phase proteins in rat liver. 982 19

The mode of cadmium-induced cell death was investigated in a rat lung epithelial cell line. Cells, grown to near confluence, were exposed to 0-30 microM CdCl2 for 0-72 h. Phase contrast microscopy and fluorescent nuclear staining showed that Cd caused morphological alterations in lung epithelial cells that are characteristic of apoptosis. These changes included cell shrinkage, detachment of the cell from its neighbors, cytoplasmic and chromatin condensation, and fragmentation of the nucleus into multiple chromatin bodies surrounded by remnants of the nuclear envelope. Apoptotic DNA degradation was validated and quantitated using a sensitive enzyme-linked immunosorbent assay (ELISA) which measures the amount of histone-bound DNA fragments in the cytosol. Using this technique, a maximum level of apoptosis (5-fold higher than control) was observed in cultures exposed for 48 h to 20 microM CdCl2. The terminal deoxyribonucleotidyl transferase mediated dUTP nick end labeling method (TUNEL) was subsequently used to determine the percentage of cells that contained Cd-induced DNA strand breaks. After 48 h, approximately 54% of the cells exposed to 20 microM Cd were TUNEL positive compared to less than 2% for control cells. Although the mechanisms by which Cd initiates apoptosis in these cells are presently not known, reactive oxygen species are likely to play a role. This possibility is supported by the finding that the first morphological features indicative of apoptosis were preceded by the up-regulation of oxidant stress genes (glutathione S-transferase-alpha, gamma-glutamylcysteine synthetase, and metallothionein-1), activation of redox sensitive transcription factors (AP-1 and NF-kappaB), and changes in various forms of glutathione (reduced, oxidized, and protein-bound).
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PMID:Characterization of cadmium-induced apoptosis in rat lung epithelial cells: evidence for the participation of oxidant stress. 1041 93

The expression of glutathione (GSH)-dependent enzymes and cytochrome P450 (P450) proteins in freshly isolated proximal tubular cells from human kidney (hPT), and the effect of primary culture on these enzymes, were determined. Freshly isolated hPT cells had relatively high activities of gamma-glutamyltransferase, gamma-glutamylcysteine synthetase, glutathione S-transferase (GST), glutathione disulfide reductase, and GSH peroxidase. Cytochrome P450 4A11 was detected in freshly isolated hPT cells, whereas CYP2E1 was not. Freshly isolated hPT cells also expressed GSTA, GSTP, and GSTT but not GSTM. Primary cultures of hPT cells maintained their epithelial-like nature and diploid status, based on measurements of morphology, cytokeratin expression, and flow cytometric analysis. hPT cells retained GSH-dependent enzyme activities during primary culture, whereas cells that had undergone subsequent passage exhibited a loss of activities of most GSH-dependent enzymes and no longer expressed P450s or GSTs. CYP4A11 expression in primary cultures of hPT cells was significantly increased after treatment for 48 h with either ethanol (50 mM) or dexamethasone (7 nM). GSTA, GSTP, and GSTT contents, although still detectable, were decreased compared with those of freshly isolated hPT cells. Our data show that hPT cells express enzymes involved in xenobiotic disposition, and that they thus provide a model suitable for studies of human renal drug metabolism. Furthermore, primary cultures of hPT cells may afford the opportunity to study factors regulating P450 enzyme expression in human kidney.
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PMID:Expression of glutathione-dependent enzymes and cytochrome P450s in freshly isolated and primary cultures of proximal tubular cells from human kidney. 1077 44

TER286 [gamma-glutamyl-alpha-amino-beta(2-ethyl-N,N,N', N'-tetrakis(2-chloroethyl)phosphorodiamidate)-sulfonyl-propionyl-( R)- (-) phenylglycine] is a novel nitrogen mustard prodrug that is preferentially activated by glutathione S-transferase P1-1 (GSTP1-1). A human promyelocytic leukemia /TER286-resistant cell line was selected by chronic, long-term exposure to the prodrug. Although resistance was not readily achieved, eventually a 5-fold resistant clone was isolated. Cross-resistance to melphalan occurred, but not to doxorubicin (Adriamycin), taxol, and gamma-glutamyl-S-(benzyl)cysteinyl-R(-)-phenyl glycine diethyl ester, a GSTP1-1 inhibitor. The protein and transcript levels and enzymatic activity of GSTP1-1 were reduced significantly in the selected resistant line. GSTalpha levels were unchanged, and GSTmu was undetectable. Although glutathione levels were elevated in human promyelocytic leukemia/TER286 cells, no changes in the expression of thiol-related genes including gamma-glutamylcysteine synthetase, gamma-glutamyl transpeptidase, or multidrug resistance protein were found. A 7-fold increase in catalase expression in the resistant cell line indicated an adaptive response to oxidative and electrophilic stress, and this was also reflected in the lower prevalence of drug-induced DNA single-strand breaks in the resistant cells. Mouse embryo fibroblast GSTP1-1(-/-) cells exhibited 2-fold resistance to TER286 compared with GSTP1-1(+/+) cells. NIH3T3 cells transfected with combinations of gamma-GCS and multidrug resistance protein exhibited enhanced resistance to TER286, although the degree of resistance was impaired by cotransfection of GSTP1-1. These results are consistent with responses in the TER286-resistant cells indicative of GSTP1-1-mediated mechanism of activation. In consequence, these data support the rationale that tumors expressing high levels of GSTP1-1 will be more sensitive to the cytotoxic effects of the drug.
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PMID:Cellular response to a glutathione S-transferase P1-1 activated prodrug. 1086 Sep 39

Glutathione (GSH), glutathione S-transferases (GSTs), and the multidrug resistance-associated protein 1 (MRP1) have been independently studied for their contributions to drug resistance. Single cDNA transfection experiments have provided inconsistent and disparate conclusions with respect to the importance of GSH and GST in conferring a resistant phenotype. Because these three proteins can act as a concerted coordinated pathway, we reasoned that equivalent increases may be required for enhanced resistance to be expressed. We have assembled these proteins together, or in various combinations, to determine whether they show cooperativity in determining drug response. Increased expression through single cDNA transfection of GSTpi, gamma-glutamylcysteine synthetase (gamma-GCS) (regulatory plus catalytic subunits), or MRP1 enhanced resistance to a number of anticancer drugs. Cotransfection of GSTpi and GCS, gave higher resistance to doxorubicin, etoposide, and vincristine than with either alone. Resistance toward chlorambucil and ethacrynic acid was similar in cells overexpressing either component or overexpressing GST alone. Coexpression of GSTpi with MRP1 conferred significant resistance above that seen for MRP1 alone to chlorambucil, etoposide, ethacrynic acid, and vincristine. The combination of GCS and MRP1 did not afford additional resistance above MRP1 alone. When all three were transfected, significantly higher levels of resistance were found for doxorubicin and etoposide. These results support the concept that coordinate enhancement of focal thiol elements of detoxification pathways provides a more efficient protective phenotype than do single components alone.
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PMID:The influence of coordinate overexpression of glutathione phase II detoxification gene products on drug resistance. 1090 Feb 22

Apoptosis involves a series of genetically programmed events associated with endonucleolytic cleavage of DNA. This process is triggered by a variety of agents, including oxidants such as hydrogen peroxide (H(2)O(2)) and it plays a key role in eliminating pre-neoplastic cells from the lung. Failure to do so could favor tumor promotion. The current study demonstrated that alveolar epithelial cells, adapted to cadmium (CdCl(2)) by repeated in vitro exposure, exhibit lower levels of H(2)O(2)-induced apoptosis than similarly challenged non-adapted cells. An immunologic assay, measuring cytoplasmic histone-associated DNA fragments, indicated maximal apoptosis 24 h after exposure to 400 microM H(2)O(2). Non-adapted cells showed a 13-fold increase in oxidant-induced apoptosis while Cd-adapted cells had only a 4-fold elevation. A terminal deoxyribonucleotidyl transferase mediated dUTP nick end labeling (TUNEL) method was used to assess the percentage of cells with DNA breaks consistent with apoptosis. Cd-adapted and non-adapted cells that were not exposed to H(2)O(2) did not differ in TUNEL positivity. However, after H(2)O(2) treatment, the percentage of TUNEL positive cells was 4-fold higher in non-adapted cultures than in adapted ones. Suppression of oxidant-induced apoptosis is due, in part, to up-regulation in the gene expression of several resistance factors including metallothioneins (MT-1 and MT-2), glutathione S-transferases (GST-alpha and GST-pi), and gamma-glutamylcysteine synthetase catalytic subunit (gamma-GCS). These steady-state mRNA changes, determined by Northern blotting, were accompanied by increased levels of MT and gamma-GCS protein, GST activity, and glutathione (GSH). Suppressed oxidant-induced apoptosis, resulting at least in part from these response modifications, could leave pre-neoplastic or neoplastic cells alive, favor clonal expansion, and ultimately lead to cancer development.
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PMID:Suppressed oxidant-induced apoptosis in cadmium adapted alveolar epithelial cells and its potential involvement in cadmium carcinogenesis. 1092 3

Certain dietary constituents can protect against chemically induced carcinogenesis in rodents. A principal mechanism by which these chemopreventive compounds exert their protective effects is likely to be via induction of carcinogen detoxification. This can be mediated by conjugation with glutathione, which is synthesized by the sequential actions of glutamate-cysteine ligase (GLCL) and glutathione synthetase. We have demonstrated that dietary administration of the naturally occurring chemopreventive agents, ellagic acid, coumarin or alpha-angelicalactone caused an increase in GLCL activity of between approximately 3- and 5-fold in rat liver. Treatment with the synthetic antioxidant ethoxyquin or the classic inducer phenobarbital caused < 2-fold induction of GLCL activity in rat liver, which was not found to be significant. The increases in GLCL activity were accompanied by increases (between 2- and 4-fold) in levels of both the catalytic heavy subunit (GLCLC) and regulatory light subunit (GLCLR). No substantial induction of GLCL was observed in rat kidney. The glutathione S-transferase (GST) subunits A1, A3, A4, A5, P1 and M1 were all found to be inducible in rat liver by most of the agents. The greatest levels of induction were observed for GST P1, following treatment with coumarin (20-fold), alpha-angelicalactone (10-fold) or ellagic acid (6-fold), and GST A5, following treatment with coumarin (7-fold), alpha-angelicalactone (6-fold) and ethoxyquin (6-fold). Glutathione synthetase was induced approximately 1.5-fold by coumarin, alpha-angelicalactone, ellagic acid and ethoxyquin. The expression of glutathione-related enzymes was also examined in preneoplastic lesions induced in rat liver by aflatoxin B(1). The majority of gamma-glutamyltranspeptidase (GGT)-positive preneoplastic foci contained increased levels of GLCLC relative to the surrounding tissue. This was usually found to be accompanied by an increase in GLCLR. Cells in the inner cortex of rat kidney were found to contain the highest levels of both GLCLC and GLCLR. The same cells showed the strongest staining for GGT activity.
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PMID:Regulation of rat glutamate-cysteine ligase (gamma-glutamylcysteine synthetase) subunits by chemopreventive agents and in aflatoxin B(1)-induced preneoplasia. 1102 40

Primary cultures of renal proximal (PT) and distal tubular (DT) cells from control and uninephrectomized (NPX) Sprague-Dawley rats were established to characterize factors that are responsible for the altered susceptibility to nephrotoxicants that occurs after compensatory renal cellular hypertrophy. Cells were grown in serum-free, hormonally defined medium and parameters were measured on days 1, 3, and 5 of primary culture. PT and DT cells from control and NPX rats appeared to maintain epithelial characteristics in culture, as shown by cytokeratin staining, morphology, protein and DNA content, and enzyme activities. Activities of several glutathione-dependent enzymes, including gamma-glutamyltransferase, glutathione S-transferase, glutathione peroxidase, and gamma-glutamylcysteine synthetase, were significantly greater in PT cells from NPX rats than in PT cells from control rats when factored by protein content. Rates of alpha-methylglucose uptake across the basolateral and brush-border membranes and sodium-dependent uptake of glutathione across the basolateral membrane were 2- to 3-fold higher in PT cells from NPX rats than in PT cells from control rats. These results are consistent with the hypertrophied phenotype being maintained in primary cultures of PT cells from NPX rats. The marked alterations in transport may play central roles in the delivery of nephrotoxicants to the target cell, and thus, increases the probability of chemically induced injury or death. These findings also suggest that these cell cultures may be useful for the study of biochemical processes associated with compensatory renal cellular hypertrophy.
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PMID:Biochemical and functional characteristics of cultured renal epithelial cells from uninephrectomized rats: factors influencing nephrotoxicity. 1116 Jun 4


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