Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

This article is a summary of laboratory methods for the hepatic drug metabolizing enzymes which are reliable, sensitive, and reasonably straightforward to perform. Assay conditions are given for which the enzyme rate determinations are linear with respect to time and protein concentration for hepatic tissue preparations from Charles River Sprague Dawley CD male rats. In selecting these particular assay methods, factors such as disposal of radioactive wastes, safety of laboratory personnel, and cost of required equipment were considered. Thus 9 of the 10 hepatic parameters utilize simple spectrophotometric techniques; the remaining assay (ethoxyresorufin O-deethylase) requires a spectrophotofluorometer. The hepatic toxification/detoxification assays are cytochrome P-450 and reduced glutathione content, NADPH-cytochrome C reductase, aminopyrine N-demethylase, ethoxyresorufin O-deethylase, glutathione S-transferase (3 substrates) and UDP-glucuronyltransferase (2 substrates).
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PMID:Laboratory methods for ten hepatic toxification/detoxification parameters. 642 71

Isoenergetic diets containing 20% corn oil, 20% beef tallow, or an equal mixture of 10% corn oil and 10% beef tallow (mixed fat) were fed to 30 rats per diet for 28 weeks following weaning. DMBA [7,12-dimethylbenz(a)anthracene] was administered (1.75 mg/100 g body weight) in a single oral dose after 4 weeks of feeding. After 28 weeks, 70% of the rats fed corn oil had mammary tumors versus 47% for mixed fat and 30% for tallow. Diet had no effect on the number of tumors per tumor-bearing rat or the proportion of tumors that were adenocarcinomas. Other rats assigned to each of the three diets were killed at the time corresponding to DMBA administration for examination of hepatic mixed-function oxidase activity. NADPH cytochrome c reductase activity and cytochrome P-450 content were higher in rats fed corn oil or mixed fat rather than tallow. However, no significant differences in aryl hydrocarbon hydroxylase, glutathione transferase, and uridine-diphosphoglucuronide transferase activities were observed. The effects of dietary fat saturation on enzyme activity failed to show a clear association with DMBA carcinogenesis. In other rats assigned to the three dietary treatments for 4 or 16 weeks, lipid saturation did not change serum prolactin (PRL) concentrations during diestrus or proestrus. PRL secretion was examined following a provocative stimulus (perphenazine) in rats fed the experimental diets for 4 or 10-22 weeks. Although perphenazine increased serum PRL and depleted the pituitary of PRL, differences in dietary lipid saturation caused no significant changes in these indices. These data show that the incidence of mammary tumors in rats fed high fat diets (20% by weight) was greater in those fed corn oil compared to beef tallow. The effect of dietary lipid source on tumorigenesis was not associated with changes in carcinogen-metabolizing enzyme activity or PRL secretion.
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PMID:Effects of dietary lipid saturation on prolactin secretion, carcinogen metabolism and mammary carcinogenesis in rats. 643 76

Glutathione peroxidase (glutathione: hydrogen-peroxide oxidoreductase, EC 1.11.1.9) was purified approximately 600-fold from rainbow trout liver soluble fraction and its activity in the NADPH microsomal lipid peroxidation system tested. The enzyme has an approximate molecular weight of 100 000, contains four subunits and four atoms of selenium per mol protein. No selenium-independent glutathione peroxidase activity could be attributed to glutathione S-transferase (EC 2.5.1.18) in trout liver. Glutathione peroxidase together with glutathione (GSH) did not provide any additional protection in the in vitro liver microsomal lipid peroxidation system over and above that provided by GSH alone. Microsomal lipid peroxidation was, however, reduced by a partially purified glutathione S-transferase together with GSH. The protection provided by dialysed liver cytosol in this system was not GSH-dependent, showing that other factors in addition to glutathione S-transferase are involved. Of other possible factors, vitamin E reduced lipid peroxidation in this system. Concentrations of vitamin E in microsomes before and after peroxidation in vitro indicated that protective cytosolic factor(s) act prior to the termination of the free radical chain reactions effected by vitamin E. A GSH-dependent protective factor was present in microsomal protein, malondialdehyde formation in the in vitro microsomal system being markedly reduced in the presence of 5 mM GSH but not significantly lowered by 1 mM GSH.
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PMID:Rainbow trout liver microsomal lipid peroxidation. The effect of purified glutathione peroxidase, glutathione S-transferase and other factors. 646 1

Coincubation of 0.4 mM [3H]glutathione with 4 mM isaxonine , an NADPH-generating system, glutathione S-transferase and mouse liver microsomes, followed by thin-layer chromatography of the incubation mixture, resulted in the appearance of a 3H-labeled peak with characteristics consistent with a glutathione- isaxonine metabolite adduct: this peak was absent if either isaxonine or the NADPH-generating system was omitted and was decreased if the transferase was omitted. In vivo, the concentrations of hepatic glutathione and glutathione disulfide were markedly decreased 2.5 hr after administration of isaxonine (4 mmol X kg-1 i.p.); this depletion of glutathione was prevented essentially by pretreatment with piperonyl butoxide. In vitro, addition of 4 mM glutathione decreased markedly the amount of [14C] isaxonine metabolite that bound to microsomal proteins during incubation of 1 mM [2-14C] isaxonine with hepatic microsomes and an NADPH-generating system. In vivo, pretreatment with diethylmaleate decreased further hepatic glutathione concentration and markedly increased the amount of [14C] isaxonine metabolite covalently bound to hepatic proteins, 2.5 hr after administration of [2-14C] isaxonine (4 mmol X kg-1 i.p.). Administration of isaxonine (4 mmol X kg-1 i.p.) decreased hepatic cytochrome P-450 concentration, but failed to produce liver cell necrosis, even in mice pretreated with phenobarbital, 3-methylcholanthrene or diethylmaleate, despite high levels of in vivo covalent binding in pretreated animals. We conclude that the reactive metabolite of isaxonine may be conjugated with glutathione or may covalently bind to hepatic proteins. The metabolite, however, has limited hepatotoxic potential in mice.
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PMID:Mechanism for isaxonine hepatitis. II. Protective role of glutathione and toxicological studies in mice. 654 80

The effects of the synthetic dibromo-pyrethroid insecticide deltamethrin on some hepatic phase I and II enzyme activities were studied in rat liver. The animals were treated with daily doses of 5 and 10 mg/kg of both pure insecticide or its commercial formulation (Decis), administered i.p. in corn oil for 7 days. The following enzyme activities were studied: NADPH-cytochrome-P450 reductase, aryl-hydrocarbon hydroxylase, aminopyrine N-demethylase, glutamyl cysteine synthetase, glutathione S-transferase, glutathione peroxidase, peroxisomal acyl-CoA oxidase, catalase, and urate oxidase. Both deltamethrin and its commercial formulation were effective in modifying the activities of several of these hepatic xenobiotic-metabolizing enzymes. However, some differences in enzyme modifications were found between treatment with pure or commercial deltamethrin, the latter being more active. This effect could be ascribed to additives, solvents, and chemical intermediates present in the Decis formulation. These results suggest that exposure to this deltamethrin commercial formulation could be more dangerous than exposure to deltamethrin alone, both in terms of its hepatotoxicity and/or alterations in the hepatic biotransformation of other occupational/environmental xenobiotics.
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PMID:Studies on hepatic xenobiotic-metabolizing enzymes in rats treated with insecticide deltamethrin. 747 74

Transfection of murine NIH3T3 fibroblasts and human MCF7 breast carcinoma cells with a pSV2-derived eukaryotic expression vector for human cytosolic glutathione peroxidase resulted in clones with increased glutathione peroxidase activity. This heterologous expression indicates that murine cells recognize the human "selenocysteine insertion sequence" in the 3' untranslated region of the mRNA which facilitates insertion of selenocysteine directed by the opal codon. Though most clones from both cell lines eventually lost their enhanced glutathione peroxidase activity despite continuous selection on G418, some NIH3T3 clones retained enhanced enzyme activity without continuous G418 exposure. Transfection of MCF7 cells with an Epstein-Barr virus (EBV)-derived episomally replicating expression vector carrying the glutathione peroxidase gene also revealed increased glutathione peroxidase activity. These MCF7 cells, however, all required exposure to G418 to maintain enhanced glutathione peroxidase activity. Detailed biochemical analysis of a stably expressing NIH3T3 clone and MCF7 expressing cells revealed no alterations in activities of copper-zinc superoxide dismutase, manganese superoxide dismutase, catalase, phospholipid-glutathione peroxidase, glutathione reductase, glutathione transferase, or NADPH-P450 reductase. Both pSV2- and EBV-derived glutathione peroxidase-expressing clones exhibited enhanced resistance to paraquat as well as to peroxides.
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PMID:Heterologous expression of selenium-dependent glutathione peroxidase affords cellular resistance to paraquat. 748 71

The peroxisome proliferators perfluorooctanoic acid (PFOA; 0.02% w/w), perfluorodecanoic acid (PFDA; 0.02%, w/w), nafenopin (0.125%, w/w), clofibrate (0.5%, w/w), and acetylsalicylic acid (ASA; 1%, w/w) were administered to male C57 BL/6 mice in their diet for two weeks. Parameters for Fe3+ ADP, NADPH or ascorbic acid-initiated lipid peroxidation in vitro were measured. Approximately a twofold increase in susceptibility to lipid peroxidation was obtained for all the peroxisome proliferators tested. Cotreatment of mice with the peroxisome proliferator ASA (1%, w/w) and a catalase inhibitor, 3-amino-1,2,4-triazole (AT; 0.4%, w/w) for 7 days resulted in little inhibition of peroxisome proliferation, an elevated level of H2O2 in vivo, and total inhibition of the increased susceptibility to lipid peroxidation in vitro. No increase in lipid peroxidation in vivo was observed. Certain antioxidant enzymes (DT-diaphorase, superoxide dismutase, glutathione transferase, glutathione peroxidase, and glutathione reductase) and components (ubiquinone and alpha-tocopherol) were also measured. The results showed that there was some induction of these antioxidant enzymes and components by ASA or aminotriazole, except for glutathione peroxidase and superoxide dismutase, which were inhibited. The possible involvement of oxidative stress in the carcinogenicity of peroxisome proliferators is discussed.
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PMID:Hepatic oxidative stress and related defenses during treatment of mice with acetylsalicylic acid and other peroxisome proliferators. 756 57

1. The metabolism of a nitrate ester-substituted dihydropyridine derivative (NND) in vitro was characterized with rabbit hepatic microsomes and cytosol. 2. Denitration activity was located in both the microsomal and cytosolic fractions, whereas oxidation to the pyridine analogue was solely located in the microsomal fraction. 3. Oxidation to the pyridine analogue required NADPH and was inhibited by carbon monoxide, miconazole and SKF-525A, suggesting that oxidation was catalysed by P450. 4. Denitration activity in the microsomes required either NADPH or GSH. Together with these results, responses to various inhibitors indicate participation of both P450 and glutathione S-transferase (GST). 5. Denitration activity in cytosol was activated by glutathione (GSH), and by dithiothreitol (DTT) to a greater extent. GSH-dependent denitration was inhibited by S-hexyl GSH, an inhibitor of GST, but DTT-dependent denitration was not. Moreover, the formation patterns of the mono-denitrated metabolites, M1 and M2, were shown to be different in each incubation condition. 6. These results suggest that the denitration of NND in cytosol could be catalysed by a GSH-independent enzyme as well as the GSH-dependent enzyme, GST.
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PMID:Metabolism of a nitrate ester, dihydropyridine derivative in rabbit hepatic microsomes and cytosol. 761 54

It has been shown previously that the anticonvulsant agent, sodium valproate, induces certain cytochrome P-450 monooxygenase activities and decreases glutathione S-transferase activity. We have used Western blotting, RNase protection assays and Northern blot hybridization to determine the effects of valproate on the abundance of individual components of the cytochrome P-450 monooxygenase and of glutathione S-transferase subunits. Due to the short half-life of the drug in rats we have used an in vitro experimental system comprised of rat hepatocytes co-cultured with rat primitive biliary epithelial cells. Valproate was shown to be a potent inducer of two members of the cytochrome P-450 (CYP)2B subfamily, CYP2B1 and 2B2. The induction of the proteins was mediated at the level of the mRNAs, with the mRNA for CYP2B1 being more highly induced than that for CYP2B2. The drug also induced, but to a much lesser extent, two important components of the cytochrome-P-450-mediated monooxygenase system, NADPH-dependent cytochrome P-450 reductase and cytochrome b5, and their corresponding mRNAs. Thus, the effects of valproate on cytochromes P-450 and other components of the cytochrome-P450-mediated monooxygenase system mimic those of another, structurally diverse, antiepileptic drug, phenobarbital. However, in contrast to phenobarbital, which induces glutathione S-transferase subunits 1, 2, 3, 4 and 7, valproate selectively decreases the abundance of subunits 3 and/or 4. It has been shown previously that CYP2B1 is involved in the production of metabolites of valproate implicated in hepatotoxicity. The induction of this protein by valproate would thus contribute substantially to the hepatotoxic effects associated with the drug.
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PMID:Effects of the anticonvulsant, valproate, on the expression of components of the cytochrome-P-450-mediated monooxygenase system and glutathione S-transferases. 763 45

Age-associated alternations in activation and deactivation of benzo[a]pyrene (BP), furylfuramide (AF2), and 2-nitrofluorene (NF) in rat liver were investigated. A modified Ames mutagenicity test system used liver 9000 g supernatant (S-9) from male Fischer 344 rats aged 3, 6, 12, and 24 months fortified with NADPH generating system alone or together with cofactors of conjugating enzymes. The numbers of revertant colonies due to mutagenic activation of BP during preincubation were markedly high in young rats and decreased with aging. They were decreased by the addition of UDP-glucuronic acid (15 mM) or glutathione (30 mM), the cofactors of UDP-glucuronyl transferase and glutathione S-transferase, respectively, in the preincubation mixture. The difference in the BP activation by liver S-9 from different age groups almost disappeared by the addition of reduced glutathione. A direct mutagen, AF2, was not metabolized during preincubation in the absence of cofactors of conjugating enzymes, but detoxified up to about 50% by the addition of glutathione to the preincubation mixture containing liver S-9 from rats of any age group. Another direct mutagen, NF, was partly detoxified during preincubation by liver S-9 from 3-month-old rats more than by that from 24-month-old rats. It is suggested that incidence of chemical carcinogenesis may increase along with aging due to the altered xenobiotics metabolism.
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PMID:Alterations in activation and deactivation of mutagens in aging rat liver. 767 Oct 22


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