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)

The P450-catalyzed hydroxylation of tamoxifen to give alpha-hydroxytamoxifen [(E)-4-{4-[2-(dimethylamino)ethoxy]phenyl}-3,4-diphenyl-3-buten-2- ol] and subsequent formation of reactive sulfate esters which alkylate DNA has been proposed to be a potential carcinogenic pathway for tamoxifen. In the present study, the ability of alpha-hydroxytamoxifen analogs to form GSH and sulfate conjugates was investigated in order to understand the structural features influencing reactivity. The para oxo analogs 1 [1-(4-methoxyphenyl)-3-hydroxy-1-butene], 2 [1-(4-hydroxyphenyl)-3-hydroxy-1-butene], and 4 [1-(4-hydroxyphenyl)-1-phenyl-3-hydroxy-1-butene] reacted with GSH instantaneously under strong acidic conditions to yield GSH conjugates in greater than 90% yields. Interestingly, the meta phenolic analogs 3 [1-(3-hydroxyphenyl)-3-hydroxy-1-butene] and 5 [1-(3-hydroxyphenyl)-1-phenyl-3-hydroxy-1-butene] did not react with GSH to any significant extent under similar conditions. Characterization of the GSH conjugates with 1H-NMR, electrospray mass spectrometry, and UV showed that all of the conjugates resulted from attack of GSH at the alpha-position of the substrates with displacement of the hydroxyl group. The formation of a single pair of diastereomeric conjugates strongly supported adduct formation to proceed through a direct S(N)2 displacement mechanism and not through a quinone methide (4-alkyl-2,5-cyclohexadien-1-one) intermediate. At physiological pH and temperature only the para hydroxy analogs 2 and 4 gave GSH conjugates, a reaction which seems to be catalyzed by isoforms of glutathione S-transferase. Similar substituent effects were observed in the sulfotransferase-mediated formation of alpha-hydroxy sulfate esters in that only the para hydroxy analogs formed conjugates at the aliphatic hydroxyl group. Finally, the present investigation showed a remarkable difference in the reactivities of para and meta phenolic analogs of alpha-hydroxybutenylbenzenes toward GSH and sulfate conjugation reactions.
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PMID:Oxo substituents markedly alter the phase II metabolism of alpha-hydroxybutenylbenzenes: models probing the bioactivation mechanisms of tamoxifen. 928 38

Myelin oligodendrocyte glycoprotein (MOG), a putative autoantigen in multiple sclerosis (MS), is a quantitatively minor component of the CNS. In view of the difficulties associated with the purification of MOG from brain tissues, the extracellular domain of human MOG corresponding to the N-terminal 121 amino acids was expressed in Escherichia coli as a glutathione sulfotransferase fusion protein. The expressed protein was localized to inclusion bodies, and varying the growth parameters resulted in the solubilization of small amounts of GST-MOG that could be affinity purified on glutathione agarose columns. The fusion protein found in the inclusion bodies could be solubilized with urea. The solubilized fusion protein was cleaved with thrombin, and the extracellular domain was purified by CM Sephadex 50 chromatography to homogeneity. Injection of recombinant human MOG into different strains of mice resulted in the induction of an MS-like disease, characterized by severe neurological impairment and extensive CNS demyelinated lesions. Recombinant MOG produced in E. coli should prove to be useful as a highly purified biological reagent for immunological, pathological, functional, and structural studies.
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PMID:Expression, purification, and encephalitogenicity of recombinant human myelin oligodendrocyte glycoprotein. 952 76

The phase I and phase II drug-metabolizing capacity of freshly isolated and cryopreserved parenchymal cells (PC) from human, rat, and mouse liver held in suspension at 37 degrees C for up to 120 min after thawing was compared. Although 7-ethoxycoumarin-O-deethylase activity was strongly reduced in freshly isolated as well as in cryopreserved PC from human, rat, and mouse liver after 120 min, 7-ethoxyresorufin-O-deethylase activity as well as the profile and formation rates of hydroxylated testosterone metabolites in general remained constant throughout the 2-h incubation period in cryopreserved PC from all three species and was similar to that measured in freshly isolated PC. The activity of glutathione S-transferase (GST) and that of UDP-glucuronosyltransferase (UDP-GT) toward 4-methylumbelliferone significantly decreased, whereas the activities of UDP-GT activity toward 4-hydroxybiphenyl and sulfotransferase in cryopreserved human PC were similar to those measured in freshly isolated PC. The activities of GST, UDP-GT toward 4-methylumbelliferone, and sulfotransferase in cryopreserved rat PC showed a significant decrease when compared with the activities in freshly isolated PC. The phase II enzyme activities in cryopreserved mouse PC proved to be far more stable, being similar to the activities of freshly isolated mouse PC at all four time points measured with the exception of GST, which showed a decay from t = 60 min onward. In conclusion, phase I drug-metabolizing enzyme activities in cryopreserved human, rat, and mouse PC are very similar to those of freshly isolated PC, whereas phase II enzyme activities are affected by cryopreservation.
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PMID:Drug metabolizing capacity of cryopreserved human, rat, and mouse liver parenchymal cells in suspension. 1057 22

The use of primary hepatocytes is now well established for both studies of drug metabolism and enzyme induction. Cryopreservation of primary hepatocytes decreases the need for fresh liver tissue. This is especially important for research with human hepatocytes because availability of human liver tissue is limited. In this review, we summarize our research on optimization and validation of cryopreservation techniques. The critical elements for successful cryopreservation of hepatocytes are (1) the freezing protocol, (2) the concentration of the cryoprotectant [10% dimethyl-sulfoxide (DMSO)], (3) slow addition and removal of DMSO, (4) carbogen equilibration during isolation of hepatocytes and before cryopreservation, and (5) removal of unvital hepatocytes by Percoll centrifugation after thawing. Hepatocytes of human, monkey, dog, rat, and mouse isolated and cryopreserved by our standard procedure have a viability > or = 80%. Metabolic capacity of cryopreserved hepatocytes determined by testosterone hydroxylation, 7-ethoxyresorufin-O-de-ethylase (EROD), 7-ethoxycoumarin-O-deethylase (ECOD), glutathione S-transferase, UDP-glucuronosyl transferase, sulfotransferase, and epoxide hydrolase activities is > or = 60% of freshly isolated cells. Cryopreserved hepatocytes in suspension were successfully applied in short-term metabolism studies and as a metabolizing system in mutagenicity investigations. For instance, the complex pattern of benzo[a]pyrene metabolites including phase II metabolites formed by freshly isolated and cryopreserved hepatocytes was almost identical. For the study of enzyme induction, a longer time period and therefore cryopreserved hepatocyte cultures are required. We present a technique with cryopreserved hepatocytes that allows the induction of testosterone metabolism with similar induction factors as for fresh cultures. However, enzyme activities of induced hepatocytes and solvent controls were smaller in the cryopreserved cells. In conclusion, cryopreserved hepatocytes held in suspension can be recommended for short-term metabolism or toxicity studies. Systems with cryopreserved hepatocyte cultures that could be applied for studies of enzyme induction are already in a state allowing practical application, but may be further optimized.
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PMID:Cryopreserved primary hepatocytes as a constantly available in vitro model for the evaluation of human and animal drug metabolism and enzyme induction. 1071 8

2-Amino-alpha-carboline (A alpha C) is a mutagenic and carcinogenic heterocyclic amine present in foods cooked at high temperature and in cigarette smoke. The mutagenic activity of A alpha C is dependent upon metabolic activation to N-hydroxy-A alpha C (N-OH-A alpha C); however, the metabolism of N-OH-A alpha C has not been studied. We have synthesized 2-nitro-alpha-carboline and N-OH-A alpha C and have examined in vitro bioactivation of N-OH-A alpha C by human and rodent liver cytosolic sulfotransferase(s) and acetyltransferase(s) and by recombinant human N-acetyltransferases, NAT1 and NAT2. The sulfotransferase-dependent bioactivation of N-OH-A alpha C by human liver cytosol exhibited large inter-individual variation (0.5-75, n = 14) and was significantly higher than bioactivation of N-hydroxy-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (N-OH-PhIP). Correlation and inhibition studies suggested that the isoform of sulfotransferase primarily responsible for bioactivation of N-OH-A alpha C in human liver cytosol is SULT1A1. O-Acetyltransferase-dependent bioactivation of N-OH-A alpha C by human liver cytosol also exhibited large inter-individual variation (16-192, n = 18). In contrast to other N-hydroxy heterocyclic amines, which are primarily substrates only for NAT2, both NAT1 and NAT2 catalyzed bioactivation of N-OH-A alpha C. The rate of bioactivation of N-OH-A alpha C by both NAT1 and NAT2 was significantly higher than that for N-OH-PhIP. In rat and mouse liver cytosols, the level of sulfotransferase-dependent bioactivation of N-OH-A alpha C was similar to the level in the high sulfotransferase activity human liver cytosol. The level of O-acetyltransferase-dependent bioactivation of N-OH-A alpha C in rat liver cytosol was also comparable with that in the high acetyltransferase activity human liver cytosol. However, the level of O-acetyltransferase-dependent bioactivation of N-OH-A alpha C in mouse liver cytosol was comparable with that in the low acetyltransferase activity human liver cytosol. In contrast to N-OH-PhIP, bioactivation of N-OH-A alpha C was not inhibited by glutathione S-transferase activity; however, DNA binding of N-acetoxy-A alpha C was inhibited 20% in the presence of GSH. These results suggest that bioactivation of N-OH-A alpha C may be a significant source of DNA damage in human tissues after dietary exposure to AalphaC and that the relative contribution of each pathway to bioactivation or detoxification of N-OH-A alpha C differs significantly from other N-hydroxy heterocyclic or aromatic amines.
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PMID:In vitro bioactivation of N-hydroxy-2-amino-alpha-carboline. 1087 13

Intestinal N-acetylglucosamine 6-O-sulfotransferase (I-GlcNAc6ST, GST-4alpha) and corneal N-acetylglucosamine 6-O-sulfotransferases (C-GlcNAc6ST, GST-4beta) are two highly homologous GlcNAc 6-O-sulfotransferase isozymes encoded by two intronless open reading frames that reside approximately 50 kb apart on human chromosome 16q23.1. I-GlcNAc6ST has been shown to catalyze 6-O-sulfation of the endothelial mucin GlyCAM-1. C-GlcNAc6ST catalyzes 6-O-sulfation of GlcNAc in keratan sulfate and null-mutations in its encoding gene cause human macular corneal dystrophy. We show here that C-GlcNAc6ST efficiently catalyzes sulfation of GlyCAM-1 when coexpressed with the latter in COS-7 cells. We have further compared expression in human of both enzymes by Northern analysis with isozyme-specific probes. While I-GlcNAc6T is expressed mostly in intestinal tissue, larger C-GlcNAc6ST transcripts are found predominantly in the brain.
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PMID:Sulfation of endothelial mucin by corneal keratan N-acetylglucosamine 6-O-sulfotransferase (GST-4beta). 1135 40

Total RNA differential display (DD) using random primers was performed for rat orthotopic liver transplantation (OLT) models. DA (RT1a) donor livers were transplanted into DA, PVG (RT1c), and LEW (RT1l) recipients: (1) syngeneic OLT (DA-DA): no rejection occurs; (2) allogeneic OLT (DA-PVG): rejection occurs, but is naturally overcome without immunosuppression; (3) allogeneic OLT (DA-LEW): animals die of acute rejection within 14 days. cDNA was isolated from selected bands, re-amplified for sequencing, and confirmed by Northern blots. Two down-regulated genes were observed in day-7 allogeneic OLT livers (DA-PVG, DA-LEW), while they were consistently expressed in day-7 syngeneic OLT (DA-DA) livers. These two genes were identified as alpha-glutathione sulfotransferase (alpha-GST) Ya gene and estrogen sulfotransferase (EST), respectively. Northern blots confirmed that their expression was down-regulated in OLT (DA-PVG) livers on days 7-26 and gradually restored. The mRNA expression of GST and EST may be good markers to predict rejection or induction of tolerance.
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PMID:Identification of two down-regulated genes in rat liver allografts by mRNA differential display. 1149 4

Drug-metabolizing enzymes are involved in the metabolic activation or detoxification of carcinogens. To evaluate animals developed as models for alternative carcinogenicity testing, we investigated whether or not a gene manipulation including the transgene of ras and the knocking out of a tumor suppressor gene such as p53 or XPA could alter the expression of representative drug-metabolizing enzymes directly or indirectly. Expression of several isoforms of cytochrome P450 (CYP) in the liver of rasH2, p53 (+/-), Tg.AC, and XPA (-/-) mice with or without treatment of prototype inducer. phenobarbital or 3-methylcholanthrene, was analyzed by Western immunoblotting in comparison with their parental strains of mice. In addition, the activities of 3 major phase II enzymes, UDP-glucronosyltransferase, sulfotransferase, and glutathione S-transferase, were compared between the gene-manipulated and the corresponding parental strains of mice. Results demonstrate that XPA gene knockout appeared to increase constitutive expression of CYP2B and CYP3A isoforms. Overexpression of human c-Ha-ras gene or p53 gene knockout appeared to increase constitutive UGT activity toward 4-nitrophenol. The content or activities of almost all other enzymes examined in the present study do not appear to be affected by the gene manipulation.
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PMID:Comparison of the levels of enzymes involved in drug metabolism between transgenic or gene-knockout and the parental mice. 1169 53

Susceptibility to colorectal cancer, one of the most common forms of cancer in the Western world, has been associated with several environmental and dietary risk factors. Dietary exposure to food derived heterocyclic amine carcinogens and polycyclic aromatic hydrocarbons have been proposed as specific risk factors. Many polymorphic Phase I and Phase II drug metabolizing enzymes are responsible for the metabolism and disposition of these compounds and it is therefore possible that inheritance of specific allelic variants of these enzymes may influence colorectal cancer susceptibility. In a multicenter case-control study, 490 colorectal cancer patients and 593 controls (433 matched case-control pairs) were genotyped for common polymorphisms in the cytochrome P450 (CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2C9, CYP2C19 and CYP2D6), glutathione S-transferase (GSTM1, GSTP1 and GSTT1), sulfotransferase (SULT1A1 and SULT1A2), N-acetyl transferase 2 (NAT2), NAD(P)H:quinone oxidoreductase (NQO1), methylenetetrahydrofolate reductase (MTHFR), and microsomal epoxide hydrolase (EPHX1) genes. Matched case-control analysis identified alleles associated with higher colorectal cancer risk as carriage of CYP1A1*2C (OR = 2.15, 95% CI 1.36-3.39) and homozygosity for GSTM1*2/*2 (OR = 1.53, 95% CI 1.16-2.02). In contrast, inheritance of the CYP2A6*2 (OR = 0.51, 95% CI 0.28-1.06), CYP2C19*2 (OR = 0.72, 95% CI 0.52-0.98) and the EPHX1(His113) alleles were associated with reduced cancer risk. We found no association with colorectal cancer risk with NAT2 genotype or any of the other polymorphic genes associated with the metabolism and disposition of heterocyclic amine carcinogens. This data suggests that heterocyclic amines do not play an important role in the aetiology of colorectal cancer but that exposure to other carcinogens such as polycyclic aromatic hydrocarbons may be important determinants of cancer risk.
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PMID:A pharmacogenetic study to investigate the role of dietary carcinogens in the etiology of colorectal cancer. 1241 32

The ability of cattle and deer liver to catalyse xenobiotic conjugation reactions was investigated and compared with that of the rat. Marked differences in the activity of these enzymes were noted between the domestic animals and rats. Hepatic microsomal epoxide hydrolase activity in cattle and deer, determined using benzo[a]pyrene 4,5-oxide as substrate, was nearly twice that of the rat. In contrast, glutathione S-transferase activity in hepatic cytosol, determined with 1-chloro-2,4-dinitrobenzene as substrate, was significantly lower in the cattle and deer. When 1,2-dichloro-4-nitrobenzene served as the accepting substrate, no activity was detectable in the cattle and deer. Similarly, glutathione reductase activity and total glutathione levels were markedly lower in the cattle and deer compared with the rat. Cytosolic sulfotransferase activity, monitored using 2-naphthol as substrate, was higher in cattle compared with the rat. Finally, microsomal UDP-glucuronosyl transferase activity, determined using 1-napththol as substrate, did not differ significantly among the three species.
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PMID:Xenobiotic conjugation systems in deer compared with cattle and rat. 1252 29


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