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

Many toxic effects are not caused by the administered compound itself, but are due to metabolites. All cell types express some xenobiotic-metabolizing enzymes, but levels and patterns are very variable. Critical metabolic steps may occur within the target cell and/or at other sites. This complex situation is difficult to mimic in vitro. The further problem is that cells that are taken into culture tend to rapidly cease the expression of important xenobiotic-metabolizing enzymes. Part of the problem may be solved by the addition of exogenous metabolizing systems, for example, in the form of freshly isolated hepatocytes, crude subcellular preparations, or purified enzymes. In these systems, the plasma membrane of the target cell may act as a barrier for the active metabolite and thereby lead to false negative results. The alternative is the use of metabolically active target cells. We therefore screened 18 cell lines for monooxygenase, cytochrome P-450 reductase, epoxide hydrolase, glutathione transferase, and UDP-glucuronosyl transferase activities. In further studies, IEC-17, IEC-18, and HuFoe-15 cells showed their capabilities of activating a broad spectrum of structurally heterogenous promutagens, as indicated by the induction of micronuclei. These cells, however, were not suited for the study of a more relevant genetic end point, the induction of hereditary functional changes (gene mutations), implying that a compromise had to be made on the level of the toxicodynamics. In the second approach, cDNAs encoding the rat cytochromes P-450IA1 and P-450IIB1, set under the control of a constitutive promoter, were transfected into V79 Chinese hamster cells, which do not express cytochromes P-450 but are ideal target cells for gene mutation assays. The resulting substrains (XEM1, XEM2, XEM3; SD1) stably expressed cytochromes P-450IA1 and P-450IIB1, respectively, and showed the corresponding monooxygenase activities. Aflatoxin B1, cyclophosphamide, dibutylnitrosamine, and benzo[a]pyrene mutated SD1 and/or XEM1 and XEM2 cells, but were inactive in parental V79 cells. The mutagenicity of benzo[a]pyrene 7,8-trans-dihydrodiol was about 1000 times more potent in XEM1 and XEM2 cells than in SD1 and V79 cells. Other promutagens were inactive in V79 as well as in the genetically engineered daughter lines. This system therefore is not yet optimal in general screening for the detection of new mutagens, but appears ideal in the identification of critical xenobiotic-metabolizing enzymes for a given mutagen.
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PMID:Search for cell culture systems with diverse xenobiotic-metabolizing activities and their use in toxicological studies. 315

Aflatoxin B1 (AFB1) is activated to AFB1-8,9-oxide (AFBO), a potent mutagenic and carcinogenic metabolite of AFB1. In the mouse, AFBO has been shown to be most efficiently detoxified by a specific isozyme of alpha-class glutathione S-transferase (GST), mGSTA3-3 (mGST-Yc). A hamster V79 cell line (V79MZr2B1, originally designated V79/SD1) previously transfected with the rat cytochrome P450-2B1 was stably transfected with an mGSTA3-3 expression vector, to study the chemopreventive role of GST in protecting against cytotoxicity or genotoxicity of AFBO. Immunoblotting demonstrated strong expression of an alpha-class GST in the mGSTA3-3 transfected cell line, whereas no detectable alpha-class GST protein was observed in the control (empty vector-transfected) cells. Previous studies with the V79MZr2B1 cell line indicated that it can activate AFB1 to a mutagenic metabolite via a transfected rat P450-2B1 stably expressed in the cells. We examined the ability of the expressed mGSTA3-3 to protect against AFB1-induced cytotoxicity or [3H]-covalent adduct formation in cellular nucleic acids. Exposure of empty vector-transfected control cells and mGSTA3-3 expressing cells to up to 600 nM [3H]-AFB1 indicated that a 70-80% reduction in DNA and RNA adducts was afforded by the expression of mGSTA3-3 in the transfected cells. Clonogenic survival assays showed that the mGSTA3-3 cell line was 4.6-fold resistant to AFB1 cytotoxicity as compared with the empty vector-transfected control SD1 cells, with IC50 values of 69 and 15 microM, respectively. The results of these studies demonstrate that mGSTA3-3 confers substantial protection against nucleic acid covalent modification and cytotoxicity by AFB1 in this transgenic cell model system.
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PMID:Expression of stably transfected murine glutathione S-transferase A3-3 protects against nucleic acid alkylation and cytotoxicity by aflatoxin B1 in hamster V79 cells expressing rat cytochrome P450-2B1. 1035 98