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Monooxygenase-mediated activation of chlorotrianisene (TACE) in covalent binding to rat hepatic microsomal proteins.
Drug Metab Dispos. 1987 Nov-Dec; 15(6):786-93.DM

Abstract

Chlorotrianisene is a therapeutic estrogen and contaminant of the pesticide methoxychlor. Incubation of [3H]chlorotrianisene with rat liver microsomes, supplemented with NADPH, yielded covalent binding of radiolabeled metabolite(s) to microsomal components. This binding was dramatically stimulated when microsomes from methylcholanthrene-treated rats were used. However, microsomes from phenobarbital-treated animals did not enhance binding. Analysis of solubilized microsomes by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed radiolabeled bands in the 45- to 66-kDa range. Furthermore, these bands were sensitive to protease degradation, indicating that the recipient macromolecules were proteins and possibly cytochrome P-450(s). Selective inhibition of binding to microsomes prepared from control, phenobarbital-, and methylcholanthrene-treated rats by inhibitors of monooxygenase activity [beta-diethylaminoethyl diphenylpropylacetate (SKF-525A) and metyrapone], by alternate substrates (ethylmorphine and benzo[a]pyrene), and by oxygen exclusion indicated that the binding was dependent upon monooxygenase activity and that a specific P-450 may be involved. Compounds containing free sulfhydryls markedly inhibited covalent binding, suggesting that the reactive intermediate is an epoxide or a free radical. The epoxide hydratase inhibitor (1,1,1-trichloropropane oxide) failed to enhance covalent binding, suggesting that an epoxide of chlorotrianisene was not the reactive intermediate. By contrast, free radical scavengers (propyl gallate, N,N'-diphenylenediamine, and ascorbic acid) markedly inhibited covalent binding, indicating that binding was mediated via a free radical. Since both methylcholanthrene and phenobarbital did not enhance demethylation of chlorotrianisene and methylcholanthrene increased covalent binding, it appears that demethylation products are not involved in covalent binding or that demethylation is not the rate-limiting step. A possible pathway for the metabolism and covalent binding of chlorotrianisene is presented.

Authors+Show Affiliations

Worcester Foundation for Experimental Biology, Shrewsbury, MA 01545.No affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, U.S. Gov't, P.H.S.

Language

eng

PubMed ID

2893703

Citation

Juedes, M J., et al. "Monooxygenase-mediated Activation of Chlorotrianisene (TACE) in Covalent Binding to Rat Hepatic Microsomal Proteins." Drug Metabolism and Disposition: the Biological Fate of Chemicals, vol. 15, no. 6, 1987, pp. 786-93.
Juedes MJ, Bulger WH, Kupfer D. Monooxygenase-mediated activation of chlorotrianisene (TACE) in covalent binding to rat hepatic microsomal proteins. Drug Metab Dispos. 1987;15(6):786-93.
Juedes, M. J., Bulger, W. H., & Kupfer, D. (1987). Monooxygenase-mediated activation of chlorotrianisene (TACE) in covalent binding to rat hepatic microsomal proteins. Drug Metabolism and Disposition: the Biological Fate of Chemicals, 15(6), 786-93.
Juedes MJ, Bulger WH, Kupfer D. Monooxygenase-mediated Activation of Chlorotrianisene (TACE) in Covalent Binding to Rat Hepatic Microsomal Proteins. Drug Metab Dispos. 1987 Nov-Dec;15(6):786-93. PubMed PMID: 2893703.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Monooxygenase-mediated activation of chlorotrianisene (TACE) in covalent binding to rat hepatic microsomal proteins. AU - Juedes,M J, AU - Bulger,W H, AU - Kupfer,D, PY - 1987/11/1/pubmed PY - 1987/11/1/medline PY - 1987/11/1/entrez SP - 786 EP - 93 JF - Drug metabolism and disposition: the biological fate of chemicals JO - Drug Metab Dispos VL - 15 IS - 6 N2 - Chlorotrianisene is a therapeutic estrogen and contaminant of the pesticide methoxychlor. Incubation of [3H]chlorotrianisene with rat liver microsomes, supplemented with NADPH, yielded covalent binding of radiolabeled metabolite(s) to microsomal components. This binding was dramatically stimulated when microsomes from methylcholanthrene-treated rats were used. However, microsomes from phenobarbital-treated animals did not enhance binding. Analysis of solubilized microsomes by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed radiolabeled bands in the 45- to 66-kDa range. Furthermore, these bands were sensitive to protease degradation, indicating that the recipient macromolecules were proteins and possibly cytochrome P-450(s). Selective inhibition of binding to microsomes prepared from control, phenobarbital-, and methylcholanthrene-treated rats by inhibitors of monooxygenase activity [beta-diethylaminoethyl diphenylpropylacetate (SKF-525A) and metyrapone], by alternate substrates (ethylmorphine and benzo[a]pyrene), and by oxygen exclusion indicated that the binding was dependent upon monooxygenase activity and that a specific P-450 may be involved. Compounds containing free sulfhydryls markedly inhibited covalent binding, suggesting that the reactive intermediate is an epoxide or a free radical. The epoxide hydratase inhibitor (1,1,1-trichloropropane oxide) failed to enhance covalent binding, suggesting that an epoxide of chlorotrianisene was not the reactive intermediate. By contrast, free radical scavengers (propyl gallate, N,N'-diphenylenediamine, and ascorbic acid) markedly inhibited covalent binding, indicating that binding was mediated via a free radical. Since both methylcholanthrene and phenobarbital did not enhance demethylation of chlorotrianisene and methylcholanthrene increased covalent binding, it appears that demethylation products are not involved in covalent binding or that demethylation is not the rate-limiting step. A possible pathway for the metabolism and covalent binding of chlorotrianisene is presented. SN - 0090-9556 UR - https://www.unboundmedicine.com/medline/citation/2893703/Monooxygenase_mediated_activation_of_chlorotrianisene__TACE__in_covalent_binding_to_rat_hepatic_microsomal_proteins_ L2 - http://dmd.aspetjournals.org/cgi/pmidlookup?view=long&pmid=2893703 DB - PRIME DP - Unbound Medicine ER -