RT Journal Article
SR Electronic
T1 Monooxygenase-mediated activation of chlorotrianisene (TACE) in covalent binding to rat hepatic microsomal proteins.
JF Drug Metabolism and Disposition
JO Drug Metab Dispos
FD American Society for Pharmacology and Experimental Therapeutics
SP 786
OP 793
VO 15
IS 6
A1 M J Juedes
A1 W H Bulger
A1 D Kupfer
YR 1987
UL http://dmd.aspetjournals.org/content/15/6/786.abstract
AB 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.