Covalent binding of [14C]methoxychlor metabolite(s) to rat liver microsomal components

[14C]Methoxychlor was incubated with NADPH-fortified liver microsomes from male rats, and covalent binding to microsomal components was determined. The binding process was markedly enhanced when microsomes from phenobarbital-treated rats were employed. However, when microsomes from methylcholanthrene-treated rats were used the level of binding was not significantly affected. Incubation in the presence of glutathione, cysteine, or ascorbate markedly diminished binding. Metyrapone and SKF 525-A, inhibitors of hepatic cytochrome P-450-linked monooxygenase activity, inhibited the binding. Also, ethylmorphine and hexobarbital, alternate substrates of the monooxygenase system, inhibited binding. There was no binding to microsomal components in the absence of NADPH or oxygen. TCPO (1,1,1-trichloropropane-2,3-oxide), an inhibitor of epoxide hydrase activity, failed to enhance the binding process. However, N,N'-diphenyl-p-phenylenediamine (NDP) and n-propyl gallate (PG), both free radical scavengers, decreased binding at micromolar concentrations without altering the extent of formation of polar [14C]methoxychlor metabolites. It was concluded that methoxychlor undergoes a hepatic microsomal monooxygenase(s)-mediated activation and that the resultant reactive metabolites (possibly free radicals) bind covalently to microsomal components. By contrast, the binding resulting from the incubation of an impure mixture of polar [14C]methoxychlor metabolites with liver microsomes did not require NADPH and O2 and was not affected by NDP, Pg, ascorbate, or heat-treatment of microsomes. This finding suggested that the binding subsequent to the initial metabolic activation of methoxychlor does not require further enzymatic transformation. However, whether the binding with metabolites represents the same chemical species as the binding with [14C]methoxychlor remains to be established.