Studies on the formation of methoxychlor-protein adduct in rat and human liver microsomes. Is demethylation of methoxychlor essential for cytochrome P450 catalyzed covalent binding?
Previous studies demonstrated that liver microsomal monooxygenases metabolize the pesticide methoxychlor into phenolic estrogenic derivatives. Additionally, methoxychlor is activated by the hepatic cytochrome P450 monooxygenase to bind covalently to microsomal proteins (Bulger WH, Temple JE and Kupfer D, Toxicol Appl Pharmacol 68: 367-374, 1983). The current study examines, in liver microsomes from control and phenobarbital-treated rats and humans, whether demethylation of methoxychlor is essential for covalent binding and whether demethylated methoxychlor metabolites are on the pathway of formation of the reactive intermediate and protein adduct. Using 3H-methoxyl-labeled and 14C-ring-labeled methoxychlor, it was demonstrated that demethylation is not essential for covalent binding. Namely, the major portion of the methoxychlor moiety in the protein adduct was found to contain intact methoxyls. Nevertheless, in the absence of methoxychlor, both the mono- and bis-demethylated methoxychlor metabolites could undergo monooxygenase-mediated covalent binding to proteins. This was demonstrated in incubations of purified 14C-labeled mono- and bis-demethylated methoxychlor metabolites with liver microsomes, in the presence of NADPH. Additionally, the dehydrochlorinated metabolite of methoxychlor, containing a double bond, underwent covalent binding, which exhibited characteristics similar to those of methoxychlor. These findings demonstrated that the protein adduct from relatively brief incubation periods contains a methoxychlor derivative with intact methoxyls. The possibility that the activation of methoxychlor involves modification of the side chain, which is the active site that binds to proteins, is discussed.
Bulger
W H
WH
Worcester Foundation for Experimental Biology, Shrewsbury, MA 01545.
Kupfer
D
D
D000818:Animals; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D008297:Male; D008731:Methoxychlor / Q000378:metabolism*; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D008995:Monoamine Oxidase / Q000378:metabolism; D010634:Phenobarbital / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains
D008995:Monoamine Oxidase; D008731:Methoxychlor; D010634:Phenobarbital