Reversible and time‐dependent inhibition of the hepatic cytochrome P450 steroidal hydroxylases by the proestrogenic pesticide methoxychlor in rat and human
Authors: Li Huan‐Chen , Mani Chitra , Kupfer David 
Published: October 2006

Methoxychlor, a currently used pesticide, is demethylated and hydroxylated by several hepatic microsomal cytochrome P450 enzymes. Also, methoxychlor undergoes metabolic activation, yielding a reactive intermediate (M*) that binds irreversibly and apparently covalently to microsomal proteins. The study investigated whether methoxychlor could inhibit or inactivate certain liver microsomal P450 enzymes.

The regioselective and stereoselective hydrox‐ylation of testosterone and the 2‐hydroxylation of estradiol (E2) were utilized as markers of the P450 enzymes inhibited by methoxychlor. Both reversible and time‐dependent inhibition were examined. Coincubation of methoxychlor and testosterone with liver microsomes from phenobarbital treated (PB‐microsomes) male rats, yielded marked diminution of 2α‐ and 16α‐testosterone hydroxylation, indicating strong inhibition of P4502C11 (P450h). Methoxychlor moderately inhibited 2β‐, 7α‐, 15α‐, 15β‐, and 16β‐hydroxylation and androstenedi‐one formation. There was only a weak inhibition of 6β‐ydroxylation of testosterone. The methox‐ychlor‐mediated inhibition of 6β‐hydroxylation was competitive. By contrast, when methoxychlor was permitted to be metabolized by PB‐microsomes or by liver microsomes from pregnenolone‐16α‐car‐bonitrile treated rats (PCN‐microsomes) prior to addition of testosterone, a pronounced time‐dependent inhibition of 6β‐hydroxylation was observed, suggesting that methoxychlor inactivates the P450 3A isozyme(s). The di‐demethylated methoxychlor (bis‐OH‐M) and the tris‐hydroxy (ca‐techol) methoxychlor metabolite (tris‐OH‐M) inhibited 6β‐hydroxylation in PB‐microsomes competitively and noncompetitively, respectively; however, these methoxychlor metabolites did not exhibit a time‐dependent inhibition. Methoxychlor inhibited competitively the formation of 7α‐hydroxytestosterone (7α‐OH‐T) and 16α‐hydroxy‐testosterone (16α‐OH‐T) but exhibited little or no time‐dependent inhibition of generation of these metabolites, indicating that P450s 2A1, 2B1/B2, and 2C11 were inhibited but not inactivated. Methoxychlor inhibited in a time‐dependent fashion the 2‐hydroxylation of E2 in PB‐microsomes. However, bis‐OH‐M exhibited solely reversible inhibition of the 2‐hydroxylation, supporting our conclusion that the inactivation of P450s does not involve participation of the demethylated metabolites. Both competitive inhibition and time‐dependent inactivation of human liver P450 3A (6β‐hydroxylase) by methoxychlor, was observed. As with rat liver microsomes, the human 6β‐hydroxylase was inhibited by bis‐OH‐M and tris‐OH‐M competitively and noncompetitively, respectively.

Testosterone and estradiol strongly inhibited the irreversible binding of methoxychlor to microsomal proteins. This might explain the “clean” competitive inhibition by methoxychlor of the 6β‐OH‐T formation when the compounds were coin‐cubated. Glutathione (GSH) has been shown to interfere with the irreversible binding of methoxychlor to PB‐microsomal proteins. The finding that the coincubation of GSH with methoxychlor partially diminishes the time‐dependent inhibition of 6β‐hydroxylation provides supportive evidence that the inactivation of P450 3A isozymes by methoxychlor is related to the formation of M*.