Reversible and time-dependent inhibition of the hepatic cytochrome P450 steroidal hydroxylases by the proestrogenic pesticide methoxychlor in rat and human.
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 hydroxylation 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 2 alpha- and 16 alpha-testosterone hydroxylation, indicating strong inhibition of P4502C11 (P450h). Methoxychlor moderately inhibited 2 beta-, 7 alpha-, 15 alpha-, 15 beta-, and 16 beta-hydroxylation and androstenedione formation. There was only a weak inhibition of 6 beta-hydroxylation of testosterone. The methoxychlor-mediated inhibition of 6 beta-hydroxylation was competitive. By contrast, when methoxychlor was permitted to be metabolized by PB-microsomes or by liver microsomes from pregnenolone-16 alpha-carbonitrile treated rats (PCN-microsomes) prior to addition of testosterone, a pronounced time-dependent inhibition of 6 beta-hydroxylation was observed, suggesting that methoxychlor inactivates the P450 3A isozyme(s). The di-demethylated methoxychlor (bis-OH-M) and the tris-hydroxy (catechol) methoxychlor metabolite (tris-OH-M) inhibited 6 beta-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 7 alpha-hydroxytestosterone (7 alpha-OH-T) and 16 alpha-hydroxytestosterone (16 alpha-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 (6 beta-hydroxylase) by methoxychlor, was observed. As with rat liver microsomes, the human 6 beta-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 6 beta-OH-T formation when the compounds were coincubated.(ABSTRACT TRUNCATED AT 400 WORDS)
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Worcester Foundation for Experimental Biology, Shrewsbury, MA 01545.
Mani
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Kupfer
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D000818:Animals; D019363:Cytochrome P-450 CYP1A1; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D005260:Female; D006801:Humans; D006900:Hydroxylation / Q000187:drug effects; D066298:In Vitro Techniques; D008297:Male; D008731:Methoxychlor / Q000378:metabolism / Q000633:toxicity*; D008862:Microsomes, Liver / Q000187:drug effects* / Q000201:enzymology; D011485:Protein Binding / Q000187:drug effects; D051381:Rats; D017207:Rats, Sprague-Dawley; D013250:Steroid Hydroxylases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D013739:Testosterone / Q000378:metabolism; D013997:Time Factors
D065607:Cytochrome P-450 Enzyme Inhibitors; D013739:Testosterone; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D019363:Cytochrome P-450 CYP1A1; C020120:estrogen 2-hydroxylase; D008731:Methoxychlor