title: Metabolism of the proestrogenic pesticide methoxychlor by hepatic P450 monooxygenases in rats and humans. Dual pathways involving novel ortho ring-hydroxylation by CYP2B.
abstract: Previous studies demonstrated that methoxychlor [1,1,1-trichloro-2,2-bis-(4-methoxyphenyl)ethane] is a proestrogen and is toxic to mammalian reproductive processes. Mammalian liver microsomes sequentially demethylate methoxychlor (I), yielding two estrogenic metabolites, mono-OH-M (II) and bis-OH-M (III). Liver microsomes from phenobarbital (PB)-treated rats (PB microsomes) additionally formed a catechol product, tris-OH-M (VII) (Kupfer et al., Chem. Res. Toxicol. 3, 8-16, 1990). This study shows that, in addition to compounds II, III and VII, male and female rat PB microsomes catalyze the formation of a novel ring-hydroxylated methoxychlor metabolite, ring-OH-M (IV). Liver microsomes from male rats treated with pregnenolone-16 alpha-carbonitrile formed the same metabolites as PB microsomes, but the ring-OH-M was formed only in minute amounts, and there was no tris-OH-M. Liver microsomes from methylchlolanthrene-treated and control male rats demethylated methoxychlor, but did not form ring-hydroxylated products. Similarly, human liver microsomes exhibited demethylation but not ring-hydroxylation. Incubation of mono-OH-M (II) with control rat liver microsomes yielded only bis-OH-M (III), whereas incubation of ring-OH-M (IV) resulted in monodemethylated (dihydroxy) compounds V/VI and didemethylated ring-hydroxylated compound, tris-OH-M (VII). Incubation of (IV) with PB microsomes led to compounds V and/or VI and tris-OH-M (VII), whereas incubation of mono-OH-M (II) yielded bis-OH-M (III) and tris-OH-M (VII). The evidence indicates that ring-hydroxylation is catalyzed by CYP2B: a) induction of CYP2B was required for ring-hydroxylation; b) antibodies against CYP2B1/2 strongly inhibited the formation of the ring-hydroxylated products by PB microsomes; c) incubation of methoxychlor with reconstituted CYP2B1 yielded both the hydroxylated (IV and VII) and the demethylated (II and III) metabolites; and d) reconstituted CYP2B1 converted mono-OH-M into bis-OH-M and tris-OH-M, whereas bis-OH-M was converted into tris-OH-M. Human CYP2B6 exhibits ring-hydroxylation, indicating that this reaction is catalyzed by several CYP2B isozymes. In addition, this study demonstrates that the formation of the catechol tris-OH-M involves two metabolic pathways: via O-demethylation followed by ring-hydroxylation and via ring-hydroxylation and subsequent O-demethylation.
authors.affiliation: Worcester Foundation for Experimental Biology, Shrewsbury, MA 01545.
authors.forename: S S
authors.initials: SS
authors.lastname: Dehal
authors.forename: D
authors.initials: D
authors.lastname: Kupfer
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001189:Aryl Hydrocarbon Hydroxylases; D002851:Chromatography, High Pressure Liquid; D003577:Cytochrome P-450 Enzyme System / Q000502:physiology*; D005260:Female; D006801:Humans; D006900:Hydroxylation; D008297:Male; D008731:Methoxychlor / Q000378:metabolism*; D010105:Oxygenases / Q000502:physiology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013250:Steroid Hydroxylases / Q000502:physiology*
chemical_list: D000911:Antibodies, Monoclonal; D003577:Cytochrome P-450 Enzyme System; D010105:Oxygenases; D013250:Steroid Hydroxylases; D001189:Aryl Hydrocarbon Hydroxylases; C025619:steroid 16-beta-hydroxylase; D008731:Methoxychlor