title: Metabolism of the endocrine disruptor pesticide-methoxychlor by human P450s: pathways involving a novel catechol metabolite.
abstract: The metabolism of methoxychlor, a proestrogenic pesticide (endocrine disruptor), was investigated with cDNA expressed human cytochrome P450s and liver microsomes (HLM). In addition to 1,1,1-trichloro-2-(4-hydroxyphenyl)-2-(4-methoxyphenyl)ethane (mono-OH-M), 1,1,1-trichloro-2, 2-bis(4-hydroxyphenyl)ethane (bis-OH-M), and 1,1,1-trichloro-2-(4-hydroxyphenyl)-2-(3, 4-dihydroxyphenyl)ethane (tris-OH-M), a new metabolite was identified as 1,1,1-trichloro-2-(4-methoxyphenyl)-2-(3, 4-dihydroxyphenyl)ethane (catechol-M; previously assumed to be ring-OH-M) and as a key metabolic intermediate. A novel metabolic route was proposed involving methoxychlor O-demethylation to mono-OH-M, followed by bifurcation of the pathway, both leading to the same final product tris-OH-M: pathway a, mono-OH-M is demethylated to bis-OH-M, followed by ortho-hydroxylation forming tris-OH-M and pathway b, mono-OH-M is ortho-hydroxylated forming catechol-M that is O-demethylated forming tris-OH-M. Among the human cDNA-expressed P450s examined, CYP1A2, 2A6, 2C8, 2C9, 2C19, and 2D6 exhibited mainly O-demethylation, with CYP2C19 being the most catalytically competent. CYP3A4, 3A5, and rat 2B1 catalyzed primarily ortho-hydroxylation of mono-OH-M (CYP3A4 being catalytically the most active) but were weak in O-demethylation. CYP1A1, 1B1, 2E1, and 4A11 demonstrated little or no catalytic activity. CYP2B6 appeared unique, catalyzing effectively both O-demethylation and ortho-hydroxylation. Thus, CYP2B6 demethylated methoxychlor to mono-OH-M and ortho-hydroxylated the mono-OH-M forming catechol-M; however, 2B6 did not appreciably demethylate mono-OH-M or ortho-hydroxylate bis-OH-M, suggesting a narrow substrate specificity. CYP2C19-catalyzed demethylation of methoxychlor, mono-OH-M and catechol-M, demonstrating relatively good substrate affinity (K(m) = 0.23 - 0.41 microM). However, the 3A4 ortho-hydroxylation of mono-OH-M and bis-OH-M exhibited lower affinity, K(m) = 12 and 25 microM, respectively. Thus, a phenolic group seems essential for efficient ortho-hydroxylation, forming catechol-M and tris-OH-M. Inhibition studies with HLM and P450s indicate that CYP2C9 and likely 2C19 are catalysts of methoxychlor-mono-demethylation.
authors.affiliation: Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, 364 Plantation St., Worcester, Massachusetts 01605-2324, USA.
authors.forename: Yiding
authors.initials: Y
authors.lastname: Hu
authors.forename: David
authors.initials: D
authors.lastname: Kupfer
mesh_terms: D002396:Catechols / Q000378:metabolism*; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D018076:DNA, Complementary / Q000235:genetics; D045166:Estradiol Congeners / Q000494:pharmacology*; D006801:Humans; D006900:Hydroxylation; D066298:In Vitro Techniques; D007306:Insecticides / Q000378:metabolism*; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008731:Methoxychlor / Q000378:metabolism*; D008862:Microsomes, Liver / Q000378:metabolism
chemical_list: D002396:Catechols; D018076:DNA, Complementary; D045166:Estradiol Congeners; D007306:Insecticides; D007527:Isoenzymes; D003577:Cytochrome P-450 Enzyme System; D008731:Methoxychlor