CYP2C subfamily, primarily CYP2C9, catalyses the enantioselective demethylation of the endocrine disruptor pesticide methoxychlor in human liver microsomes: use of inhibitory monoclonal antibodies in P450 identification.
1. The endocrine disruptor pesticide methoxychlor undergoes O-demethylation by mammalian liver microsomes forming chiral mono-phenolic (1,1,1-trichloro-2-(4-hydroxyphenyl)-2-(4-methoxyphenyl)ethane, i.e. mono-OH-M) and achiral bis-phenolic oestrogenic metabolites. Human liver microsomes (HLM) generated primarily the S-mono-OH-M. 2. Inhibitory monoclonal antibodies (MAb) identified those P450s catalysing the enantioselective O-demethylation of methoxychlor. In HLM, O-demethylation was inhibited by MAb anti-2C9 (30-40%), diminishing the per cent of S-mono-OH-M from about 80 to 55-60%. MAb anti-CYP1A2, 2A6, 2B6, 2C8, 2C19, 2D6 and 3A4 did not affect the demethylation rate in HLM. Nevertheless, MAb anti-CYP1A2 decreased the formation of R-mono-OH-M from 21-23 to 10-17%, indicating that CYP1A2 exhibits a role in generating the R-enantiomer. 3. Among cDNA-expressed human P450s (supersomes), CYP2C19 was the most active in demethylation, but in HLM, CYP2C19 appeared inactive (no inhibition by MAb anti-CYP2C19). There was a substantial difference in the per cent inhibition of demethylation by MAb anti-CYP2C9 and anti-rat CYP2C (MAb inhibiting all human CYP2C forms) and in altering the enantioselectivity, suggesting that demethylation by combined CYP2C8, 2C18 and 2C19 was significant (20-30%). 4. Polymorphism of methoxychlor demethylation was examined with supersomes and HLM-expressing CYP2C9 allelic variants. CYP2C9*1 and 2C9*2 were highly active; however, CYP2C9*3 appeared inactive.
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Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA 01605, USA.
Krausz
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Gelboin
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Kupfer
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D000483:Alleles; D019138:Antibodies, Blocking / Q000494:pharmacology*; D000911:Antibodies, Monoclonal / Q000494:pharmacology*; D001189:Aryl Hydrocarbon Hydroxylases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*; D001711:Biotransformation; D002384:Catalysis; D019388:Cytochrome P-450 CYP1A2 / Q000378:metabolism; D065609:Cytochrome P-450 CYP1A2 Inhibitors; D065729:Cytochrome P-450 CYP2C9; D018076:DNA, Complementary / Q000096:biosynthesis / Q000235:genetics; D003640:Dealkylation; D004702:Endocrine Glands / Q000187:drug effects; D006801:Humans; D066298:In Vitro Techniques; D007306:Insecticides / Q000378:metabolism* / Q000633:toxicity; D008731:Methoxychlor / Q000378:metabolism* / Q000633:toxicity; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology*; D013237:Stereoisomerism
D019138:Antibodies, Blocking; D000911:Antibodies, Monoclonal; D065609:Cytochrome P-450 CYP1A2 Inhibitors; D018076:DNA, Complementary; D007306:Insecticides; C450260:CYP2C9 protein, human; D065729:Cytochrome P-450 CYP2C9; D001189:Aryl Hydrocarbon Hydroxylases; D019388:Cytochrome P-450 CYP1A2; D008731:Methoxychlor