Glucuronidation of the oxidative cytochrome P450-mediated phenolic metabolites of the endocrine disruptor pesticide: methoxychlor by human hepatic UDP-glucuronosyl transferases.
Methoxychlor, a currently used pesticide, is a proestrogen exhibiting estrogenic activity in mammals in vivo. Methoxychlor undergoes oxidative metabolism by cytochromes P450, yielding 1,1,1-trichloro-2-(4-hydroxyphenyl)-2-(4-methoxyphenyl)ethane (mono-OH-M) and 1,1,1-trichloro-2,2-bis(4-hydroxyphenyl)ethane (bis-OH-M) as main metabolites. Since humans may be exposed to these estrogenic metabolites, which are potential substrates of UDP-glucuronosyltransferases (UGTs), their glucuronide conjugation was investigated with human liver preparations and individual UGTs. Incubation of both mono-OH-M and bis-OH-M with human liver microsomes formed monoglucuronides. The structures of the glucuronides were identified by liquid chromatography/tandem mass spectometry. Examination of cDNA-expressed recombinant human hepatic UGTs revealed that several catalyze glucuronidation of both compounds. Among the cDNA-expressed UGT1A enzymes, UGT1A9 seemed to be the main catalyst of formation of mono-OH-M-glucuronide, whereas UGT1A3 seemed to be the most active in bis-OH-M-glucuronide formation. Furthermore, the chiral selectivity of mono-OH-M glucuronidation was examined. The results of the incubation of single enantiomers generally agreed with the chiral analyses of mono-OH-M derived from the glucuronidase digestion of the glucuronides of the racemic mono-OH-M. There was a relatively slight but consistent enantioselective preference of individual UGT1A1, UGT1A3, UGT1A9, and UGT2B15 enzymes for glucuronidation of the S- over the R-mono-OH-M, whereas in human liver microsomes differences were observed among donors in generating the respective R/S-mono-OH-M ratio. Since it was previously shown that human liver microsomes demethylate methoxychlor mainly into S-mono-OH-M, the observation that UGT1A isoforms preferentially glucuronidate the S-mono-OH-M suggests a suitable mechanism for eliminating this major enantiomer. This enantiomeric preference, however, is not extended to all samples of human liver microsomes that we tested.
Hazai
Eszter
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Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Room 815, 364 Plantation St., Worcester, MA 01605-2324, USA.
Gagne
Peter V
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Kupfer
David
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D015153:Blotting, Western; D002853:Chromatography, Liquid; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D014453:Glucuronosyltransferase / Q000378:metabolism*; D006801:Humans; D006843:Hydrocarbons, Chlorinated / Q000737:chemistry / Q000378:metabolism*; D066298:In Vitro Techniques; D007306:Insecticides / Q000378:metabolism*; D013058:Mass Spectrometry; D008731:Methoxychlor / Q000378:metabolism* / Q000031:analogs & derivatives; D008862:Microsomes, Liver / Q000378:metabolism; D010084:Oxidation-Reduction; D010636:Phenols / Q000737:chemistry / Q000378:metabolism*; D013237:Stereoisomerism; D013997:Time Factors
D006843:Hydrocarbons, Chlorinated; D007306:Insecticides; D010636:Phenols; D003577:Cytochrome P-450 Enzyme System; D014453:Glucuronosyltransferase; C404910:2,2-bis(4-hydroxyphenyl)-1,1,1-trichloroethane; D008731:Methoxychlor; C495657:1,1,1-trichloro-2-(4-hydroxyphenyl)-2-(4-methoxyphenyl)ethane