Abstract
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.
Footnotes
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↵1 It should be noted that, since bilirubin is a substrate of UGT1A1, the absence of inhibition by bilirubin might merely reflect that mono-OH-M is a better substrate than bilirubin; however, the mRNA levels of UGT isoforms in human liver indicated that UGT1A1 is one of the least abundant UGT isoforms. Therefore, this finding and our observation that UGT1A1 is not highly active in mono-OH-M glucuronidation suggest that UGT1A1 plays a minor role in mono-OH-M glucuronidation in human liver microsomes.
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This work was supported by a National Institutes of Health Grant ES00834 from the National Institute of Environmental Health Sciences (NIEHS). Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIEHS.
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ABBREVIATIONS: mono-OH-M, 1,1,1-trichloro-2-(4-hydroxyphenyl)-2-(4-methoxyphenyl)ethane; bis-OH-M, 1,1,1-trichloro-2,2-bis(4-hydroxyphenyl)ethane; UGT, UDP-glucuronosyltransferase; HPLC, high-performance liquid chromatography; LC/MS, liquid chromatography/mass spectometry; MS/MS, tandem mass spectometry; ER, estrogen receptor.
- Received July 24, 2003.
- Accepted March 9, 2004.
- The American Society for Pharmacology and Experimental Therapeutics
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