Role of CYP2C19 and CYP1A2 inO-Demethylation
Abstract
1,1,1-Trichloro-2,2-bis(4-methoxyphenyl)ethane (methoxychlor) is a widely used pesticide that is pro-estrogenic. We have elucidated the human cytochrome P450 enzymes responsible for conversion of methoxychlor into its major metabolite, the mono-O-demethylated derivative (mono-OH-M) that is estrogenic. Incubation of methoxychlor with microsomes from insect cells overexpressing either CYP1A2, CYP2C18, or CYP2C19 yielded mono-OH-M with turnover numbers of 14.9, 15.5, and 39.1 nmol/min/nmol of P450, respectively. CYP2B6 and CYP2C9 were much less active. Incubations with purified CYP2C19 and CYP2C18 resulted in formation of mono-OH-M, and also the bis-demethylated metabolite. Co-incubation of liver microsomes with methoxychlor and various P450 isoform-selective inhibitors suggested involvement of several P450s in mono-O-demethylation, including CYP1A2, CYP2A6, CYP2C9, and CYP2C19. A role for CYP2C19, CYP1A2, and CYP2A6 was also indicated by multivariate regression analysis of the mono-O-demethylase activity in a panel of human liver microsomes characterized for isoform-specific catalytic activities (R2= 0.96). Based on the totality of the evidence, CYP2C19 appears to be the major catalyst of methoxychlor mono-O-demethylation. However, in individuals lacking functional CYP2C19 (e.g.the “poor metabolizer” phenotype), CYP1A2 may play the predominant role. CYP2A6, CYP2C9, and CYP2B6 probably contribute to a lesser extent. Although CYP2C18 is an efficient methoxychlor demethylase, its expression in liver is reportedly low or absent, suggesting a negligible role for this enzyme in methoxychlor metabolism. Lengthy incubations of liver microsomes with methoxychlor produced other secondary and tertiary metabolites. Efficient conversion of methoxychlor to estrogenic mono-OH-M by liver microsomes suggests that methoxychlor has the potential to be estrogenic in humans, as observed in several animal species.
Footnotes
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Send reprint requests to: Dr. David Kupfer, Department of Pharmacology and Molecular Toxicology, University of Massachusetts Medical Center, 55 Lake Ave. North, Worcester, MA 01655.
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This work was supported by Grants ES00834 and ES05737 from the National Institute of Environmental Health Sciences, National Institutes of Health. The contents of this article are solely the responsibility of the authors and do not necessarily represent the official view of the National Institute of Environmental Health Sciences, National Institutes of Health. This manuscript is dedicated to the memory of William H. Bulger, who provided the data for fig. 4.
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Portions of this work were presented at the Annual Meeting of the Society of Toxicology, Seattle, WA, on March 1–5, 1998, and appeared in abstract form in ToxicolSci42(1-S), Abstract 453, 1998.
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↵2 Although tolbutamide is considered a selective substrate for CYP2C9 methyl hydroxylation, CYP2C19 was recently reported to be active in catalyzing this reaction, suggesting that tolbutamide could be an effective competitive inhibitor of CYP2C19 in liver microsomes (Shimada et al., 1997).
- Abbreviations used are::
- methoxychlor
- [1,1,1-trichloro-2,2-bis(4-methoxyphenyl)ethane]
- 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]
- tris-OH-M
- [1,1,1-trichloro-2(3,4-dihydroxyphenyl)-2(4-hydroxyphenyl)ethane]
- CYP
- cytochrome P450
- TAO
- troleandomycin
- DDTC
- diethyldithiocarbamate
- Received March 16, 1998.
- Accepted May 5, 1998.
- The American Society for Pharmacology and Experimental Therapeutics
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