Short Communication Mechanism of Induction of Cytochrome P450 Enzymes by the Proestrogenic Endocrine Disruptor Pesticide-Methoxychlor: Interactions of Methoxychlor Metabolites with the Constitutive Androstane Receptor System

Methoxychlor, a structural analog of the DDT pesticide, was previously shown to induce rat hepatic CYP2B and -3A mRNAs and the corresponding proteins [J Biochem Mol Toxicol 1998;12:315– 323], Additionally, methoxychlor was found to activate the constitutive androstane receptor (CAR) system and induce CYP2B6 (J Biol Chem 1999;274:6043–6046), suggesting a mechanism for methoxychlor-mediated cytochrome P450 (P450) 2B induction. However, it has not been established whether CAR activation and P450 induction was due to methoxychlor per se and/or due to its metabolites. Also, a possible link between the estrogenic potency of methoxychlor metabolites and CAR activation or P450 induction was not investigated. The current study explores the ability of methoxychlor and its metabolites to activate CAR and whether their potency of CAR activation correlates with their respective estrogenicity. Methoxychlor and its metabolites {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]; ringOH-M [1,1,1-trichloro-2(4-methoxyphenyl)-2*-(3-hydroxy-4-methoxyphenyl)ethane]; and tris-OH-M [1,1,1-trichloro-2(4-hydroxyphenyl)-2*(3,4-dihydroxyphenyl)ethane]} were found to be potent activators of CAR. Dose response curves indicated that tris-OH-M is a more potent CAR activator than methoxychlor, mono-OH-M, and bis-OH-M. Since tris-OH-M is a much weaker estrogen receptor-a agonist than monoOH-M and bis-OH-M, it seems that estrogenicity is not a significant factor in CAR activation. These findings indicate that alteration of methoxychlor-benzene rings, i.e., generation of phenolic constituents, does not appreciably alter CAR activation and suggest that a common structural motif in the methoxychlor class of compounds controls CAR activation. Studies are needed to identify the structural motif necessary for CAR activation and CYP2B induction. Methoxychlor, structurally analogous to DDT, is used as a substitute for DDT, which has been banned in the industrially developed countries (Metcalf, 1976). The increased usage of methoxychlor is attributed to its facile biodegradability, its relatively short half-life in animals, and its low toxicity in mammals as compared with DDT (Metcalf, 1976; Kupfer, 1982). However, despite the favorable characteristics, there is considerable concern for the possible toxicity of methoxychlor in animals and humans. This concern stems from observations that the major methoxychlor metabolites exhibit estrogenic activity (Bulger et al., 1978, 1985; Kupfer and Bulger, 1980a,b; Osterhout et al., 1981; Kupfer, 1982) and that certain methoxychlor metabolites display antiandrogenic activity (Maness et al., 1998; Gaido et al., 2000). Additionally, methoxychlor elicits reproductive toxicity in mammals (Bal and Mungkorn, 1978; Cummings and Gray, 1989; Cummings and Laskey, 1993), apparently because of its hormonal activity. Consequently, methoxychlor has been undergoing extensive toxicity studies as a prototype endocrine disruptor (Chapin et al., 1997; Cummings, 1997). Since DDT induces hepatic cytochrome P450 (P450) (CYP2B and CYP3A) enzymes in rats, mimicking phenobarbital (PB)-type inducers (Hart and Fouts, 1965; Lubet et al., 1992; Li et al., 1995) and because methoxychlor is a structural analog of DDT (Fig. 1A), it was assumed that methoxychlor will induce the same P450s as DDT. Indeed, the administration of methoxychlor to rats induced hepatic CYP2B and -3A enzymes (Li et al., 1995). Additionally, Northern blot analyses of the mRNAs encoding these enzymes and nuclear run-on experiments indicated that the induction was attributable to increased rate of transcription (Li and Kupfer, 1998). The profile of the P450 isoforms induced by methoxychlor in rats suggested that methoxychlor is a PB-type inducer. Support for that notion resides in the demonstration that several PB-type inducers activate the artificially repressed nuclear receptor CAR system from HepG2 human hepatoma cell line and induce NR1 luciferase reporter activity (Sueyoshi et al., 1999). CAR is an orphan nuclear hormone This study was supported by National Institute of Environmental Health Sciences Grant ES00834 (to D.K.). 1 Abbreviations used are: methoxychlor, 1,1,1-trichloro-2,29-bis(4-methoxyphenyl)ethane; P450, cytochrome P450; TLC, thin-layer chromatography; monoOH-M, 1,1,1-trichloro-2 (4-hydroxyphenyl)-29-(4-methoxyphenyl)ethane; bis-OH-M, 1,1,1-trichloro-2,29-bis(4-hydroxyphenyl)ethane, often referred to as HPTE; trisOH-M, 1,1,1-trichloro-2(4-hydroxyphenyl)-29-(3,4-dihydroxyphenyl)ethane; ringOH-M, 1,1,1-trichloro-2(4-methoxyphenyl)-29-(3-hydroxy-4-methoxyphenyl)ethane; DDT, 1,1,1-trichloro-2,29-bis (4-chlorophenyl)ethane; diphenyl-DDT, 1,1,1-trichloro2,29-bis(phenyl)ethane; PB, phenobarbital; CAR, constitutively active receptor or constitutive androstane receptor; TCPOBOP, 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene; ER, estrogen receptor; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin. Send reprint requests to: Dr. David Kupfer, Dept. of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, 55 Lake Ave., North, Worcester, MA 01655. E-mail: [email protected] 0090-9556/01/2906-781–785$3.00 DRUG METABOLISM AND DISPOSITION Vol. 29, No. 6 Copyright © 2001 by The American Society for Pharmacology and Experimental Therapeutics 261/905107 DMD 29:781–785, 2001 Printed in U.S.A. 781 at A PE T Jornals on Jne 0, 2017 dm d.aspurnals.org D ow nladed from