Introduction

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1,1,1-Trichloro-2,2-bis(4-methoxyphenyl)ethane (methoxychlor¹) (fig. 1) is a 1,1-bis(p-chlorophenyl)-2,2,2-trichloroethane (DDT) substitute preferred as an insecticide because of its limited persistence in the biosphere (Gardner and Bailey, 1975). In addition, it exhibits low acute toxicity in mammals (Hodge et al., 1950). However, methoxychlor elicits broad endocrine toxicities related to its estrogen-like behavior including adverse developmental, reproductive, and behavioral effects (Chapin et al., 1997; reviewed in Cummings, 1997). Moreover, methoxychlor may induce endocrine cancers in rodents (Reuber, 1980).

View larger version (7K): [in this window] [in a new window]   Fig. 1.   Structure of methoxychlor.

The in vivo estrogenic effects of methoxychlor are attributed to the action of its metabolites, or, additionally, in the case of technical grade preparation, to its phenolic impurities (Bulger and Kupfer, 1985). Indeed, both the mono- and bis-demethylated metabolites (mono-OH-M and bis-OH-M, respectively) exhibit estrogenic activity in vitro, whereas the parent compound is essentially inactive (Bulger et al., 1978; Ousterhout et al., 1981). The cytochrome P450 isoforms that catalyze demethylation (Kishimoto and Kurihara, 1996) and ortho-hydroxylation (Dehal and Kupfer, 1994; Stresser et al., 1996) have been identified in rats, but little information is available regarding its metabolism by human P450s (Stresser and Kupfer, 1997, 1998). In this report, we identify the major P450s expected to catalyze conversion of methoxychlor to its single major metabolite, the mono-O-demethylated derivative.

	Materials and Methods

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Chemicals. Glucose-6-phosphate, glucose-6-phosphate dehydrogenase, NADPH, coumarin, troleandomycin, tranylcypromine, sulfaphenazole, diethyldithiocarbamate, EDTA disodium salt, and activated charcoal (untreated powder) were purchased from Sigma Chemical Co. (St. Louis, MO). Furafylline was obtained from Salford Ultrafine Chemicals and Research (Manchester, United Kingdom). Tolbutamide and ketoconazole were purchased from Research Biochemials International (Natick, MA). S-Mephenytoin was obtained from Gentest Corp. (Woburn, MA). Quinidine and -naphthoflavone were obtained from Aldrich Chemical Co. (Milwaukee, WI). Bis-OH-M was kindly provided by Dr. T. Fujita (Kyoto University, Kyoto, Japan) and Dr. J. Sanborn (Illinois Natural History Survey, Urbana, IL). Methoxychlor was purchased from Chem Service (West Chester, PA). [ring-UL-¹⁴C]Methoxychlor was obtained from Sigma (5.85 mCi/mmol) or California Bionuclear (La Brea, CA; 1.8 mCi/mmol). [ring-UL-¹⁴C]Mono-OH methoxychlor was prepared biosynthetically by incubation of [ring-UL-¹⁴C]methoxychlor with human liver microsomes as described below.

Microsome Preparation. Portions of human livers were obtained from National Disease Research Interchange (Philadelphia, PA) or Prof. Urs Meyer (Basel, Switzerland) and kept frozen at 70^oC until use. Livers were thawed and homogenized in 0.25 M sucrose (5 ml sucrose/g liver), using a Potter-Elvehjem homogenizer and a Teflon pestle (Eberbach Corp., Ann Arbor, MI), and microsomes were prepared by differential centrifugation (Burstein and Kupfer, 1971). The resulting microsomal pellets were resuspended in 1.15% KCl solution, followed by centrifugation at 105,000g for 1hr. The microsomal pellet, after discarding the supernatant, was covered with 1.15% KCl (nominally 2 ml) and stored at 70^oC until use. Protein concentration was determined by the method of Lowry et al. (1951), using bovine serum albumin as a standard. Minor modifications were incorporated into the assay that improve the range of linearity with protein concentration (Stauffer, 1975). Additional human liver microsomes, characterized for P450 isoform catalytic activities, were purchased from International Institute for the Advancement of Medicine (Exton, PA). Microsomes from insect cells engineered to express single human P450 isoforms were purchased from Gentest, Inc. (Woburn, MA).

Incubation of Microsomes with [¹⁴C]Methoxychlor or [¹⁴C]Mono-OH-M. Incubations with human and rat liver microsomes were conducted in a 100- or 250-µl volume in a shaking water bath at 37°C. The assay mixture contained the following components at the concentrations indicated: sodium phosphate (70 mM, pH 7.4), MgCl₂ (10 mM), EDTA (1 mM), microsomal protein (0.05-1.0 mg/ml), KCl (30 mM), and substrate (usually 1 or 25 µM) delivered in ethanol (1% v/v). After a 2-min preincubation period at 37°C, the reaction was initiated with an NADPH-regenerating system [glucose 6-phosphate (10 mM), glucose-6-phosphate dehydrogenase (2.0 units/ml), and NADPH (0.5 mM)]. Control incubations were conducted in the absence of the cofactor NADPH. The reaction was terminated at various times by the addition of 10 volumes of ice-cold ethanol. After centrifugation, the supernatant was transferred to borosilicate tubes and evaporated under nitrogen gas. The residue was dissolved in 50 µl of ethanol, and a portion was applied to a TLC plate (normal phase, 9:1 chloroform:acetone or reversed phase TLC (C₁₈ TLC) in a solvent system of methanol:water:acetic acid (75:24:1, v/v) (10). To obtain sufficient [¹⁴C]mono-OH-M for use as a substrate, 10 incubations that contained human liver microsomes (1 mg/ml), [¹⁴C]methoxychlor (53 µM) and an NADPH-regenerating system in a final volume of 5 ml were conducted for 2 hr at 37°C. Additional components were similar to that described above. After addition of ethanol to terminate the reaction and centrifugation, the supernatants were combined, evaporated to dryness, and resuspended in a small volume of ethanol. After TLC analysis, the zone containing the [¹⁴C]mono-OH-M (verified by co-migration of authentic [³H]mono-OH-M) was recovered by scraping the plate followed by ethanol elution. Incubations with insect cell microsomes containing cDNA-expressed P450s were conducted in a similar manner, except that samples were not agitated during the incubation period.

Incubations With Heterologously Expressed Purified P450s. Purified CYP2C9, 2C18, 2C19, 2D6, NADPH cytochrome P450 reductase, and cytochrome b₅, expressed in Escherichia coli, were obtained from Drs. Toby Richardson and Eric Johnson (The Scripps Research Institute, La Jolla, CA). Components, added in order, were 10 pmol P450, 0.2 units NADPH-P450 reductase, 40 pmol cytochrome b₅ (when present), and 1.5 µg L--dilauroyl-phosphatidylcholine, sonicated in H₂O. When cytochrome b₅ was omitted from the reaction, buffer only was used in its place. These components were allowed to incubate on ice for 25 min before the addition of sodium phosphate buffer, MgCl₂, KCl, and substrate. The samples were then incubated at 37°C for 2 min, before initiating the reaction by the addition of an NADPH regenerating system consisting of glucose 6-phosphate (2.5 µmol), NADPH (0.125 µmol), and glucose-6-phosphate dehydrogenase (0.5 IU) in 25 µl H₂O. The final volume of the mixture was 250 µl.

Chemical Inhibition Experiments. Incubations with human P450 isoform-selective inhibitors were conducted in a manner similar to that described above. The concentrations of inhibitors and conditions used to maximize selectivity were based on the data of Newton et al. (1995) or Chauret et al. (1997). Inhibitors and the concentrations used were furafylline (20 µM), sulfaphenazole (100 µM), quinidine (10 µM), TAO (50 µM), tranylcypromine (50 µM), -naphthoflavone (2.5 µM), DDTC (30 µM), S-(+)-mephenytoin (500 µM), tolbutamide (500 µM), coumarin (200 µM), and ketoconazole (1 µM). All inhibitors were dissolved in ethanol, and the final concentration of ethanol in all incubations was 1%. Two inhibition protocols were followed. With TAO, DDTC, and furafylline, all components except methoxychlor were incubated for 15 min at 37°C. After the preincubation period, methoxychlor was added and the incubation was continued for 10 min before being terminated by the addition of ethanol. With all other inhibitors, the preincubation period was omitted.

Regression Analysis. Univariate or stepwise multiple linear regression analysis was performed to compare methoxychlor mono-O-demethylase activity (dependent variable) with one or more P450 isoform specific marker activities [independent variable(s)] in panels of human liver microsomes using Instat Version 3 (GraphPad Software, Inc. San Diego, CA). Analysis of variance was used to determine whether the model was statistically significant (e.g. p values of 0.05). Michaelis-Menten parameters were determined from nonlinear, least squares regression analysis using the equation for a single-enzyme model with CYP1A2 or CYP2C19 catalysis, v = (V_max[S])/(K_m + [S]), or with human liver microsomes, using the equation for a two-enzyme model, v = [(V_max1[S])/(K_m1 + [S])]/[(V_max2[S])/(K_m2 + [S])] (Origin Version 5.0, Microcal Software, Inc., Northampton, MA).

	Results

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Demethylation of Methoxychlor. Liver microsomes pooled from 3 individuals were incubated with saturating levels of methoxychlor (25 µM). A single major metabolite, the mono-O-demethylated derivative is observed (fig. 2). Formation of mono-OH-M was proportional to protein concentration up to 2 mg/ml (fig. 3) and with time up to 20 min (fig. 4). With longer incubation times, lesser products of secondary and tertiary metabolism are detected, including bis-OH-M and tris-OH-M (fig. 4). Low level catalysis to additional ring-hydroxylated metabolites (Stresser and Kupfer, 1998) and a metabolite(s) that binds covalently to liver microsomal protein have also been demonstrated previously (Bulger and Kupfer, 1989). Similarly, incubation of 25 µM methoxychlor for 10 min with microsomes from four different human livers yielded mono-OH-M, whereas bis-OH-M, tris-OH-M, or other metabolites were not detected or was produced only in small amounts (table 1). In contrast, rat liver microsomes are efficient in catalyzing methoxychlor conversion to both mono-OH-M and bis-OH-M (table 1). Accordingly, for three of the four human liver samples shown in table 1, longer incubation times of 60 min were necessary to generate quantifiable bis-OH-M when mono-OH-M was used as the substrate. With rat liver microsomes, nearly complete conversion of mono-OH-M to bis-OH-M was observed. The mono-demethylase activity was assessed in a panel of 26 samples of human liver microsomes overall and was found to vary 23-fold (data not shown), ranging from 91 to 2056 pmol/min/mg protein (mean ± SD, 354 ± 392; median, 304). To assess metabolism at concentration that might be more relevant to environmental exposures, incubations were also conducted at 1 µM methoxychlor using livers from 14 individuals. Rates of mono-O-demethylation varied 80-fold (data not shown), ranging from 14 to 1130 pmol/min/mg protein (mean ± SD, 169 ± 283; median, 101).

View larger version (14K): [in this window] [in a new window]   Fig. 2.   Radioscan of TLC of aliquot from an incubation of pooled human liver microsomes, N = 3 livers (1.0 mg/ml, 4-min incubation) with 1.75 µM [14C]methoxychlor (solid line) or of authentic standards of bis-OH-M, mono-OH-M and methoxychlor (dotted lines), with migration distances of 9.2, 14.0, and 15.6 cm, respectively. The origin was at 3.7 cm, and the solvent front was at 18 cm.

View larger version (16K): [in this window] [in a new window]   Fig. 3.   Effect of human liver microsomal protein concentration on the formation of mono-OH-M. Each value represents the mean of duplicate incubations. Substrate (methoxychlor) concentration was 25 µM. Incubation times were 10 min for the protein concentrations of 0.1 mg/1.0 ml to 0.75 mg/ml and 5 min for 1.0 mg/ml or higher (initial rate conditions).

View larger version (22K): [in this window] [in a new window]   Fig. 4.   Time course of the formation of mono-OH-M, bis-OH-M, tris-OH-M, and total metabolism (measured by disappearance of parent methoxychlor) by pooled human liver microsomes (N = 3 livers). The protein concentration was 0.87 mg/ml, and the substrate concentration was 25 µM.

Effect of Chemical Inhibitors. The effect of chemical inhibitors on human liver mono-O-demethylation of methoxychlor at two different substrate concentrations is shown in fig. 5A (gray bars, 1 µM methoxychlor; black bars, 25 µM methoxychlor). Only moderate inhibition was observed, by several inhibitors using 25 µM methoxychlor. Tranylcypromine, an inhibitor of CYP2C19-catalyzed S-(+)-mephenytoin 4'-hydroxylase (Inaba et al., 1985) and CYP2A6-catalyzed 7-hydroxylation of coumarin (Draper et al., 1997), inhibited demethylation to the greatest extent, followed by furafylline and sulfaphenazole. At 1 µM substrate, strong inhibition was observed by CYP2C19 inhibitors tranylcypromine and S-mephenytoin; tolbutamide, furafylline, TAO, sulfaphenazole, and coumarin exhibited moderate inhibition. At both substrate concentrations, quinidine, ketoconazole, and DDTC were found, overall, to be weak inhibitors. These data suggested that multiple isoforms could catalyze mono-O-demethylation of methoxychlor. To explore this possibility in greater detail, we examined the effect of selected inhibitors on individual preparations of human liver microsomes that exhibited varied isoform-specific catalytic activity (fig. 5B). As expected, inhibitor effects varied substantially, depending on the liver, confirming multiple isoform involvement in methoxychlor demethylation.

View larger version (42K): [in this window] [in a new window]   Fig. 5.   Effect of P450 isoform-selective inhibitors on methoxychlor O-demethylation by human liver microsomes. Data are expressed as percentage of inhibition compared with a control incubation containing the ethanol vehicle only (concentration of ethanol = 1%) and represent the mean of two to four determinations. A, effect of inhibitors on the rate of methoxychlor O-demethylation catalyzed by liver microsomes pooled from three individuals. Rates were determined using 25 µM methoxychlor (black bars) or 1 µM methoxychlor (gray bars). The range or standard error was 10% of the mean. The uninhibited activities, expressed as pmol/min/mg of protein were 157 and 170 for mechanism-based and competitive inhibitors, respectively. B, effect of inhibitors on the rate of methoxychlor O-demethylation (1 µM methoxychlor) catalyzed by liver microsomes from five individuals. The range or standard error was 9% of the means. The uninhibited rates, expressed as pmol/min/mg protein were 1189 (H225), 167 (H051), 172 (H205), 24 (H140), and 25 (H094). The concentrations of the inhibitors used were 100 µM (sulfaphenazole), 2.5 µM (ANF, or -naphthoflavone), 200 µM (coumarin), and 500 µM (S-(+)-mephenytoin).

Correlation Studies with P450 Isoform-Specific Catalytic Activities. Methoxychlor mono-demethylase activity was measured in a panel of 13 (using 1 µM methoxychlor) or 16 (using 25 µM methoxychlor) human liver microsomal preparations that had been characterized previously by the vendor for several P450-isoform selective activities (table 2). With 1 µM methoxychlor, mono-demethylation correlated significantly with phenacetin O-deethylase (R² = 0.54), a marker of CYP1A2 activity (Distlerath et al., 1985) and S-(+)-mephenytoin 4'-hydroxylase activity (R² = 0.79), a marker of CYP2C19 content (Wrighton et al., 1993; Goldstein et al., 1994). Similar, but slightly less significant, correlations were observed using 25 µM substrate concentration. Additionally, at this concentration of methoxychlor, mono-demethylation correlated significantly with total P450 content of the microsomes. Stepwise multiple regression analysis indicated that, in addition to CYP1A2 and 2C19 marker activities, CYP2A6-catalyzed 7-hydroxylation of coumarin (Yamano et al., 1990; Miles et al., 1990) could account for the variability in the data at 1 µM substrate concentration.

Studies With Recombinant P450 Isoforms. Insect cell microsomes containing various, singly expressed P450 isoforms were found to catalyze methoxychlor mono-O-demethylation (fig. 6). The most active isoform at 25 µM was CYP2C19, supporting our previous results with recombinant P450s expressed in lymphoblastoid cells (Stresser and Kupfer, 1998). Slightly less active were CYP1A2 and 2C18. Mono-OH-M was formed also by CYP2B6 and CYP2C9, but was not detected when CYP3A4, CYP3A5, or control cell microsomes lacking expressed P450 were used. Similar results were observed using 1 µM methoxychlor as substrate, although CYP2C18 was the most active at this concentration.

View larger version (17K): [in this window] [in a new window]   Fig. 6.   Mono-O-demethylation of methoxychlor by single, recombinant P450 isoforms in microsomes from insect cells. The concentration of P450 within incubations was 4 nM or 8 nM when 1 µM (gray bars) or 25 µM (black bars) substrate concentration was used. The incubation period was 10 min.

Kinetic Studies With Recombinant CYP2C19 and 1A2 and Human Liver Microsomes. Kinetic analysis of mono-demethylation was performed with microsomes from insect cells overexpressing CYP1A2 or CYP2C19 (fig. 7) and human liver microsomes (fig. 8). Nonlinear regression analysis revealed the data for CYP1A2 and CYP2C19 followed standard Michaelis-Menten kinetics for a one-enzyme model, whereas that for human liver microsomes fit a two-enzyme model. Kinetic parameters are shown in table 4. Both P450 isoforms exhibited similar apparent Michaelis-Menten constants of approximately 0.5-0.6 µM. CYP2C19 exhibited a 2-fold higher V_max and therefore displayed a greater value for V_max/K_M, a measure of in vitro intrinsic clearance (Rane et al., 1977). Human liver microsomes, pooled from the livers of three individuals, exhibited a low K_M component of 0.35 µM and a high K_M component of 12 µM. Despite relatively large errors associated with these parameters, the Eadie-Hofstee plot clearly shows involvement of more than one enzyme.

View larger version (19K): [in this window] [in a new window]   Fig. 7.   Effect of substrate concentration on the rate of mono-O-demethylation of methoxychlor catalyzed by CYP1A2 or CYP2C19 in microsomes from insect cells. The concentration of P450 in the reaction was 4 nM. Incubation periods were 2-5 min.

View larger version (19K): [in this window] [in a new window]   Fig. 8.   Eadie-Hofstee plot for the mono-O-demethylation of methoxychlor catalyzed by liver microsomes pooled from three human livers. The concentration of protein in the reaction was 0.5 mg/ml. Incubation periods were 2-5 min.

	Discussion

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The biotransformation of methoxychlor by animals has been investigated extensively (Chapin et al., 1997; Kishimoto and Nurihara, 1996; Dehal and Kupfer, 1994; Kishimoto et al., 1995; Bulger et al., 1985; Schlenk et al., 1997; Kapoor et al., 1970; Davison et al., 1982), but relatively few studies have been conducted with human tissues (Stresser and Kupfer, 1998; Bulger and Kupfer, 1989; Kupfer et al., 1990). Human liver microsomes catalyze primarily mono-O-demethylation of this insecticide. Secondary and tertiary metabolism occur during longer incubation periods, resulting in a second demethylation and ortho-hydroxylation. There is low level metabolism to other ortho-hydroxylated metabolites and this may occur without prior O-demethylation. In addition, methoxychlor undergoes activation to a reactive metabolite of unknown structure that binds covalently to microsomal protein (Bulger and Kupfer, 1989). The estrogenicity of methoxychlor mono- and bis-phenolic metabolites has been established (Bulger et al., 1978, 1985; Ousterhout et al., 1981), but whether catechol or other metabolites exhibit estrogenic or anti-estrogenic effects has not yet been determined. The estrogenic action of methoxychlor in animals has prompted concern that human exposure to methoxychlor may elicit endocrine disrupting effects (Chapin et al., 1997; Cummings, 1997).

In this study, we have characterized the human P450 isoforms that are the primary catalysts in the major, initial metabolic pathway, namely that leading to formation of mono-OH-M. An Eadie-Hofstee plot of methoxychlor mono-O-demethylation by human liver microsomes indicated biphasic enzyme kinetics, suggesting the involvement of multiple isoforms in this reaction. Indeed, using a panel of recombinant P450 isoforms expressed in E. coli or insect cells in the present study, and based on previous data with recombinant P450s expressed in lymphoblast cells (Stresser and Kupfer, 1998), multiple isoforms were shown capable of methoxychlor mono-O-demethylation. CYP2C18, CYP2C19, and CYP1A2 appear to be the most active in this catalysis, whereas CYP2C9 displayed variable activity and 2A6 and 2B6 exhibited overall lesser activity. CYP1A1, CYP1B1, CYP2D6, CYP2E1, CYP3A4, and CYP3A5 were found incapable of catalyzing demethylation of methoxychlor. Although CYP2C18 was the most active at low substrate concentration using the insect cell expressed enzyme, its expression in human liver is believed to be minuscule or absent (Jung et al., 1997), and consequently it is unlikely to be a significant methoxychlor demethylase in liver microsomes.

The primary involvement of CYP1A2 and CYP2C19 in demethylation of methoxychlor is supported by correlation analysis. Univariate regression analysis indicated a significant correlation of mono-demethylase activity with CYP2C19 catalyzed S-(+)-mephenytoin 4'-hydroxylase activity, and CYP1A2-catalyzed phenacetin O-deethylase activity, at both subsaturating (1 µM) and saturating (25 µM) methoxychlor concentrations. Stepwise multivariate regression analysis indicated that, in addition to CYP2C19 and CYP1A2 catalyzed marker activities, CYP2A6-catalyzed coumarin 7-hydroxylase activity significantly correlated with mono-demethylation using 1 µM methoxychlor.

Based on the above findings, we pursued further the role of CYP1A2 and CYP2C19 in mono-O-demethylation of methoxychlor by kinetic analysis. Both enzymes exhibited single-enzyme Michaelis-Menten kinetics and displayed similar apparent K_M values of 0.5-0.6 µM, consistent with the high affinity component observed in human liver microsomes. CYP2C19 exhibited a V_max of more than 2-fold that calculated for CYP1A2. This is consistent with our previous findings where recombinant CYP2C19 expressed in lymphoblast cells was found to be significantly more active than similarly expressed CYP1A2 (Stresser and Kupfer, 1998) under saturating substrate conditions.

We demonstrated that cDNA-expressed CYP2C19, purified from E. coli lysates, catalyzed methoxychlor mono-demethylation at rates more than 4-fold over CYP2C9 and CYP2C18. The effects of cytochrome b₅ in an appropriately reconstituted system on rates of catalysis differed depending on the CYP2C isoform. Whereas cytochrome b₅ inhibited CYP2C9 mono-OH-M formation and CYP2C19 bis-OH-M formation, it stimulated production of both metabolites by CYP2C18. The differential effects of cytochrome b₅ on CYP2C isoforms are consistent with previous studies by others (Rodrigues et al., 1996; Raucy et al., 1994).

Inhibition by chemicals reported to exhibit selectivity for certain P450 isoforms are supportive of a role for CYP2C19 (tranylcypromine, S-mephenytoin, tolbutamide²), CYP1A2 (-naphthoflavone, furafylline), CYP2A6 (tranylcypromine, coumarin) and CYP2C9 (sulfaphenazole, tolbutamide) in the mono-demethylation of methoxychlor in human liver. Lack of potent inhibition by any inhibitor in these experiments is consistent with the conclusion that multiple isoforms are involved. As expected, most competitive inhibitors exhibited greater inhibition at subsaturating concentrations of methoxychlor. When individual liver microsomal preparations were examined for their catalytic activity in the presence of selected inhibitors, variable inhibition of methoxychlor mono-O-demethylation was observed, a finding that probably depended on the content of a particular P450 isoform in a given liver. The highly variable and idiosyncratic nature of P450 isoform expression is well documented in human livers (Shimada et al., 1994).

A second demethylation of methoxychlor also occurs, forming bis-OH-M, a more potent estrogen than mono-OH-M (Ousterhout et al., 1981; Bulger et al., 1985). Unlike rats, humans are inefficient at forming bis-OH-M in vitro, and lengthy incubation times are usually required to detect this metabolite. The identity of the human P450s responsible for this reaction has not been fully characterized, although purified and reconstituted CYP2C18 and CYP2C19 clearly can catalyze this reaction. CYP2B6 and CYP1A2 catalyze the ortho-hydroxylation of methoxychlor (Stresser and Kupfer, 1997), whereas CYP3A4 is most active in catalyzing ortho-hydroxylation of mono- or bis-demethylated methoxychlor (Stresser and Kupfer, 1997, 1998).

	Conclusions

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We have found that multiple P450 isoforms contribute to methoxychlor metabolism in human liver microsomes. The sum of the evidence indicates that polymorphically expressed CYP2C19 is the major methoxychlor mono-demethylase, but may be only slightly more involved than CYP1A2. In those individuals lacking or expressing only low levels of CYP2C19, other isoforms, in particular CYP1A2, but also CYP2A6, CYP2C9, and CYP2B6, are likely to be major contributors to this reaction. Because methoxychlor exhibits adverse developmental and reproductive effects in various animal model systems that have been attributed to its estrogenic phenolic metabolites, exposure to this insecticide may pose similar health risks in humans, the magnitude of which may be related to P450 isoform expression.