Validation of cytochrome P450 time-dependent inhibition assays: a two-time point IC50 shift approach facilitates kinact assay design.

1. Recent guidance from the US Food and Drug Administration (USFDA) has advocated testing of time-dependent inhibition of cytochrome P450 (CYP), which can be addressed by performing IC(50) shift as well as K(I)/k(inact) determinations. 2. Direct (IC(50), K(i)) and time-dependent inhibition (IC(50) shift, K(I)/k(inact)) assays were validated in human liver microsomes with liquid chromatography-tandem mass spectrometry (LC/MS/MS) analysis for the following enzyme/substrate/inhibitor combinations: CYP1A2/phenacetin/alpha-naphthoflavone/furafylline, CYP2C8/amodiaquine/montelukast/gemfibrozil-1-O-beta-glucuronide, CYP2C9/diclofenac/sulfaphenazole/tienilic acid, CYP2C19/S-mephenytoin/S-benzylnirvanol/S-fluoxetine, CYP2D6/dextromethorphan/quinidine/paroxetine, and CYP3A4/midazolam/testosterone/ketoconazole/azamulin/verapamil/diltiazem. IC(50) shift assays were performed with two pre-incubation time points (10 and 30 min) to facilitate k(inact) assay design. 3. Data obtained show good agreement with literature values. For rapid acting inhibitors, such as azamulin/CYP3A4 and tienilic acid/CYP2C9, the IC(50) shifts were similar at both time points suggesting a short maximum pre-incubation time with closely spaced time points for the K(I)/k(inact) assay. Slow acting inhibitors (such as verapamil/CYP3A4 or S-fluoxetine/CYP2C19) showed an increase in IC(50) shift between 10 and 30 min suggesting a longer maximum pre-incubation time with wider spacing of time points for K(I)/k(inact). 4. The two-time point IC(50) shift experiment proved to be an excellent method for the selection of appropriate K(I)/k(inact) assay parameters and is suitable for the routine analysis of P450 inhibition by drug candidates.

CYP2D6 catalyzes tamoxifen 4-hydroxylation in human liver.

The major metabolites of tamoxifen (tam) formed by animal and human liver microsomes are mono-N-demethylated tam, 4-hydroxy-tam (4-OH-tam), and tam-N-oxide. The N-desmethylated-tam and 4-OH-tam are formed by P450s, whereas the N-oxide is primarily formed by flavin-containing monooxygenase. Because 4-OH-tam is a highly potent antiestrogen (and possibly is the active anticancer tam metabolite) and is on the path of formation of the reactive intermediate that binds covalently to proteins and DNA, it was of importance to identify the P450(s) catalyzing its formation. In the current study, three different preparations of expressed human P450s in Escherichia coli, lymphoblastoma cells, and insect cell line and livers from several human donors were used to identify the P450 isoform catalyzing the 4-hydroxylation (preliminary results were reported by Dehal et al., Eleventh International Symposium on Microsomes and Drug Oxidations, p. 71. Los Angeles, 1996). Tam metabolism was examined with human CYP2C8, 2C9, 2C18, 2C19, and 2D6 expressed in E. coli. These P450s were reconstituted with P450 reductase and lipid and were incubated with 50 microM [3H]tam and NADPH at 37 degrees C for 60 min. Essentially all of the recombinant P450s catalyzed the N-demethylation to various degrees; however, only 2D6 yielded detectable levels of 4-OH-tam. The inclusion of cytochrome b5 in the reconstituted system of 2D6 and 2C9 did not significantly affect the rate of 4-hydroxylation, indicating that b5 is not essential for this activity. Tam metabolism by CYP1A1, 1A2, 2A6, 2B6, 2C8, 2C9, 2D6, 2E1, and 3A4, expressed in lymphoblastoma cells, revealed that only 2D6 significantly catalyzed the 4-hydroxylation. Tam metabolism by CYP2D6 coexpressed with P450 reductase in a baculovirus infected insect cell line ("supersomes") exhibited marked tam 4-hydroxylation. In an experiment with human liver microsomes, the inclusion of quinidine, a specific 2D6 inhibitor, resulted in approximately 50% inhibition of tam 4-hydroxylation without affecting N-demethylation. Polyclonal antibodies raised against 2D6 moderately inhibited (approximately 30%) the 4-hydroxylation in human liver microsomes. These results demonstrate a significant contribution by CYP2D6 to the catalysis of tam-4-hydroxylation by human liver.

Evidence that the catechol 3,4-Dihydroxytamoxifen is a proximate intermediate to the reactive species binding covalently to proteins.

Metabolism of tamoxifen by rat and human hepatic microsomal cytochrome P450s (CYPs) forms a reactive intermediate that irreversibly binds to microsomal proteins (C. Mani and D. Kupfer, Cancer Res., 51: 6052-6058, 1991.). The current study examines the nature of the tamoxifen metabolite that is proximate to the reactive intermediate(s). The rate of covalent binding of tamoxifen metabolites, tamoxifen N-oxide, N-desmethyltamoxifen, and tamoxifen N-oxide-epoxide was approximately equal to or less than that of tamoxifen. By contrast, covalent binding of 4-hydroxytamoxifen (4-OH-tam) was 3-5-fold higher than that of tamoxifen, indicating that among the metabolites examined, 4-OH-tam or its metabolite(s) is most proximate to the reactive intermediate(s). Incubation of 4-OH-tam with liver microsomes from PCN-treated rat yielded three detectable metabolites. One was identified as 4-OH-tam N-oxide via its facile reduction back to 4-OH-tam by titanium(III) chloride. Another metabolite of 4-OH-tam, assumed to be 3,4-dihydroxytamoxifen (3,4-di-OH-tam) catechol, was demonstrated by its monomethylation with [3H]S-adenosyl-L-methionine ([3H]SAM) in presence of endogenous catechol-O-methyltransferase. Monomethylated catechol from 4-OH-tam was formed at a 3-4-fold higher rate than from tamoxifen. It was reasoned that if the catechol is most proximate metabolite to the reactive intermediate, then its methylation would reduce the formation of the reactive intermediate and result in lower rate of covalent binding. In fact, addition of radioinert SAM to incubations of tamoxifen inhibited covalent binding by 17-23%. By contrast, inclusion of 1.0 mM S-adenosyl-L-homocysteine, a potent inhibitor of catechol-O-methyltransferase-mediated methylation of 3,4-di-OH-tam, essentially overcame the inhibition of the covalent binding by SAM. Additionally, ascorbic acid and glutathione, inhibitors of covalent binding of tamoxifen, produced an elevation of methylated catechol. These findings collectively indicate that 3,4-di-OH-tam is proximate to the ultimate reactive intermediate that results in covalent binding to microsomal proteins.

Steroidal alpha-methylene delta-lactones as potential antitumor agents.

Four novel steroidal alpha-methylene delta-lactones have been synthesized and shown to be active against human nasopharyngeal carcinoma (KB) cells in culture. The syntheses involved the use of known alpha-methylenation procedures. In addition, the lactone 6 was directly methylenated by reaction with CH2O/KOH or Et2NH/CH2O/Et2NH.HCl. The formation of a cysteine adduct (15) with the alpha-methylene lactone 10 is reported.

Partial purification and characterization of two sesquiterpene cyclases from sage (Salvia officinalis) which catalyze the respective conversion of farnesyl pyrophosphate to humulene and caryophyllene.

Humulene cyclase and caryophyllene cyclase, two enzymes which catalyze the cyclization of farnesyl pyrophosphate to the respective sesquiterpene olefins, have been partially purified from the supernatant fraction of a sage (Salvia officinalis) leaf epidermis extract and separated from each other by a combination of hydrophobic interaction, gel filtration, and ion-exchange chromatography. The molecular weight of both cyclases was estimated by gel filtration to be 57,000 and both cyclases exhibited a pH optimum of 6.5 and preferred Mg2+ (Km approximately 1.5 mM) as the required divalent metal cation. Both enzymes possessed a Km of about 1.7 microM for farnesyl pyrophosphate, were strongly inhibited by p-hydroxymercuribenzoate, and exhibited comparable sensitivities to a variety of other potential inhibitors. The properties of the two sesquiterpene olefin cyclases, which are the first from a higher plant source to be examined in detail, were very similar to each other and to other monoterpene, sesquiterpene, and diterpene cyclases previously described.

Metabolism of monoterpenes: specificity of the dehydrogenases responsible for the biosynthesis of camphor, 3-thujone, and 3-isothujone.

Sage (Salvia officinalis) is shown to contain two electrophoretically distinct dehydrogenases for the respective oxidations of (+)-borneol to (+)-camphor, and of (+)-cis-sabinol to (+)-sabinone en route to (-)-3-isothujone. Similarly, tansy (Tanacetum vulgare) is shown to contain two electrophoretically distinct dehydrogenases for the respective oxidations of (-)-borneol to (-)-camphor and of (+)-cis-sabinol to (+)-sabinone en route to (+)-3-thujone. These results demonstrate that separate dehydrogenases are responsible for the biosynthesis of camphor from borneol and of the thujyl ketones via cis-sabinol, and they also indicate that the previously reported oxidations of various thujanols by the borneol dehydrogenases are only coincidental activities not relevant to the formation of 3-thujone and 3-isothujone.

Metabolism of the proestrogenic pesticide methoxychlor by hepatic P450 monooxygenases in rats and humans. Dual pathways involving novel ortho ring-hydroxylation by CYP2B.

Previous studies demonstrated that methoxychlor [1,1,1-trichloro-2,2-bis-(4-methoxyphenyl)ethane] is a proestrogen and is toxic to mammalian reproductive processes. Mammalian liver microsomes sequentially demethylate methoxychlor (I), yielding two estrogenic metabolites, mono-OH-M (II) and bis-OH-M (III). Liver microsomes from phenobarbital (PB)-treated rats (PB microsomes) additionally formed a catechol product, tris-OH-M (VII) (Kupfer et al., Chem. Res. Toxicol. 3, 8-16, 1990). This study shows that, in addition to compounds II, III and VII, male and female rat PB microsomes catalyze the formation of a novel ring-hydroxylated methoxychlor metabolite, ring-OH-M (IV). Liver microsomes from male rats treated with pregnenolone-16 alpha-carbonitrile formed the same metabolites as PB microsomes, but the ring-OH-M was formed only in minute amounts, and there was no tris-OH-M. Liver microsomes from methylchlolanthrene-treated and control male rats demethylated methoxychlor, but did not form ring-hydroxylated products. Similarly, human liver microsomes exhibited demethylation but not ring-hydroxylation. Incubation of mono-OH-M (II) with control rat liver microsomes yielded only bis-OH-M (III), whereas incubation of ring-OH-M (IV) resulted in monodemethylated (dihydroxy) compounds V/VI and didemethylated ring-hydroxylated compound, tris-OH-M (VII). Incubation of (IV) with PB microsomes led to compounds V and/or VI and tris-OH-M (VII), whereas incubation of mono-OH-M (II) yielded bis-OH-M (III) and tris-OH-M (VII). The evidence indicates that ring-hydroxylation is catalyzed by CYP2B: a) induction of CYP2B was required for ring-hydroxylation; b) antibodies against CYP2B1/2 strongly inhibited the formation of the ring-hydroxylated products by PB microsomes; c) incubation of methoxychlor with reconstituted CYP2B1 yielded both the hydroxylated (IV and VII) and the demethylated (II and III) metabolites; and d) reconstituted CYP2B1 converted mono-OH-M into bis-OH-M and tris-OH-M, whereas bis-OH-M was converted into tris-OH-M. Human CYP2B6 exhibits ring-hydroxylation, indicating that this reaction is catalyzed by several CYP2B isozymes. In addition, this study demonstrates that the formation of the catechol tris-OH-M involves two metabolic pathways: via O-demethylation followed by ring-hydroxylation and via ring-hydroxylation and subsequent O-demethylation.

Cytochrome P-450 3A and 2D6 catalyze ortho hydroxylation of 4-hydroxytamoxifen and 3-hydroxytamoxifen (droloxifene) yielding tamoxifen catechol: involvement of catechols in covalent binding to hepatic proteins.

Earlier study suggested that 3,4-dihydroxytamoxifen (tam catechol), a tamoxifen metabolite, is proximate to the reactive intermediate that binds covalently to proteins and possibly to DNA (). The current study demonstrates that rat and human hepatic cytochrome P-450s (CYPs) catalyze tam catechol formation from tamoxifen (tam), 3-hydroxy-tam (Droloxifene), and 4-hydroxy-tam (4-OH-tam). Higher levels of catechol were formed from 4-OH-tam and 3-hydroxy-tam than from tam. Evidence that human hepatic CYP3A4 and 2D6 catalyze the formation of tam catechol from 4-OH-tam and supportive data that the catechol is proximate to the reactive intermediate, was obtained: 1) There was a good correlation (r = 0.82; p

Metabolism of Monoterpenes : Metabolic Fate of (+)-Camphor in Sage (Salvia officinalis).

The bicyclic monoterpene ketone (+)-camphor undergoes lactonization to 1,2-campholide in mature sage (Salvia officinalis L.) leaves followed by conversion to the beta-d-glucoside-6-O-glucose ester of the corresponding hydroxy acid (1-carboxymethyl-3-hydroxy-2,2,3-trimethyl cyclopentane). Analysis of the disposition of (+)-[G-(3)H]camphor applied to midstem leaves of intact flowering plants allowed the kinetics of synthesis of the bis-glucose derivative and its transport from leaf to root to be determined, and gave strong indication that the transport derivative was subsequently metabolized in the root. Root extracts were shown to possess beta-glucosidase and acyl glucose esterase activities, and studies with (+)-1,2[U-(14)C]campholide as substrate, using excised root segments, revealed that the terpenoid was converted to lipid materials. Localization studies confirmed the radiolabeled lipids to reside in the membranous fractions of root extracts, and analysis of this material indicated the presence of labeled phytosterols and labeled fatty acids (C(14) to C(20)) of acyl lipids. Although it was not possible to detail the metabolic steps between 1,2-campholide and the acyl lipids and phytosterols derived therefrom because of the lack of readily detectable intermediates, it seemed likely that the monoterpene lactone was degraded to acetyl CoA which was reincorporated into root membrane components via standard acyl lipid and isoprenoid biosynthetic pathways. Monoterpene catabolism thus appears to represent a salvage mechanism for recycling mobile carbon from senescing oil glands on the leaves to the roots.

Induction of the hepatic CYP2B and CYP3A enzymes by the proestrogenic pesticide methoxychlor and by DDT in the rat. Effects on methoxychlor metabolism.

In earlier investigations, methoxychlor treatment did not elicit induction of hepatic P450 monooxygenases in rats, apparently due to the short half-life of methoxychlor in vivo. The current study demonstrates that multiple bidaily doses of methoxychlor to female rats produce a marked induction of the hepatic microsomal P450 2B1/2B2 and 3A proteins. There was no increase in CYP1A1 or CYP2E1 proteins, demonstrating selectivity of induction by methoxychlor. Similarly, treatment with DDT, a methoxychlor analog, increased CYP2B and 3A proteins but had no effect on CYP1A1 and 2E1. Methoxychlor moderately elevated the enzymatic activity corresponding to CYP2B and 3A catalysis. In immature rats, only the higher dose of methoxychlor (300 mg/kg), produced elevation of testosterone hydroxylation at the 16 alpha position (major product) that was statistically significant, indicative of increased catalysis by CYP2B1/2B2. Both the low (150 mg/kg) and high dose (300 mg/kg) of methoxychlor increased the 6 beta hydroxylation (major product) and 2 beta and 15 beta hydroxylation (minor products) of testosterone, indicative of increased catalysis by CYP3A. In mature female rats, both methoxychlor and DDT treatment elevated the 16 alpha and 6 beta hydroxylation and androstenedione formation. Additional indication of methoxychlor- and DDT-mediated induction of CYP2B enzymatic activity in mature and immature rats was evident from increased ring hydroxylation of methoxychlor, an activity attributed to CYP2B. These findings indicate that methoxychlor and DDT belong to the phenobarbital type of inducers and that exposure to methoxychlor can affect its own metabolism. The methoxychlor-mediated increase in CYP2B and 3A proteins was considerably larger than the increase in the corresponding enzymatic activities. The possible reasons for the lack of correlation between P450 levels and their enzymatic activities and the potential relevance of induction by methoxychlor to its metabolism and toxicity are discussed.

Ring hydroxylation of [o-3H]methoxychlor as a probe for liver microsomal CYP2B activity: potential for in vivo CYP2B assay.

An in vitro radiometric assay selective for inducible CYP2B activity is described. The assay is based on the quantification of 3H2O release that occurs during o-ring hydroxylation of [o-3H]methoxychlor by liver microsomes in the presence of NADPH. 3H2O is isolated by removing > 99.9% of the parent compound and organic metabolites by facile charcoal extraction and filtration. There was no evidence for an NIH shift during ring hydroxylation, and there was little or no isotope effect. Selectivity for CYP2B was demonstrated using liver microsomes prepared from rats and mice treated with inducers of different CYP isoforms. Ring hydroxylation of [o-3H]methoxychlor was elevated 11.4-fold over control values in liver microsomes from male rats treated with phenobarbital. With mice, phenobarbital treatment elevated liver microsomal ring hydroxylation 7.1-fold. Clofibrate, 3-methylcholanthrene, or beta-naphthoflavone treatment of male rats or pyridine treatment of female rats did not elevate liver microsomal ring-hydroxylase activity, indicating that CYP4A, 1A, and 2E1 do not support this reaction. In female rats, dexamethasone and pregnenolone-16 alpha-carbonitrile treatment elevated ring hydroxylation up to 5.5- and 3.2-fold, respectively, an activity that may be attributed to CYP2B induction in those animals. Incubation of liver microsomes from phenobarbital-treated males with monospecific anti-CYP2B monoclonal antibodies (Mab) inhibited ring-hydroxylase activity up to 86%, demonstrating predominantly CYP2B-mediated catalysis. An 86% inhibition by these Mabs was also observed using liver microsomes from male mice treated with phenobarbital, indicating the assay is not limited to rats. The CYP2B mechanism-based inhibitor orphenadrine caused a 76% decline in activity, providing further evidence for CYP2B involvement. Unlike other CYP2B-selective assays, this method may be readily adapted to in vivo studies, by measuring urinary excretion of 3H2O as an indication of total body CYP2B activity.

The aromatase inactivator 4-hydroxyandrostenedione (4-OH-A) inhibits tamoxifen metabolism by rat hepatic cytochrome P-450 3A: potential for drug-drug interaction of tamoxifen and 4-OH-A in combined anti-breast cancer therapy.

Tamoxifen (tam), an anti-breast cancer agent, is metabolized into tam-N-oxide by the hepatic flavin-containing monooxygenase and into N-desmethyl- and 4-hydroxy-tam by cytochrome P-450s (CYPs). Additionally, tam is metabolically activated by hepatic CYP3A, forming a reactive intermediate that binds covalently to proteins. Tam and 4-hydroxyandrostenedione (4-OH-A) are currently used to treat breast cancer, and it has been contemplated that 4-OH-A be given concurrently with tam to contravene potential tumor resistance to tam. Because alterations in tam metabolism may influence its therapeutic efficacy, the effect of 4-OH-A on tam metabolism was examined. Incubation of tam with liver microsomes from phenobarbital-treated rats, in the presence of 4-OH-A (10-100 microM), resulted in marked inhibition of tam-N-demethylation and tam covalent binding and in decreased tam-N-oxide accumulation; however, there was no inhibition of the formation of 4-hydroxy-tam and of 3,4-dihydroxytamoxifen. These findings indicate that 4-OH-A inhibits CYP3A, but not P-450(s) that catalyze tam 4-hydroxylation. The diminished tam-N-oxide accumulation could be due to decreased N-oxide formation and/or due to increased N-oxide reduction. Incubation of tam-N-oxide with liver microsomes containing heat-inactivated flavin-containing monooxygenase demonstrated that 4-OH-A increases the accumulation of tam, possibly by diminishing its P-450-mediated metabolism. Kinetic studies indicate that 4-OH-A is a competitive inhibitor of CYP3A, but not a time-dependent inactivator. Consequently, the concurrent treatment of tam and 4-OH-A may result in increased tam half-life and thus could potentiate the therapeutic efficacy of tam and diminish the potential side effects of tam by inhibiting its covalent binding to proteins and possibly to DNA.

Concurrent flavin-containing monooxygenase down-regulation and cytochrome P-450 induction by dietary indoles in rat: implications for drug-drug interaction.

Our laboratory has previously shown that dietary administration of indole-3-carbinol (I3C) to male Fischer 344 rats has the very unusual property of inducing hepatic levels of a number of cytochrome P450s (CYPs), especially CYP1A1, while markedly inhibiting the levels of flavin-containing monooxygenase (FMO) 1 protein and its catalytic activity. We hypothesized that rats fed I3C or 3,3'-diindolylmethane (DIM), one of its major acid condensation products formed in vivo, should exhibit a marked shift in the metabolic profiles of drugs or xenobiotics that are substrates for both monooxygenase systems. Male rats were fed AIN-76A powdered diets containing 0, 1000, or 2500 ppm I3C or DIM for 4 weeks. Dietary I3C and DIM reduced FMO1 protein levels (8% reduction with I3C and 84% with DIM at 1000 ppm, and 90% reduction with I3C and 97% with DIM at 2500 ppm) in hepatic microsomes. The ratio of FMO (N-oxygenation)- to CYP (N-demethylation)-mediated metabolism of N,N-dimethylaniline decreased in liver microsomes from I3C- or DIM-fed rats from near unity to 0.02 at the highest dietary doses. FMO-mediated N-oxygenation (nicotine N-1'-oxide) was decreased, whereas CYP-mediated (nornicotine and nicotine delta (1,5)-iminium ion) metabolism of nicotine was unchanged in liver microsomes from rats fed I3C or DIM. Similarly, the ratio of FMO to CYP metabolites of tamoxifen decreased due to a reduction in N-oxygenation. This study demonstrates alteration of FMO- and CYP-mediated drug metabolism in vitro by dietary I3C or DIM and suggests the potential for altered toxicity of tamoxifen and nicotine in vivo.

CYP2D6-mediated catalysis of tamoxifen aromatic hydroxylation with an NIH shift: similar hydroxylation mechanism in chicken, rat and human liver microsomes.

1. 4-Tritiated-tamoxifen (4-[(3)H]-tamoxifen) and 4-deuterated-tamoxifen (4-[(2)H]-tamoxifen) were synthesized to examine tamoxifen metabolism by human P450 (CYP) forms and also for the possibility of determining tamoxifen-4-hydroxylation in humans in vivo. 2. Liver microsomes from several species and cDNA-expressed human P450s were incubated with 4-[(3)H]-tamoxifen and the reaction monitored by assaying 4-hydroxytamoxifen (4-OH-tam) and (3)H(2)O formed. However, tamoxifen-4-hydroxylation did not generate stoichiometric amounts of (3)H(2)O and the expected unlabelled 4-OH-tam but instead yielded radiolabelled 4-OH-tam, apparently from [(3)H]-migration to the ortho-position, referred to as the NIH shift. 3. CYP2D6 was the prime catalyst of tam-4-hydroxylation, whereas CYP2B6, 2C9 and 2C19 yielded only low levels of 4-OH-tam; nevertheless, in all cases the 4-OH-tam was radioactive, apparently resulting from reactions involving an NIH shift. 4. Chicken liver microsomal preparation, being catalytically the most active in tamoxifen-4-hydroxylation, was incubated with deuterated tamoxifen (4-[(2)H]-tamoxifen) in order to determine whether an NIH shift occurs. Ion-trap mass-spectrometry of the HPLC-purified 4-OH-tam, from that incubation, indicated about 60% of [(2)H]-retention in 4-OH-tam, signifying an NIH shift. These findings indicate that the aromatic hydroxylation of tamoxifen does not entail hydroxyl insertion with an Sn2-displacement of hydrogen or a hydrogen isotope ((2)H or (3)H), but apparently involves epoxidation followed by migration of the (3)H, (2)H or (1)H to the ortho-position, and dissociation of the (1)H in preference to (3)H or (2)H, i.e. retention of the hydrogen isotope appears to be related to the bond strengths: C-(3)H>C-(2)H>C-(1)H.

Flavin-containing monooxygenase isoform specificity for the N-oxidation of tamoxifen determined by product measurement and NADPH oxidation.

The Km value for tamoxifen is 1.2 mM for mouse FMO1 (human FMO1 is not expressed in adults) and 1.4 mM for human FMO3, with no detectable activity being expressed toward tamoxifen by FMO5 from either mouse or human. These data are derived from experiments using 3H-tamoxifen as substrate in which the product, tamoxifen N-oxide, was measured directly. It was not possible to derive meaningful data from the measurement of NADPH consumption because Escherichia coli preparations, in the presence of tamoxifen, regardless of whether the E. coli was expressing an FMO isoform, consumed large amounts of NADPH without the appearance of tamoxifen N-oxide or other discernable product.

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]; ring-OH-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-alpha agonist than mono-OH-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.

Interaction of methoxychlor and related compounds with estrogen receptor alpha and beta, and androgen receptor: structure-activity studies.

We previously demonstrated differential interactions of the methoxychlor metabolite 2,2-bis(p-hydroxyphenyl)-1,1, 1-trichloroethane (HPTE) with estrogen receptor alpha (ERalpha), ERbeta, and the androgen receptor (AR). In this study, we characterize the ERalpha, ERbeta, and AR activity of structurally related methoxychlor metabolites. Human hepatoma cells (HepG2) were transiently transfected with human ERalpha, ERbeta, and AR plus an appropriate steroid-responsive luciferase reporter vector. After transfection, cells were treated with various concentrations of HPTE or structurally related compounds in the presence (for detecting antagonism) and absence (for detecting agonism) of 17beta-estradiol and dihydrotestosterone. The monohydroxy analog of methoxychlor, as well as monohydroxy and dihydroxy analogs of 2, 2-bis(p-hydroxyphenyl)-1,1-dichloroethylene, had ERalpha agonist activity and ERbeta and AR antagonist activity similar to HPTE. The trihydroxy metabolite of methoxychlor displayed only weak ERalpha agonist activity and did not alter ERbeta or AR activities. Replacement of the trichloroethane or dichloroethylene group with a methyl group resulted in a compound with ERalpha and ERbeta agonist activity that retained antiandrogenic activities. This study identifies some of the structural requirements for ERalpha and ERbeta activity and demonstrates the complexity involved in determining the mechanism of action of endocrine-active chemicals that simultaneously act as agonists or antagonists through one or more hormone receptors.

Cytochrome P450 catalyzed covalent binding of methoxychlor to rat hepatic, microsomal iodothyronine 5'-monodeiodinase, type I: does exposure to methoxychlor disrupt thyroid hormone metabolism?

The insecticide methoxychlor is estrogenic in birds and mammals and interferes with sexual development and reproduction, but it is not known whether this toxicity is due solely to its estrogenicity. We now have found that during hepatic, microsomal metabolism of [ring-14C]- or [3H-OCH3]methoxychlor, their metabolite primarily binds to iodothyronine 5'-monodeiodinase, type I (5'-ID1). The purified, radiolabeled protein reacted with antibodies against protein disulfide isomerase, isoform Q5, which is highly homologous to 5'-ID1. Sequencing of the radiolabeled tryptic peptide indicated that methoxychlor bound to cysteine 372 or 375 or to lysine 376 of 5'-ID1. Treatment of rats with methoxychlor for 4 days decreased hepatic, microsomal 5'-ID1 activity from 2.94 to 2.20 nmol/min-mg prot (P < 0.02). Since 5'-ID1 catalyzes thyroxine conversion to the biologically active triiodothyronine, these data suggest that methoxychlor may interfere with thyroid hormone metabolism. This may be an additional factor in its environmental toxicity.