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PubMed1995

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.

Animals↗
PubMed1990

Studies on the formation of methoxychlor-protein adduct in rat and human liver microsomes. Is demethylation of methoxychlor essential for cytochrome P450 catalyzed covalent binding?

Previous studies demonstrated that liver microsomal monooxygenases metabolize the pesticide methoxychlor into phenolic estrogenic derivatives. Additionally, methoxychlor is activated by the hepatic cytochrome P450 monooxygenase to bind covalently to microsomal proteins (Bulger WH, Temple JE and Kupfer D, Toxicol Appl Pharmacol 68: 367-374, 1983). The current study examines, in liver microsomes from control and phenobarbital-treated rats and humans, whether demethylation of methoxychlor is essential for covalent binding and whether demethylated methoxychlor metabolites are on the pathway of formation of the reactive intermediate and protein adduct. Using 3H-methoxyl-labeled and 14C-ring-labeled methoxychlor, it was demonstrated that demethylation is not essential for covalent binding. Namely, the major portion of the methoxychlor moiety in the protein adduct was found to contain intact methoxyls. Nevertheless, in the absence of methoxychlor, both the mono- and bis-demethylated methoxychlor metabolites could undergo monooxygenase-mediated covalent binding to proteins. This was demonstrated in incubations of purified 14C-labeled mono- and bis-demethylated methoxychlor metabolites with liver microsomes, in the presence of NADPH. Additionally, the dehydrochlorinated metabolite of methoxychlor, containing a double bond, underwent covalent binding, which exhibited characteristics similar to those of methoxychlor. These findings demonstrated that the protein adduct from relatively brief incubation periods contains a methoxychlor derivative with intact methoxyls. The possibility that the activation of methoxychlor involves modification of the side chain, which is the active site that binds to proteins, is discussed.

Animals↗
PubMed2001

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.

Androstanes↗
PubMedDate not supplied

Interactions of methoxychlor, methoxychlor base-soluble contaminant, and 2,2-bis(p-hydroxyphenyl)-1,1,1-trichloroethane with rat uterine estrogen receptor.

Laboratory grade methoxychlor (99% pure), base-washed methoxychlor, and a metabolite of methoxychlor, 2,2-bis(p-hydroxyphenyl)-1,1,1-trichloroethane (HPTE), were tested for their ability to compete with [3 H] estradiol-17 beta ([3 H]E2) for specific binding to the estrogen receptor from immature rat uterine cytosol. The binding was determined on 10--30% sucrose gradients and by a dextran-coated charcoal assay and subsequent Scatchard plot analysis. On gradients, laboratory grade methoxychlor, but not base-washed methoxychlor, suppressed [3 H]E2 binding to the 8S estrogen receptor. However, the base-soluble fraction from washing of laboratory grade methoxychlor caused suppression o[3 H]E2 binding on sucrose gradients at a concentration as low as 3.6 ppm. Scatchard plot analysis indicated that the inhibition of binding observed with laboratory grade methoxychlor was competitive in nature and not caused by receptor destruction. It was concluded that laboratory grade methoxychlor contained a contaminant that was potentially estrogenic. HPTE, an in vivo metabolite of methoxychlor, caused a marked suppression of [3 H]E2 binding in the 85 region of the gradients. Analysis by Scatchard plot indicated that the effect of HPTE was not to decrease the number of E2 binding sites but merely to alter the affinity of binding to the receptor, presumably in a competitive manner. The low K1 value for HPTE suggested an extremely high affinity for uterine cytosolic E2 receptors.

Animals↗
PubMed1975

Residues of methoxychlor and other chlorinated hydrocarbons in water, sand, and selected fauna following injections of methoxychlor black fly larvicide into the Saskatchewan River, 1972.

In May 1972, 0.309 ppm methoxychlor black fly larvicide was applied in a single test on the North Saskatchewan River. Eight to nine days later residues of 0.05-0.10 ppm methoxychlor occurred in sand 21-22 km downstream from the point of injection. Methoxychlor was not detected in water, insect larvae, shellfish, or muscle tissues of three fish species on the same sampling date. Perhaps because of relatively high oil content in goldeye fish, methoxychlor residues in muscle tissues were 1.0-1.5 ppm in 8 percent of those sampled, 0.21-0.99 in 21 percent, and 0.02-0.20 in 37 percent. In 34 percent of the goldeye fish no residues were detected. Goldeye and other fish collected before or 17 weeks after this injection did not contain detectable levels of methoxychlor. River water in two samples of the injected slug of water collected 6.5 km downstream from the point of injection contained 0.14 and 0.16 ppm methoxychlor. The suspended solids filtered from these sample contained 40 and 47 percent of this methoxychlor (437 and 892 ppm, respectively). Thus methoxychlor may act selectively against filter-feeding species, especially black fly larvae.

Animals↗
PubMed2006

Demethylation of the pesticide methoxychlor in liver and intestine from untreated, methoxychlor-treated, and 3-methylcholanthrene-treated channel catfish (Ictalurus punctatus): evidence for roles of CYP1 and CYP3A family isozymes.

Exposure to the organochlorine pesticide methoxychlor (MXC) is associated with endocrine disruption in several species through biotransformation to mono-desmethyl-MXC (OH-MXC) and bis-desmethyl-MXC (HPTE), which interact with estrogen receptors. The biotransformation of [14C]methoxychlor was examined in channel catfish (Ictalurus punctatus), a freshwater species found in the southern United States. Hepatic microsomes formed OH-MXC and HPTE, assessed by comigration with authentic standards. The Km for OH-MXC formation by control liver microsomes was 3.8 +/- 1.3 microM (mean +/- S.D., n = 4), and Vmax was 131 +/- 53 pmol/min/mg protein. These values were similar to those of catfish pretreated with 2 mg/kg methoxychlor i.p. for 6 days (Km 3.3 +/- 0.8 microM and Vmax 99 +/- 17 pmol/min/mg) but less (p < 0.05) than the kinetic parameters for catfish treated with 3-methylcholanthrene (3-MC), which had Km of 6.0 +/- 1.1 microM and Vmax of 246 +/- 6 pmol/min/mg protein. Liver microsomes from 3-MC-treated fish produced significantly more of the secondary metabolite and more potent estrogen, HPTE. Intestinal microsomes formed OH-MXC at lower rates than liver. Methoxychlor pretreatment significantly reduced intestinal metabolite formation from 32 +/- 4 to 15 +/- 6 pmol/min/mg (mean +/- S.D., n = 4), whereas 3-MC treatment significantly increased OH-MXC production to 72 +/- 22 pmol/min/mg. Ketoconazole, clotrimazole, and alpha-naphthoflavone all decreased the production of OH-MXC in liver microsomes, whereas alpha-naphthoflavone stimulated HPTE formation, suggesting that CYP1 and CYP3 family isozymes demethylated methoxychlor. The results suggest that the formation of estrogenic metabolites from methoxychlor would be more rapid in catfish coexposed to CYP1 inducers.

Animals↗
PubMed1995

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.

Amino Acid Sequence↗
PubMed1997

Influence of beta-naphthoflavone and methoxychlor pretreatment on the biotransformation and estrogenic activity of methoxychlor in channel catfish (Ictalurus punctatus).

The organochlorine pesticide methoxychlor [1,1,1-trichloro-2,2-bis(4-methoxyphenyl) ethane] (MXC) has been classified as a proestrogen in mammals and fish, requiring demethylation prior to eliciting estrogenic activity or binding to the estrogen receptor. While microsomal demethylation occurs readily in the liver of fish, little is known about the enzyme(s) responsible or the effect of cytochrome P450 (CYP) inducers, other than those of CYP1A and CYP2K, on biotransformation. Consequently, male channel catfish were pretreated with MXC or beta-naphthoflavone (BNF), alone and in combination, to determine their effects on CYP protein expression, MXC biotransformation by hepatic microsomes, microsomal protein binding, and MXC estrogenic activity as determined by serum vitellogenin and 17beta-estradiol. Liver microsomes of both treated and untreated mature male catfish catalyzed formation of monodemethylated MXC, bisdemethylated MXC, as well as ring-hydroxylated metabolites. Pretreatment with BNF did not affect MXC metabolite profiles, overall rates of MXC biotransformation, or microsomal proteins recognized by anti-trout CYP2K1, but had the expected effect of inducing CYP1A and associated ethoxyresorufin O-deethylase activity. By contrast, pretreatment with MXC, alone or in combination with BNF, significantly reduced rates of MXC biotransformation and binding to liver microsomal protein. MXC/BNF cotreatment followed by MXC significantly induced serum vitellogenin, whereas MXC treatment alone led to a nonsignificant increase in vitellogenin and a significant increase in serum 17beta-estradiol. Thus, estrogenic activity elicited by cotreatment with MXC and BNF can occur despite diminished capacity of liver microsomes to catalyze formation of estrogenic demethylated metabolites or metabolites that bind microsomal protein. Possible mechanisms of MXC-induced attenuation of CYP-dependent metabolism are discussed.

Animals↗
PubMed2005

Comparative in vitro metabolism of methoxychlor in male and female rats: metabolism of demethylated methoxychlor metabolites by precision-cut rat liver slices.

The in vitro metabolism of demethylated methoxychlor (MXC) metabolites, mono-OH-MXC (including (R)- and (S)-isomers) and bis-OH-MXC (mono- and bis-demethylated MXC, respectively), was conducted using precision-cut liver slices to understand the sex-dependent metabolism of MXC in rats. In the study with bis-OH-MXC, the substrate underwent extensive conjugation producing its glucuronide and glucuronide/sulphate diconjugate, and no significant sex differences were found. On the contrary, the metabolism of mono-OH-MXC appeared to exhibit the sex differences in the metabolic profiles. The bis-OH-MXC glucuronide and glucuronide/sulphate diconjugate were major metabolites in male rat, whereas the mono- and bis-OH-MXC glucuronides predominated in the female. The per cent distribution of the demethylated products (sum of bis-OH-MXC derivatives) was approximately 90% for the male (for both isomers) and 81 (R-) to 56% (S-) for the female. The metabolic profiles in (S)-mono-OH-MXC, which is the predominant enantiomer preferentially produced in MXC metabolism in rats, showed a similar pattern to that of MXC compared with the (R)-isomer. The results indicate that the sex differences in oxidative demethylation of the intermediate, (S)-mono-OH-MXC, could be one of the probable reasons for the sex-dependent metabolism of MXC in rats, and the stereo-structural preference of the contributing demethylase enzymes appear to be involved.

Animals↗
PubMed1993

Reversible and time-dependent inhibition of the hepatic cytochrome P450 steroidal hydroxylases by the proestrogenic pesticide methoxychlor in rat and human.

Methoxychlor, a currently used pesticide, is demethylated and hydroxylated by several hepatic microsomal cytochrome P450 enzymes. Also, methoxychlor undergoes metabolic activation, yielding a reactive intermediate (M*) that binds irreversibly and apparently covalently to microsomal proteins. The study investigated whether methoxychlor could inhibit or inactivate certain liver microsomal P450 enzymes. The regioselective and stereoselective hydroxylation of testosterone and the 2-hydroxylation of estradiol (E2) were utilized as markers of the P450 enzymes inhibited by methoxychlor. Both reversible and time-dependent inhibition were examined. Coincubation of methoxychlor and testosterone with liver microsomes from phenobarbital treated (PB-microsomes) male rats, yielded marked diminution of 2 alpha- and 16 alpha-testosterone hydroxylation, indicating strong inhibition of P4502C11 (P450h). Methoxychlor moderately inhibited 2 beta-, 7 alpha-, 15 alpha-, 15 beta-, and 16 beta-hydroxylation and androstenedione formation. There was only a weak inhibition of 6 beta-hydroxylation of testosterone. The methoxychlor-mediated inhibition of 6 beta-hydroxylation was competitive. By contrast, when methoxychlor was permitted to be metabolized by PB-microsomes or by liver microsomes from pregnenolone-16 alpha-carbonitrile treated rats (PCN-microsomes) prior to addition of testosterone, a pronounced time-dependent inhibition of 6 beta-hydroxylation was observed, suggesting that methoxychlor inactivates the P450 3A isozyme(s). The di-demethylated methoxychlor (bis-OH-M) and the tris-hydroxy (catechol) methoxychlor metabolite (tris-OH-M) inhibited 6 beta-hydroxylation in PB-microsomes competitively and noncompetitively, respectively; however, these methoxychlor metabolites did not exhibit a time-dependent inhibition. Methoxychlor inhibited competitively the formation of 7 alpha-hydroxytestosterone (7 alpha-OH-T) and 16 alpha-hydroxytestosterone (16 alpha-OH-T) but exhibited little or no time-dependent inhibition of generation of these metabolites, indicating that P450s 2A1, 2B1/B2, and 2C11 were inhibited but not inactivated. Methoxychlor inhibited in a time-dependent fashion the 2-hydroxylation of E2 in PB-microsomes. However, bis-OH-M exhibited solely reversible inhibition of the 2-hydroxylation, supporting our conclusion that the inactivation of P450s does not involve participation of the demethylated metabolites. Both competitive inhibition and time-dependent inactivation of human liver P450 3A (6 beta-hydroxylase) by methoxychlor, was observed. As with rat liver microsomes, the human 6 beta-hydroxylase was inhibited by bis-OH-M and tris-OH-M competitively and noncompetitively, respectively. Testosterone and estradiol strongly inhibited the irreversible binding of methoxychlor to microsomal proteins. This might explain the "clean" competitive inhibition by methoxychlor of the 6 beta-OH-T formation when the compounds were coincubated.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed2002

Combined effects of dietary phytoestrogen and synthetic endocrine-active compound on reproductive development in Sprague-Dawley rats: genistein and methoxychlor.

Humans and wildlife are frequently exposed to mixtures of endocrine active-compounds (EAC). The objective of the present study was to investigate the potential of the phytoestrogen genistein to influence the reproductive developmental toxicity of the endocrine-active pesticide methoxychlor. Three levels of genistein (0, 300, or 800 ppm) and two levels of methoxychlor (0 or 800 ppm) were used in this study. Sprague-Dawley rats were exposed to the two compounds, either alone or in combinations, through dietary administration to dams during pregnancy and lactation and to the offspring directly after weaning. Both compounds, methoxychlor in particular, were associated with reduced body growth at 800 ppm, but pregnancy outcome was not affected by either treatment. An acceleration of vaginal opening (VO) in the exposed female offspring was the only observed effect of genistein at 300 ppm. Exposure to 800 ppm genistein or 800 ppm methoxychlor caused accelerated VO and also altered estrous cyclicity toward persistent estrus in the female offspring. The estrogenic responses to genistein and methoxychlor administered together were apparently accumulative of the effects associated with each compound alone. Methoxychlor, but not genistein, delayed preputial separation (PPS) in the male rats. When administered with methoxychlor, genistein at 800 ppm enhanced the effect of methoxychlor on delaying PPS. Genistein and methoxychlor treatment did not change gender-specific motor activity patterns in either sex. To explore possible mechanisms for interaction between the two compounds on development, we performed estrogen receptor (ER)- and androgen receptor (AR)-based in vitro transcriptional activation assays using genistein and the primary methoxychlor metabolite 2,2-bis-(p-hydroxyphenyl)-1,1,1-trichloroethane (HPTE). While the in vitro assays supported the estrogenic effects of genistein and methoxychlor and the antiandrogenic effects of methoxychlor, the reactivity of these compounds with ERs alpha and beta could not predict the greater in vivo estrogenic potency of methoxychlor over genistein; nor could the potentiation of the methoxychlor effect on PPS by genistein be predicted based on in vitro HPTE and genistein reactions with the AR. Data from this study indicate that phytoestrogens are capable of altering the toxicological behaviors of other EACs, and the interactions of these compounds may involve complexities that are difficult to predict based on their in vitro steroid receptor reactivities.

Animals↗
PubMed1985

Role of hepatic monooxygenases in generating estrogenic metabolites from methoxychlor and from its identified contaminants.

Previous investigations demonstrated that methoxychlor [1,1,1-trichloro-2,2-bis(4-methoxyphenyl)ethane] contains estrogenic contaminants and that methoxychlor per se is not an estrogen but is a proestrogen being metabolized in vivo into estrogenic products. The present study examined structurally identified methoxychlor contaminants as to their estrogenic or proestrogenic properties. Also, the estrogenic activity of demethylated metabolites of methoxychlor and of one contaminant was determined. To examine these properties, we utilized an assay developed by us that monitors whether a given compound, incubated with isolated rat uteri, can diminish the uterine cytosolic estrogen receptor and elevate the nuclear estrogen receptor and whether metabolic intervention by hepatic microsomal monooxygenase(s) is required by the respective compound for this cellular redistribution of the receptor. Of the 15 compounds examined which constitute with methoxychlor 99.5% of total technical grade methoxychlor, two compounds, 1,1-dichloro-2-(4-hydroxyphenyl)-2-(4-methoxyphenyl)ethene (mono-OH-MDDE) and 1,1,1-trichloro-2-(4-hydroxyphenyl)-2-(4-methoxyphenyl)ethane (mono-OH-methoxychlor), were active per se and two compounds, 1,1-dichloro-2,2-bis(4-methoxyphenyl)ethene (MDDE) and methoxychlor, required metabolic transformation for estrogenic activity to be manifested. Subsequently, it was shown that the mono- and bis-OH metabolites of MDDE and of methoxychlor were active estrogens and that the order of activity, either by the above procedure or in terms of relative binding affinity to rat uterine cytosolic receptor, was as follows: bis-OH-MDDE much greater than bis-OH-methoxychlor greater than mono-OH-MDDE greater than mono-OH-methoxychlor. Following the in vitro observations, the activity of MDDE and bis-OH-MDDE was determined in vivo in immature rats. It appears that both compounds are estrogenic, yielding marked elevation in ornithine decarboxylase (EC 4.1.1.17) levels and moderate increase in uterine weight. A comparison with methoxychlor and bis-OH-methoxychlor [1,1,1-trichloro-2,2-bis(p-hydroxyphenyl)ethane] demonstrates that the order of potencies is similar to that observed in the in vitro determinations. These studies demonstrate the usefulness of the in vitro assay for determining the estrogenic and proestrogenic properties of compounds of which limited quantities are available, often insufficient for in vivo determination. Also, whereas the in vitro assay is simple and rapid, a lengthy investigation might be required to determine in vivo whether a given compound is an estrogen or a proestrogen.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMedDate not supplied

Actions of the endocrine disruptor methoxychlor and its estrogenic metabolite on in vitro embryonic rat seminiferous cord formation and perinatal testis growth.

The current study examines the actions of methoxychlor and its estrogenic metabolite, 2, 2-bis-(p-hydroxyphenyl)-1, 1, 1-trichloroethane (HPTE), on seminiferous cord formation and growth of the developing rat testis. The developing testis in the embryonic and early postnatal period is likely more sensitive to hormonally active agents than at later stages of development. Embryonic day 13 (E13) testis organ cultures were treated with either 0.2, 2, or 20 microM methoxychlor or 1, 3, 6, 15, 30, or 60 microM HPTE to examine effects on cord formation. No concentration of methoxychlor completely inhibited cord formation. However, cord formation was abnormal with the presence of a reduced number of cords and appearance of "swollen" cords at the 2 and 20 microM concentrations of methoxychlor. The swollen cords were due to an increase in the number of cells in a cord cross section and reduction of interstitial cell numbers between cords. Treatment of embryonic day 13 (E13) testes with HPTE caused abnormal cord formation at the 3 microM and 6 microM concentrations, and completely inhibited cord formation at the 15, 30, and 60 microM concentrations. In addition to the estrogenic metabolite HTPE, methoxychlor can also be metabolized into anti-androgenic compounds. Therefore, to determine the spectrum of potential actions of methoxychlor on testis development, different concentrations of estradiol, testosterone, and an anti-androgen (flutamide) were utilized to determine their effects on E13 testis organ culture morphology. Estradiol (1 microM) and flutamide (0.1microM) both inhibited seminiferous cord formation in E13 testis organ cultures. Therefore, methoxychlor may be acting through the androgen and/or estrogen receptors to elicit its actions on seminiferous cord formation. Reverse transcription polymerase chain reaction (PCR) (RT-PCR) confirmed the presence of estrogen receptor alpha (ERalpha) mRNA from embryonic day 14 (E14) through postnatal day 5 (P5) while estrogen receptor beta (ERbeta) mRNA did not appear until approximately E16 of testis development. Androgen receptor (AR) expression was present from E14 through P5 of testis development, but at apparently reduced levels at E14 and E16. Immunohistochemical analysis localized ERalpha to the cells of the seminiferous cords at E14 though P5 while ERbeta was present in cells of the interstitium at E16 and P0. Androgen receptor was localized to germ and interstitial cells. The effects of methoxychlor, HPTE, estradiol, and testosterone on cell growth of perinatal testes was determined with a thymidine incorporation assay in postnatal day zero (P0) testis cell cultures. Methoxychlor (0.002, 0.02, and 0.2 microM) and HPTE (2 and 20 microM) stimulated thymidine incorporation in P0 testis cell cultures in a similar manner to estradiol (0.01, 0.1, and 1 microM). In addition, testosterone (0.1 microM) also stimulated thymidine incorporation in P0 testis cultures. Observations suggest that methoxychlor and its metabolite HPTE can alter normal embryonic testis development and growth. The actions of methoxychlor and HPTE are likely mediated in part through the steroid receptors confirmed to be present in the developing testis.

Animals↗
PubMed2001

Effect of methoxychlor on antioxidant system of goat epididymal sperm in vitro.

AIM: To evaluate the effect of methoxychlor on the antioxidant system of goat epididymal sperm. METHODS: Epididymis of adult goat was obtained from local slaughter houses and sperm were collected by chopping the epididymis in modified Ringer' s phosphate solution (RPS). After several washings, the sperm samples were dispersed in RPS and incubated with methoxychlor (1 micromol/L, 10 micromol/L and 100 micromol/L) and methoxychlor + vitamin C (100 micromol/L each) for 3 h at 32 degrees C. After incubation, the sperm motility and viability were assessed. An aliquot of sperm sample was homogenized, centrifuged and used for the assay of superoxide dismutase, glutathione peroxidase, glutathione reductase and lipid peroxidation. RESULTS: In methoxychlor-incubated sperm and in sperm co-incubated with methoxychlor and vitamin C, the sperm motility and viability showed no significant changes as compared to the corresponding controls. In methoxychlor-incubated sperm the activity of superoxide dismutase, glutathione reductase and glutathione peroxidase were decreased while lipid peroxidation was increased in a dose-dependent manner. Co-incubation of sperm with methoxychlor and vitamin C showed no changes in the activity of superoxide dismutase, glutathione reductase and glutathione peroxidase and in the level of lipid peroxidation. CONCLUSION: Methoxychlor induced oxidative stress in epididymal sperm of goats by decreasing the levels of antioxidant enzymes. Co-incubation of sperm with methoxychlor and vitamin C, a natural antioxidant, reversed the effect of methoxychlor.

Animals↗
PubMed1998

Human cytochrome P450-catalyzed conversion of the proestrogenic pesticide methoxychlor into an estrogen. Role of CYP2C19 and CYP1A2 in O-demethylation.

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.

Animals↗
PubMed2002

Effect of methoxychlor on the antioxidant system in mitochondrial and microsome-rich fractions of rat testis.

Methoxychlor, an environmental contaminant, which is widely used as a pesticide in many countries, has been shown to induce reproductive abnormalities in male rats. The precise nature and mechanism of action of methoxychlor on the male reproductive system is not clear. In the present study, we have sought to investigate the induction of oxidative stress in the testis of rat after exposure to methoxychlor. Methoxychlor (1, 10, and 100 mg kg(-1) body weight per day) was administered orally to the rats for 45 days. After 24 h of the last treatment the animals were killed using anesthetic ether. The body weight of the animals administered with methoxychlor did not show any significant change. The weights of the testis, epididymis, seminal vesicles and ventral prostate decreased significantly in 100 mg dose but remained unchanged in 1 and 10 mg doses. Mitochondrial and microsome-rich fractions of the testis were obtained by the method of differential centrifugation. The activities of antioxidant enzymes such as superoxide dismutase, catalase, glutathione reductase and glutathione peroxidase decreased significantly in the animals treated with methoxychlor in a dose-dependent manner in the mitochondrial and microsome-rich fractions of rat testis. The levels of hydrogen peroxide generation (H(2)O(2)) and lipid peroxidation increased in mitochondrial and microsome-rich fractions of the testis of the rats treated with methoxychlor. The results suggested that the low to medium doses of methoxychlor elicit depletion of antioxidant enzymes and concomitant increase in the levels of H(2)O(2) and lipid peroxidation differentially in mitochondrial and microsome-rich fractions of rat testis. In conclusion, the adverse effect of methoxychlor on male reproduction could be due to the induction of oxidative stress in testis.

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