Impact of endocrine-active compounds on adrenal androgen production in pigs during neonatal period.

This study investigated the impact of neonatal exposure to endocrine-active compounds (EACs): flutamide (antiandrogen), 4-tert-octylphenol (an estrogenic compound), and methoxychlor (an organochlorine insecticide exhibiting estrogenic, antiestrogenic and antiandrogenic activities) on androgen production within porcine adrenal glands. The expression of genes related to androgen synthesis and the level of androgen production were analyzed (i) in the adrenal glands of piglets exposed to EACs during the first 10 days of life (in vivo study), and (ii) in adrenal explants from sow-fed or formula-fed 10-day-old piglets incubated with EACs (ex vivo study). EACs affected the expression of genes linked to adrenal androgen biosynthesis. The prominent effect of methoxychlor on downregulation of StAR, CYP11A1 and HSD3B and upregulation of CYP17A1 and SULT2A1 were demonstrated. Furthermore, our study revealed divergent response to EACs between sow-fed and formula-fed piglets, suggesting that natural feeding may provide protection against adverse EACs effects, particularly those interfering with estrogens action.

Transcriptional networks associated with the immune system are disrupted by organochlorine pesticides in largemouth bass (Micropterus salmoides) ovary.

Largemouth bass (Micropterus salmoides) inhabiting Lake Apopka, Florida are exposed to high levels of persistent organochlorine pesticides (OCPs) and dietary uptake is a significant route of exposure for these apex predators. The objectives of this study were to determine the dietary effects of two organochlorine pesticides (p, p'-dichlorodiphenyldichloroethylene; p, p' DDE and methoxychlor; MXC) on the reproductive axis of largemouth bass. Reproductive bass (late vitellogenesis) were fed one of the following diets: control pellets, 125ppm p, p'-DDE, or 10ppm MXC (mg/kg) for 84days. Due to the fact that both p,p' DDE and MXC have anti-androgenic properties, the anti-androgenic pharmaceutical flutamide was fed to a fourth group of largemouth bass (750ppm). Following a 3 month exposure, fish incorporated p,p' DDE and MXC into both muscle and ovary tissue, with the ovary incorporating 3 times more organochlorine pesticides compared to muscle. Endpoints assessed were those related to reproduction due to previous studies demonstrating that these pesticides impact the reproductive axis and we hypothesized that a dietary exposure would result in impaired reproduction. However, oocyte distribution, gonadosomatic index, plasma vitellogenin, and plasma sex steroids (17ß-estradiol, E2 and testosterone, T) were not different between control animals and contaminant-fed largemouth bass. Moreover, neither p, p' DDE nor MXC affected E2 or T production in ex vivo oocyte cultures from chemical-fed largemouth bass. However, both pesticides did interfere with the normal upregulation of androgen receptor that is observed in response to human chorionic gonadotropin in ex vivo cultures, an observation that may be related to their anti-androgenic properties. Transcriptomics profiling in the ovary revealed that gene networks related to cell processes such as leukocyte cell adhesion, ossification, platelet function and inhibition, xenobiotic metabolism, fibrinolysis, and thermoregulation were altered by p, p' DDE, MXC, and flutamide. Interestingly, immune-related gene networks were suppressed by all three chemicals. The data suggest that p, p' DDE and flutamide affected more genes in common with each other than either chemical with MXC, consistent with studies suggesting that p, p' DDE is a more potent anti-androgen than MXC. These data demonstrate that reproductive health was not affected by these specific dietary treatments, but rather the immune system, which may be a significant target of organochlorine pesticides. The interaction between the reproductive and immune systems should be considered in future studies on these legacy and persistent pesticides.

Hepatocytes as in vitro test system to investigate metabolite patterns of pesticides in farmed rainbow trout and common carp: Comparison between in vivo and in vitro and across species.

In vitro tools using isolated primary fish hepatocytes have been proposed as a useful model to study the hepatic metabolism of xenobiotics in fish. In order to evaluate the potential of in vitro fish hepatocyte assays to provide information on in vivo metabolite patterns of pesticides in farmed fish, the present study addressed the following questions: Are in vitro and in vivo metabolite patterns comparable? Are species specific differences of metabolite patterns in vivo reflected in vitro? Are metabolite patterns obtained from cryopreserved hepatocytes comparable to those from freshly isolated cells? Rainbow trout and common carp were dosed orally with feed containing the pesticide methoxychlor (MXC) for 14days. In parallel, in vitro incubations using suspensions of freshly isolated or cryopreserved primary hepatocytes obtained from both species were performed. In vivo and in vitro samples were analyzed by thin-layer chromatography with authentic standards supported by HPLC-MS. Comparable metabolite patterns from a qualitative perspective were observed in liver in vivo and in hepatocyte suspensions in vitro. Species specific differences of MXC metabolite patterns observed between rainbow trout and common carp in vivo were well reflected by experiments with hepatocytes in vitro. Finally, cryopreserved hepatocytes produced comparable metabolite patterns to freshly isolated cells. The results of this study indicate that the in vitro hepatocyte assay could be used to identify metabolite patterns of pesticides in farmed fish and could thus serve as a valuable tool to support in vivo studies as required for pesticides approval according to the EU regulation 1107.

Interactions of endosulfan and methoxychlor involving CYP3A4 and CYP2B6 in human HepaRG cells.

Humans are usually exposed to several pesticides simultaneously; consequently, combined actions between pesticides themselves or between pesticides and other chemicals need to be addressed in the risk assessment. Many pesticides are efficient activators of pregnane X receptor (PXR) and/or constitutive androstane receptor (CAR), two major nuclear receptors that are also activated by other substrates. In the present work, we searched for interactions between endosulfan and methoxychlor, two organochlorine pesticides whose major routes of metabolism involve CAR- and PXR-regulated CYP3A4 and CYP2B6, and whose mechanisms of action in humans remain poorly understood. For this purpose, HepaRG cells were treated with both pesticides separately or in mixture for 24 hours or 2 weeks at concentrations relevant to human exposure levels. In combination they exerted synergistic cytotoxic effects. Whatever the duration of treatment, both compounds increased CYP3A4 and CYP2B6 mRNA levels while differently affecting their corresponding activities. Endosulfan exerted a direct reversible inhibition of CYP3A4 activity that was confirmed in human liver microsomes. By contrast, methoxychlor induced this activity. The effects of the mixture on CYP3A4 activity were equal to the sum of those of each individual compound, suggesting an additive effect of each pesticide. Despite CYP2B6 activity being unchanged and increased with endosulfan and methoxychlor, respectively, no change was observed with their mixture, supporting an antagonistic effect. Altogether, our data suggest that CAR and PXR activators endosulfan and methoxychlor can interact together and with other exogenous substrates in human hepatocytes. Their effects on CYP3A4 and CYP2B6 activities could have important consequences if extrapolated to the in vivo situation.

Increased sensitivity of estrogen receptor alpha overexpressing antral follicles to methoxychlor and its metabolites.

Methoxychlor (MXC), an organochlorine pesticide, and its metabolites, mono-hydroxy MXC (MOH) and bis-hydroxy MXC (HPTE) are known ovarian toxicants and can cause inhibition of antral follicle growth. Since these chemicals bind to estrogen receptor alpha (ESR1), we hypothesized that ovaries overexpressing ESR1 (ESR1 OE) would be more susceptible to toxicity induced by MXC and its metabolites because the chemicals can bind to more ESR1 in the antral follicles. We cultured antral follicles from controls and ESR1 OE mouse ovaries with either the vehicle dimethylsulfoxide (DMSO), MXC, MOH, or HPTE. The data show that at 96 h, the cultured antral follicles from ESR1 OE antral follicles are more susceptible to toxicity induced by MXC, MOH, and HPTE because low doses of these chemicals cause follicle growth inhibition in ESR1 OE mice but not in control mice. On comparing gene expression levels of nuclear receptors in the cultured antral follicles of ESR1 OE and control follicles, we found differential messenger RNA (mRNA) expression of Esr1, estrogen receptor beta (Esr2), androgen receptor (Ar), progesterone receptor (Pr), and aryl hydrocarbon receptor (Ahr) between the genotypes. We also analyzed mRNA levels of Cyp3a41a, the enzyme metabolizing MOH and HPTE, in the cultured follicles and found that Cyp3a41a was significantly lower in DMSO-treated ESR1 OE follicles compared with controls. In ESR1 OE livers, we found that Cyp3a41a levels were significantly lower compared with control livers. Collectively, these data suggest that MXC and its metabolites cause differential gene expression in ESR1 OE mice compared with controls. The results also suggest that the increased sensitivity of ESR1 OE mouse ovaries to toxicity induced by MXC and its metabolites is due to low clearance of the metabolites by the liver and ovary.

Metabolism of methoxychlor by the P450-monooxygenase CYP6G1 involved in insecticide resistance of Drosophila melanogaster after expression in cell cultures of Nicotiana tabacum.

Cytochrome P450 monooxygenase CYP6G1 of Drosophila melanogaster was heterologously expressed in a cell suspension culture of Nicotiana tabacum. This in vitro system was used to study the capability of CYP6G1 to metabolize the insecticide methoxychlor (=1,1,1-trichloro-2,2-bis(4-methoxyphenyl)ethane, 1) against the background of endogenous enzymes of the corresponding non-transgenic culture. The Cyp6g1-transgenic cell culture metabolized 96% of applied methoxychlor (45.8 microg per assay) within 24 h by demethylation and hydroxylation mainly to trishydroxy and catechol methoxychlor (16 and 17%, resp.). About 34% of the metabolism and the distinct formation of trishydroxy and catechol methoxychlor were due to foreign enzyme CYP6G1. Furthermore, methoxychlor metabolism was inhibited by 43% after simultaneous addition of piperonyl butoxide (458 microg), whereas inhibition in the non-transgenic culture amounted to 92%. Additionally, the rate of glycosylation was reduced in both cultures. These results were supported by the inhibition of the metabolism of the insecticide imidacloprid (6; 20 microg, 24 h) in the Cyp6g1-transgenic culture by 82% in the presence of piperonyl butoxide (200 microg). Due to CYP6G1 being responsible for imidacloprid resistance of Drosophila or being involved in DDT resistance, it is likely that CYP6G1 conveys resistance to methoxychlor (1). Furthermore, treating Drosophila with piperonyl butoxide could weaken the observed resistance phenomena.

Methoxychlor induces apoptosis via mitochondria- and FasL-mediated pathways in adult rat testis.

In the past few years, there has been much concern about the adverse health effects of environmental contaminants in general and organochlorine in particular. Studies have shown the repro-toxic effects of long-term exposure to methoxychlor, a member of the organochlorine family. However, the insight into the mechanisms of gonadal toxicity induced by methoxychlor is not well known. In the present study we sought to elucidate the mechanism(s) underpinning the gonadal effects within hours of exposure to methoxychlor. Experimental rats were divided into six groups of four each. Animals were orally administered with a single dose of methoxychlor (50mg/kg body weight) and killed at 0, 3, 6, 12, 24, and 72h post-treatment. The levels and time-course of induction of apoptosis-related proteins like cytochorome C, caspase 3 and procaspase 9, Fas-FasL and NF-kappaB were determined to assess sequential induction of apoptosis in the rat testis. DNA damage was assessed by TUNEL assay and flowcytometry. Administration of methoxychlor resulted in a significant increase in the levels of cytosolic cytochrome c and procaspase 9 as early as 6h following exposure. Time-dependent elevations in the levels of Fas, FasL, pro- and cleaved caspase 3 were observed. The DNA damage was measured and showed time-dependent increase in the TUNEL positive cells, and also by flowcytometry of testicular cells. The study demonstrates induction of testicular apoptosis in adult rats following exposure to a single dose of methoxychlor.

Anaerobic biodegradation of organochlorine pesticides in contaminated soil - significance of temperature and availability.

Anaerobic biodegradation of the pesticides: gamma-hexachlorocyclohexane, methoxychlor, o,p'- and p,p'-DDT in field polluted soil was tested at 12, 22 and 30 degrees C, using methanogenic granular sludge as inoculum. The contaminants were removed quite effectively at all temperatures and their removal rates increased 1.2-1.7 times with the increase in temperature. In most cases pesticide concentrations after an initial substantial decline remained almost constant until the end of experiment. These residual concentrations were also temperature dependent and they were 1.4-8.2 times higher at 12 degrees C than at 30 degrees C. DDT was degraded via DDD and accumulation of this metabolite was lower (19-64%) than the corresponding amount of removed DDT, especially at higher temperatures. Further transformation of DDD was confirmed by formation of p,p'-dichlorobenzophenone. Additional experiment demonstrated that removal was limited to readily desorbing fractions of pesticides, while their desorption-resistant fractions persisted in the soil. However, DDD metabolite was only partially removed despite its good desorbability.

Effect of the methoxychlor metabolite HPTE on the rat ovarian granulosa cell transcriptome in vitro.

Ovarian granulosa cells play a central role in steroidogenesis, which is critical for female reproduction. Follicle-stimulating hormone (FSH) promotes cyclic adenosine monophosphate (cAMP)-mediated signaling to regulate granulosa cell steroidogenesis. We have shown previously that 2,2-bis-(p-hydroxyphenyl)-1,1,1-trichloroethane (HPTE) inhibits FSH- and dibutyryl cAMP-stimulated steroidogenesis and affects the messenger RNA levels of steroidogenic pathway enzymes in rat granulosa cells. However, HPTE showed a differential effect in FSH- and cAMP-stimulated cells in that HPTE more completely blocked FSH- when compared to cAMP-driven steroidogenesis. The objective of this study was to analyze the effects of HPTE on global gene expression profiles in untreated granulosa cells and those challenged with FSH or cAMP. Granulosa cells from immature rats were cultured with 0, 1, 5, or 10 microM HPTE in the presence or absence of either 3 ng FSH/ml or 1mM cAMP for 48 h. Total RNA was isolated for real-time quantitative PCR and microarray analysis using the GeneChip Rat Genome 230 2.0 and ArrayAssist Microarray Suite. An investigation of changes in gene expression across all HPTE treatments showed that HPTE altered more genes in FSH- (approximately 670 genes) than in cAMP-stimulated cells (approximately 366 genes). Analysis confirmed that HPTE more effectively inhibited FSH- than cAMP-induced steroid pathway gene expression and steroidogenesis. Furthermore, expression patterns of novel genes regulating signal transduction, transport, cell cycle, adhesion, differentiation, motility and growth, apoptosis, development, and metabolism were all altered by HPTE. This study further established that HPTE exerts differential effects within the granulosa cell steroidogenic pathway and revealed that these effects include broader changes in gene expression.

Reductive dechlorination of methoxychlor and DDT by human intestinal bacterium Eubacterium limosum under anaerobic conditions.

Methoxychlor [1,1,1-trichloro-2,2-bis(p-methoxyphenyl)ethane], a substitute for 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT), is a compound of environmental concern because of potential long-term health risks related to its endocrine-disrupting and carcinogenic potency. In order to determine the metabolic fate of methoxychlor and DDT in the human intestinal gut, Eubacterium limosum (ATCC 8486), a strict anaerobe isolated from the human intestine that is capable of O-demethylation toward O-methylated isoflavones, was used as a model intestinal microbial organism. Under anaerobic incubation conditions, E. limosum completely transformed methoxychlor and DDT in 16 days. Based on gas chromatography-mass chromatography analyses, the metabolites produced from methoxychlor and DDT by E. limosum were confirmed to be 1,1-dichloro-2,2-bis(p-methoxyphenyl)ethane (methoxydichlor) and 1,1-dichloro-2,2-bis(p-chlorophenyl)ethane (DDD), respectively. This study suggests that E. limosum in the human intestinal gut might be a participant in the reductive dechlorination of methoxychlor to the more antiandrogenic active methoxydichlor.

The methoxychlor metabolite, HPTE, directly inhibits the catalytic activity of cholesterol side-chain cleavage (P450scc) in cultured rat ovarian cells.

Exposure to the pesticide methoxychlor in rodents is linked to impaired steroid production, ovarian atrophy and reduced fertility. Following in vivo administration, it is rapidly converted by the liver to 2,2-bis-(p-hydroxyphenyl)-1,1,1-trichloroethane (HPTE), the reported active metabolite. Both methoxychlor and HPTE have weak estrogenic and antiandrogenic activities, and these effects are thought to be mediated through the estrogen and androgen receptors, respectively. Previous in vivo studies on methoxychlor exposure to female animals have demonstrated decreased progesterone production but no change in serum estrogen levels. We recently showed that HPTE specifically inhibits the P450 cholesterol side-chain cleavage (P450scc, CYP11A1) step resulting in decreased androgen production by cultured rat testicular Leydig cells. The current studies examined the mechanism of action of HPTE on progesterone production by cultured ovarian cells (granulosa and theca-interstitial) from pregnant mare serum gonadotropin-primed immature rats. In addition, we evaluated whether the effects of HPTE on rat ovarian cell progesterone biosynthesis were mediated through the estrogen or androgen receptors. Exposure to HPTE (0, 10, 50 or 100nM) alone progressively inhibited progesterone formation in cultured theca-interstitial and granulosa cells and the P450scc catalytic activity in theca-interstitial cells in a dose-dependent manner with significant declines starting at 50nM. However, HPTE did not change mRNA levels of the P450scc system (P450scc, adrenodoxin reductase and adrenodoxin) as well as P450scc protein levels. Of interest, estradiol, xenoestrogens (bisphenol-A or 4-tert-octylphenol), a pure antiestrogen (ICI 182,780), or antiandrogens (4-hydroxyflutamide or the vinclozolin metabolite M-2), had no effect on progesterone production even at 1000nM. Co-treatment of HPTE with ICI 182,780 did not block the effect of HPTE on progesterone formation. These studies suggest that the decline in progesterone formation following exposure to HPTE in cultured ovarian cells is associated with the inhibition of catalytic activity of P450scc at least in theca-interstitial cells. This action does not appear to be mediated through the estrogen or androgen receptor signaling pathways, and other chemicals exhibiting estrogenic, antiestrogenic or antiandrogenic properties do not mimic its effect on ovarian steroid production.

Biodegradation of methoxychlor and its metabolites by the white rot fungus Stereum hirsutum related to the inactivation of estrogenic activity.

The white rot fungus Stereum hirsutum was used to degrade methoxychlor [2,2,2-trichloro-1,1-bis(4-methoxyphenyl)ethane] in culture and the degraded products were extensively determined. The estrogenic activity of the degraded products of methoxychlor was examined using cell proliferation and pS2 gene expression assays in MCF-7 cells. S. hirsutum showed high resistance to methoxychlor 100 ppm, and the mycelial growth was fully completed within 8 days of incubation at 30 degrees C. Methoxychlor in liquid culture medium was gradually converted into 2,2-dichloro-1,1-bis(4-methoxyphenyl)ethane, 2,2-dichloro-1,1-bis(4-methoxyphenyl)ethylene, 2-chloro-1,1-bis(4-methoxyphenyl) ethane, 2-chloro-1,1-bis(4-methoxyphenyl) ethylene, and 1,1-bis(4-methoxyphenyl)ethylene, indicating that methoxychlor is dominantly degraded by dechlorination and dehydrogenation. MCF-7 cells were demonstrated to proliferate actively at the 10-5 M concentration of methoxychlor. However, cell proliferation was significantly inhibited by the incubation with methoxychlor culture media containing S. hirsutum. In addition, the expression level of pS2 mRNA was increased at the concentration (10-5 M) of methoxychlor. The reductive effect of S. hirsutum for methoxychlor was clear but not significant as in the proliferation assay.

The methoxychlor metabolite, 2,2-bis-(p-hydroxyphenyl)-1,1,1-trichloroethane, inhibits steroidogenesis in rat ovarian granulosa cells in vitro.

The exquisitely balanced hormonal mechanisms that control female fertility can be affected by several internal and external factors including pathogens, genetic maladies, and environmental agents. In the latter group are natural and synthetic agents known as endocrine disruptors. One such compound, 2,2-bis-(p-hydroxyphenyl)-1,1,1-trichloroethane (HPTE), is the predominant metabolite of the pesticide methoxychlor. The effects of HPTE on ovarian steroidogenesis have not been previously reported and were investigated in the present study. Granulosa cells harvested from immature rats were treated with follicle-stimulating hormone (FSH) or N(6),2'-O-dibutyryladenosine 3',5'-cyclic monophosphate (db-cAMP) in the presence or absence of HPTE. After 48h, progesterone (P4) and estradiol-17beta (E2) concentrations were measured in the culture media. Steady-state levels of the mRNAs encoding steroidogenic acute regulatory protein (StAR), P450 side-chain cleavage (P450scc), 3beta-hydroxysteroid dehydrogenase type 1 (3beta-HSD), and P450 aromatase (P450arom) were examined using real-time PCR. Both FSH- and db-cAMP-stimulated P(4) accumulation were impaired by HPTE. In contrast, FSH-, but not db-cAMP-stimulated, E2 content was suppressed by HPTE. The FSH-dependent increase in the abundance of P450scc, 3beta-HSD, and P450arom mRNAs was blocked by HPTE; however, StAR expression was not altered. Although db-cAMP-dependent P450arom was moderately reduced by HPTE, the levels of db-cAMP-dependent StAR, P450scc, and 3beta-HSD mRNAs were increased in the presence of HPTE. These data collectively show that HPTE can disrupt P4 and E2 production in granulosa cells, with implications for sites of action both preceding and following the generation of cAMP. The steroid-modulatory effects of HPTE in granulosa cells appear to involve the general suppression of the FSH-dependent expression of mRNAs encoding steroid pathway proteins, whereas the disparate effects of HPTE on cAMP-dependent mRNA content in this regard suggest a broader and more complex mechanism of action.

David Kupfer: a career retrospective.

David Kupfer's research career spanned 50 years and he authored or co-authored over 160 papers and book chapters. Although best known for his work centering on cytochrome P450 metabolism of prostaglandins, steroids, and proestrogenic compounds, David's research also contributed key advances in the areas of P450 induction and catalytic mechanism, breast cancer therapy, and analytical methodology. His research is reviewed here.

David Kupfer, Ph.D. A mentor and a scientist.

I worked with the late Dr. David Kupfer for nearly nine years at the Worcester Foundation/University of Massachusetts Medical School, Worcester, MA. I was involved in the metabolism of methoxychlor and tamoxifen, the areas of research close to David's heart. We demonstrated the metabolic pathways of these compounds in rats and humans, and the covalent binding to microsomal proteins that could result in long-term toxic manifestations. I learned a lot from David, who was a mentor and friend/colleague. His death has left a void in my heart and he will be sorely missed.

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.

The reported active metabolite of methoxychlor, 2,2-bis(p-hydroxyphenyl)-1,1,1-trichloroethane, inhibits testosterone formation by cultured Leydig cells from neonatal rats.

Methoxychlor (MC) is an insecticide that is presently used on agricultural crops, especially after the ban on the use of 2,2-bis(p-chlorophenyl)-1,1,1-trichloroethane (DDT) in the United States. Following administration in vivo, MC is converted to 2,2-bis(p-hydroxyphenyl)-1,1,1-trichloroethane (HPTE), which is thought to be the active agent. However, both MC and HPTE have been reported to have weak estrogenic and antiandrogenic activities, and they are thought to exert their potential adverse (endocrine disruptive) effects through the estrogen and androgen receptors, respectively. In a recent study, HPTE was shown to inhibit both basal and hCG-stimulated testosterone production by cultured Leydig cells from immature and adult rats, and these effects were reported to be mediated through the estrogen receptor. Because fetal Leydig cells represent a separate population from adult Leydig cells and many of the reported adverse actions of endocrine disruptors are thought to have their effects during gestational exposure, the present studies examined the effects of HPTE on testosterone formation by cultured fetal Leydig cells from neonatal rats to determine whether these cells are sensitive to HPTE. Our studies demonstrated that HPTE inhibited both basal and hCG-stimulated testosterone formation in a dose-dependent manner. Significant declines in testosterone were observed at about 100nM HPTE, and this effect was detected as early as 1h after exposure. The main effects of HPTE appeared to be localized to the cholesterol side-chain cleavage step which converts cholesterol to pregnenolone. In addition, this effect did not appear to be mediated through the estrogen receptor as a weak estrogen or the androgen receptor as an antiandrogen, which are the currently proposed modes of action of MC and HPTE.

The effects of the reported active metabolite of methoxychlor, 2,2-bis(p-hydroxyphenyl)-1,1,1-trichloroethane, on testosterone formation by cultured Leydig cells from young adult rats.

Methoxychlor (MC) is an insecticide that is currently used on a variety of agricultural crops, especially following the ban of 2,2-bis(p-chlorophenyl)-1,1,1-trichloroethane (DDT) use in the United States. Following in vivo administration, MC is converted to 2,2-bis(p-hydroxyphenyl)-1,1,1-trichloroethane (HPTE), which is proposed to be the active agent. Both MC and HPTE have been demonstrated to exhibit weak estrogenic and antiandrogenic activities, and they are thought to exert their effects through estrogen or androgen receptors, respectively. A recent study reported that HPTE inhibited both basal and hCG-stimulated testosterone formation by immature and adult cultured rat Leydig cells and that this effect was mediated through the estrogen receptor. In the current studies, we examined the effects of HPTE on basal and hCG-stimulated testosterone formation by cultured Leydig cells from young adult rats. In addition, we evaluated whether the effects of HPTE on rat Leydig cell testosterone biosynthesis were mediated through the estrogen receptor as an estrogen agonist or the androgen receptor as an antiandrogen. The current studies demonstrated that HPTE inhibited both basal and hCG-stimulated testosterone formation in a dose-dependent manner with significant declines in testosterone being observed at approximately 100 nM. The effects of HPTE were localized to the cholesterol side-chain cleavage step; however, these effects were not mediated through the classic estrogen receptor or by its acting as an antiandrogen, the currently recognized modes of action of MC and HPTE.

Cross-talk between endocrine-disrupting chemicals and cytokine signaling through estrogen receptors.

STAT3 mainly acts as a signal transducer of IL-6 family cytokines and transcriptionally activates specific target genes. STAT3 has also been demonstrated to mediate cellular transformation and is found in numerous cancers. Endocrine-disrupting chemicals (EDCs) are a diverse group of chemicals that bind to estrogen receptors (ERs), mimic estrogenic actions, and may have adverse effects on human health. In our previous study, we demonstrated that estrogens suppressed the STAT3-mediated transcription activity through ERs. In this study, we examined the effects of EDCs on STAT3-mediated signaling through ERs. Surprisingly, some of EDCs enhanced STAT3-mediated transcription activity through ERs. This finding strongly suggests that EDCs may play an important role in the endocrine functions by mimicking cytokine activity by stimulating STAT3 actions through ERs.

Enantioselective recognition of mono-demethylated methoxychlor metabolites by the estrogen receptor.

Metabolites of methoxychlor such as 2-(p-hydroxyphenyl)-2-(p-methoxyphenyl)-1,1,1-trichloroethane (mono-OH-MXC) and 2,2-bis(p-hydroxyphenyl)-1,1,1-trichloroethane (bis-OH-MXC), have estrogenic activity. Mono-OH-MXC is a chiral compound in which the carbon atom bridging two benzene rings is the chiral centre. In previous studies the estrogenic activity of racemic mono-OH-MXC has been measured, and the activity of each enantiomer of this compound has not yet been elucidated. In this study, we evaluated the estrogen receptor-binding activity of each enantiomer of mono-OH-MXC to clarify the enantioselective recognition by the estrogen receptor. (S)-mono-OH-MXC showed 3-fold higher binding activity than that of the (R) enantiomer. The activity of bis-OH-MXC was only 1.7-fold higher than that of (S)-mono-OH-MXC. This result suggests that the one hydroxy group and the orientation of the CCl3 group of mono- and bis-OH-MXCs are important for the interaction with the estrogen receptor. The result also points out the estrogenic activity of methoxychlor after metabolic activation in vivo, which predominantly produces the (S)-mono-OH-MXC, may be higher than estimated from the in vitro activity of racemic mixtures.