Pregnenolone co-treatment partially restores steroidogenesis, but does not prevent growth inhibition and increased atresia in mouse ovarian antral follicles treated with mono-hydroxy methoxychlor.

Mono-hydroxy methoxychlor (mono-OH MXC) is a metabolite of the pesticide, methoxychlor (MXC). Although MXC is known to decrease antral follicle numbers, and increase follicle death in rodents, not much is known about the ovarian effects of mono-OH MXC. Previous studies indicate that mono-OH MXC inhibits mouse antral follicle growth, increases follicle death, and inhibits steroidogenesis in vitro. Further, previous studies indicate that CYP11A1 expression and production of progesterone (P4) may be the early targets of mono-OH MXC in the steroidogenic pathway. Thus, this study tested whether supplementing pregnenolone, the precursor of progesterone and the substrate for HSD3B, would prevent decreased steroidogenesis, inhibited follicle growth, and increased follicle atresia in mono-OH MXC-treated follicles. Mouse antral follicles were exposed to vehicle (dimethylsulfoxide), mono-OH MXC (10 µg/mL), pregnenolone (1 µg/mL), or mono-OH MXC and pregnenolone together for 96 h. Levels of P4, androstenedione (A), testosterone (T), estrone (E1), and 17ß-estradiol (E2) in media were determined, and follicles were processed for histological evaluation of atresia. Pregnenolone treatment alone stimulated production of all steroid hormones except E2. Mono-OH MXC-treated follicles had decreased sex steroids, but when given pregnenolone, produced levels of P4, A, T, and E1 that were comparable to those in vehicle-treated follicles. Pregnenolone treatment did not prevent growth inhibition and increased atresia in mono-OH MXC-treated follicles. Collectively, these data support the idea that the most upstream effect of mono-OH MXC on steroidogenesis is by reducing the availability of pregnenolone, and that adding pregnenolone may not be sufficient to prevent inhibited follicle growth and survival.

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.

Mono-hydroxy methoxychlor alters levels of key sex steroids and steroidogenic enzymes in cultured mouse antral follicles.

Methoxychlor (MXC) is an organochlorine pesticide that reduces fertility in female rodents by decreasing antral follicle numbers and increasing follicular death. MXC is metabolized in the body to mono-hydroxy MXC (mono-OH). Little is known about the effects of mono-OH on the ovary. Thus, this work tested the hypothesis that mono-OH exposure decreases production of 17ß-estradiol (E2) by cultured mouse antral follicles. Antral follicles were isolated from CD-1 mice (age 35-39 days) and exposed to dimethylsulfoxide (DMSO), or mono-OH (0.1-10 µg/mL) for 96 h. Media and follicles were collected for analysis of sex steroid levels and mRNA expression, respectively. Mono-OH treatment (10 µg/mL) decreased E(2) (DMSO: 3009.72±744.99 ng/mL; mono-OH 0.1 µg/mL: 1679.66±461.99 ng/mL; 1 µg/mL: 1752.72±532.41 ng/mL; 10 µg/mL: 45.89±33.83 ng/mL), testosterone (DMSO: 15.43±2.86 ng/mL; mono-OH 0.1µg/mL: 17.17±4.71 ng/mL; 1 µg/mL: 13.64±3.53 ng/mL; 10 µg/mL: 1.29±0.23 ng/mL), androstenedione (DMSO: 1.92±0.34 ng/mL; mono-OH 0.1 µg/mL: 1.49±0.43ng/mL; 1 µg/mL: 0.64±0.31 ng/mL; 10 µg/mL: 0.12±0.06 ng/mL) and progesterone (DMSO: 24.11±4.21 ng/mL; mono-OH 0.1µg/mL: 26.77±4.41 ng/mL; 1 µg/mL: 20.90±3.75 ng/mL; 10 µg/mL: 9.44±2.97 ng/mL) levels. Mono-OH did not alter expression of Star, Hsd3b1, Hsd17b1 and Cyp1b1, but it did reduce levels of Cyp11a1, Cyp17a1 and Cyp19a1 mRNA. Collectively, these data suggest that mono-OH significantly decreases levels of key sex steroid hormones and the expression of enzymes required for steroidogenesis.

Methoxychlor-induced alteration in the levels of HSP70 and clusterin is accompanied with oxidative stress in adult rat testis.

Methoxychlor, an organochlorine pesticide, has been reported to induce abnormalities in male reproductive tract. However, the insight into the mechanisms of gonadal toxicity induced by methoxychlor is not well known. We investigated whether treatment with methoxychlor would alter the levels of stress proteins, heat shock proteins (HSP), and clusterin (CLU), and oxidative stress-related parameters in the testis of adult male rats. Animals were exposed to a single dose of methoxychlor (50 mg/kg body weight) orally and were terminated at various time points (0, 3, 6, 12, 24, and 72 h) using anesthetic ether. The levels of HSP70, CLU, and the activities of superoxide dismutase (SOD), catalase, and lipid peroxidation levels were evaluated in a 10% testis homogenate. A sequential reduction in the activities of catalase and SOD with concomitant increase in the levels of thiobarbituric acid reactive substance (TBARS) was observed. These changes elicited by methoxychlor were very significant between 6-12 h of posttreatment. Immunoblot analysis of HSP revealed the expression of HSP72, an inducible form of HSP, at certain time points (3-24 h) following exposure to methoxychlor. Similarly, the levels of secretory CLU (sCLU) were also found to be elevated between 3-24 h of treatment. The present data demonstrate methoxychlor-elicited increase in the levels of inducible HSP72 and sCLU, which could be a part of protective mechanism mounted to reduce cellular oxidative damage.

Stimulation of transactivation of the largemouth bass estrogen receptors alpha, beta-a, and beta-b by methoxychlor and its mono- and bis-demethylated metabolites in HepG2 cells.

The purpose of this study was to determine the mechanisms by which the pesticide, methoxychlor (MXC), acts as an environmental endocrine disruptor through interaction with the three largemouth bass (Micropterus salmoides) estrogen receptors (ERs) alpha, betaa, and betab. MXC is a less-environmentally persistent analog of DDT that behaves as a weak estrogen. Using transient transfection assays in HepG2 cells, we have previously shown that each receptor is responsive to the endogenous ligand 17beta-estradiol (E(2)) in a dose-dependent manner. The parent compound, MXC, showed dose-dependent stimulation of transcriptional activation through all three ERs. In addition to the parent molecule, each of the metabolites was also estrogenic with all three ERs. The order of potency for ERalpha and ERbetab was HPTE>OH-MXC>MXC, while the opposite order was seen for ERbetaa. HepG2 cells did not substantially metabolize MXC to the active metabolites, thus the activity of MXC was not due to metabolism. When examining the effects of increasing concentrations of MXC at a fixed concentration of E(2), all three ERs show increased activity compared to that with E(2) alone, showing that the effects of MXC and E(2) are additive. However, when this experiment was repeated with increasing concentrations of HPTE at a fixed concentration of E(2), the activity of ERalpha was decreased, that of ERbetab was increased, while that of ERbetaa was unaffected compared to E(2) alone. These experiments suggest that HPTE functions as an E(2) antagonist with ERalpha, an E(2) agonist with ERbetab and does not perturb E(2) stimulation of ERbetaa. While it is clear the ERbeta subtypes are the products of different genes (due to a gene duplication in teleosts) the differences in their responses to MXC and its metabolites indicate that their functions diverge, both in their in vivo molecular response to E(2), as well as in their interaction with endocrine disrupting compounds found in the wild.

Methoxychlor metabolites may cause ovarian toxicity through estrogen-regulated pathways.

The pesticide methoxychlor (MXC) is a reproductive toxicant that targets antral follicles of the mammalian ovary. Cytochrome P450 enzymes metabolize MXC to mono-OH MXC (1,1,1-trichloro-2-(4-hydroxyphenyl)-2-(4-methoxyphenyl)ethane [mono-OH]) and bis-OH MXC (1,1,1-trichloro-2,2-bis(4-hydroxyphenyl)ethane [HPTE]), two compounds that are proposed to be more toxic than the parent compound, can interact with the estrogen receptor (ER), and are proposed to be responsible for ovarian toxicity. Thus, this work tested the hypothesis that MXC metabolites may be responsible for inducing antral follicle-specific toxicities in the ovary and that this toxicity may be mediated through ER-regulated pathways. Mouse antral follicles were isolated and exposed to mono-OH (0.01-10 microg/ml), HPTE (0.01-10 microg/ml), or MXC (100 microg/ml) alone or in combination with ICI 182,780 (ICI; 1 microM) or 17beta-estradiol (E2; 10 and 50 nM) for 96 h. Follicle diameters were measured at 24-h intervals. After culture, follicles were morphologically evaluated for atresia. Both mono-OH and HPTE (10 microg/ml) inhibited follicle growth and increased follicle atresia. The antiestrogen, ICI, did not protect antral follicles from MXC or metabolite toxicity in regard to follicle growth or atresia, but E2 decreased MXC- and mono-OH-induced atresia in small antral follicles. These data suggest that MXC metabolites inhibit follicle growth and induce atresia and that ER-regulated pathways may mediate the ovarian toxicity of MXC and its metabolites.

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.

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)

Photochemical transformation of the DDT and methoxychlor degradation products, DDE and DMDE, by sunlight.

DDE and DMDE, degradation products of the pesticides DDT and methoxychlor, rapidly undergo an unusual photoisomerization in solution when exposed to sunlight. The isomerization involves the exchange of a vinyl chlorine and an ortho aromatic hydrogen. Other photoproducts identified were corresponding benzophenones and 1,1-diaryl-2-chloroethylenes. Quantum yields for the reactions were measured and then used to compute sunlight photolysis half-lives for DMDE and DDE. Although both compounds absorb only the short-wavelength ultraviolet component of sunlight, their photolysis was found to be surprisingly rapid. During summer at latitude 40 degrees N, the photolysis half-lives near the surface of a water body are one hour and one day for dissolved DMDE and DDE, respectively. Photolysis of the DDE photoisomers is about an order of magnitude slower than that of DDE, suggesting that they may accumulate under environmental conditions. The DDE photoisomers photocyclize to form chlorinated dibenzofulvene and dichlorofluorenone. Neither DDE nor its photoisomers photoreact in solution to form PCB's. The environmental significance of these results is discussed, and its is suggested that the persistence of DDE in inland surface waters may be related to its tendency to sorb onto sediments and biota where not light is present.

Physicochemical-activity relationships in asymmetrical analogues of methoxychlor.

Compounds of the general formula 2-aryl-2-(p-methoxyphenyl)-1,1,1-trichloroethane have been prepared and tested for toxicity toward houseflies, pretreated for 1 h with 2mug of piperonyl butoxide. The majority of the compounds synthesized were chosen with the aid of computer programs designed to ensurewell-spread sets of minimally correlated physicochemical parameter values. A nonlinear two-dimensional representation was used to map the active region of physiochemical parameter space and a regression equation was obtained relating the observed toxicity to a combination of these physicochemical parameters. The equation indicates that toxicity increases with the hydrophobicity of the molecules but is decreased markedly by the introduction of bulky substituents into the ortho positions of the benzene ring and less markedly by bulky substituents in the meta and para positions. Substituents which donate electrons to the benzene ring by the "resonance" effect favor high toxicity. The equation performs well in forecasting the toxicity of further members of the series.