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Epidermal growth factor (EGF) and its receptor (EGF-R) have been implicated as mediators for estrogen induced cellular growth. This study examines whether the action of the estrogenic pesticide methoxychlor (MXC) parallels the action of 17 beta-estradiol (E2) on uterine EGF-R. Administration of 20 micrograms E2/sexually immature female rat increased 125I-EGF binding to membranes extracted from whole uteri 175% over endogenous levels, while 500 mg MXC/kg led to a 156% increase. E2 in both 20 and 40 micrograms/rat doses and 500 mg MXC/kg led to maximal stimulation over endogenous levels, 12-h posttreatment. Rats were treated with E2, MXC, or vehicle plus 100 micrograms actinomycin-D (ACT-D) or 100 micrograms cycloheximide (CYCLO) per rat to determine if mRNA transcription and translation are involved in the increased EGF-R binding following estrogenic treatment. Only ACT-D inhibited the estrogenic stimulation of EGF-R binding, resulting in a 44% decrease when given concurrently with E2 or MXC, suggesting transcription is required. Additionally, ACT-D decreased endogenous receptor levels by 55%. No other differences were detected. When EGF-R binding data were analyzed by the method of Scatchard, both E2 and MXC, at maximal dosages, elevated uterine EGF-R binding sites by over 200% after 12 h as measured by maximal binding (Bmax) with no significant difference in dissociation constant (Kd) values. These results demonstrate that both E2 and MXC can stimulate the number of EGF-R binding sites without significantly altering the receptor binding affinity (Kd). Further, this stimulation is time dependent and is affected by dose.

Endometriosis is a disease of women where endometrial tissue is found growing at ectopic sites. While evidence suggesting a role for the ovarian hormones in endometriosis exists, no complete studies of the roles of estrogen and progesterone have heretofore been performed. Also, if estrogen has a role in the growth and/or maintenance of endometriosis, it is likely that the proestrogenic pesticide, methoxychlor (MXC), might also have such an effect. Sixty rats underwent surgery on Day 0 to induce endometriosis. On Day 21, all rats were ovariectomized. During surgery, the diameters of all endometriotic implants (which were fully developed) were measured. Starting on Day 21, groups of rats were treated daily, for 3 weeks, with (a) vehicle (b) estrone, 1 micrograms/rat, E;(c) progesterone, 2 mg/rat, P; (d) E + P, 1 micrograms + 2 mg; (e) MXC, 250 mg/kg; or (f) MXC + P, 250 mg/kg + 2 mg/rat. On Day 42, all rats were killed, and the diameters of all endometriotic sites were measured. While no differences in diameter were found across groups prior to ovariectomy, ovariectomy plus treatment altered the growth of endometriosis tissue. Progesterone and vehicle treatments produced results that were identical: regression of endometriotic sites. Both estrogen and MXC treatments maintained endometriotic site size at a level greater than that in the vehicle-treated group. The combination of progesterone with either estrone or MXC did not alter the effect of either chemical. We conclude that while estrogen promotes the growth of endometriosis, progesterone either produces regression or fails to maintain the sites. MXC, at a relatively high dose, supports the development of endometriosis. Concurrent progesterone treatment does not modulate the effects of estrone or MXC. These results suggest that exposure of women to high doses of MXC may exacerbate the development of endometriosis or contribute to its recurrence.

Methoxychlor (MXC), a proestrogenic pesticide, has adverse effects on fertility and uterine function in rodents. MXC is converted to an estrogenic substance, 2,2-bis-(p-hydroxyphenyl)-1,1,1-trichloroethane (HPTE), which binds to the estrogen receptor. We examined the similarities in mechanism between MXC (500 mg/kg) and estrogen (10 micrograms/rat) actions using the estrogen-induced protein, IP, also known as creatine kinase. Immature, female rats were treated with MXC or estradiol (E2). Concurrent treatment included actinomycin D (100 micrograms), cycloheximide (100 micrograms), or progesterone (0.5 mg). Uterine proteins were labeled in vitro with 3H for treated rats and with 14C for controls. The uteri were combined, cytosol was isolated, non-denaturing (ND) gels were run, and dpm/gel slice were plotted. In a follow-up study, labeled cytosols from MXC- and E2-treated rats were immunoprecipitated with a monoclonal antibody to creatine kinase. The immunoprecipitate was run on SDS gels. The data show that both MXC and E2 treatments result in ND gels with peaks in (a) induced protein and (b) the 3H/14C ratio, in the same position. The induction of IP by MXC is time- and dose-dependent. Concurrent treatment with MXC plus actinomycin D or cycloheximide blocked IP synthesis, a result parallel to E2 action signifying the necessity of RNA and protein synthesis for IP induction. Progesterone did not block either MXC or E2 induction of IP synthesis. Immunoprecipitation of creatine kinase revealed a single peak at a molecular weight of approximately 49,000. SDS gels of cytosol after MXC or E2 treatment also yielded protein and ratio peaks at molecular weights of approximately 49,000. This estimate is near the published estimated molecular weight of creatine kinase of 46,000. We conclude that MXC action parallels that of estradiol on the induction and regulation of the estrogen-induced protein in immature rat uterus.

Methoxychlor (MXC) has adverse effects on fertility and rat uteri via its active metabolite HPTE (2,2-bis(p-hydroxyphenyl)-1,1,1-trichloroethane). Uterine peroxidase, a marker of estrogen action, was used to probe potential mechanisms of MXC's adverse effects. Specifically, our objective was to compare the regulation of the effects of estrogen and MXC on uterine peroxidase. Immature female rats were treated with MXC (250 mg/kg; gavage) 24 h prior to the measurement of uterine peroxidase activity, with or without concurrent treatment with actinomycin D, cycloheximide, progesterone, or tamoxifen. MXC alone produced an increase in peroxidase activity. The prior and/or concurrent treatment with the compounds listed blocked the MXC-induced stimulation of peroxidase. These data show similarities between the mechanisms of estrogen MXC action. Both estrogen and MXC act to stimulate uterine peroxidase activity via increased RNA and protein synthesis and this stimulation can be blocked by progesterone and tamoxifen.