Methoxychlor Regulates Rat Uterine Estrogen-Induced Protein

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 μg/rat) actions using the estrogen-induced protein, IP, also known as creatine kinase. Immature, female rats were treated with MXC or estradiol (E 2). Concurrent treatment included actinomycin D (100 μg), cycloheximide (100 μg), 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, nondenaturing (ND) gels were run, and dpm/gel slice were plotted. In a follow-up study, labeled cytosols from MXC- and E 2 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 E 2 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 E 2 action signifying the necessity of RNA and protein synthesis for IP induction. Progesterone did not block either MXC or E 2 induction of IP synthesis. Immunoprecipitation of creatine kinase revealed a single peak at a molecular weight of ~49,000. SDS gels of cytosol after MXC or E 2 treatment also yielded protein and ratio peaks at molecular weights of ~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.