This proposal examines the involvement of metabolism of methoxychlor (a current pesticide) and of structurally related contaminants of methoxychlor in the formation of products with biological (toxicological) activity in mammals. Two monooxygenase-mediated pathways will be studied. The first pathway producing demethylated products which elicit estrogenic or antiestrogenic activity and the second yielding a reactive intermediate (M*) which covalently binds to proteins and may elicit a toxic response. The question whether the same P-450 isozymes are involved in both pathways will be examined with the help of antibodies specific for given isozymes. Also, when appropriated, reconstituted P-450 systems will be used to address that question. Several methods will be utilized to determine whether methoxychlor and its contaminant 1,1-dichloro-2,2-bis(p-methoxyphenyl)ethene [MDDE] are proestrogens or proantiestrogens. We will expand our current in vitro method to determine whether metabolism of these compounds leads to the activation of the uterine estrogen receptor causing an increase in gene expression [progesterone receptor and ornithine decarboxylase (ODC)]. The MCF7 cell line and chick oviduct will be employed to distinguish between estrogenic and antiestrogenic activities. In both systems, the ability of an estrogen to increase and of an antiestrogen to have no effect or to inhibit the formation of estrogen-responsive marker proteins, will be examined. In the chick oviduct, the induction of ODC will be used to distinguish between estrogens and antiestrogens. The similarity between the activity of chlorinated hydrocarbons and classical antiestrogens in the desensitization of induction of uterine ODC will be explored. With the help of antibodies to ODC and with ODC-cDNA we'll determine whether the desensitization is due to inhibition of enzyme synthesis at the translational or transcriptional level. Also we'll determine whether the initial induction of ODC is necessary for the subsequent desensitization. The understanding of the desensitization, only marginal with estradiol, should shed light on the mechanism of antiestrogen action. The study of the second pathway of metabolism of methoxychlor will involve identification of M* via characterization of the adducts formed with N-acetylcysteine and with proteins. Using hepatocytes and in vivo experiments, we'll determine whether formation of these adducts in vitro reflects the in vivo situation. Finally, the relation between covalent binding of methoxychlor and hepatotoxicity will be examined at the cellular and whole animal levels.
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