This study will examine the interaction of environmental pollutants, primarily pro-estrogenic/estrogenic pesticides and structurally related compounds (e.g., methoxychlor and chlorotrianisene) with several mammalian enzyme systems and determine the molecular mechanisms of how such interactions could produce subtle or overt toxicities. Methoxychlor (M), a pesticide with pro-estrogenic activity, contains approx. 50 contaminants, among these chlorotrianisene (TACE), a triphenylethylene derivative that exhibits estrogenic/anti-estrogenic characteristics. Tamoxifen, a TACE analog, with anti-breast cancer therapeutic activity, will serve as a model for triphenylethylene compounds. In rodent and human, M and TACE are demethylated by hepatic cytochrome P450s (CYP) into estrogenic products and M & TACE are metabolically activated, forming reactive intermediates (RI) that bind covalently to hepatic proteins. The potency of the estrogenic metabolites of M and TACE will be determined with respect to binding and activation of the two isoforms of estrogen receptor (ER-alpha and -beta). This may explain the dilemma that certain compounds are estrogenic in one tissue and anti-estrogenic or inactive in another tissue. The structures of the RI of M &TACE will be deduced from their glutathione or N-acetylcysteine adducts. The major M-binding protein in liver, protein disulfide isomerase (PDI) is a chaperone enzyme that catalyzes the proper folding of proteins. The covalent binding of M to PDI and treatment of rats with M appears to diminish hepatic PDI activity. Such decrease in PDI activity is of utmost importance, since malfolded proteins may elicit significant toxicity. The mechanisms of the effects of metabolic activation of methoxychlor, TACE and Tamoxifen on PDI activity in vitro and the effect of treatment with these compounds on PDI in vivo, will be explored. The mechanism by which M and TACE diminish hepatic steroidal 5-alpha-reductase, an enzyme converting testosterone into DHT (an active male hormone and important for reproduction in females), will be determined. Whether M-metabolites are anti-androgenic in vitro and in vivo will be explored. If affirmative, it would suggest that the mechanism of M-mediated lowering of 5-alpha-reductase is due to the anti-androgenic metabolites. The mechanism of hydroxylation of phenolic M-metabolites in particular and of phenols in general (forming catechols) by CYP3A4, the major human P450, will be investigated. CYP3A4 catalyzes the metabolism of the majority of drugs and is involved in drug-drug interaction. Understanding the mechanism of 3A4 activity would be useful in predicting drug interactions and drug:drug-metabolite interactions.
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