(Taken from application) Certain chlorinated hydrocarbon compounds (OCs), including PCBs, DDT, and chlordane, have been reported to exert estrogenic action. This project will assess a newly identified cellular and molecular mechanism of estrogenicity of organochlorines found in sediments in the lower Hudson River. Our hypothesis, which is based on studies of similar compounds in cancer cell lines and in aquatic organisms, is that a mechanism of estrogenic action of organochlorines is inhibition of MDR efflux pumps in cell membranes. We hypothesize that inhibition of the MDR pump by organochlorines increases intracellular accumulation of endogenous hormones (estradiol), with resultant adverse effects on endocrine function and development that are associated with elevated estrogen exposures. This is a novel pathway through which OCs may act as hormone mimics. In the previous project period, we observed a range of estrogenic and progestagenic responses with individual OC compounds (DDT, chlordane, PCBs, PAHs) and mixtures that mimic Hudson River sediment extracts, in tumor cell lines (Ishikawa, Var1, MCF7, T47D) as well as in the hER-yeast assay. In the next period, we propose to employ the hER yeast assay to assess the effect on the MDR pump of OCs found in the Hudson River watershed. By taking advantage of the presence of the homologous Pdr5 transporter in yeast plasma membranes, we will be able to characterize the effects of gene deletion/overexpression of membrane pumps on hormonal activity. Hormone binding and the ?-gal reporter assays (optimized in the previous project period) will be used to measure resulting estrogenic activity. Then, the capacity of chlorinated hydrocarbons to suppress the efflux of estrogenic compounds will be compared by spectroscopically monitoring the rate of efflux of rhodamine 6G (a well known pump substrate and fluorescent probe) as a function of OC concentration. In addition to xenobiotics, the molecular chaperone Hsp 90 (whose expression levels are very sensitive to the presence of xenobiotics) may regulate pump activity. We will examine the effects of changing the intracellular concentration of Hsp90, by overexpression under stress (chemical and thermal), inhibition (using the chemical geldanamycin) and gene modification.
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