Project Summary (2001-2006)

Certain chlorinated hydrocarbon compounds (OCs), including PCBs, DDT, and chlordane, have been reported to exert estrogenic action. This project is assessing a newly identified cellular and molecular mechanism of estrogenicity of organochlorines found in sediments in the lower Hudson River. The 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. Researchers 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.

Previously, project investigators 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, Varl, MCF7, T47D) as well as in the hER-yeast assay. Researchers are now employing 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, the scientists are characterizing the effects of chemical inhibition and gene deletion/overexpression of membrane pumps on hormonal activity of Superfund contaminants. Hormone binding and the b -gal reporter assays (optimized in the previous project period) are being 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 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 xenobiotics) may regulate pump activity. Project investigators are examining the effects of changing the intracellular concentration of Hsp90, by overexpression under stress (chemical and thermal), inhibition (using the chemical geldanamycin) and gene modification.

Several advances may result from this research. First, if xenobiotics are good substrates for MDR pumps, they may increase the cellular accumulation of endogenous and exogenous hormones, a finding that will change our view of single dose-effect relationships and complex mixtures. Second, data from this study will characterize a critical defense mechanism that may prevent the accumulation and thus reduce toxicity of synthetic chemicals. Lastly, understanding the determinant chemical structure of pump blockers would provide a rational basis for the identification of new potential endocrine disruptors that act by inhibition of efflux pumps.