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RECOMBINANT ANDROGEN RECEPTOR (AR) BINDING ACROSS VERTEBRATE SPECIES: COMPARISON OF BINDING OF ENVIRONMENTAL COMPOUNDS TO HUMAN, RAINBOW TROUT AND FATHEAD MINNOW AR.
Citation:
WILSON, V. S., M. C. CARDON, L. E. GRAY, AND P. C. HARTIG. RECOMBINANT ANDROGEN RECEPTOR (AR) BINDING ACROSS VERTEBRATE SPECIES: COMPARISON OF BINDING OF ENVIRONMENTAL COMPOUNDS TO HUMAN, RAINBOW TROUT AND FATHEAD MINNOW AR. Presented at SETAC, Montreal, QC, CANADA, November 05 - 09, 2006.
Description:
In vitro screening assays designed to identify androgen mimics or antagonists typically use mammalian (rat, human) androgen receptors (AR). Although the amino acid sequences of receptors from nonmammalian vertebrates are not identical to the mammalian receptors, it is uncertain whether these differences affect interactions of potential endocrine disrupting chemicals (EDC) with the receptors. Limited evidence exists that fish steroid hormone receptors not only differ from mammalian receptors structurally but also in their binding affinities for some steroids and environmental chemicals. In the current work, competitive binding assays were conducted using full length recombinant AR from fathead minnow (fhAR), rainbow trout (rtAR) and human (hAR) in COS cells. In this system, the binding affinities of individual receptors can be investigated without the potentially confounding effects of other steroid receptors which are present in tissue extracts. Saturation binding and Scatchard analysis using [3H]R1881, a synthetic androgen, revealed a dissociation constant (Kd) of 0.24 nM for the rtAR, 1.8 nM for the fhAR and 0.83 nM for the hAR. To date about twenty chemicals have been tested with each of the three receptors, including natural and synthetic androgens and environmental compounds such as the AR agonists testosterone, androstenedione, 17á- and 17â-trenbolone, 11-ketotestosterone, AR antagonists such as p,p'-DDE, linuron, and vinclozolin and its metabolites M1 and M2 and pesticides such as atrazine. While the affinity of individual compounds for the three receptors varies quantitatively among these species, so far no species specific AR ligands have been identified. In future studies, we plan to expand the structural and chemical domain of compounds tested and to include AR from additional non-mammalian species as well.