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(267) Species Differences in Androgen and Estrogen Receptor Structure and Function Among Vertebrates and Invertebrates: Interspecies Extrapolations regarding Endocrine Disrupting Chemicals .
Wilson, Vickie*,1, Ankley, Gerald2, Gooding, Meredith1,3, Reynolds, Philip1,4, Noriega, Nigel, Cardon, Mary1, Hartig, Phillip1, Lambright, Christy1, Thornton, Joseph5, Gray, Leon1, 1 USEPA, ORD, NHEERL, RTD, RTP, NC, USA2 USEPA, ORD, NHEERL, MED, DULUTH, MN, USA3 NORTH CAROLINA STATE UNIVERSITY, RALEIGH, NC, USA4 UNIVERSITY OF NORTH CAROLINA, CHAPEL HILL, NC, USA5 COLUMBIA UNIVERSITY, NY, NY, USA
ABSTRACT- In vitro screening assays designed to identify hormone mimics or antagonists typically use mammalian estrogen (ER) and androgen receptors (AR). However, there is uncertainty concerning species differences in binding of toxicants to steroid receptors because gene and protein structures are not identical among animal species. To test the hypothesis that chemicals interact with ER or AR among vertebrates and invertebrates similarly, the binding affinities of selected chemicals for these receptors are being assessed in vitro. Since the last presentation of this project at SETAC, considerable progress has been made in several areas, including completion of cDNA libraries for alligator testis (Alligator mississippenisis), fathead minnow (fh) viscera (Pimephales promelas), Northern leopard frog liver (Rana pipens), mud snail (Ilyanassa obsoleta) and Daphnia magna. The libraries of the alligator, minnow, frog and snail are being screened for AR and/or ER via a standard hybridization approach. To date, fhAR and fhER alpha have been isolated and sequenced and the sequences were used for phylogenetic analysis. Binding assays with the fhAR transiently expressed in COS cells demonstrate that this receptor is functional. In addition, the affinity of the rainbow trout (rt)AR for chemicals is being compared to affinity of the human (h)AR. Saturation ligand binding and Scatchard analysis using the synthetic androgen [3H]R1881 revealed a Kd of 0.28 nM for rtAR. Competitive binding studies with [3H]R1881 were conducted in COS cells using several steroids and xenobiotics known to bind hAR. The relative order of binding affinities of androgens for rtAR and hAR were: R1881> trenbolone> 11-ketotestosterone> dihydrotestosterone> testosterone> androstenedione. We have yet to identify a chemical that displays a significant preference for either rtAR or hAR, Some of our results differ from reports in the literature which assessed fish AR binding in tissue cytosolic preparations. These studies will facilitate the identification of chemicals that affect reproduction and development in animals via steroid hormone receptors, and ultimately affect the design of screening batteries for endocrine disrupting chemicals. This is an abstract of a proposed presentation and does not necessarily reflect USEPA policy.
Key words: androgen receptor, estrogen receptor, interspecies extrapolation, steroid binding assays
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