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GENOMIC AND PROTEOMIC BASIS FOR INTERSPECIES EXTRAPOLATIONS BASED UPON ESTROGEN AND ANDROGEN RECEPTORSTRUCTURE AND FUNCTION AMONG ANIMALS
Citation:
Wilson, V S., G T. Ankley, K L. Bobseine, M C. Cardon, M. P. Gooding, L. E. GRAY, JR., L. J. GUILLETTE JR., P. C. HARTIG, G. A. HELD, J. J. KORTE, G. A. LEBLANC, P. REYNOLDS, J. E. WELCH, AND E. M. WILSON. GENOMIC AND PROTEOMIC BASIS FOR INTERSPECIES EXTRAPOLATIONS BASED UPON ESTROGEN AND ANDROGEN RECEPTORSTRUCTURE AND FUNCTION AMONG ANIMALS. Presented at TestSmart--Endocrine Disruptors, Fairfax, VA, February 25-26, 2002.
Description:
Most in vitro hazard assessments for the screening and identification of endocrine disrupting compounds (EDCs), including those outlined in the EDSP Tier 1 Screening (T1S) protocols, use mammalian steroid hormone receptors. There is uncertainty, however, concerning differences that may exist in the binding affinities of toxicants for steroid receptors from other species. For example, metabolites of the fungicide vinclozolin have been shown to bind the androgen receptor from humans and rats, but the results of binding assays in fish are contradictory. The goal of this work is to conduct an in-depth comparison of androgen (AR) and estrogen receptor (ER) structure and function across selected species. The cDNA sequences for the rainbow trout androgen (rtAR) and estrogen (rtER) receptors were obtained from published sources. The rtAR has been transfected into and the protein expressed in COS cells. Scatchard analysis with [3H]R1881 has been completed and competitive binding assays are currently being conducted in this system. Preliminary data indicates that the binding affinity of rtAR for some chemicals appears to differ with some ligands from that of hAR. The cDNA sequence for the rtER has been subcloned into a Baculovirus expression vector and semi-purified. Competitive binding assays comparing the rtER to the hER are planned. Tissues have been collected and cDNA libraries completed for alligator testis (Alligator mississippenisis), fathead minnow viscera (Pimephales promelas), Northern Leopard frog liver (Rana pipens), mud snail body (Ilyanassa obsoleta), and Daphnia magna. Tissues have yet to be collected for Japanese quail and African reed frog. The ER and AR from the fathead minnow have both been isolated and sequences confirmed. The full coding region of the ER was isolated from the library using the rtER as a probe and full length AR was isolated using a fathead AR fragment supplied by scientists from the US EPA lab in Duluth. The alligator cDNA library is currently being screened for the ER by traditional methods using the rtER as a probe and for the AR by rtPCR with primers designed from the canary AR sequence. The frog library is currently being screened with probes targeting the most highly conserved regions of the AR and work has also begun to screen the mud snail library for any steroid hormone receptor. As receptors are identified and sequenced, future studies will include detailed comparisons of receptor sequences to known mammalian sequence information, development of expressions systems for proteins to study functionality in comparison to mammalian receptors, and development of additional in vitro assays that incorporate receptors from these species. These investigations will help resolve some of the across-species extrapolation issues associated with EDCs and, ultimately, may have a major impact on future risk assessment protocols.