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Substitution of synthetic chimpanzee androgen receptor for human androgen receptor in competitive binding and transcriptional activation assays for EDC screening
Citation:
Hartig, P. C., M. C. Cardon, C. R. Lambright, K. L. Bobseine, V. S. Wilson, AND L. E. Gray, Jr. Substitution of synthetic chimpanzee androgen receptor for human androgen receptor in competitive binding and transcriptional activation assays for EDC screening. TOXICOLOGY LETTERS. Elsevier Science Ltd, New York, NY, 174(1-3):89-97, (2007).
Impact/Purpose:
The United States Environmental Protection Agency (USEPA) Office of Coordination and Policy (OSCP) requested that we develop a nonhuman mammalian receptor binding assay for possible use in their Endocrine Disruptor Screening Program (EDSP). Here we report a novel approach for the synthesis and expression of the chimpanzee androgen receptor (chAR). This receptor is free of all patent restrictions and available to all researchers. Furthermore, we have provided it in a form which can be used in well accepted testing protocols and in doing so reduce or eliminate the need for animals. We demonstrate that these new reagents can be used in standard a whole cell binding assay, transcriptional activation assay, cell free binding assays. Use of these assays in screening by EPA and OECD was recently proposed to the international committee that is working on the validation of new ER and AR binding assays and, upon clearance, this report will be submitted to the appropriate EPA and OECD committee members for consideration. This will have a major impact on their efforts as they are about to begin an international validation effort on the AR binding assay with a recombinant receptor. The tools described here will be freely distributed throughout the scientific community and should aid in the identification and evaluation of environmental EDCs and be applicable to human, companion animal and other mammalian/wildlife species.
Description:
The potential effect of receptor-mediated endocrine modulators across species is of increasing concern. In attempts to address these concerns we are developing androgen and estrogen receptor binding assays using recombinant hormone receptors from a number of species across different vertebrate classes. The United States Environmental Protection Agency (USEPA) Office of Coordination and Policy (OSCP) requested that we develop a nonhuman mammalian receptor binding assay for possible use in their Endocrine Disruptor Screening Program (EDSP). Since the chimpanzee androgen receptor is very similar to that of humans and thus possesses properties which could be exploited in future endocrine studies, we synthesized and expressed this gene in eukaryotic expression plasmids, baculovirus expression vectors and replication deficient adenovirus. In all ligand binding and transcriptional activation assays tested, the chimpanzee receptor performed essentially identically to the human receptor. This suggests that the chimpanzee gene could substitute for the human gene in endocrine screening assays.
