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CLONING AND IN VITRO EXPRESSION AND CHARACTERIZATION OF THE ANDROGEN RECEPTOR AND ISOLATION OF ESTROGEN RECEPTOR α FROM THE FATHEAD MINNOW (PIMEPHALES PROMELAS)
Wilson, V S., M C. Cardon, J. Thornton, J J. Korte, G T. Ankley, J. Welch, L E. Gray Jr., AND P C. Hartig. CLONING AND IN VITRO EXPRESSION AND CHARACTERIZATION OF THE ANDROGEN RECEPTOR AND ISOLATION OF ESTROGEN RECEPTOR α FROM THE FATHEAD MINNOW (PIMEPHALES PROMELAS). ENVIRONMENTAL SCIENCE & TECHNOLOGY. American Chemical Society, Washington, DC, 38(23):6314-6321, (2004).
to better identify hormone mimics or antagonists
In vitro screening assays designed to identify hormone mimics or antagonists typically use mammalian (rat, human) estrogen (ER) and androgen receptors (AR). Although we know that the amino acid sequences of steroid receptors in nonmammalian vertebrates are not identical to the mammalian receptors, a great deal of uncertainty exists as to whether these differences impact binding of potential endocrine disrupting chemicals (EDCs) to the receptors. This work details preparation of a cDNA library from a commonly utilized test species, the fathead minnow (Pimephales promelas). The cDNA library was used to isolate an AR and ER α, both of which were sequences. In addition the fathead minnow AR was expressed and characterized with respect to function using saturation and competitive binding assays in COS monkey kidney cells. Saturation experiments determined that the Kd of the fathead (fh) AR for the potent synthetic androgen R1881 was 1.8 nM, which is comparable to that for the human AR in the same assay system. In COS whole cell competitive binding assays, potent androgens such as dihydrotestosterone and 11-ketotestosterone also were shown to be high affinity ligands for the fhAR. Future plans include comparison of binding affinities of the fhAR to those of the human AR using a range of EDCs. Use of the COS cells allows comparison of binding affinities of the two receptors within the same system.
Record Details:Record Type: DOCUMENT (JOURNAL/PEER REVIEWED JOURNAL)
Organization:U.S. ENVIRONMENTAL PROTECTION AGENCY
OFFICE OF RESEARCH AND DEVELOPMENT
NATIONAL HEALTH AND ENVIRONMENTAL EFFECTS RESEARCH LABORATORY
MID-CONTINENT ECOLOGY DIVISION
TOXIC EFFECTS CHARACTERIZATION RESEARCH