Cloning and in vitro expression and characterization of the androgen receptor and isolation of estrogen receptor alpha from the fathead Minnow (Pimephales promelas).
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 affect interactions of potential endocrine-disrupting chemicals (EDC) with the receptors. This leads to substantial uncertainty with respect to the utility of mammalian-based screening assays to predict possible effects of EDCs in nonmammalian wildlife. This paper describes preparation of a cDNA library from a small fish model commonly used in ecological risk assessments, the fathead minnow (Pimphales promelas). The cDNA library was subsequently used to isolate and sequence both AR and ERalpha. In addition, the fathead minnow (fh)AR was expressed and characterized with respect to function using saturation and competitive binding assays in COS monkey kidney cells. Saturation experiments along with subsequent Scatchard analysis determined that the Kd of the fhAR 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 were also shown to be high affinity ligands for the fhAR. We also report affinity of the receptor for a number of environmental contaminants including the AR agonists androstenedione and 17a- and 17beta-trenbolone;AR antagonists such as p,p'-DDE, linuron, and vinclozolin; and the ER agonist 17beta-estradiol. Future plans include comparison of binding affinities of the fhAR to those of the human AR, also expressed in COS cells, using a range of EDCs.
Wilson
Vickie S
VS
US Environmental Protection Agency, ORD, NHEERL, Reproductive Toxicology Division, North Carolina 27711, USA. wilson.vickie@epa.gov
Cardon
Mary C
MC
Thornton
Joseph
J
Korte
Joseph J
JJ
Ankley
Gerald T
GT
Welch
Jeffery
J
Gray
L Earl
LE
Hartig
Phillip C
PC
D000726:Androgen Antagonists / Q000494:pharmacology; D000818:Animals; D001667:Binding, Competitive; D001681:Biological Assay; D019556:COS Cells; D019976:Cloning, Organism; D003530:Cyprinidae / Q000378:metabolism* / Q000502:physiology; D004702:Endocrine Glands / Q000187:drug effects; D004965:Estrogen Antagonists / Q000494:pharmacology; D047628:Estrogen Receptor alpha / Q000302:isolation & purification* / Q000378:metabolism; D015870:Gene Expression / Q000187:drug effects; D015723:Gene Library; D000882:Haplorhini; D008024:Ligands; D011944:Receptors, Androgen / Q000235:genetics* / Q000378:metabolism
D000726:Androgen Antagonists; D004965:Estrogen Antagonists; D047628:Estrogen Receptor alpha; D008024:Ligands; D011944:Receptors, Androgen