Estrogen Receptor Binding of Xenoestrogens and Phytoestrogens in Japanese Quail (Coturnix japonica)

Abstract

Estrogenic compounds must bind to estrogen receptors (ER) and modulate estrogen-sensitive gene expression. However, various in vivo and in vitro assays have established that xenoestrogens and phytoestrogens are rather weak estrogens, with a 5,000- to 10,000-fold lower binding affinity to ER than estradiol-17β (E2). The purpose of this study was to determine the binding affinity of various estrogen-like chemicals to bacterially expressed quail ERα. The first-strand cDNA was synthesized from total RNA isolated from mature female quail liver with oligo-dT primed reverse transcription. The cDNA included the hinge region, the ligand-binding domain, and the C-terminal domain of quail ERα. It was amplified by PCR, and the PCR product was ligated into GST fusion protein expression vector, and transfected to the E. coli DH5α strain. A binding assay using the supernatant of the cell lysate was performed by incubation with [3H] E2 and increasing concentrations of competitor at 4°C for 18h. Unbound steroids were removed by the addition of dextran-coated charcoal, followed by centrifugation for 15min. The radioactivity of the supernatant was determined with a liquid scintillation counter. Quail ERα expressed bacterially showed binding affinity to E2 with a dissociation constant of 1.74±0.34 X10-10M. The competition studies indicated that the relative binding affinities for the synthetic estrogens, diethylstilbestrol and ethynylestradiol, are very high, but that of the xenoestrogens, bisphenol A and nonylphenol, are very low. The phytoestrogens, coumestrol and genistein, can compete with E2 with a significant binding affinity.