Binding competition between estrogen and xenobiotic chemicals with estrogen receptors in the porcine endometrium
2007
Okano, A.(National Inst. of Livestock and Grassland Science, Tsukuba, Ibaraki (Japan)) | Takahashi, H. | Takahashi, M. | Geshi, M.
Evidences for xenobiotic chemical (XBC) induced reproductive disorders have been reported in wild animals and human, because XBCs act as the disruptor of the estrogen receptor (ER) in agonistic or antagonistic manner. There is no report about the binding competition of estrogen and XBCs to ER in the porcine endometrium. Therefore, we characterized and quantified the binding competition to ER between estrogen and six XBCs (diethylstilbestrol: DES, p,p-isopropylidenediphenol: Bis, p-n-nonylphenol: Non, daidzein:Da, coumestrol :Coum, bis (2-ethylhexyl) phthalate: Ph). To clarify ER characteristics, we carried out radio-receptor assay (RRA) of ER in the endometrium. Cytosolic and nuclear fractions were prepared from endometrial tissue with hypotonic 10mM Tris-HCl with 1.5mM EDTA and 1mM dithiothreitol. To choose optimum conditions for RRA, both fractions were incubated with radioactive ligand (sup(3)H-estradiol-17beta:sup(3)H-Esub(2)) at 4, 20 or 37 deg C for 0.5, 2, 4, 8 or 24h. ERS were identified in both fractions of the endometrium. Binding of sup(3)H-Esub(2) depended on the protein amount, and reacted to the maximal at 24h and 2h after incubation, respectively at 4 deg C and 37 deg C. Secondly, binding affinities were examined by Scachard analysis. Binding characteristics were varied on the stages of estrous cycle and the range of Kd and Bmax were 0.12-0.49nM and 0.12-0.015nmol/mg protein, respectively. Binding competitions between sup(3)H-Esub(2) and Esub(2) or each of XBC were examined. Competition studies demonstrated that ER bound Esub(2) and XBCs with several degrees of binding affinity. Higher binding affinities of binding were found in Des, followed by Esub(2) and Non. Binding competitions of Coum, Bis, Da and Ph to Esub(2) were relatively low.
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