The Center for Alternatives to Animal Testing is an academic center affiliated with the Division of Toxicological Sciences in the Department of Environmental Health Sciences of the Johns Hopkins University Bloomberg School of Public Health.

 

Johns Hopkins School of Public Health

Abstract for TestSmart -- Endocrine Disruptors

The Use of Polarized Fluorescence to Assay Receptor/Ligand/ERE Binding

Georgi N. Nikov, Mathewos Eshete, Rajendram V. Rajnarayanan, Stephen Boue, Nancy E. Hopkins, and William L. Alworth
Department of Chemistry, Tulane University and Tulane/Xavier Center for Bioenvironmental Research, Tulane University Medical School, New Orleans, LA

Epidemiologic and experimental model studies support the hypothesis that dietary estrogens from plant sources (phytoestogens) may play a role in the prevention of breast and prostate cancer. The molecular mechanisms of such chemopreventative effects are still unclear.

Polarized fluorescence is an ideal technique to investigate the binding of chemical ligands to receptor proteins and we have used this technique to investigate the possiblility that phytoestrogens may bind to the estrogen receptor proteins in humans (ERa and ERβ) in a manner that differentially affects interactions of the ligand-ER complexes with different DNA sequences (EREs). Polarized flourescence was used to measure the binding affinities of genistein, coumestrol, daidzein, glyceollin, and zearalenone for human ERa and ERβ. Competitive binding experiments revealed higher affinity of the phytoestrogens for ERβ than for ERa. Genistein, with a median inhibitory concentration of 12 nM, is the most potent of the phytoestrogens tested and has the same relative binding affinity for ERβ as 17β-estradiol. Polarized fluorescence was also used to measure the effect of these phytoestogens on the ability of ERa and ERβ to associate with specific EREs. The direct binding of recombinant human estrogen receptors to fluorescein-labeled EREs indicates that phytoestrogen ligands cause conformational changes in both human Ers which result in differentiated affinities of the ligand-ER complexes for EREs from the Xenopus vitellogenin A2 gene and from the human pS2 gene. The most intriguing finding is that the molecular volume of coumestrol-ERβ and genistein-ERβ complexes when bound to human pS2 ERE are 1.5 times higher as measured by the polarized fluorescence than the molecular volume of 17beta-estradiol-ERβ complex when bound to the pS2 ERE. We conclude that ligand binding differentially affects the physical properties of ER-ERE complexes and that these changes can be detected and measured with the technique of polarized fluorescence.

We have also used polarized fluorescence to measure the binding of diethylstilbestrol (DES), 4-hydroxytamoxifen, and a new class of synthetic estrogens, adamantylphenols, to human ERa and ERβ and the effect of these estrogen agonists and antagonists on the ability of human recombinant estrogen receptors to bind to the Xenopus vit A2 and the human pS2 EREs.

Our goal is to use the data obtained from in vitro measurements of the changes in polarized fluorescence resulting from binding different classes of ligands to human ERa and ERβ, and of the binding of the ligand ER complexes to different EREs, to classify compounds as potentially harmful estrogen agonists (DES) or potentially protective estrogen antagonists (4-hydroxytamoxifen and phytoestrogens).