Interaction of Estrogenic Chemicals and Phytoestrogens with Estrogen Receptor β

Abstract

The rat, mouse and human estrogen receptor (ER) exists as two subtypes, ER alpha and ER beta, which differ in the C-terminal ligand-binding domain and in the N-terminal transactivation domain. In this study, we investigated the estrogenic activity of environmental chemicals and phytoestrogens in competition binding assays with ER alpha or ER beta protein, and in a transient gene expression assay using cells in which an acute estrogenic response is created by cotransfecting cultures with recombinant human ER alpha or ER beta complementary DNA (cDNA) in the presence of an estrogen-dependent reporter plasmid. Saturation ligand-binding analysis of human ER alpha and ER beta protein revealed a single binding component for [3H]-17beta-estradiol (E2) with high affinity [dissociation constant (Kd) = 0.05 - 0.1 nM]. All environmental estrogenic chemicals [polychlorinated hydroxybiphenyls, dichlorodiphenyltrichloroethane (DDT) and derivatives, alkylphenols, bisphenol A, methoxychlor and chlordecone] compete with E2 for binding to both ER subtypes with a similar preference and degree. In most instances the relative binding affinities (RBA) are at least 1000-fold lower than that of E2. Some phytoestrogens such as coumestrol, genistein, apigenin, naringenin, and kaempferol compete stronger with E2 for binding to ER beta than to ER alpha. Estrogenic chemicals, as for instance nonylphenol, bisphenol A, o, p'-DDT and 2',4',6'-trichloro-4-biphenylol stimulate the transcriptional activity of ER alpha and ER beta at concentrations of 100-1000 nM. Phytoestrogens, including genistein, coumestrol and zearalenone stimulate the transcriptional activity of both ER subtypes at concentrations of 1-10 nM. The ranking of the estrogenic potency of phytoestrogens for both ER subtypes in the transactivation assay is different; that is, E2 >> zearalenone = coumestrol > genistein > daidzein > apigenin = phloretin > biochanin A = kaempferol = naringenin > formononetin = ipriflavone = quercetin = chrysin for ER alpha and E2 >> genistein = coumestrol > zearalenone > daidzein > biochanin A = apigenin = kaempferol = naringenin > phloretin = quercetin = ipriflavone = formononetin = chrysin for ER beta. Antiestrogenic activity of the phytoestrogens could not be detected, except for zearalenone which is a full agonist for ER alpha and a mixed agonist-antagonist for ER beta. In summary, while the estrogenic potency of industrial-derived estrogenic chemicals is very limited, the estrogenic potency of phytoestrogens is significant, especially for ER beta, and they may trigger many of the biological responses that are evoked by the physiological estrogens.

Comparison of in Vivo and in Vitro Reporter Gene Assays for Short-Term Screening of Estrogenic Activity

“…A. Gustafsson, Karolinska Institute, Huddinge, Sweden. Transactivation of these receptor constructs by (xeno)estrogens was tested in the human embryonic kidney (293HEK) cell line, which was acquired from the American Type Culture Collection (ATCC, Rockville, MD), essentially as described elsewhere (11). Briefly, the 293HEK cells were plated at a density of 35000 cells/cm 2 in 24 well plates (Costar).…”

Combating Malaria with Plant Molecules: A Brief Update

“…Phytoestrogens are a group of chemical compounds derived from plants that share the ability to bind to the estrogen receptors (ERs) and to trigger estrogen dependent transcription [5]. These compounds are synthesized in plants starting from phenylpropanoids and simple phenols [6].…”

“…The difference of the effects achieved by phytoestrogens in comparison to E2 is mainly due to their lower ERα binding affinity. Phytoestrogens generally have a higher relative binding affinity for ERβ than ERα and a very weak binding affinity for ERα in comparison to E2 [5, 127-129]. The distinct ER binding capacities affect gene transcription and ER signaling.…”

“…Solid phase binding assays show a higher binding affinity (20- to 30-fold more) of ERβ for genistein, apigenin and kaempferol. This different affinity is probably linked to the position and number of the hydroxyl substituents on the flavone or isoflavone molecule [5]. Genistein binds to ERβ in a cavity between H3 and H11, adopting a similar binding mode to the one of E2.…”

“…Indeed, by eliminating one hydroxyl group (daidzein, biochanin A) or two hydroxyl groups (formononetin) it is possible to observe a great loss in ERβ ligand binding affinity. The flavone apigenin has moderate affinity for both ER subtypes and addition of hydroxyl groups (kaempferol, quercetin) does not increase but decreases the binding affinities [5]. …”