Endocrinology. 1998 Oct;139(10):4252-63.
Interaction of estrogenic chemicals and phytoestrogens with estrogen receptor beta.
Kuiper GG, Lemmen JG, Carlsson B, Corton JC, Safe SH, van der Saag PT, van der Burg B, Gustafsson JA.
Center for Biotechnology and Department of Medical Nutrition, Karolinska Institute, Huddinge, Sweden.
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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.

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Introduction

The steroid hormone estrogen influences the growth, differentiation, and functioning of many target tissues. These include tissues of the female and male reproductive systems such as mammary gland, uterus, vagina, ovary, testes, epididymis, and prostate (1). Estrogens also play an important role in bone maintenance, in the central nervous system and in the cardiovascular system where estrogens have certain cardioprotective effects (1, 2, 3, 4). Estrogens diffuse in and out of cells but are retained with high affinity and specificity in target cells by an intranuclear binding protein, termed the estrogen receptor (ER). Once bound by estrogens, the ER undergoes a conformational change allowing the receptor to interact with chromatin and to modulate transcription of target genes (5, 6, 7). We have cloned a novel ER cDNA from rat prostate (8), named ERß, different from the previously cloned ER cDNA (consequently renamed ER). Rat ERß cDNA encodes a protein of 485 amino acid residues with a calculated molecular weight of 54200. Rat ERß protein is highly homologous to rat ER protein, particularly in the DNA binding domain (95% amino acid identity) and in the C-terminal ligand binding domain (55% homology). In addition, recently a variant rat ERß cDNA was cloned that has an in-frame insertion of 54 nucleotides that results in the predicted insertion of 18 amino acids within the ligand-binding domain (9, 10). Mouse (11, 12) and human homologs (13, 14) of rat ERß have been cloned, and similar homologies in the various domains of the subtypes were found. Expression of ERß was investigated by Northern blotting, RT-PCR, and in situ hybridization; prominent expression was found in prostate, ovary, epididymis, testis, bladder, uterus, lung, thymus, colon, small intestine, vessel wall, pituitary, hypothalamus, cerebellum, and brain cortex (4, 10, 11, 12, 13, 14, 15, 16). Saturation ligand binding experiments revealed high affinity and specific binding of 17ß-estradiol (E2) by ERß protein, and ERß is able to stimulate transcription of an estrogen response element containing reporter gene in an E2-dependent manner (10, 11, 12, 13, 15). More extensive studies showed that some synthetic estrogens and naturally occurring steroidal ligands have different relative affinities for ER vs. ERß, although most ligands (including various antiestrogens) bind with very similar affinity to both ER subtypes (15).

There is increasing concern over the putative effects of various chemicals released into the environment on the reproduction of humans and other species. Threats to the reproductive capabilities of birds, fish, and reptiles have become evident and similar effects in humans have been proposed (17, 18, 19, 20, 21). In the past 50 yr, the incidence of testicular cancer and developmental male reproductive tract abnormalities appear to have increased in some developed countries (19). Several reports have also provided evidence for a decline in semen quality and/or sperm count over the same period, although this change may not be universal (19 and references therein). Male offspring born to mothers who were given diethylstilbestrol (DES), a very potent synthetic estrogen, to prevent miscarriages have an increased incidence of undescended testes, urogenital tract abnormalities, and reduced semen quality compared with those from mothers who did not take DES (22 and references therein). In mice injected with DES between days 9 and 16 of gestation, there is an increased risk of intraabdominal testes, sterility, and abnormalities in the urogenital tract of the offspring (22 and references therein). The similarities between the observations made in DES offspring and the abnormalities being observed in the general population have led to the hypothesis that one potential cause of the rise in male reproductive tract abnormalities might be inappropriate exposure to estrogens or suspected environmental estrogenic chemicals (from pesticides, components of plastics, hand creams, etc.) especially during fetal and/or neonatal life (17, 18, 19, 20, 21). Examples of suspected environmental estrogenic chemicals include OH-PCBs (polychlorinated hydroxybiphenyls), DDT and derivatives, certain insecticides and herbicides as Kepone and methoxychlor, certain plastic components as bisphenol A, and some components of detergents and their biodegradation products as, for instance, alkylphenols (17, 18, 19, 20, 21, 23, 24, 25, 26, 27, 28, 29). All these compounds bind weakly to the ERα protein extracted from rat uterus or human breast tumor cells or with recombinant ERα protein (23, 24, 25, 26, 27, 28, 29). No data are yet available on the potential interaction of estrogenic chemicals with ERß, and interactions of xenoestrogens with this subtype may be related to some recent observations. In the rat and mouse prostate, ERß messenger RNA (mRNA) is highly expressed in the secretory epithelial cells (8, 30), and it has been shown that fetal or neonatal exposure to E2/DES or estrogenic chemicals causes not only permanent changes in the size of the prostate but also in the expression level of certain genes (30, 31, 32). In the fetal rat testis, ERß is expressed in Sertoli cells and gonocytes (33), and maternal exposure to DES or 4-octylphenol alters the expression of steroidogenic factor I (SF-1) in Sertoli cells of the fetal rat testis (34). In the human mid-gestational fetus, high amounts of ERß mRNA are present in the testes, but the cellular localization is unknown (35).

Human diet contains several plant-derived, nonsteroidal weakly estrogenic compounds (1). They are either produced by plants themselves (phytoestrogens), or by fungi that infect plants (mycoestrogens). Chemically, the phytoestrogens can be divided into three main classes: flavonoids (flavones, isoflavones, flavanones and chalcones) such as genistein, naringenin, and kaempferol; coumestans (such as coumestrol); and lignans (such as enterodiol and enterolactone). Mycoestrogens are mainly zearalenone (resorcylic acid lactone) or derivatives thereof, which have been associated with estrogenizing syndromes in cattle fed with mold-infected grain (1). Phytoestrogens and mycoestrogens act as weak mitogens for breast tumor cells in vitro, compete with 17ß-estradiol for binding to ERα protein, and induce activity of estrogen-responsive reporter gene constructs in the presence of ER protein (36, 37, 38). Intake of phytoestrogens is significantly higher in countries where the incidence of breast and prostate cancers is low, suggesting that they may act as chemopreventive agents (39). The chemopreventive effect of dietary soy, which is rich in phytoestrogens, has been demonstrated on the development of chemically or irradiation-induced mammary tumors in mice (39 and references therein), and as a delayed development of dysplastic changes in the prostate of neonatally estrogenized mice (40). The expression of ERß in rat, mouse, and human prostate might be of importance in this regard. Phytoestrogens are believed to exert their chemopreventive action by interacting with estrogen receptors, although alternative mechanisms, most notably inhibition of protein tyrosine kinase activity, have been proposed (39, 41).

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Results

Table 1. RBA of suspected environmental endocrine disruptors for ERα and ERß from solid-phase (Scintistrip) competition experiments

Table 2. Binding affinity of various phytoestrogens for ERα and ERß

Table 3. Relative transactivation activity¹ of various compounds for ERα and ERß

Discussion

The ER binds a large number of compounds that exhibit remarkably diverse structural features. In fact, the estrogen receptor is probably unique among the steroid receptors in its ability to interact with a wide variety of compounds. This is true for the ERα subtype but also for the ERß subtype. Binding studies have provided a description of the ligand structure-estrogen receptor binding affinity relationships and a model for the ligand binding site (61). This model indicated that the whole E2 skeleton, that is; the aromatic A-ring, the B- and C-rings, and the OH-group in the D-ring contribute significantly to receptor binding. It was also predicted that the receptor-bound ligand is completely surrounded by the receptor with minimal exposure to solvent. The recently determined crystal structure of the ERα ligand-binding domain complexed with E2 provided important confirmation for this model (62). The phenolic hydroxyl group of the A-ring of E2 nestles between two α-helices and makes several direct hydrogen bonds. This pincer-like arrangement around the A-ring imposes an absolute requirement on ligands to contain an aromatic ring, whereas the remainder of the binding pocket can accept a number of different hydrophobic groups. The overall promiscuity of the ER can be attributed to the size of the binding cavity, which has a volume almost twice that of the E2 molecular volume. The length and the width of the E2 skeleton is very well matched by the receptor, but there are large unoccupied cavities opposite the B-ring and the C-ring of E2 (62). Obviously, several phytoestrogens (coumestrol, genistein) fit very well into the available space, certainly for the ERß protein. It is difficult to understand why other phytoestrogens do not exhibit higher binding affinities because the orientation of the nonsteroidal ligands within the binding pocket is unknown.

Although most of the estrogenic chemicals examined in this study contain at least one aromatic ring with a hydroxyl group, their relative affinities are generally 1000- to 10,000-fold lower than E2. The complexes formed with the ER are probably very unstable, as shown for various alkylphenols (63), and it is likely that these compounds do not completely enter the ligand-binding pocket. The observed radioligand competition might reflect blockade of E2 entrance to the binding site or interaction with another low affinity site that causes a change in the high affinity E2 binding site. If this is true, it will be difficult to use quantitative-structure activity relationship (QSAR) models developed using ligands that bind with high affinity to predict those chemical structures from compound libraries that might disrupt development and reproduction in wildlife, as has been proposed recently (64). Despite their very low binding affinities, several of the suspected endocrine disruptors exhibit estrogenic activities in the transactivation assay system with ERα as well as ERß, albeit only at a potency that is more than 1000-fold lower than that of E2. Obviously, these compounds can induce at least partially the conformational changes involved in the formation of a transcriptionally competent activation function in the ligand-binding domain (62). No striking differences in the relative binding affinities for the tested compounds between ERα and ERß could be detected. Both ER subtypes could therefore be involved in the described developmental and reproductive effects of estrogenic chemicals, depending on their fetal tissue distribution pattern (17, 18, 19, 20, 21, 22, 30, 31, 32, 33, 34, 35).

The relatively low estrogenic potencies of suspected endocrine disruptors suggests that these chemicals alone are unlikely to produce adverse effects during fetal development (21). These compounds occur as mixtures in the environment and diet, and synergistic transcriptional activation of binary mixtures of weakly estrogenic chemicals have been described (65). However, in subsequent detailed studies these synergistic interactions for ER ligand-binding or transactivation could not be confirmed (65, 66). Some suspected endocrine disruptors have been shown to interact not only with the ER but also with the androgen receptor or to interfere with steroid hormone synthesis or metabolism (20). Combined effects of mixtures of endocrine disruptors with a different mode of action could in this way result in synergistic responses in vivo (20 and references therein). Most suspected endocrine disruptors have been tested in in vitro systems (radioligand competition, transactivation assays) and these tests may underestimate or overestimate their in vivo estrogenic potency. The estrogenic potency of bisphenol A in vitro is 1000- to 5000-fold lower than that of E2, but in vivo bisphenol A was rather effective in stimulating PRL release from the pituitary (57). Development of in vivo reporter systems for the assessment of the estrogenic activity of suspected endocrine disruptors might be necessary. If the ligand-binding domain of the ER is fused to a DNA-recombinase, the recombinase activity is controlled efficiently by either agonistic or antagonistic ligands (67, 68). Transgenic mice could be produced in which activation of the recombinase hybrid is detected via elimination of a disruption in a reporter gene (for instance galactosidase or lac Z), thus enabling the use of a simple histochemical reaction in mouse embryos to study the activity of suspected estrogenic chemicals. Of all the suspected endocrine disruptors tested the OH-PCB-K and OH-PCB-L compounds have the highest binding affinity (Table 1), but this is not reflected in the transcription activation potency because compounds with lower binding affinity have equally high estrogenic activity (Table 1 and Table 3 and dose-response curves not shown). The estrogenic potency of compounds is a complicated phenomenon that is the result of a number of factors, such as differential effects on the transactivation functionalities of the receptor, the particular coactivators recruited and the cell- and target gene promoter-context (62). The apparently lower transcriptional activity of ERß compared with ERα (Fig. 3) has also been reported in transient transfection experiments using different cell lines (CHO, COS, HeLa) and reporter gene constructs (11, 12, 13, 69). In contrast, in human osteosarcoma or human endometrial carcinoma cells the transcriptional activity of ERß was higher than that of ERα (70). The reason for these differences in transcriptional activity of the ER subtypes is at the moment unknown, but it might reflect differential expression of transcriptional coactivators or differential stability of the receptor proteins.

Several phytoestrogens have a higher binding affinity for the ERß protein (Fig. 2), and both ER subtype transcripts are present in prostate and breast tumor biopsies, although expression levels vary widely (14, 71). In several epidemiological studies, an inverse relation has been suggested between the risk of prostate cancer or breast cancer and the intake of soy foods or the urinary excretion of phytochemicals (39, 40, 41, 72, 73, 74), although in other studies this could not be confirmed (72). The possibility still exists that the association between reduced breast- and prostate cancer risk and phytoestrogen intake is not causal, and merely results from some other dietary characteristic. Despite the inconclusive epidemiological findings, several putative mechanisms that could account for the hypothesized chemopreventive effects of phytoestrogens have been proposed. Most prominently, phytoestrogens have been suggested to exert strong antiestrogenic effects, thereby inhibiting development of hormone-related cancers (39, 72). In our study, only zearalenone exhibited some antagonistic activity. All other phytoestrogens, including the flavonoids that are present in soy foods, showed only agonistic activity. In previous in vitro studies, involving ERα, only agonistic or at best partial antagonistic activities instead of complete antagonistic activities were reported (36, 37, 38, 75). Several other mechanisms for the proposed chemopreventive effects of flavonoids have been suggested, including induction of cancer cell differentiation, inhibition of protein tyrosine kinases, suppression of angiogenesis, and direct antioxidant effects (41, 76). These alternative mechanisms generally occur at flavonoid concentrations much higher (>5 µM) than the concentrations at which estrogenic effects are detected (<100 nM), and show a different structure-activity relationship; moreover, the effects are observed in cells in the absence of ER expression, and therefore it seems unlikely that all of these effects are ER mediated (41, 77, 78). On the other hand, because both ER subtypes are expressed in bone and the cardiovascular system (4, 79, 80, 81) and given the quite strong estrogenic activity of certain phytoestrogens, the potential beneficial effects of increased food intake of phytoestrogens in the prevention of postmenopausal osteoporosis and cardiovascular diseases should be further investigated (82).

Categories: 1998, Endocrine, Estrogen, Hormone disruptors, Soy, Phytoestrogens