Mammalian Estrogen Receptor Research Articles

Zearalenone Does Not Show Genotoxic Effects in the Drosophila melanogaster Wing Spot Test, but It Induces Oxidative Imbalance, Development, and Fecundity Alterations

Zearalenone (ZEN) is a non-steroidal mycoestrogen produced by the Fusarium genus. ZEN and its metabolites compete with 17-beta estradiol for cytosolic estrogen receptors, causing reproductive alterations in vertebrates. ZEN has also been associated with toxic and genotoxic effects, as well as an increased risk for endometrial adenocarcinomas or hyperplasia, breast cancer, and oxidative damage, although the underlying mechanisms remain unclear. Previous studies have monitored cellular processes through levels of transcripts associated with Phase I Xenobiotic Metabolism (Cyp6g1 and Cyp6a2), oxidative stress (hsp60 and hsp70), apoptosis (hid, grim, and reaper), and DNA damage genes (Dmp53). In this study, we evaluated the survival and genotoxicity of ZEN, as well as its effects on emergence rate and fecundity in Drosophila melanogaster. Additionally, we determined levels of reactive oxygen species (ROS) using the D. melanogaster flare and Oregon R(R)-flare strains, which differ in levels of Cyp450 gene expression. Our results showed that ZEN toxicity did not increase mortality by more than 30%. We tested three ZEN concentrations (100, 200, and 400 μM) and found that none of the concentrations were genotoxic but were cytotoxic. Taking into account that it has previously been demonstrated that ZEN administration increased hsp60 expression levels and apoptosis gene transcripts in both strains, the data agree with an increase in ROS and development and fecundity alterations. Since Drosophila lacks homologous genes for mammalian estrogen receptors alpha and beta, the effects of this mycotoxin can be explained by a mechanism different from estrogenic activity.

Estrogenic activity of zearalenone, α-zearalenol and β-zearalenol assessed using the E-screen assay in MCF-7 cells

Mycotoxins, including zearalenone (ZEA), can occur worldwide in cereals. They can enter the food chain and cause several health disorders. ZEA and its derivatives (α-zearalenol, α-ZOL and β-zearalenol, β-ZOL) have structural analogy to estrogen, thus they can bind to estrogen receptors (ERs). In order to characterize the estrogenic activity of ZEA, α-ZOL and β-ZOL, the proliferation of ER-positive human breast cancer cells (MCF-7) exposed to these mycotoxins was measured. After exposure at levels ranging from 6.25 to 25 µM, cell proliferation was evaluated by using the E-Screen bioassay. In accordance with previous studies, our results show the estrogenic activity of ZEA, α-ZOL and β-ZOL in MCF-7 cells. This effect is related to ZEA and its metabolites being flexible enough to bind to mammalian ERs. The relative proliferative effect (RPE) ranged from 10% to 91%. The α-ZOL induced the highest proliferative effect due to its higher affinity for the ERs compared to the other mycotoxins.

IMPACTS OF PHYTOESTROGENS ON LIVESTOCK PRODUCTION: A REVIEW

Phytoestrogens, polyphenolic compounds, are one of the major plant secondary metabolites found abundantly in animal diets. However, these phytochemicals play initial important roles in plant growth, development and maintenance. They also have many biological roles in animal body, affecting their productivity. The richest sources of phytoestrogens in animal diets are legumes, particularly, soybeans and clover. These plants often comprise substantial part of animal feed, thus presenting a potential source of phytoestrogens.The interest in these compounds was started earlier, over 50 years ago, when they have been known to affect fertility of an Australian sheep herd, resulting in an economic loss. The putative effects of phytoestrogens are based on their structural similarity to the mammalian estrogen 17β-estradiol (E2) and thus their potency to bind with mammalian estrogen receptors. More attention has been paid to these metabolites since they can act as estrogen agonists or antagonists which make their effects are not expected.. The estrogenic activity of phytoestrogens depends on many factors such as chemical structure of the compound, bioavailability, animal specie, responsive tissue and its estrogen receptor (ER) sub-type (ERα or ERβ) and metabolites resulting from their fermentation and digestion. Reports showed that phytoestrogens could affect animal fertility and growth, quality of animal products and human health. This review illustrates the impacts of phytoestrogens on the livestock production system, focusing on physiology of reproduction, as well as the knowledge obtained from research in laboratory and field scales.

The estrogen receptor fusion system in mouse models: a reversible switch.

Reversible regulatory mouse models have significantly contributed to our understanding of normal tissue and cancer biology, providing the opportunity to temporally control initiation, progression, and evolution of physiological and pathological events. The tamoxifen inducible system, one of the best-characterized "reversible switch" models, has a number of beneficial features. In this system, the hormone-binding domain of the mammalian estrogen receptor is used as a heterologous regulatory domain. Upon ligand binding, the receptor is released from its inhibitory complex and the fusion protein becomes functional. We summarize the advantages and drawbacks of the system, describe several mouse models that rely on it, and discuss potential improvements that could render it even more useful and versatile.

Estrogenic chemicals often leach from BPA-free plastic products that are replacements for BPA-containing polycarbonate products.

BackgroundXenobiotic chemicals with estrogenic activity (EA), such as bisphenol A (BPA), have been reported to have potential adverse health effects in mammals, including humans, especially in fetal and infant stages. Concerns about safety have caused many manufacturers to use alternatives to polycarbonate (PC) resins to make hard and clear, reusable, plastic products that do not leach BPA. However, no study has focused on whether such BPA-free PC-replacement products, chosen for their perceived higher safety, especially for babies, also release other chemicals that have EA.MethodsWe used two, well-established, mammalian cell-based, assays (MCF-7 and BG1Luc) to assess the EA of chemicals that leached into over 1000 saline or ethanol extracts of 50 unstressed or stressed (autoclaving, microwaving, and UV radiation) BPA-free PC-replacement products. An EA antagonist, ICI 182,780, was used to confirm that agonist activity in leachates was due to chemicals that activated the mammalian estrogen receptor.ResultsMany unstressed and stressed, PC-replacement-products made from acrylic, polystyrene, polyethersulfone, and Tritan™ resins leached chemicals with EA, including products made for use by babies. Exposure to various forms of UV radiation often increased the leaching of chemicals with EA. In contrast, some BPA-free PC-replacement products made from glycol-modified polyethylene terephthalate or cyclic olefin polymer or co-polymer resins did not release chemicals with detectable EA under any conditions tested.ConclusionsThis hazard assessment survey showed that many BPA-free PC- replacement products still leached chemicals having significant levels of EA, as did BPA-containing PC counterparts they were meant to replace. That is, BPA-free did not mean EA-free. However, this study also showed that some PC-replacement products did not leach chemicals having significant levels of EA. That is, EA-free PC-replacement products could be made in commercial quantities at prices that compete with PC-replacement products that were not BPA-free. Since plastic products often have advantages (price, weight, shatter-resistance, etc.) compared to other materials such as steel or glass, it is not necessary to forgo those advantages to avoid release into foodstuffs or the environment of chemicals having EA that may have potential adverse effects on our health or the health of future generations.

Are typical human serum BPA concentrations measurable and sufficient to be estrogenic in the general population?

Mammalian estrogen receptors modulate many physiological processes. Chemicals with structural features similar to estrogens can interact with estrogen receptors to produce biological effects similar to those caused by endogenous estrogens in the body. Bisphenol A (BPA) is a structural analogue of estrogen that binds to estrogen receptors. Exposure to BPA in humans is virtually ubiquitous in industrialized societies, but BPA is rapidly detoxified by metabolism and does not accumulate in the body. Whether or not serum concentrations of BPA in humans are sufficiently high to disrupt normal estrogen-related biology is the subject of intense political and scientific debate. Here we show a convergence of robust methods for measuring or calculating serum concentrations of BPA in humans from 93 published studies of more than 30,000 individuals in 19 countries across all life stages. Typical serum BPA concentrations are orders of magnitude lower than levels measurable by modern analytical methods and below concentrations required to occupy more than 0.0009% of Type II Estrogen Binding Sites, GPR30, ERα or ERβ receptors. Occupancies would be higher, but ≤0.04%, for the highest affinity receptor, ERRγ. Our results show limited or no potential for estrogenicity in humans, and question reports of measurable BPA in human serum.

A helminth cestode parasite express an estrogen-binding protein resembling a classic nuclear estrogen receptor

The role of an estrogen-binding protein similar to a known mammalian estrogen receptor (ER) is described in the estradiol-dependent reproduction of the helminth parasite Taenia crassiceps. Previous results have shown that 17-β-estradiol induces a concentration-dependent increase in bud number of in vitro cultured cysticerci. This effect is inhibited when parasites are also incubated in the presence of an ER binding-inhibitor (tamoxifen). RT-PCR assays using specific oligonucleotides of the most conserved ER sequences, showed expression by the parasite of a mRNA band of molecular weight and sequence corresponding to an ER. Western blot assays revealed reactivity with a 66 kDa protein corresponding to the parasite ER protein. Tamoxifen treatment strongly reduced the production of the T. crassiceps ER-like protein. Antibody specificity was demonstrated by immunoprecipitating the total parasite protein extract with anti-ER-antibodies. Cross-contamination by host cells was discarded by flow cytometry analysis. ER was specifically detected on cells expressing paramyosin, a specific helminth cell marker. Parasite cells expressing the ER-like protein were located by confocal microscopy in the subtegumental tissue exclusively. Analysis of the ER-like protein by bidimensional electrophoresis and immunoblot identified a specific protein of molecular weight and isoelectric point similar to a vertebrates ER. Sequencing of the spot produced a small fragment of protein similar to the mammalian nuclear ER. Together these results show that T. crassiceps expresses an ER-like protein which activates the budding of T. crassiceps cysticerci in vitro. To the best of our knowledge, this is the first report of an ER-like protein in parasites. This finding may have strong implications in the fields of host-parasite co-evolution as well as in sex-associated susceptibility to this infection, and could be an important target for the design of new drugs.

Differences in sensitivity but not selectivity of xenoestrogen binding to alligator versus human estrogen receptor alpha

Reproductive abnormalities in alligators exposed to contaminants in Lake Apopka, Florida, USA represent a clear example of endocrine disruption in wildlife. Several of these contaminants that are not able to bind to mammalian estrogen receptors (such as atrazine and cyanazine) have previously been reported to bind to the alligator estrogen receptor from oviductal tissue. Binding of known Lake Apopka contaminants to full length estrogen receptors alpha from human (hERalpha) and alligator (aERalpha) was assessed in a side-by-side comparison within the same assay system. Baculovirus-expressed recombinant hERalpha and aERalpha were used in a competitive binding assay. Atrazine and cyanazine were not able to bind to either receptor. p,p'-Dicofol was able to bind to aERalpha with a concentration inhibiting 50% of binding (IC50) of 4 microM, while only partially displacing 17beta-estradiol (E2) from hERalpha and yielding a projected IC50 of 45 microM. Chemicals that only partially displaced E2 from either receptor, including some dichlorodiphenyltrichloroethane (DDT) metabolites and trans-nonachlor, appeared to have higher affinity for aERalpha than hERalpha. p,p'-Dicofol-mediated transcriptional activation through aERalpha and hERalpha was assessed to further explore the preferential binding of p,p'-dicofol to aERalpha over hERalpha. p,p'-Dicofol was able to stimulate transcriptional activation in a similar manner with both receptors. However, the in vitro results obtained with p,p'-dicofol were not reflected in an in vivo mammalian model, where Kelthane (mixed o,p'- and p,p'-dicofol isomers) did not elicit estrogenic effects. In conclusion, although there was no evidence of exclusively species-specific estrogen receptor binders, some xenoestrogens, especially p,p'-dicofol, had a higher affinity for aERalpha than for hERalpha.

One-way inhibiting cross-talk between arylhydrocarbon receptor (AhR) and estrogen receptor (ER) signaling in primary cultures of rainbow trout hepatocytes

The aryl hydrocarbon receptor (AhR) and the estrogen receptor (ER) are ligand-activated transcription factors, both of which can be activated by environmental pollutants. The AhR regulates cytochrome P450 1A (CYP1A) expression and can be induced by aromatic hydrocarbons. The ER regulates vitellogenin (VTG) expression and can be induced by estrogenic substances. Both receptor responses are established biomarkers used to assess the effects of pollutants in the aquatic environment. The receptors can also be affected in situations of mixed exposure. Cross-talk between these receptor pathways has been suggested, although there are conflicting data in the literature. We investigated cross-talk between ER–VTG and AhR–CYP1A signaling pathways in primary cultures of rainbow trout hepatocytes, using quantitative PCR (qPCR) for mRNA analyses and studies of CYP1A catalytic function and protein expression. The model agonists β-naphthoflavone (BNF) and 17α-ethinylestradiol (EE 2) were used for AhR and ER activation, respectively. Combined exposure to BNF and EE 2 reduced the EE 2-mediated induction of VTG mRNA levels by about 40%, but had no effect on the BNF-mediated CYP1A mRNA levels, indicative of a one-way inhibiting AhR–ER cross-talk. However, basal levels of CYP1A mRNA were reduced 40% upon exposure to EE 2 alone, implying different cross-talk mechanism between basal and induced CYP1A mRNA levels. The mammalian ER antagonist fulvestrant (ICI) is commonly described as an absolute ER antagonist. However, ICI failed to reverse the ER activation caused by EE 2 in the present study. The CYP1A-mediated ethoxyresorufin- O-deethylase (EROD) activity was reduced by 80% in cells co-treated with BNF and EE 2, compared to cells exposed to BNF alone. In vitro inhibiting studies suggests that this reduction was a result of inhibition of the CYP1A catalyst by EE 2 since EE 2 acted as a potent inhibitor (IC 50: 4.6 μM) of the EROD activity. In addition, ICI also acted as a potent inhibitor of the EROD enzyme (IC 50: 0.6 μM). Taken together, our data supports a one-way inhibiting AhR–ER cross-talk in rainbow trout hepatocytes exposed to a mixture of BNF and EE 2.

Transcriptional activity and biological effects of mammalian estrogen receptor ligands on three hepatic estrogen receptors in Mozambique tilapia

Like other fish species, Mozambique tilapia has three forms of estrogen receptor, ERα, ERβ1, and ERβ2. A primary function of 17β-estradiol (E 2) in oviparous species is the hepatic induction of the yolk precursor protein, vitellogenin (Vg). To characterize the roles of ERs in Vg production, transactivation assays and an in vivo study were carried out utilizing agonists for mammalian ERα and ERβ, and an antagonist for mammalian ERα, propyl-pyrazole-triol (PPT), diarylpropionitrile (DPN), and methyl-piperidino-pyrazole (MPP), respectively. ERα was more sensitive and responsive to PPT than ERβ1 or ERβ2 in transactivation assays. All ER isoforms indicated equivalent responsiveness to DPN compared with E 2, although sensitivity to DPN was lower. MPP exhibited antagonistic action on transactivation of all ER isoforms and reduced the E 2 effect on Vg and ERα 48 h post-injection. DPN increased ERα and Vg expression and plasma Vg post-injection, whereas PPT was without effect; DPN seems to stimulate Vg production through activation of ERα. The ligand binding domain of all tilapia ER forms shares only 60–65% amino acid identity with human ERα and ERβ. This, together with our results, clearly indicates that agonistic or antagonistic characteristics of PPT, DPN and MPP cannot be extrapolated from mammalian to piscine ERs.

Comparison of relative binding affinities to fish and mammalian estrogen receptors: The regulatory implications

Screening and testing of chemicals binding to estrogen receptors (ERs) emerge as an important issue in several regulatory programs or frameworks. Discrepancies exist, however, whether fish ERs should be included in the regulatory programs. In view of the differences in binding affinities to ERα and ERβ and the significant contribution of ERβ to biological effects of chemicals, it remains unknown whether both types of ERs are needed for the regulatory purposes. This study collected publications on binding affinities to both mammalian and fish ERs for 65 chemicals, covering a wide range of strong, moderate, weak and non-ER binders. Systematic evaluation of the data was performed in order to compare the difference in binding affinity of chemicals to fish and mammalian ERs and to subtypes of ERs. Except the reference estrogen 17β-estradiol, all 64 chemicals have differential values of relative binding affinity (RBA), which result mostly from the inter-laboratory tests other than interspecies differences. It is concluded that ER binding in one vertebrate species or one subtype of ERs could be extrapolated to other species or subtypes of ERs for most of chemicals for the regulatory purpose. Fish ERs are likely more sensitive to some chemicals of weak binders than mammalian ERs, suggesting the importance of including fish ERs in the regulatory programs. Issues on data interpretation and testing strategy for the regulatory purpose have been discussed.

Daphnia magna responses to a vertebrate estrogen receptor agonist and an antagonist: A multigenerational study

Whereas ecological risk assessments rely on standardized aquatic toxicity tests to assess ecological hazards, these techniques have limited utility for endocrine-active compounds, including select pharmaceuticals. Due to structural similarity between of vertebrate estrogens and ecdysone, previous studies suggest that endocrine-active pharmaceuticals may interfere with invertebrate endocrine systems, while other investigations do not support these suggestions. We assessed effects of the pharmaceuticals 17 α-ethinylestradiol and faslodex, model therapeutics designed to interact with vertebrate estrogen receptors, on endocrine biomarkers and transgenerational life-history parameters of a model invertebrate, Daphnia magna. Identical studies were performed with 20-hydroxyecdysone and testosterone, which served as positive controls for ecdysteroid receptor agonism and antagonism, respectively. Results from this study at biochemical, individual and population levels suggest that a mammalian estrogen receptor agonist and antagonist did not act through the ecdysone receptor in D. magna. Acute-to-chronic ratios based on various chronic responses ranged from 2.59 to 5.18 for 17 α-ethinylestradiol and 1.29–12.9 for faslodex. Toxicity exerted by these therapeutics on D. magna likely resulted from non-endocrine-mediated responses. Mechanism-specific biomarkers, multigenerational designs and population growth models may be useful to assess organismal and population level responses to low-level exposures, which may serve to reduce uncertainty in future hazard assessments of invertebrate responses to endocrine-active pharmaceuticals in the environment.

The Mineralocorticoid Receptor-How to Get Away with Promiscuity: Evolution of Hormone-Receptor Complexity by Molecular Exploitation. Science 312: 97-101, 2006.

The Mineralocorticoid Receptor-How to Get Away with Promiscuity: Evolution of Hormone-Receptor Complexity by Molecular Exploitation. Science 312: 97-101, 2006.

Candida Albicans: The Estrogen Target for Vaginal Colonization

Estradiol (E(2)) stimulates colonization of the vagina by Candida albicans. Although this yeast expresses an estrogen-binding protein (EBP), the cellular target for estrogenic modulation of this infection is unresolved. Findings support direct E(2)-induced C. albicans growth as well as indirect effects via E(2)-induced changes in the vaginal epithelium. Our primary goal was to pursue the issue of direct versus indirect estrogen action on vaginal candidiasis using diethylstilbestrol (DES), an efficacious mammalian estrogen receptor agonist, which exhibits no detectable affinity for the EBP of C. albicans. We used both in vitro and in vivo experimentation with an EBP-positive strain of C. albicans isolated from the human vagina. Ligand-binding studies were performed with steroidal and nonsteroidal estrogens and anti-estrogens using the soluble EBP from both the yeast and the rat uterus. Mature ovariectomized rats were treated with either E(2) or DES for 7 days before and after C. albicans inoculation into the vaginas. Subsequent estrogen-sensitive colonization was quantified based on cultures of vaginal homogenates on Sabouraud dextrose (SD) agar pour plates. We confirmed that our isolate of C. albicans contained a high-affinity EBP, with no detectable affinity for DES. Vaginal colonization by C. albicans was 8.6-fold greater in response to in vivo treatment with E(2) than with the comparable dose regimen of DES. The mechanism for estrogen-sensitive vaginal colonization by C. albicans includes a functional ligand-EBP interaction within the yeast.

Cloning and In Vitro Expression and Characterization of the Androgen Receptor and Isolation of Estrogen Receptor α from the Fathead Minnow (Pimephales promelas)

In vitro screening assays designed to identify hormone mimics or antagonists typically use mammalian (rat, human) estrogen (ER) and androgen receptors (AR). Although we know that the amino acid sequences of steroid receptors in nonmammalian vertebrates are not identical to the mammalian receptors, a great deal of uncertainty exists as to whether these differences affect interactions of potential endocrine-disrupting chemicals (EDC) with the receptors. This leads to substantial uncertainty with respect to the utility of mammalian-based screening assays to predict possible effects of EDCs in nonmammalian wildlife. This paper describes preparation of a cDNA library from a small fish model commonly used in ecological risk assessments, the fathead minnow (Pimphales promelas). The cDNA library was subsequently used to isolate and sequence both AR and ERalpha. In addition, the fathead minnow (fh)AR was expressed and characterized with respect to function using saturation and competitive binding assays in COS monkey kidney cells. Saturation experiments along with subsequent Scatchard analysis determined that the Kd of the fhAR for the potent synthetic androgen R1881 was 1.8 nM, which is comparable to that for the human AR in the same assay system. In COS whole cell competitive binding assays, potent androgens such as dihydrotestosterone and 11-ketotestosterone were also shown to be high affinity ligands for the fhAR. We also report affinity of the receptor for a number of environmental contaminants including the AR agonists androstenedione and 17a- and 17beta-trenbolone;AR antagonists such as p,p'-DDE, linuron, and vinclozolin; and the ER agonist 17beta-estradiol. Future plans include comparison of binding affinities of the fhAR to those of the human AR, also expressed in COS cells, using a range of EDCs.

Estrogen regulates pulmonary alveolar formation, loss, and regeneration in mice.

Lung tissue elastic recoil and the dimension and number of pulmonary gas-exchange units (alveoli) are major determinants of gas-exchange function. Loss of gas-exchange function accelerates after menopause in the healthy aged and is progressively lost in individuals with chronic obstructive pulmonary disease (COPD). The latter, a disease of midlife and later, though more common in men than in women, is a disease to which women smokers and never smokers may be more susceptible than men; it is characterized by diminished lung tissue elastic recoil and presently irremediable alveolar loss. Ovariectomy in sexually immature rats diminishes the formation of alveoli, and estrogen prevents the diminution. In the present work, we found that estrogen receptor-alpha and estrogen receptor-beta, the only recognized mammalian estrogen receptors, are required for the formation of a full complement of alveoli in female mice. However, only the absence of estrogen receptor-beta diminishes lung elastic tissue recoil. Furthermore, ovariectomy in adult mice results, within 3 wk, in loss of alveoli and of alveolar surface area without a change of lung volume. Estrogen replacement, after alveolar loss, induces alveolar regeneration, reversing the architectural effects of ovariectomy. These studies 1) reveal estrogen receptors regulate alveolar size and number in a nonredundant manner, 2) show estrogen is required for maintenance of already formed alveoli and induces alveolar regeneration after their loss in adult ovariectomized mice, and 3) offer the possibility estrogen can slow alveolar loss and induce alveolar regeneration in women with COPD.

Import and export of nuclear proteins: focus on the nucleocytoplasmic movements of two different species of mammalian estrogen receptor.

There is a wealth of information regarding the import and export of nuclear proteins in general. Nevertheless, the available data that deals with the nucleocytoplasmic movement of steroid hormone receptors remains highly limited. Some research findings reported during the past five years have succeeded in identifying proteins related to the movement of estrogen receptor alpha from the cytoplasm to the nucleus. What is striking in these findings is the facilitatory role of estradiol in the transport process. A similar conclusion has been drawn from the studies on the plasma membrane-to nucleus movement of the alternative form of estrogen receptor, the non-activated estrogen receptor (naER). The internalization of naER from the plasma membrane takes place only in the presence of estradiol. While the gene regulatory functions of ER alpha appear to get terminated following its ubiquitinization within the nucleus, the naER, through its deglycosylated form, the nuclear estrogen receptor II (nER II) continues to remain functional even beyond its existence within the nucleus. Recent studies have indicated the possibility that the estrogen receptor that regulates the nucleo cytoplasmic transport of m RNP is the nERII. This appears to be the result of the interaction between nERII and three proteins belonging to a group of small nuclear ribonucleo proteins (snRNP). The interaction of nERII with two of this protein appears to activate the inherent Mg2+ ATPase activity of the complex, which leads to the exit of the RNP through the nuclear pore complex.

Co-expression and Sequence Determination of Estrogen Receptor Variant Messenger RNAs in Swine Uterus

Steroid hormones and their receptors play an important role in reproductive process. Estrogen is intimately involved with pregnancy and its function is mediated through the estrogen receptor which has been chosen as a candidate gene to study litter size in pigs. In this study, we report that two estrogen receptor variants, designated pER-1 and pER-2 were co-expressed in the uteri of normal cycling Lan-Yu pig (Sus vittatus; a small-ear miniature in Taiwan) with the pER-1 expression level appeared to be several times higher than that of pER-2. These receptor variants were isolated using reverse transcription-PCR from the pig uteri and their sequences were determined. The pER-1 and pER-2 sequences, which are homologous to those found in other mammalian estrogen receptors, encode putative proteins consisting of 574 and 486 amino acids, respectively. A deletion in exon I was identified in both sequences, with deletion lengths of 63 bp in pER-1 and 327 bp in pER-2. The deletion in pER-1 is internal to that in pER-2 and both deletions resulted in a truncation of the B domain, which confers the transactivating activity of estrogen receptor protein. This result describes the existence of estrogen receptor variants with a deletion in exon I and implies the possibility that physiological functioning of an estrogen receptor may not require the presence of an intact B domain.

Development of a fish reporter gene system for the assessment of estrogenic compounds and sewage treatment plant effluents

This study reports on the development and application of a fish-specific estrogen-responsive reporter gene assay. The assay is based on the rainbow trout (Oncorhynchus mykiss) gonad cell line RTG-2 in which an acute estrogenic response is created by cotransfecting cultures with an expression vector containing rainbow trout estrogen receptor a complementary DNA (rtERalpha cDNA) in the presence of an estrogen-dependent reporter plasmid and an estrogen receptor (ER) agonist. In a further approach, RTG-2 cells were stably transfected with the rtERalpha cDNA expression vector, and clones responsive to 17beta-estradiol (E2) were selected. The estrogenic activity of E2, 17alpha-ethinylestradiol, 4-nonylphenol, nonylphenoxy acetic acid, 4-tert-octylphenol, bisphenol A, o,p'-DDT, p,p'-DDT, o,p'-2,2-bis(chlorophenyl)-1,1-dichloroethylene (o,p'-DDE), p,p'-DDE, o,p'-2,2-bis(chlorophenyl)-1,1-di-chloroethane (o,p'-DDD), p,p'-DDD, and p,p'-2,2-bis(chlorophenyl)acetic acid (p,p'-DDA) was assessed at increasing concentrations. All compounds except o,p'-DDT, p,p'-DDE, and p,p'-DDA showed logistic dose-response curves, which allowed the calculation of lowest-observed-effect concentrations and the concentrations at which half-maximal reporter gene activities were reached. To check whether estrogen-responsive RTG-2 cells may be used to detect the estrogenic activity of environmental samples, an extract from a sewage treatment plant (STP) effluent was assessed and found to have estrogenic activity corresponding to the transcriptional activity elicited by 0.05 nM of E2. Dose-response curves of nonylphenol, octylphenol, bisphenol A, and o,p'-DDD revealed that the RTG-2 reporter gene assay is more sensitive for these compounds when compared to transfection systems recombinant for mammalian ERs. These differences may have an effect on the calculation of E2 equivalents when estrogenic mixtures of known constitution, or environmental samples, such as STP effluents, are assessed.

Characterisation of the molecular responses to xenoestrogens using gene expression profiling

The two estrogen receptors (ERs), ERαand ERβ, are molecular targets of the xenoestrogen class of endocrine disruptors. These receptors are ligand-activated transcription factors regulated by naturally occurring estrogens. Activation of mammalian ERs by xenoestrogens has been associated with both adverse and beneficial effects. A more informed appraisal of the likely risks and benefits of xenoestrogens requires an understanding of their biological effects at the molecular level. Receptor binding and reporter-based transient transfection assays indicate that some compounds, including the phytoestrogens genistein and coumestrol, activate ERβpreferentially, suggesting that this receptor subtype may play a predominant role in mediating the effects of these compounds. We review recent advances in our understanding of the responses of mammalian cells to estrogenic compounds and describe an approach to decipher these effects at the molecular level.