Double evaluations of chemicals using a cocktail of fused recombinant receptors

Some chemicals have multipotential as endocrine-disrupting chemicals (EDCs). For example, some chemicals act as both estrogens and antiandrogens. Numbers of such chemicals should be evaluated from many aspects; however, labor and expenses are generally limited. We have developed two expression systems for the wild type of human estrogen receptor alpha and the wild type of human androgen receptor fused with a maltose binding protein. They are soluble and have binding activities. They showed dose-responses to natural hormones and well-known potential EDCs. After we established each assay condition for a competitive binding assay using each receptor, we found that two assay systems can be carried out simultaneously under limited and harmonized conditions. Under harmonized conditions using a cocktail of two types of receptors, we could estimate natural hormones and potential EDCs. Interference between two assay systems was not observed under these conditions. We believe that some competitive binding assays can be carried out using a cocktail of receptors at the same time if interference among different assay systems can be avoided by choosing ideal conditions.     

Structural and functional evidences for the interactions between nuclear hormone receptors and endocrine disruptors at low doses

Endocrine-disrupting chemicals (EDCs) represent a broad class of exogenous substances that cause adverse effects in the endocrine system mainly by interacting with nuclear hormone receptors (NRs). Humans are generally exposed to low doses of pollutants, and current researches aim at deciphering the mechanisms accounting for the health impact of EDCs at environmental concentrations. Our correlative analysis of structural, interaction and cell-based data has revealed a variety of, sometimes unexpected, binding modes, reflecting a wide range of EDC affinities and specificities. Here, we present a few representative examples to illustrate various means by which EDCs achieve high-affinity binding to NRs. These examples include the binding of the mycoestrogen α-zearalanol to estrogen receptors, the covalent interaction of organotins with the retinoid X- and peroxisome proliferator-activated receptors, and the cooperative binding of two chemicals to the pregnane X receptor. We also discuss some hypotheses that could further explain low-concentration effects of EDCs with weaker affinity towards NRs.     

Endocrine disruptors: present issues, future directions

A variety of natural products and synthetic chemicals, known collectively as endocrine-disrupting compounds (EDCs), mimic or interfere with the mechanisms that govern vertebrate reproductive development and function. At present, research has focused on (i) the morphological and functional consequences of EDCs; (ii) identifying and determining the relative potencies of synthetic and steroidal compounds that have endocrine-disrupting effects; (iii) the mechanism of action of EDCs at the molecular level; and (iv) the recognition that in "real life," contamination usually reflects mixtures of EDCs. Future research must examine (i) the interactive nature of EDCs, particularly whether the threshold concept as developed in traditional toxicological research applies to these chemicals; (ii) when and how EDCs act at the physiological level, particularly how they may organize the neural substrates of reproductive physiology and behavior; (iii) the various effects these compounds have on different species, individuals, and even tissues; and (iv) how adaptations may evolve in natural populations with continued exposure to EDCs. Several predictions are offered that reflect these new perspectives. Specifically, (i) the threshold assumption will be found not to apply to EDCs because they mimic the actions of endogenous molecules (e.g., estrogen) critical to development; hence, the threshold is automatically exceeded with exposure. (ii) Behavior can compound and magnify the effects of EDCs over successive generations; that is, bioaccumulated EDCs inherited from the mother not only influence the morphological and physiological development of the offspring but also the offsprings' reproductive behavior as adults. This adult behavior, in turn, can have further consequences on the sexual development of their own young. (iii) The sensitivity of a species or an individual to a compound is related to species (individual)-typical concentrations of circulating gonadal steroid hormones. Related to this is the recent finding that alternate forms of the putative receptors are differentially distributed, thereby contributing to the different effects that have been observed. (iv) Except in extraordinary situations, populations often continue to exist in contaminated sites. One possible explanation for this observation that needs to be considered is that animals can rapidly adapt to the nature and level of contamination in their environment. It is unlikely that successive generations coincidentally become insensitive to gonadal steroid hormones fundamentally important as biological regulators of development and reproduction. Rather, adaptive alterations in the genes that encode steroid receptors may occur with chronic exposure to EDCs, allowing the sex hormone receptor to discriminate natural steroids from EDCs.     

An assessment of the presence and health risks of endocrine-disrupting chemicals in the drinking water treatment plant of Wu Chang,China

An assessment of the presence and health risks of endocrine-disrupting chemicals in the drinking water treatment plant (DWTP) of Wu Chang was performed. A recombinant yeast assay was used to assess the endocrine disrupting activity of the effluents of the DWTP. Agonistic activities of the estrogen receptor and androgen receptor were not detected in any of the effluent samples. However, anti-estrogenic and anti-androgenic activities were observed. In addition, the removal rates of the DWTP for the anti-androgenic activities were limited. A health risk assessment was performed on the basis of the results of the recombinant yeast assay, and the total daily production of hormones was used to evaluate the health risks of these types of endocrine-disrupting chemicals. The predicted effects of the anti-estrogenic and anti-androgenic disrupting activities were below 1.5%. This study suggested that the combined toxicity bioassays with health risk assessment could provide an available method to assess endocrine-disrupting chemicals and to evaluate the potential adverse effects on human health for aquatic environmental samples.     

Development of methods for extraction and in vitro quantification of estrogenic and androgenic activity of wastewater samples

Chemicals released into the environment by anthropogenic activities have been linked to estrogenic or androgenic effects in exposed wildlife, and there is a need to develop and validate rapid and cost-effective methods to quantify the total estrogenic and androgenic activity of environmental water samples. In this study, estrogen receptors (ER) were isolated from sheep (Ovis aries) uteri and rainbow trout (Oncorhynchus mykiss) livers and androgen receptors (AR) were isolated from rainbow trout brains. The isolated receptors were used in competitive receptor binding assays to test the affinity of known estrogenic and androgenic chemicals for the receptor binding site, and results were compared with literature values for the rat uterine ER binding assay and the E-Screen. The relative binding affinities of the tested compounds to ER from different species were similar, and binding to the ER was a more responsive endpoint than the cellular effect measured in the E-Screen. Using the sheep ER binding assay in combination with solid-phase extraction, the estrogenic activity in a raw sewage sample from a municipal treatment plant in Brisbane (Queensland, Australia) was measured at 51-73 ng/L estradiol equivalents (EEq).     

Identification of an estrogenic hormone receptor in Caenorhabditis elegans

Changes in both behavior and gene expression occur in Caenorhabditis elegans following exposure to sex hormones such as estrogen and progesterone, and to bisphenol A (BPA), an estrogenic endocrine-disrupting compound. However, only one steroid hormone receptor has been identified. Of the 284 known nuclear hormone receptors (NHRs) in C. elegans, we selected nhr-14, nhr-69, and nhr-121 for analysis as potential estrogenic hormone receptors, because they share sequence similarity with the human estrogen receptor. First, the genes were cloned and expressed in Escherichia coli, and then the affinity of each protein for estrogen was determined using a surface plasmon resonance (SPR) biosensor. All three NHRs bound estrogen in a dose-dependent fashion. To evaluate the specificity of the binding, we performed a solution competition assay using an SPR biosensor. According to our results, only NHR-14 was able to interact with estrogen. Therefore, we next examined whether nhr-14 regulates estrogen signaling in vivo. To investigate whether these interactions actually control the response of C. elegans to hormones, we investigated the expression of vitellogenin, an estrogen responsive gene, in an nhr-14 mutant. Semi-quantitative RT-PCR showed that vitellogenin expression was significantly reduced in the mutant. This suggests that NHR-14 is a C. elegans estrogenic hormone receptor and that it controls gene expression in response to estrogen.     

Glutamate antagonism fails to reverse mitochondrial dysfunction in late phase of experimental neonatal asphyxia in rats

Because estrogen plays important neurotrophic and neuroprotective roles in the brain by activating estrogen receptors (ERs), disruption of normal estrogen signaling can leave neurons vulnerable to a variety of insults, including β-amyloid peptide (Aβ). Aroclor1254 (A1254) belongs to the endocrine-disrupting chemical (EDC) polychlorinated biphenyls and has anti-estrogenic properties. In the present study, we evaluated the effect of A1254 on the protective activity of estrogen against Aβ toxicity in differentiated cholinergic SN56 cells. Aged Aβ25-35 causes apoptotic cell death in differentiated SN56 cells, and the cytotoxic evidences are effectively rescued by estrogen. We found that A1254 abolishes the neuroprotective activity of estrogen against Aβ toxicity, and attenuates the suppressive effect of estrogen on Aβ-induced tau phosphorylation and JNK activation. The effects of A1254 on the neuroprotective effects of estrogen in Aβ toxicity are very similar to the effects of the estrogen receptor antagonist ICI182,780. Thus, exposure to EDCs that have anti-estrogenic activity might interfere with normal estrogen-activated neuroprotective signaling events and leave neurons more vulnerable to dangerous stimuli. Our present results provide new understanding of the mechanisms contributing to the harmful effects of EDCs on the function and viability of neurons, and the possible relevance of EDCs in the pathogenesis of neurodegenerative diseases such as Alzheimer’s disease.     

Molecular mechanism(s) of endocrine‐disrupting chemicals and their potent oestrogenicity in diverse cells and tissues that express oestrogen receptors

Endocrine‐disrupting chemicals (EDCs) are natural or synthetic compounds present in the environment which can interfere with hormone synthesis and normal physiological functions of male and female reproductive organs. Most EDCs tend to bind to steroid hormone receptors including the oestrogen receptor (ER), progesterone receptor (PR) and androgen receptor (AR). As EDCs disrupt the actions of endogenous hormones, they may induce abnormal reproduction, stimulation of cancer growth, dysfunction of neuronal and immune system. Although EDCs represent a significant public health concern, there are no standard methods to determine effect of EDCs on human beings. The mechanisms underlying adverse actions of EDC exposure are not clearly understood. In this review, we highlighted the toxicology of EDCs and its effect on human health, including reproductive development in males and females as shown in in vitro and in vivo models. In addition, this review brings attention to the toxicity of EDCs via interaction of genomic and non‐genomic signalling pathways through hormone receptors.     

Effects of municipal effluents on serotonin and dopamine levels in the freshwater mussel Elliptio complanata

Sex differentiation and gametogenesis represent critical steps in the reproductive process and are subject to hormonal control by serotonin, dopamine and steroids such as estradiol-17beta and testosterone. The purpose of this study sought to examine the endocrine-disrupting activity that a primary-treated municipal effluent might have on the metabolism of biogenic amine levels. First, serotonin receptors transfected in Chinese hamster ovary (CHO) cells were used to screen for the presence of serotonin receptor agonist or antagonist. Second, one group of Elliptio complanata mussels were exposed to single compounds likely to be found in municipal wastewaters and another group was exposed in situ to the municipal effluent plume for 90 days in experimental cages. Results showed that solid phase C-8 extracts of surface water downstream a municipal effluent could activate the transport of serotonin by receptors at a distance of at least 5 km from its outfall thereby indicating the presence of serotonin mimics in the effluent dispersion plume. Levels of serotonin and monoamine oxidase (MAO) activity in nerve ganglia of mussels exposed for 90 days to the municipal effluent were, respectively, reduced and increased at a distance 10-km downstream. Injections of estradiol-17beta and nonylphenol in mussels decreased the levels of serotonin and dopamine, but increased MAO activity in the gonad and nerve ganglia. Exposure to estrogenic chemicals present in municipal effluents may therefore alter the normal metabolism of serotonin and dopamine, both of which are involved in sexual differentiation in bivalves and fish. Chemicals acting through E2 receptor-mediated pathways and serotonin receptors are likely to cause the observed effects.     

Nonylphenol modulates expression of androgen receptor and estrogen receptor genes differently in gender types of the hermaphroditic fish Rivulus marmoratus

To uncover the effect of estrogenic chemicals [4-nonylphenol (NP) and bisphenol A (BisA)] on the expression of androgen receptor (AR) and estrogen receptors (ERalpha and ERbeta) in the hermaphroditic fish Rivulus marmoratus, we cloned the full length of the cDNAs encoding AR, ERalpha, and ERbeta from gonadal tissue of R. marmoratus and analyzed the modulation of expression of these genes following exposure to estrogenic chemicals using real-time RT-PCR. R. marmoratus AR, ERalpha, and ERbeta genes showed a high similarity to the relevant fish species on amino acid residues, respectively. Rm-ERalpha and Rm-ERbeta cDNAs included a serine-rich region when compared to other teleost fish ER genes. Tissue-specific expression of Rm-AR and Rm-ERbeta mRNAs in adult hermaphrodite R. marmoratus was high in the gonad, while Rm-ERalpha mRNA was high in the liver based on real-time RT-PCR. In addition, Rm-AR and Rm-ERalpha mRNAs increased along with developmental stage from stage 3 (5 dpf) to hatching, while Rm-ERbeta mRNA increased from stage 2 (2 dpf). To uncover the effect of estrogenic chemicals on R. marmoratus, we exposed the fish to NP (300 microg/l) and BisA (600 microg/l) for 96 h. Significant down-regulation of Rm-AR, Rm-ERalpha, and Rm-ERbeta mRNA was observed in gonadal tissue after exposure to NP but not BisA. In the liver, there were gender differences in gene expression after EDC exposure. These results demonstrate that expression patterns of the Rm-AR, Rm-ERalpha, and Rm-ERbeta genes in the hermaphroditic fish, R. marmoratus, vary according to tissue and developmental stage as well as the specificity of environmental estrogenic chemicals. These genes can be useful as molecular biomarkers in assessing the potential impact of estrogenic compounds using this species as a model system.     

Exposure to Paper Mill Effluent at a Site in North Central Florida Elicits Molecular-Level Changes in Gene Expression Indicative of Progesterone and Androgen Exposure

Endocrine disrupting compounds (EDCs) are chemicals that negatively impact endocrine system function, with effluent from paper mills one example of this class of chemicals. In Florida, female Eastern mosquitofish (Gambusia holbrooki) have been observed with male secondary sexual characteristics at three paper mill-impacted sites, indicative of EDC exposure, and are still found at one site on the Fenholloway River. The potential impacts that paper mill effluent exposure has on the G. holbrooki endocrine system and the stream ecosystem are unknown. The objective of this study was to use gene expression analysis to determine if exposure to an androgen receptor agonist was occurring and to couple this analysis with in vitro assays to evaluate the presence of androgen and progesterone receptor active chemicals in the Fenholloway River. Focused gene expression analyses of masculinized G. holbrooki from downstream of the Fenholloway River paper mill were indicative of androgen exposure, while genes related to reproduction indicated potential progesterone exposure. Hepatic microarray analysis revealed an increase in the expression of metabolic genes in Fenholloway River fish, with similarities in genes and biological processes compared to G. holbrooki exposed to androgens. Water samples collected downstream of the paper mill and at a reference site indicated that progesterone and androgen receptor active chemicals were present at both sites, which corroborates previous chemical analyses. Results indicate that G. holbrooki downstream of the Fenholloway River paper mill are impacted by a mixture of both androgens and progesterones. This research provides data on the mechanisms of how paper mill effluents in Florida are acting as endocrine disruptors.     

MCX based solid phase extraction combined with liquid chromatography tandem mass spectrometry for the simultaneous determination of 31 endocrine-disrupting compounds in surface water of Shanghai

A novel analytical method employing MCX (mixed-mode cationic exchange) based solid phase extraction (SPE) coupled with liquid chromatography tandem mass spectrometry (LC-MS/MS) was developed to detect 31 endocrine-disrupting compounds (EDCs) in surface water samples simultaneously. The target EDCs belong to five classes, including seven estrogens, eight androgens, six progesterones, five adrenocortical hormones and five industrial compounds. In order to simultaneously concentrate the target EDCs and eliminate matrix interferences in the water samples, MCX SPE cartridges were employed for SPE, and then followed by a simple and highly efficient three-step sequential elution procedure. Two electrospray ionization (ESI) detection modes, positive (ESI+) and (ESI-), were optimized for HPLC-MS/MS analysis to obtain the highest sensitivity for all the EDCs. The limits of detection (LODs) were 0.02-1.9 ng L(-1), which are lower than or comparable to these reported in references. Wide linear ranges (LOD-100 ng L(-1) for ESI+ mode, and LOD-200 ng L(-1) for ESI- mode) were obtained with determination coefficients (R(2)) higher than 0.99 for all the compounds. With five internal standards, good recoveries (84.4-103.0%) of all the target compounds were obtained in selected surface water samples. The developed method was successfully applied to investigate the EDCs occurrence in the surface water of Shanghai by analyzing surface water samples from 11 sites. The results showed that nearly all the target compounds (30 in 31) were present in the surface water samples of Shanghai, of which three industrial compounds (4-t-OP, BPA, and BPF) showed the highest concentrations (median concentrations were 11.88-23.50 ng L(-1)), suggesting that industrial compounds were the dominating EDCs in the surface water of Shanghai, and much more attention should be paid on these compounds. Our present research demonstrated that SPE with MCX cartridges combined with HPLC-MS/MS was convenient, efficient and reliable for multiclass analysis of EDCs in surface water.     

Homology-modeled ligand-binding domains of medaka estrogen receptors and androgen receptors: A model system for the study of reproduction

Estrogen and androgen and their receptors play critical roles in physiological processes such as sexual differentiation and development. Using the available structural models for the human estrogen receptors alpha and beta and androgen receptor as templates, we designed in silico agonist and antagonist models of medaka estrogen receptor (meER) alpha, beta-1, and beta-2, and androgen receptor (meAR) alpha and beta. Using these models, we studied (1) the structural relationship between the ligand-binding domains (LBDs) of ERs and ARs of human and medaka, and (2) whether medaka ER and AR can be potential models for studying the ligand-binding activities of various agonists and antagonists of these receptors by docking analysis. A high level of conservation was observed between the sequences of the ligand-binding domains of meERα and huERα, meERβ1 and huERβ, meERβ2, and huERβ with 62.8%, 66.4%, and 65.1% identity, respectively. The sequence conservation between meARα and huAR, meARβ, and huAR was found with 70.1% and 61.0% of identity, respectively. Thirty-three selected endocrine disrupting chemicals (EDCs), including both agonists and antagonists, were docked into the LBD of ER and AR, and the corresponding docking score for medaka models and human templates were calculated. In order to confirm the conservation of the overall geometry and the binding pocket, the backbone root mean square deviation (RMSD) for Cα atoms was derived from the structure superposition of all 10 medaka homology models to the six human templates. Our results suggested conformational conservation between the ERs and ARs of medaka and human, Thus, medaka could be highly useful as a model system for studies involving estrogen and androgen interaction with their receptors.