Potential roles of nuclear receptors in mediating neurodevelopmental toxicity of known endocrine‐disrupting chemicals in ascidian embryos

Endocrine Disrupting Chemicals (EDCs) are molecules able to interfere with the vertebrate hormonal system in different ways, a major one being the modification of the activity of nuclear receptors (NRs). Several NRs are expressed in the vertebrate brain during embryonic development and these NRs are suspected to be responsible for the neurodevelopmental defects induced by exposure to EDCs in fishes or amphibians and to participate in several neurodevelopmental disorders observed in humans. Known EDCs exert toxicity not only on vertebrate forms of marine life but also on marine invertebrates. However, because hormonal systems of invertebrates are poorly understood, it is not clear whether the teratogenic effects of known EDCs are because of endocrine disruption. The most conserved actors of endocrine systems are the NRs which are present in all metazoan genomes but their functions in invertebrate organisms are still insufficiently characterized. EDCs like bisphenol A have recently been shown to affect neurodevelopment in marine invertebrate chordates called ascidians. Because such phenotypes can be mediated by NRs expressed in the ascidian embryo, we review all the information available about NRs expression during ascidian embryogenesis and discuss their possible involvement in the neurodevelopmental phenotypes induced by EDCs.     

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.     

An extended LXXLL motif sequence determines the nuclear receptor binding specificity of TRAP220

The interaction of coactivators with the ligand-binding domain of nuclear receptors (NRs) is mediated by amphipathic alpha-helices containing the signature motif LXXLL. TRAP220 contains two LXXLL motifs (LXM1 and LXM2) that are required for its interaction with NRs. Here we show that the nuclear receptor interaction domain (NID) of TRAP220 interacts weakly with Class I NRs. In contrast, SRC1 NID binds strongly to both Class I and Class II NRs. Interaction assays using nine amino acid LXXLL core motifs derived from SRC1 and TRAP220 revealed no discriminatory NR binding preferences. However, an extended LXM1 sequence containing amino acids -4 to +9, (where the first conserved leucine is +1) showed selective binding to thyroid hormone receptor and reduced binding to estrogen receptor. Replacement of either TRAP220 LXXLL motif with the corresponding 13 amino acids of SRC1 LXM2 strongly enhanced the interaction of the TRAP220 NID with the estrogen receptor. Mutational analysis revealed combinatorial effects of the LXM1 core and flanking sequences in the determination of the NR binding specificity of the TRAP220 NID. In contrast, a mutation that increased the spacing between TRAP220 LXM1 and LXM2 had little effect on the binding properties of this domain. Thus, a 13-amino acid sequence comprising an extended LXXLL motif acts as the key determinant of the NR binding specificity of TRAP220. Finally, we show that the NR binding specificity of full-length TRAP220 can be altered by swapping extended LXM sequences.     

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.     

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.     

Zebrafish (Danio rerio) androgen receptor: Sequence homology and up-regulation by the fungicide vinclozolin

Steroid hormones regulate gene expression in organisms by binding to receptor proteins. These hormones include the androgens, which signal through androgen receptors (ARs). Endocrine disrupters (EDCs) are chemicals in the environment that adversely affect organisms by binding to nuclear receptors, including ARs. Vinclozolin, a fungicide used on fruit and vegetable crops, is a known anti-androgen, a type of EDC that blocks signals from testosterone and its derivatives. In order to better understand the effects of EDCs, further research on androgen receptors and other hormone signaling pathways is necessary. In this study, we demonstrate the evolutionary conservation between the genomic structure of the human and zebrafish ar genes and find that ar mRNA expression increases in zebrafish embryos exposed to vinclozolin, which may be evolutionarily conserved as well. At 48 and 72 h post-fertilization, vinclozolin-treated embryos express ar mRNA 8-fold higher than the control level. These findings suggest that zebrafish embryos attempt to compensate for the presence of an anti-androgen by increasing the number of androgen receptors available.     

Monitoring Endocrine Disrupter Compounds in the Tunisian Hamdoun River using In Vitro Bioassays

Anthropogenic chemicals occurring in the environment, namely endocrine-disrupting chemicals (EDCs), have generated growing concern over their potential adverse effects on human wildlife health and ecosystem processes. This interest resulted particularly from their abilities to mimic the effect of endogenous hormones. In this study, we used stable transfected reporter cell lines to investigate the endocrine-disrupting profile of water as well as sediment samples. Samples are collected from up- and downstream of an industrial wastewater discharge point at the Hamdoun River in the vicinity of an industrial zone located at the center of Tunisia. The analysis of estrogen, androgen, and xenobiotic (pregnane X and dioxin) ligands receptors expressed by chimeric cell lines indicated that while the water and sediment samples from upstream sites have lower levels of estrogenic activity, those from downstream exhibited stronger estrogenic, aryl hydrocarbon receptor (AhR), and Pregnane X Receptor (PXR) activities. Moreover, collected samples have shown hormonal activity in terms of all tested receptors except the androgenic ones. In vitro recombinant estrogen receptor competitive binding assays revealed that while the estrogenic activities of the downstream water sample compounds had a strong affinity for estrogen receptor α (ERα), those present in the sediment samples showed a weaker one. These findings were consolidated by subsequent chemical analysis (high-performance liquid chromatography with UV detectors). Our results indicate that the water and sediment discharges at the Hamdoun River represent a major sink for EDCs from natural and industrial effluents, particularly those of the textile industry, with pernicious potential to disrupt normal endocrine functions.     

Non-traditional targets of endocrine disrupting chemicals: the roots of hormone signaling

The topic of endocrine disruption and the broad range of physiologicaleffects caused by endocrine disrupting chemicals (EDCs) canonly be meaningfully framed within an ecological and evolutionarycontext. Environmental pollutants and EDCs operate by disruptingthe "chemical communication" that coordinates signaling withinan organism. Here we discuss how EDCs are also able to disruptthe chemical communication between plants and soil bacterianecessary for initiating nitrogen-fixing symbiosis. We alsoexamine, through examples of pollutant-related impacts on awide range of invertebrates, the need for identifying emergingtargets of EDCs. We suggest broadening the defined field ofendocrine disruption to encompass the effects of synthetic chemicalsthat interfere with signaling and communication, not only withinan organism, but also between organisms and linking ecosystems.The ecological consequences of failing to recognize novel targetsof chemical pollutants and EDCs may be a net loss of biologicaldiversity and a further imbalance of the global nitrogen cycle.     

Neuroendocrine and behavioral implications of endocrine disrupting chemicals in quail

Studies in our laboratory have focused on endocrine, neuroendocrine, and behavioral components of reproduction in the Japanese quail. These studies considered various stages in the life cycle, including embryonic development, sexual maturation, adult reproductive function, and aging. A major focus of our research has been the role of neuroendocrine systems that appear to synchronize both endocrine and behavioral responses. These studies provide the basis for our more recent research on the impact of endocrine disrupting chemicals (EDCs) on reproductive function in the Japanese quail. These endocrine active chemicals include pesticides, herbicides, industrial products, and plant phytoestrogens. Many of these chemicals appear to mimic vertebrate steroids, often by interacting with steroid receptors. However, most EDCs have relatively weak biological activity compared to native steroid hormones. Therefore, it becomes important to understand the mode and mechanism of action of classes of these chemicals and sensitive stages in the life history of various species. Precocial birds, such as the Japanese quail, are likely to be sensitive to EDC effects during embryonic development, because sexual differentiation occurs during this period. Accordingly, adult quail may be less impacted by EDC exposure. Because there are a great many data available on normal development and reproductive function in this species, the Japanese quail provides an excellent model for examining the effects of EDCs. Thus, we have begun studies using a Japanese quail model system to study the effects of EDCs on reproductive endocrine and behavioral responses. In this review, we have two goals: first, to provide a summary of reproductive development and sexual differentiation in intact Japanese quail embryos, including ontogenetic patterns in steroid hormones in the embryonic and maturing quail. Second, we discuss some recent data from experiments in our laboratory in which EDCs have been tested in Japanese quail. The Japanese quail provides an excellent avian model for testing EDCs because this species has well-characterized reproductive endocrine and behavioral responses. Considerable research has been conducted in quail in which the effects of embryonic steroid exposure have been studied relative to reproductive behavior. Moreover, developmental processes have been studied extensively and include investigations of the reproductive axis, thyroid system, and stress and immune responses. We have conducted a number of studies, which have considered long-term neuroendocrine consequences as well as behavioral responses to steroids. Some of these studies have specifically tested the effects of embryonic steroid exposure on later reproductive function in a multigenerational context. A multigenerational exposure provides a basis for understanding potential exposure scenarios in the field. In addition, potential routes of exposure to EDCs for avian species are being considered, as well as differential effects due to stage of the life cycle at exposure to an EDC. The studies in our laboratory have used both diet and egg injection as modes of exposure for Japanese quail. In this way, birds were exposed to a specific dose of an EDC at a selected stage in development by injection. Alternatively, dietary exposure appears to be a primary route of exposure; therefore experimental exposure through the diet mimics potential field situations. Thus, experiments should consider a number of aspects of exposure when attempting to replicate field exposures to EDCs.     

In Vivo Screening Using Transgenic Zebrafish Embryos Reveals New Effects of HDAC Inhibitors Trichostatin A and Valproic Acid on Organogenesis

The effects of endocrine disrupting chemicals (EDCs) on reproduction are well known, whereas their developmental effects are much less characterized. However, exposure to endocrine disruptors during organogenesis may lead to deleterious and permanent problems later in life. Zebrafish (Danio rerio) transgenic lines expressing the green fluorescent protein (GFP) in specific organs and tissues are powerful tools to uncover developmental defects elicited by EDCs. Here, we used seven transgenic lines to visualize in vivo whether a series of EDCs and other pharmaceutical compounds can alter organogenesis in zebrafish. We used transgenic lines expressing GFP in pancreas, liver, blood vessels, inner ear, nervous system, pharyngeal tooth and pectoral fins. This screen revealed that four of the tested chemicals have detectable effects on different organs, which shows that the range of effects elicited by EDCs is wider than anticipated. The endocrine disruptor tetrabromobisphenol-A (TBBPA), as well as the three drugs diclofenac, trichostatin A (TSA) and valproic acid (VPA) induced abnormalities in the embryonic vascular system of zebrafish. Moreover, TSA and VPA induced specific alterations during the development of pancreas, an observation that was confirmed by in situ hybridization with specific markers. Developmental delays were also induced by TSA and VPA in the liver and in pharyngeal teeth, resulting in smaller organ size. Our results show that EDCs can induce a large range of developmental alterations during embryogenesis of zebrafish and establish GFP transgenic lines as powerful tools to screen for EDCs effects in vivo.     

Direct measure of fluorescence intensity for efficient receptor-binding assay: conjugates of ethinylcarboxyestradiol and 5(and 6)-carboxyfluorescein via alpha, omega-diaminoalkanes as a tracer for estrogen receptor

Steroidal nuclear receptors (NRs) have been acknowledged as a target binding protein of so-called endocrine disruptors. It is therefore necessary to develop an efficient assay system for screening these endocrine-disrupting chemicals. We here describe the first exemplification of a direct measure of fluorescence intensity for a binding assay of NRs. We designed and synthesized a series of conjugates of 17alpha-ethinylcarboxyestradiol with carboxyfluorescein, both carboxyl groups of which were cross-linked with alpha,omega-diaminoalkanes. The resulting fluorescein-linked estradiol derivatives E2(n)cF (n=2, 4, 6, 8, 10 and 12) were evaluated for their fluorescence and receptor-binding characteristics. E2(4)cF and E2(8)cF exhibited the sufficient binding affinity to the recombinant estrogen receptor (ER) in the radiolabel binding assay using [(3)H]17beta-estradiol, and showed excellent fluorescent characteristics in the fluorescence measurements with and without ER. They exhibited sufficiently large specific binding characteristics with adequate K(d)- and B(max)-values. When these fluorescent ligands were used as a tracer for the binding assay against the ER, assay data of various compounds were shown to be compatible with those obtained from the ordinary binding assay using [(3)H]17beta-estradiol. The present study clearly shows that measurement of fluorescence intensity, instead of fluorescence polarization, affords an adequate receptor-binding assay system.     

Effects of possible endocrine disruptors on MyD88-independent TLR4 signaling

Endocrine disrupting chemicals (EDCs) may potentially worsen infectious diseases because EDCs disturb human immune function by interfering with endocrine balance. To evaluate the influence of EDCs on the innate immune function of macrophages, we investigated the effects of 37 possible EDCs on lipopolysaccharide-induced activation of the IFN-beta promoter. Alachlor, atrazine, benomyl, bisphenol A, carbaryl, diethyl phthalate, dipropyl phthalate, kelthane, kepone, malathion, methoxychlor, octachlorostyrene, pentachlorophenol, nonyl phenol, p-octylphenol, simazine and ziram all inhibited the activation. Kepone and ziram showed strong inhibitory effects. Aldicarb, amitrole, benzophenone, butyl benzyl phthalate, 2,4-dichlorophenoxy acetic acid, dibutyl phthalate, 2,4-dichlorophenol, dicyclohexyl phthalate, diethylhexyl adipate, diethylhexyl phthalate, dihexyl phthalate, di-n-pentyl phthalate, methomyl, metribuzin, nitrofen, 4-nitrotoluene, permethrin, trifluralin, 2,4,5-trichlorophenoxyacetic acid and vinclozolin had no significant effects at 100 muM. These results indicate that some agrochemicals and resin-related chemicals may potentially inhibit macrophage function, which suggests that endocrine disruptors may influence the development of infectious diseases.     

Endocrine Toxicants and Reproductive Success in Fish

There is compelling evidence on a global scale for compromised growth and reproduction, altered development, and abnormal behaviour in feral fish that can be correlated or in some cases causally linked with exposure to endocrine disrupting chemicals (EDCs). Attributing cause and effect relationships for EDCs is a specific challenge for studies with feral fish as many factors including food availability, disease, competition and loss of habitat also affect reproduction and development. Even in cases where there are physiological responses of fish exposed to EDCs (e.g., changes in reproductive hormone titres, vitellogenin levels), the utility of these measures in extrapolating to whole animal reproductive or developmental outcomes is often limited. Although fish differ from other vertebrates in certain aspects of their endocrinology, there is little evidence that fish are more sensitive to the effects of EDCs. Therefore, to address why endocrine disruption seems so widespread in fish, it is necessary to consider aspects of fish physiology and their environment that may increase their exposure to EDCs. Dependence on aquatic respiration, strategies for iono-osmotic regulation, and maternal transfer of contaminants to eggs creates additional avenues by which fish are exposed to EDCs. This paper provides an overview of responses observed in feral fish populations that have been attributed to EDCs and illustrates many of the factors that need consideration in evaluating the risks posed by these chemicals.