Transgenic Zebrafish Reveal Tissue-Specific Differences in Estrogen Signaling in Response to Environmental Water Samples

Background: Environmental endocrine disruptors (EEDs) are exogenous chemicals that mimic endogenous hormones such as estrogens. Previous studies using a zebrafish transgenic reporter demonstrated that the EEDs bisphenol A and genistein preferentially activate estrogen receptors (ERs) in the larval heart compared with the liver. However, it was not known whether the transgenic zebrafish reporter was sensitive enough to detect estrogens from environmental samples, whether environmental estrogens would exhibit tissue-specific effects similar to those of BPA and genistein, or why some compounds preferentially target receptors in the heart.Methods: We tested surface water samples using a transgenic zebrafish reporter with tandem estrogen response elements driving green fluorescent protein expression (5xERE:GFP). Reporter activation was colocalized with tissue-specific expression of ER genes by RNA in situ hybridization.Results: We observed selective patterns of ER activation in transgenic fish exposed to river water samples from the Mid-Atlantic United States, with several samples preferentially activating receptors in embryonic and larval heart valves. We discovered that tissue specificity in ER activation was due to differences in the expression of ER subtypes. ERα was expressed in developing heart valves but not in the liver, whereas ERβ2 had the opposite profile. Accordingly, subtype-specific ER agonists activated the reporter in either the heart valves or the liver.Conclusion: The use of 5xERE:GFP transgenic zebrafish revealed an unexpected tissue-specific difference in the response to environmentally relevant estrogenic compounds. Exposure to estrogenic EEDs in utero was associated with adverse health effects, with the potentially unanticipated consequence of targeting developing heart valves.Citation: Gorelick DA, Iwanowicz LR, Hung AL, Blazer VS, Halpern ME. 2014. Transgenic zebrafish reveal tissue-specific differences in estrogen signaling in response to environmental water samples. Environ Health Perspect 122:356–362; http://dx.doi.org/10.1289/ehp.1307329     

Endocrine-Disrupting Chemical Exposure and the American Alligator: A Review of the Potential Role of Environmental Estrogens on the Immune System of a Top Trophic Carnivore

Endocrine-disrupting chemicals (EDCs) alter cellular and organ system homeostasis by interfering with the body’s normal physiologic processes. Numerous studies have identified environmental estrogens as modulators of EDC-related processes in crocodilians, notably in sex determination. Other broader studies have shown that environmental estrogens dysregulate normal immune function in mammals, birds, turtles, lizards, fish, and invertebrates; however, the effects of such estrogenic exposures on alligator immune function have not been elucidated. Alligators occupy a top trophic status, which may give them untapped utility as indicators of environmental quality. Environmental estrogens are also prevalent in the waters they occupy. Understanding the effects of these EDCs on alligator immunity is critical for managing and assessing changes in their health and is thus the focus of this review.     

Chemical communication threatened by endocrine-disrupting chemicals

Communication on a cellular level--defined as chemical signaling, sensing, and response--is an essential and universal component of all living organisms and the framework that unites all ecosystems. Evolutionarily conserved signaling "webs," existing both within an organism and between organisms, rely on efficient and accurate interpretation of chemical signals by receptors. Therefore, endocrine-disrupting chemicals (EDCs), which have been shown to disrupt hormone signaling in laboratory animals and exposed wildlife, may have broader implications for disrupting signaling webs that have yet to be identified as possible targets. In this article, I explore common evolutionary themes of chemical signaling (e.g., estrogen signaling in vertebrates and phytoestrogen signaling from plants to symbiotic soil bacteria) and show that such signaling systems are targets of disruption by EDCs. Recent evolutionary phylogenetic data have shown that the estrogen receptor (ER) is the ancestral receptor from which all other steroid receptors have evolved. In addition to binding endogenous estrogens, ERs also bind phytoestrogens, an ability shared in common with nodulation D protein (NodD) receptors found in Rhizobium soil bacteria. Recent data have shown that many of the same synthetic and natural environmental chemicals that disrupt endocrine signaling in vertebrates also disrupt phytoestrogen-NodD receptor signaling in soil bacteria, which is necessary for nitrogen-fixing symbiosis. Bacteria-plant symbiosis is an unexpected target of EDCs, and other unexpected nontarget species may also be vulnerable to EDCs found in the environment.     

Health impacts of estrogens in the environment, considering complex mixture effects

BACKGROUND: Environmental estrogens in wastewater treatment work (WwTW) effluents are well established as the principal cause of reproductive disruption in wild fish populations, but their possible role in the wider health effects of effluents has not been established. OBJECTIVES: We assessed the contribution of estrogens to adverse health effects induced in a model fish species by exposure to WwTW effluents and compared effects of an estrogen alone and as part of a complex mixture (i.e., spiked into effluent). METHODS: Growth, genotoxic, immunotoxic, metabolic, and endocrine (feminized) responses were compared in fathead minnows (Pimephales promelas) exposed for 21 days to a potent estrogenic effluent, a weakly estrogenic effluent before and after spiking with a steroidal estrogen [17 alpha-ethinyl-estradiol (EE2)], and to EE2 alone. RESULTS: In addition to endocrine disruption, effluent exposure induced genotoxic damage, modulated immune function, and altered metabolism; many of these effects were elicited in a sex-specific manner and were proportional to the estrogenic potencies of the effluents. A key finding was that some of the responses to EE2 were modified when it was present in a complex mixture (i.e., spiked into effluent), suggesting that mixture effects may not be easily modeled for effluent discharges or when the chemicals impact on a diverse array of biological axes. CONCLUSION: These data reveal a clear link between estrogens present in effluents and diverse, adverse, and sex-related health impacts. Our findings also highlight the need for an improved understanding of interactive effects of chemical toxicants on biological systems for understanding health effects of environmental mixtures.     

斑马鱼及其在环境毒理学中的应用

斑马鱼是一种热带淡水鱼,饲养简单,管理方便,易于获取.斑马鱼常年产卵,世代周期短,一般3～4个月就能达到性成熟.其胚胎透明,并且在体外发育,易于观察.长期以来斑马鱼一直被广泛地运用于发育生物学和毒理学的研究中.近年来,众多研究显示了斑马鱼应用于环境毒理学研究中有其独特的优势,并已逐渐成为环境毒理学家们的新宠.利用转基因斑马鱼对污染物进行快速监测已成为目前研究的热点.     

An environmentally relevant concentration of estrogen induces arrest of male gonad development in zebrafish, Danio rerio

The aim of the present study was to elucidate how full life-cycle exposure to estrogens impacts zebrafish development and reproduction, compared to partial life-cycle exposure only, and whether the estrogen-induced effects in zebrafish are reversible or irreversible. Zebrafish were exposed in a flow-through system to an environmentally relevant concentration (3 ng/L) of the synthetic estrogen 17alpha-ethinylestradiol (EE2) either from fertilization until the all-ovary stage of gonad development (i.e., 42 d postfertilization [DPF] in our experiment) or from fertilization until the reproductive stage (i.e., 118 DPF). Reversibility of the estrogen-induced effects was assessed after 58 d of depuration in EE2-free water until 176 DPE Early life exposure led to a lasting induction of plasma vitellogenin (VTG) in adult females but altered neither the sex ratio nor the reproductive capabilities. Full life-cycle exposure resulted in elevated VTG concentrations and caused gonadal feminization in 100% of exposed fish and thus inhibited reproduction. Two types of ovaries were observed in continuously exposed adult fish, immature ovaries with primary growth stage oocytes only and mature ovaries containing the full range of all oocyte maturation stages. Fish with immature ovaries had plasma VTG levels like control males, while fish with mature ovaries had female-like VTG levels. The effects of full life cycle exposure were at least partly reversible, and 26% of fish of the previous all-female cohort developed fully differentiated testes. These findings suggest that continuous estrogen exposure had arrested the developmental transition of the gonads of genetic males from the early all-ovary stage to functional testes. After the exposure had ceased, however, these males apparently were able to accomplish testicular differentiation.     

Phytoestrogen signaling and symbiotic gene activation are disrupted by endocrine-disrupting chemicals

Some organochlorine pesticides and other synthetic chemicals mimic hormones in representatives of each vertebrate class, including mammals, reptiles, amphibians, birds, and fish. These compounds are called endocrine-disrupting chemicals (EDCs). Similarly, hormonelike signaling has also been observed when vertebrates are exposed to plant chemicals called phytoestrogens. Previous research has shown the mechanism of action for EDCs and phytoestrogens is as unintended ligands for the estrogen receptor (ER). Although pesticides have been synthesized to deter insects and weeds, plants produce phytoestrogens to deter herbivores, as attractant cues for insects, and as recruitment signals for symbiotic soil bacteria. Our data present the first evidence that some of the same organochlorine pesticides and EDCs known to disrupt endocrine signaling through ERs in exposed wildlife and humans also disrupt the phytoestrogen signaling that leguminous plants use to recruit Sinorhizobium meliloti soil bacteria for symbiotic nitrogen fixation. Here we report that a variety of EDCs and pesticides commonly found in agricultural soils interfere with the symbiotic signaling necessary for nitrogen fixation, suggesting that the principles underlying endocrine disruption may have more widespread biological and ecological importance than had once been thought.     

The role of estrogens in cardiovascular disease in the aftermath of clinical trials

The effects of estrogens on reproductive tissues and climacteric symptoms are unambiguous. However, their effects on other tissues and, in particular, the cardiovascular system remain controversial. In general, premenopausal women are protected from coronary heart disease (CHD) compared with aged-matched men but this ;female protection' appears to be lost after menopause, suggesting beneficial effects of female sex hormones on the cardiovascular system. This view has been supported by observational studies showing that estrogen replacement therapy (ERT) is associated with a 30% to 50% decrease in CHD risk in postmenopausal women. Nevertheless, randomized clinical trials in postmenopausal women, with or without pre-existing CHD, have found no benefits to combined hormone replacement therapy (HRT). A possible explanation for these apparently contradictory findings may relate to the divergent effects of estrogens depending on the state of the vascular endothelium. It has been suggested that estrogens may prevent the development and early progression of atherosclerosis by contributing to the maintenance of endothelial health but may have a neutral or negative effect on a dysfunctional endothelium or on advanced atheromatous lesions. Furthermore, it is possible that estrogens have adverse effects on other cardiovascular risk factors such as thrombosis and inflammation. It is also conceivable that a decline in vascular estrogen receptor expression with advancing age, through methylation of the estrogen receptor promoter, prevents postmenopausal women from deriving the beneficial cardiovascular effects of estrogens. In conclusion, estrogens possibly prevent the development of atherosclerosis through favourable effects on an intact endothelium, but once the vascular endothelium is damaged, the prothrombotic and possibly proinflammatory effects of estrogens are likely to predominate and prove harmful.     

Oxidative stress in zebrafish embryos induced by short-term exposure to bisphenol A, nonylphenol, and their mixture

Bisphenol A (BPA) and nonylphenol (NP) are well-known endocrine-disrupting chemicals (EDCs) present in the aquatic environment, but little is known about their oxidative stress effects on fish embryos. In the present study, we examined the oxidative stress indices and antioxidant parameters of zebrafish embryos after a short-term exposure to various concentrations of BPA, NP, and their mixture (BPA-NP) for 4 h postfertilization (hpf) to 168 hpf. Exposure to the chemicals was found to enhance the production of hydroxyl radicals and lipid peroxidation in a concentration-dependent manner. The content of total glutathione (TG), reduced glutathione (GSH), and oxidized glutathione (GSSH), as well as the activity of antioxidant enzymes including catalase, superoxide dismutase, glutathione peroxidase, glutathione reductase, and glutathione-S-transferase were all significantly inhibited after exposure to BPA, NP, and BPA-NP, indicating the occurrence of oxidative stress. Coexposure to BPA-NP resulted in an additive effect on some antioxidant parameters. In addition, the alkaline phosphatase activity was also significantly inhibited after exposure to BPA, NP, and their mixtures. Our results demonstrated that BPA, NP, and BPA-NP in aquatic systems can affect antioxidant responses in zebrafish embryos.     

Identification of endocrine-disrupting effects in aquatic vertebrates and invertebrates: report from the European IDEA project

The EU-funded project IDEA aimed to evaluate (a) what parameters and endpoints allow the detection of endocrine-mediated developmental and reproductive effects of (xeno)estrogens in life cycle- and life stage-specific toxicity tests with the zebrafish Danio rerio, a small laboratory fish used in many ecotoxicity test guidelines, and (b) whether substances that act as estrogens in vertebrates may also adversely affect the development, differentiation, and reproduction of aquatic invertebrates. The invertebrate species investigated included Hydra vulgaris, Gammarus pulex, Chironomus riparius, Hyalella azteca, and Lymnaea stagnalis. The animals were exposed to the model estrogenic chemicals ethynylestradiol (EE2), bisphenol A (BPA), and octylphenol (OP), which exert their endocrine activity in vertebrates through the estrogen receptor. As endpoints, developmental and reproductive parameters at the organism level as well as molecular and cellular parameters were measured. Life cycle exposure of zebrafish to (xeno)estrogens induced a specific, partly irreversible response pattern, consisting mainly of (a) induction of vitellogenin (VTG), (b) alterations of gonad differentiation, (c) delay of first spawning, and (d) reduced fertilization success. The effects of EE2 on zebrafish were expressed at environmentally realistic concentrations, while BPA and OP became effective at concentrations higher than those usually found in the environment. The vitellogenic response was equally sensitive as the reproductive parameters in the case of EE2, but VTG was more sensitive in the case of BPA. Partial life cycle exposure of zebrafish had lasting effects on fish development and reproduction only when the fish were exposed during the stage of juvenile bisexual gonad differentiation. In (partial) life cycle and multigeneration studies with invertebrates, (xeno)estrogenic impact was assessed by a range of developmental and reproductive parameters including hatching, growth, moulting, mating behavior, and egg number. Several parameters were found to be responsive to (xeno)estrogens; however, most effects were induced only at higher, probably nonphysiological concentrations. Low-dose effects were observed in full life cycle experiments, particularly in the second generation. It remains to be established whether the estrogen-induced alterations in the invertebrate species indeed do result from disturbances of the endocrine system. The findings of the present research project support the development of appropriate testing methodologies for substances with estrogenic activity.     

Current scientific understanding of the environmental biosafety of transgenic fish and shellfish

A fluorescent zebrafish was the first genetically engineered animal to be marketed, and biotechnologists are developing many transgenic fish and shellfish. Biosafety science is not sufficiently advanced to be able to draw scientifically reliable and broadly trusted conclusions about the environmental effects of these animals. The science is best developed for identifying hazards posed by environmental spread of a transgenic fish or shellfish and least developed for assessing potential ecological harms of spread. Environmental spread of certain transgenic fish or shellfish could be an indirect route of entry into the human food supply. The management of predicted environmental risks is in its infancy and has thus far focused on the first step of the risk management process, i.e. risk reduction, via a few confinement methods. There is a critical need to improve scientific methods of environmental safety assessment and management and to gather empirical data needed to substantiate biosafety conclusions and to effectively manage transgenic fish and shellfish. Scientists and potentially affected parties should participate in prioritising the knowledge gaps to be addressed.     

Endocrine disrupting compounds (EDCs) and pharmaceuticals and personal care products (PPCPs) in the aquatic environment: implications for the drinking water industry and global environmental health

Endocrine disrupting compounds (EDCs) and pharmaceuticals and personal care products (PPCPs) are a group of chemical compounds with diverse physical and chemical properties. Recent studies have indicated undesired effects of EDCs and PPCPs at their reported trace concentrations (ng l^-1 to mg l^-1). This paper reviews the current knowledge on the sources, properties, occurrence and health impacts of EDCs and PPCPs, and their removal from drinking water using ozonation and ozone/hydrogen peroxide-based advanced oxidation. The paper also examines the potential threats posed by these chemicals to drinking water and public health. While these compounds are known to have adverse effects on ecosystem health, notably in the fish population, a similar link is yet to be established between ingestion of these compounds through drinking water and human health. In addition, data on the effectiveness of existing methods for the removal of these compounds are not conclusive. Further studies are required to characterize risks, and also to evaluate and optimize existing removal processes. Also concerted international effort is urgent to cut down the risk of exposure and restrain the production and marketing of toxic chemicals.     

Estrogenic and toxic effects of methoxychlor on zebrafish (Danio rerio)

Although zebrafish (Danio rerio) have been suggested as a good candidate for screening potential endocrine disruptors, little information is available on the effects of weak estrogens on this species. We investigated the sensitivity of different life stages of zebrafish toward toxic and estrogenic properties of methoxychlor (MXC). Short-term tests with adults resulted in a sex-specific 96-h lethal concentration for 50% (LC50) of the test animals of 36 microg/L for males and 129 microg/L for females. To determine the estrogenic capacity of MXC, adult zebrafish were exposed to 0, 0.5, 5, and 50 microg MXC/L for 14 d. Induction of vitellogenin ([VTG] measured with protein electrophoresis and Western blot) in males was detected at 5 and 50 microg MXC/L. Females, however, did not exhibit higher blood VTG concentrations at the tested MXC concentrations. In a second series of experiments, juvenile zebrafish were exposed to 0, 0.05, 0.5, and 5 microg MXC/L for 33 d. Survival, length, weight, and condition of larvae were examined as indicators of toxic stress and the VTG content in whole body homogenates of juveniles was measured to determine xenoestrogenic effects. No effects of the tested concentrations of MXC were observed. Finally, the effect of MXC on zebrafish eggs, exposed to 0, 1, 10, and 32 microg MXC/L, was examined. Hatching and survival of hatched zebrafish were affected at 10 and 32 microg MXC/L. This study demonstrated that adult male zebrafish are sensitive toward the estrogenic effects of MXC. However, the use of VTG induction to detect effects of (xeno)estrogens in early life stages has to be further investigated, as low concentrations of VTG were detectable in exposed as well as unexposed juvenile fish.     

内分泌干扰物MXC对生殖和胚胎发育的影响

日趋增加的环境和工业化合物,最近已被定义为具有内分泌活性的环境内分泌干扰化合物,对人类健康产生了严重的危害.尤其是类似雌激素作用的环境内分泌干扰物引起动物生殖变异、出生缺陷和威胁人类生殖内分泌健康,在最近几年里已引起人类学家的日趋重视.甲氧滴滴涕是具有雌激素活性的非甾体杀虫剂,对胚胎和生殖发育有潜在危险.该文就甲氧滴滴涕对生殖和胚胎的毒性作用进行综述.     

Effects of endocrine-disrupting contaminants on amphibian oogenesis: methoxychlor inhibits progesterone-induced maturation of Xenopus laevis oocytes in vitro

There is currently little evidence of pollution-induced endocrine dysfunction in amphibia, in spite of widespread concern over global declines in this ecologically diverse group. Data regarding the potential effects of endocrine-disrupting contaminants (EDCs) on reproductive function in amphibia are particularly lacking. We hypothesized that estrogenic EDCs may disrupt progesterone-induced oocyte maturation in the adult amphibian ovary, and tested this with an in vitro germinal vesicle breakdown assay using defolliculated oocytes from the African clawed frog, Xenopus laevis. While a variety of natural and synthetic estrogens and xenoestrogens were inactive in this system, the proestrogenic pesticide methoxychlor was a surprisingly potent inhibitor of progesterone-induced oocyte maturation (median inhibitive concentration, 72 nM). This inhibitory activity was specific to methoxychlor, rather than to its estrogenic contaminants or metabolites, and was not antagonized by the estrogen receptor antagonist ICI 182,780, suggesting that this activity is not estrogenic per se. The inhibitory activity of methoxychlor was dose dependent, reversible, and early acting. However, washout was unable to reverse the effect of short methoxychlor exposure, and methoxychlor did not competitively displace [3H]progesterone from a specific binding site in the oocyte plasma membrane. Therefore, methoxychlor may exert its action not directly at the site of progesterone action, but downstream on early events in maturational signaling, although the precise mechanism of action is unclear. The activity of methoxychlor in this system indicates that xenobiotics may exert endocrine-disrupting effects through interference with progestin-regulated processes and through mechanisms other than receptor antagonism.     

Sorption of steroid estrogens to soils and sediments

Steroid estrogens at sub-micrograms per liter levels are frequently detected in surface water, and increasingly cause public concern of their potential impacts on ecosystems and human health. Assessing the environmental fate and risks of steroid estrogens requires accurate characterization of various physicochemical and biological processes involving these chemicals in aquatic systems. This paper reports sorption of three estrogens, 17beta-estradiol (estradiol), estrone, and 17alpha-ethinyl estradiol (EE2), by seven soil and sediment samples at both equilibrium and rate-limiting conditions. The results indicated that attainment of sorption equilibrium needs about 2 d when aqueous estrogen concentrations (C(t)s) are 25 to 50% of their solubility limits (S(W)S), but equilibrium requires 10 to 14 d when the Ct is 20 times lower than the S(W). The measured sorption isotherms are all nonlinear, with the Freundlich model parameter n ranging from 0.475 to 0.893. The observed isotherm nonlinearity correlates to a gradual increase of single-point organic carbon-normalized sorption distribution coefficient (capacity) (K(OC)) as the equilibrium estrogen concentration (Ce) decreases. At Ce = 0.5S(W), all three estrogens have log K(OC) values of 3.14 to 3.49, whereas at Ce = 0.02S(W), the log K(OC) values for estrone, EE2, and estradiol are within ranges of 3.40 to 3.81, 3.45 to 3.85, and 3.71 to 4.12, respectively. This study suggests that, when at sub-micrograms per liter levels, these estrogenic chemicals may exhibit even slower rates and greater capacities of sorption by soils and sediments.