What is an endocrine disruptor?

Endocrine disrupting chemicals (EDCs) and potential EDCs are mostly man-made found in various materials. By interfering with the body's endocrine system, endocrine disruptors produce adverse developmental, reproductive, neurological, and immune effects in humans, abnormal growth patterns and neurodevelopmental delays in children. Thus, diethylstilbestrol (DES) a non-steroidal estrogen, which is regarded as a proof of concept, induces clear cell carcinoma among young women. EDCS may be found in plastic bottles and metal food cans (BPA), medical devices (phthalates), detergents, flame retardants (polybrominated diphenyl ethers), food (BPA), toys (phthalates), cosmetics and drugs (parabens), and pesticides (alkyl phenols such as nonylphenol). The deleterious effects of endocrine disruptors constitute a real public health issue. However concerning the mechanisms of action of EDCs, many questions remain unanswered and need further investigations.     

A treatise on hazards of endocrine disruptors and tool to evaluate them

Hormones mediate a major part of our essential physiological functions. Both endogenous and exogenous compounds and their metabolites are known to act through hormone receptors leading to regulation of endocrine function. The endogenous ligands that control reproductive functions are generally steroids such as 17beta-estradiol, androgens, progesterone, pregnenolone and glucocorticoids. However, exogenous compounds that are structurally and functionally similar gain entry into animals including humans through the diet or by occupational exposures, causing endocrine disruption. In the recent decade, there is a lot of apprehension about the so-called "endocrine disruptors" which are wide spread in the environment, mainly due to unrestricted human activity. These compounds of anthropogenic or natural origin mimic the action of sex hormones and can interfere with the endocrine system. It has been hypothesized that environmental exposure to synthetic estrogenic chemicals and related endocrine active compounds may be responsible for malformations in the male reproductive tract, crytorchidism, hypospadias, decrease in sperm counts, decreased male reproductive capacity and even testicular cancers. The increasing concern in both public and scientific communities about these abnormalities have prompted the initiation of epidemiological studies to not only identify, but to also analyze the short and long term effects of endocrine disruptors. As a result, a number of assays have been developed and are undergoing validation aimed at high throughput screening of chemical agents that disrupt endocrine activity. This review consolidates the findings of epidemiological studies, particularly in relation to male reproductive disorders and brings to light the various types of in vitro and in vivo models that are available for tiered testing of suspected compounds.     

Contribution of environmental pollutants to male infertily: a working model of germ cell apoptosis induced by plasticizers

Bisphenol A [2,2-bis(4-hydroxyphenyl)propane] (BPA), 4-nonylphenol (NP) and di(2-ethylhexyl)phthalate (DEHP), and its metabolite mono-2-ethylhexyl phthalate (MEHP) are chemicals found in plastics, which act as endocrine disruptors (EDs) in animals, including human. EDs act like hormones in the endocrine system, and disrupt the physiologic function of endogenous hormones. Most people are exposed to different endocrine disruptors and concern has been raised about their true effect on reproductive organs. In the testis, they seem to preferentially attack developing testis during puberty rather than adult organs. However, the lack of information about the molecular mechanism, and the apparently controversial effect observed in different models has hampered the understanding of their effects on mammalian spermatogenesis. In this review, we critically discuss the available information regarding the effect of BPA, NP and DEHP/ MEHP upon mammalian spermatogenesis, a major target of EDs. Germ cell sloughing, disruption of the blood-testis-barrier and germ cell apoptosis are the most common effects reported in the available literature. We propose a model at the molecular level to explain the effects at the cellular level, mainly focused on germ cell apoptosis.     

Endocrine Disruption and Developmental Abnormalities of Female Reproduction

The developing organism is particularly sensitive to exposureto estrogenic chemicals during a critical period in the inductionof longterm changes in female reproductive organs, and persistentmolecular alterations induced by the perinatal estrogenic agents.The perinatal mouse model can be utilized as an indicator ofpossible longterm consequences of exposure to exogenous estrogeniccompounds including environmental endocrine disruptors. Attentionshould be paid to abnormalities in female genital organs exposedto estrogenic endocrine disruptors during fetal and early postnataldevelopment in mammals including humans.     

Estrogen- and androgen-sensitive bioassays based on primary cell and tissue slice cultures from three-spined stickleback (Gasterosteus aculeatus)

Endocrine disrupting compounds are chemicals that may interfere with the endocrine system causing severe effects in organisms. The three-spined stickleback (Gasterosteus aculeatus L.) offers a potential for the assessment of endocrine disruption caused by a) estrogenic xenobiotics through the estrogen-dependent protein vitellogenin and b) androgenic xenobiotics through the androgen-dependent protein spiggin. The stickleback is presently the only known fish species with a quantifiable androgen and anti-androgen biomarker endpoint. In the current study, hepatocyte and kidney primary cell cultures and liver and kidney tissue slice cultures were prepared and used for detecting estrogenic or androgenic activity in vitro through the action of hormones or municipal sewage water. The results indicate that stickleback male hepatocyte cultures are suitable in detecting estrogenic activity and stickleback female kidney tissue slice cultures in detecting androgenic activity. The tested sewage water showed high estrogenic activity but no significant androgenic activity. Primary cell and tissue slice cultures isolated from the three-spined stickleback will allow simultaneously screening in vitro for potential estrogenic and androgenic activity of complex samples.     

Identification of estrogen-regulated genes by microarray analysis of the uterus of immature rats exposed to endocrine disrupting chemicals

Environmental estrogenic compounds which bind to the estrogen receptor (ER) can block or alter endogenous functions of estrogen in reproductive and developmental stages. A microarray technology is a very valuable method for the prediction of hormone-responsive activities in various gene expressions. Thus, we investigated the altered gene expression by estrogen and endocrine disruptors (EDs) using microarray technology in the uterus of immature rats. In this study, the expression levels of only 555 genes (7.42%) among the 7636 genes spotted on microarray chips were enhanced by more than two-fold following treatment with estradiol (E2), suggesting that direct or rapid response to E2 is widespread at the mRNA levels in these genes. In addition, elevated expression levels of the genes (over 2-fold) were observed by diethylstilbestrol (DES; 9.01%), octyl-phenol (OP; 8.81%), nonyl-phenol (NP; 9.51%), bisphenol-A (BPA; 8.26%) or genistein (9.97%) in the uterus of immature rats. The expression levels of representative genes, i.e., calbindin-D9k (CaBP-9k; vitamin D-dependent calcium-binding protein), oxytocin, adipocyte complement related protein (MW 30 kDa), lactate dehydrogenase A and calcium binding protein A6 (S100a6; calcyclin), were confirmed in these tissues by real-time PCR. In addition, the mRNA levels of these genes by real-time PCR were increased at follicular phase when E2 level was elevated during estrous cycle of adult female rats. In conclusion, these results indicate distinct altered expression of responsive genes following exposure to E2 and estrogenic compounds, and implicate distinct effects of endogenous E2 and environmental endocrine disrupting chemicals in the uterus of immature rats.     

Xenoestrogenic compounds promote capacitation and an acrosome reaction in porcine sperm

There is growing evidence that endocrine disruptors bind to hormone receptors; since these receptors are present on the sperm membrane, sperm are potentially a useful model for examining estrogenic activities of endocrine disruptors. The objective of the present study was to compare the effects of two xenoestrogenic compounds (genistein and 4-tert-octylphenol) to those of two steroids (estrogen and progesterone) and heparin on in vitro capacitation and the acrosome reaction in a porcine sperm model. Porcine sperm were incubated with various concentrations (0.001-100 μM) of each chemical for 15 or 30 min, and then capacitation and the acrosome reaction were assessed using chlortetracycline. Estrogen and progesterone were considerably more potent than the other chemicals in stimulating capacitation. Estrogen stimulated sperm capacitation at all tested concentrations after 15 min of incubation (P < 0.05), whereas progesterone stimulated sperm capacitation at all tested concentrations after 15 and 30 min (P < 0.05). The effect of genistein on sperm capacitation was comparable with that of estrogen, and it was the most potent in stimulating the acrosome reaction. Genistein stimulated the acrosome reaction at all tested concentrations after 30 min (P < 0.05). However, 4-tert-octylphenol had the least effect on capacitation and the acrosome reaction. In summary, since all chemicals studied effectively altered capacitation and the acrosome reaction, it was concluded that porcine sperm could be a useful model for in vitro screening of potential endocrine disruptors. It was noteworthy that concurrent comparisons to steroids increased the ability to determine estrogenic characteristics of the tested chemicals.     

Establishment of a transgenic yeast screening system for estrogenicity and identification of the anti-estrogenic activity of malachite green

Endocrine disruptors refer to chemical compounds in the environment which interfere with the endocrine systems of organisms. Among them, environmental estrogens pose serious problems to aquatic organisms, in particular fish. It is therefore important and necessary to have a fast and low-cost system to screen the large number of different chemical compounds in the aquatic environment for their potential endocrine disrupting actions. In this study, a screening platform was developed to detect xenoestrogens in the aquatic environment using the fission yeast Schizosaccharomyces pombe, and applied for compound screening. The aim was to demonstrate any significant potential differences between the fish screening system and the human screening system. To this end, a yeast expression vector harboring a fish estrogen receptor alpha and a reporter vector containing the estrogen responsive element fused with the Escherichia coli LacZ gene were constructed. After transformation with these two vectors, the transformed yeast clones were confirmed by Western blotting and selected on the basis of the beta-galactosidase activity. In this transgenic yeast system, the natural estrogen (estradiol) and other known xenoestrogens such as diethylstilbestrol, bisphenol A, genistein and dichloro-diphenyl-trichloroethane exhibited dose-dependent activities. Using this system, more than 40 putative endocrine disruptors including phytoestrogens, pesticides, herbicides, industrial dyes and other industrial chemicals were screened. Ten of them were demonstrated to exhibit estrogenic actions. Industrial dyes such as malachite green (MG) that disrupt thyroid hormone synthesis are extensively used and are widely distributed in the aquatic environment. Using this system, MG did not show any estrogenic action, but was demonstrated to exhibit anti-estrogenic activity.     

Endocrine disruptors in bottled mineral water: estrogenic activity in the E-Screen

Human exposure to endocrine disruptors is well documented by biomonitoring data. However, this information is limited to few chemicals like bisphenol A or phthalate plasticizers. To account for so-far unidentified endocrine disruptors and potential mixture effects we employ bioassays to detect endocrine activity in foodstuff and consequently characterize the integrated exposure to endocrine active compounds. Recently, we reported a broad contamination of commercially available bottled water with estrogenic activity and presented evidence for the plastic packaging being a source of this contamination. In continuation of that work, we here compare different sample preparation methods to extract estrogen-like compounds from bottled water. These data demonstrate that inappropriate extraction methods and sample treatment may lead to false-negative results when testing water extracts in bioassays. Using an optimized sample preparation strategy, we furthermore present data on the estrogenic activity of bottled water from France, Germany, and Italy: eleven of the 18 analyzed water samples (61.1%) induced a significant estrogenic response in a bioassay employing a human carcinoma cell line (MCF7, E-Screen). The relative proliferative effects ranged from 19.8 to 50.2% corresponding to an estrogenic activity of 1.9-12.2 pg estradiol equivalents per liter bottled water. When comparing water of the same spring that is packed in glass or plastic bottles made of polyethylene terephthalate (PET), estrogenic activity is three times higher in water from plastic bottles. These data support the hypothesis that PET packaging materials are a source of estrogen-like compounds. Furthermore, the findings presented here conform to previous studies and indicate that the contamination of bottled water with endocrine disruptors is a transnational phenomenon.     

Effects of endocrine disrupters on sex steroid synthesis and metabolism pathways in fish

The interactions of estrogenic (nonylphenol, dicofol, atrazine), androgenic (organotins, phthalates, fenarimol) and anti-androgenic compounds (vinclozolin, diuron, p,p'-DDE) with key enzymatic activities involved in both synthesis and metabolism of sex hormones was investigated. Carp testicular microsomes incubated in the presence of androstenedione and different xenobiotics evidenced higher sensitivity of 5alpha-reductase activity than 17beta-hydroxysteroid dehydrogenase activity towards those chemicals. Dicofol, organotins and phthalates were among the most effective inhibitors. In contrast, ovarian synthesis of maturation-inducing hormones (20alpha- and 20beta-hydroxysteroid dehydrogenase activities) were enhanced by nonylphenol, dicofol, fenarimol and p,p'-DDE. Metabolic clearance pathways of hormones were also affected. Fenarimol, nonylphenol and triphenyltin inhibited the glucuronidation of testosterone and estradiol at concentrations as low as 10, 50 and 100 microM, respectively. Triphenyltin, tributyltin and nonylphenol were also inhibitors of estradiol sulfation with IC(50) values of 17, 18 and 41 microM. Overall, the data indicates the interaction of selected chemicals with key enzymatic pathways involved in both synthesis and metabolism of sex hormones. This interference might be one of the underlying mechanisms for the reported hormonal disrupting properties of the tested compounds, and might finally affect physiological processes such as gamete growth and maturation.     

Endocrine disrupting chemicals and disease susceptibility

Environmental chemicals have significant impacts on biological systems. Chemical exposures during early stages of development can disrupt normal patterns of development and thus dramatically alter disease susceptibility later in life. Endocrine disrupting chemicals (EDCs) interfere with the body's endocrine system and produce adverse developmental, reproductive, neurological, cardiovascular, metabolic and immune effects in humans. A wide range of substances, both natural and man-made, are thought to cause endocrine disruption, including pharmaceuticals, dioxin and dioxin-like compounds, polychlorinated biphenyls, DDT and other pesticides, and components of plastics such as bisphenol A (BPA) and phthalates. EDCs are found in many everyday products--including plastic bottles, metal food cans, detergents, flame retardants, food additives, toys, cosmetics, and pesticides. EDCs interfere with the synthesis, secretion, transport, activity, or elimination of natural hormones. This interference can block or mimic hormone action, causing a wide range of effects. This review focuses on the mechanisms and modes of action by which EDCs alter hormone signaling. It also includes brief overviews of select disease endpoints associated with endocrine disruption.     

环境雌激素双酚A的研究进展

环境荷尔蒙是现代生命科学领域研究的热点之一,如今越来越多的人开始关注这一话题。在我们的生活环境中充斥着各种化学有毒试剂,其中,有一类物质能够模拟或抑制内分泌激素的活动,我们称之为内分泌干扰物。内分泌干扰物有能力改变内分泌系统的结构和功能。双酚A作为一种环境雌激素,属于内分泌干扰物的一种。双酚A被广泛应用于聚碳酸酯塑料和环氧树脂的制造。双酚A具有弱雌激素效应,能够与雌激素受体结合,引起内分泌系统的应答。目前的研究表明,双酚A会透过血胎屏障影响到胚胎发育,会对神经内分泌系统、肝组织功能以及生殖器的发育造成损伤。本文主要综述了环境雌激素双酚A在小鼠发育阶段所引起的诸多不利影响,并对环境荷尔蒙未来的研究方向进行了展望。     

Developmental exposure to environmental estrogens alters anxiety and spatial memory in female mice

Humans and wildlife are exposed to numerous anthropogenic drugs and pollutants. Many of these compounds are hormonally active, and recent evidence suggests that the presence of these endocrine disruptors permanently alters normal development and physiology in a variety of vertebrate species. Here, we report on the effects of developmental exposure to two common estrogenic pollutants, bisphenol A and ethinyl estradiol on sexually dimorphic, non-reproductive behavior. Mice (Mus musculus domesticus) were exposed to environmentally relevant levels of these chemicals (2 and 200 microg/kg/day for bisphenol A and 5 microg/kg/day for ethinyl estradiol) throughout prenatal and early postnatal development. As adults, the animals were observed in a variety of tests measuring sexually dimorphic behaviors including short-term spatial memory (in a radial-arm maze and a Barnes maze) and anxiety (in an elevated-plus maze and a light/dark preference chamber). Developmental exposure to ethinyl estradiol was found to masculinize behavior in all of the assays used. Bisphenol A increased anxious behavior in a dose-dependent fashion but had no effect on spatial memory. These results indicate that non-reproductive, sexually dimorphic behavior is sensitive to endocrine disruption. In addition, these experiments suggest that both humans and wildlife are being exposed to levels of these endocrine disrupting compounds that are sufficient to disrupt the development of the nervous system and that may have permanent consequences on sexually dimorphic behaviors.     

Combined cross-linked enzyme aggregates from versatile peroxidase and glucose oxidase: production, partial characterization and application for the elimination of endocrine disruptors

Versatile peroxidase (VP) from Bjerkandera adusta was insolubilized in the form of cross-linked enzyme aggregates (CLEA®s). Of the initially applied activity 67% was recovered as CLEA®s. Co-aggregation of VP with glucose oxidase from Aspergillus niger led to an increased activity recovery of 89%. The combined CLEA®s showed higher stability against H₂O₂ and exerted VP activity upon glucose addition. The elimination of the endocrine disrupting chemicals bisphenol A, nonylphenol, triclosan, 17α-ethinylestradiol and the hormone 17β-estradiol (10 mg L⁻¹ each) and the removal of their estrogenic activity by combined CLEA®s were tested in batch experiments. Within 10 min, the combined CLEA®s were able to remove all the endocrine disruptors except triclosan (residual concentration 74%). The removal of the estrogenic activity was higher than 55% for all compounds, except triclosan. A membrane reactor continuously operated with combined CLEA®s could almost completely remove bisphenol A (10 mg L⁻¹) for 43 h.     

In vivo bioluminescence imaging to evaluate estrogenic activities of endocrine disrupters

Reporter gene technology is widely used to measure activity of hormone analogs, and bioluminescent in vitro assays have allowed rapid screening of numerous chemicals either to identify new agonists or antagonists of hormones or to detect the presence of endocrine disrupters in the environment. Stable bioluminescent cell lines have been established and they provide reproducible dose–response curves and accurate determination of in vitro efficiencies of various chemicals. In vivo, however, these molecules can be metabolized, bound by proteins, or stored in fats and thus could display efficiencies different from those observed in vitro. In vivo assays, such as the uterotrophic bioassay, require numerous sacrificed animals, and responses not only are dependent on an estrogenic action but also imply other factors. For a faster assay and to avoid the use of numerous animals, we developed an in vivo biosensor constituted of stable bioluminescent cells implanted in nude mice. MCF-7 bioluminescent cell lines were chosen since their proliferation is low in the absence of estrogen and the xenograft size can thus be stable for several weeks. Luciferase gene expression was monitored noninvasively with a cooled charge-coupled device camera. Quantitative analysis allowed us to compare in vitro and in vivo actions of different estrogenic compounds (estradiol, estrone) and endocrine disruptors (ethynylestradiol, genistein, octylphenol, and 2,4′-dichlorodiphenyldichloroethylene) in the same cell lines and to follow hormone action on a living animal as a function of time. Different administration protocols have been used and good correlation was observed for most products. However, we found that ethynylestradiol was the most efficient chemical when orally administered.     

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.     

Use and role of invertebrate models in endocrine disruptor research and testing

Historically, invertebrates have been excellent models for studying endocrine systems and for testing toxic chemicals. Some invertebrate endocrine systems are well suited for testing chemicals and environmental media because of the ease of using certain species, their sensitivity to toxic chemicals, and the broad choice of models from which to choose. Such assays will be useful in identifying endocrine disruptors to protect invertebrate populations and as screening systems for vertebrates. Hormone systems are found in all animal phyla, although the most simple animals may have only rudimentary endocrine systems. Invertebrate endocrine systems use a variety of types of hormones, including steroids, peptides, simple amides, and terpenes. The most well-studied hormone systems are the molting and juvenile hormones in insects, the molting hormones in crustaceans, and several of the neurohormones in molluscs and arthropods. These groups offer several options for assays that may be useful for predicting endocrine disruption in invertebrates. A few invertebrate phyla offer predictive capabilities for understanding vertebrate endocrine-disrupting chemicals. The echinoderms, and to a lesser extent molluscs, have closer evolutionary relationships with the vertebrates than the arthropods and these phyla. The recently identified estrogen receptor structure within the genome of the marine gastropod, Aplysia, indicates that the estrogens, and probably the basic steroid receptor, are quite old evolutionarily. This review of the recent literature confirms the effects of some endocrine-disrupting chemicals on invertebrates--tributyltin on snails, pesticides on insects and crustaceans, and industrial compounds on marine animals.     

环境激素类物质对人类健康的影响

环境激素是具有生物体内分泌激素干扰作用的一大类化学物质。环境激素在环境中普遍存在,不仅对生物的繁殖具有一定影响,而且还对生物的许多行为和感知能力产生影响。论文综述了近年来国内外有关环境激素的研究进展,综合分析了环境激素对人类健康的影响,阐述了其作用机理,并探讨了环境激素类物质控制和管理途径。     

Alteration of gene expression of G protein-coupled receptors in endocrine disruptors-caused hyperactive rats

We examined the effects of endocrine disruptors on rat behavioral and cellular responses. Single intracisternal administration of bisphenol A, p-octylphenol, nonylphenol, dibutylphthalate (DBP), dicyclohexylphthalate (DCHP), or diethylhexylphthalate (DEHP) into 5-day-old male Wistar rats caused significant hyperactivity at 4-5 weeks of age. It was about 1.3- to 1.6-fold more active in the nocturnal phase than control rats. Based on DNA macroarray analyses of the midbrain at 8 weeks of age, the endocrine disruptors altered the levels of gene expression of G protein-coupled receptors that were involved in not only dopaminergic neurotransduction but also many peptidergic neurotransduction. The gene expression of dopamine receptor D1A was decreased by nonylphenol, DBP, or DEHP by 0.23- to 0.4-fold, whereas that of dopamine D2 was increased by nonylphenol or DBP by 2- to 2.8-fold. It was notable that four of six endocrine disruptors tested, i.e. nonylphenol, DBP, DCHP, and DEHP largely downregulated the levels of gene expression of galanin receptor 2 by 0.11- to 0.28-fold. Bisphenol A, DBP or DCHP significantly decreased the levels of gene expression of dopamine transporter at 8 weeks more than 0.5-fold. Immunohistochemical analyses revealed that p-octylphenol impaired the immunoreactivity for tyrosine hydroxylase in substantia nigra pars compacta. Thus, endocrine disruptors caused hyperactivity in the rat, probably regulating the levels not only of gene expression but also of proteins of both G-protein-coupled receptors systems and dopaminergic neurotransduction system.     

Enhanced expression of laccase during the degradation of endocrine disrupting chemicals in Trametes versicolor

A putative laccase cDNA from a white-rot basidiomycete, Trametes versicolor, that consisted of 1,769 nucleotides was cloned using the rapid amplification of cDNA ends (RACE)-PCR method. The deduced amino acid sequence had 4 putative copper binding regions, which are common to fungal laccases. In addition, the sequence was 57 approximately 97 % homologous to sequences of other T. versicolor laccases. Additionally, the expression of laccase and manganese peroxidase in this fungus were both greatly increased under degrading conditions for bisphenol A, nonylphenol and two phthalic esters (benzylbutylphthalate and diethylphthalate), all of which are reportedly endocrine disrupting chemicals (EDCs). Furthermore, the estrogenic activities of the EDCs also decreased rapidly during incubation when examined in a two-hybrid yeast system. Finally, kojic acid inhibited the removal of estrogenic activities generated by bisphenol A and nonylphenol, which confirmed that laccase was involved in the degradation of EDCs in T. versicolor.