环境污染对几类水生无脊椎动物内分泌功能扰乱的研究现状

近年来，在环境毒理学这门边缘学科中又诞生了一个新的领域，即环境污染对内分泌功能的扰乱。研究发现，许多人工合成的杀虫剂和工业化合物能够扰乱脊椎动物的内分泌功能，这些化合物也存在于水环境中。近年来，这些环境有机污染物是否对水生无脊椎动物的内分泌功能同样具有扰乱作用成了环境内分泌学这个新领域的热点之一。由于近年来的研究侧重于腔肠动物、轮虫、软体动物、甲壳动物及棘皮动物，因此，本文主要介绍有关环境污染物对这几类水生无脊椎动物内分泌功能扰乱的研究进展。另外，对环境污染对水生无脊椎动物内分泌扰乱这个研究热点的现状以及今后的发展方向进行了评述。在从事环境污染对无脊椎动物内分泌功能影响的研究时，研究者必须意识到无脊椎动物和脊椎动物在内分泌机制上的差异，不可随意地在这两大类动物类群之间互相引伸研究结果。     

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.     

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.     

Purification and characterization of vitellin from the estuarine mysid Neomysis integer (Crustacea; Mysidacea)

Invertebrates account for roughly 95% of all animals, yet surprisingly, little effort has been invested to understand their value in signaling potential environmental endocrine disruption. There has been, however, much recent attention on vitellogenin induction in egg-laying invertebrates and vertebrates as indicators of exposure to estrogenic xenobiotics. Mysid shrimp (Crustacea: Mysidacea) have been put forward by several researchers and regulatory bodies (e.g., US-EPA) as suitable test organisms for the evaluation of environmental endocrine disruption. In view of developing sensitive assays to study endocrine disruption in the estuarine mysid Neomysis integer, we isolated and characterized vitellin, the major yolk protein in eggs. Vitellin was purified using gel filtration and characterized by electrophoresis using different staining procedures. Specific (as shown by Western blotting) polyclonal antibodies were produced in rabbit against the purified vitellin of N. integer. These antisera will be used to develop immunoassays to study vitellogenesis in mysids and to detect potential stimulatory or inhibitory effects of endocrine disruptors on the production of vitellin.     

Endocrine-related reproductive effects in molluscs

Research on endocrine disruption has been a major topic of the past decade. Although most studies concentrated on vertebrate species, invertebrates are now gaining more attention. In particular, data on molluscs is increasing. One of the best-documented and more relevant examples of endocrine disruption is the imposex phenomenon affecting some gastropod species. But the increasing interest is also due to the fact that molluscs, especially bivalves, are good bioindicators used for decades in environmental studies and that progress have been made in the understanding of the physiology and endocrinology of some mollusc species. Recent results suggest that molluscs can be adversely affected by compounds that alter their reproduction and that vertebrate-type sex-steroids metabolism or mechanism of action could be involved in these effects. Nevertheless, the endocrine system of molluscs appears to be dissimilar in many aspects to those of vertebrates and sex-steroids might not have the same importance in all mollusc species. This diversity constitutes an important opportunity to examine and understand new and alternative mechanisms for endocrine disruption.     

Effects of endocrine disruptors on reproduction in viviparous teleosts with intraluminal gestation

Many water systems worldwide are affected by pollutants, including potential endocrine disruptors (EDs). Most studies on the effects of EDs on fish reproduction have focused on oviparous species. However, some important groups of fishes are not oviparous and there is scarce information about how EDs affect species with alternative reproductive modes. Goodeinae is a viviparous matrotrophic subfamily with intraluminal gestation (IG), where transfer of nutrients occurs and embryos develop inside the ovarian cavity. Goodeinae is endemic to the Mexican Central Plateau, an area affected by potential EDs, including 2,4-dichlorophenoxyacetic acid (2,4-D). This review synthesizes the available information about EDs in viviparous teleosts with IG and performs a case study on the effects of 2,4-D on gonadal structure of two Goodeinae species. We hypothesized that individuals exposed to 2,4-D might show altered gonadal structure. The available information included effects on gene expression, sexual differentiation, gametogenesis, secondary sexual characteristics, and sexual behavior. Knowledge gaps persisted on the effects of EDs on viviparous teleosts with IG. Holistic approaches are needed to understand the mechanisms underlying endocrine disruption effects. Field studies are needed to evaluate the consequences of EDs on wild populations. The case study revealed histological alterations in oocytes, spermatogonia, and sperm cysts in fishes exposed to 2,4-D. Ultrastructurally, gonads exhibited alterations in oocyte mitochondrial and nuclear membranes, and in spermatid mitochondria. The observed changes could be related to 2,4-D exposure, which may affect species reproduction in their natural environment. Matrotrophic viviparous teleosts with IG may serve as models to explore endocrine disruption.     

Environmental endocrine disrupters dysregulate estrogen metabolism and Ca2+ homeostasis in fish and mammals via receptor-independent mechanisms

Xenoestrogen endocrine disrupters (EDs) in the environment are thought to be responsible for a number of examples of sexual dysfunction that have recently been reported in several species. There is growing concern that these compounds may also cause abnormalities of the male reproductive tract and reduced spermatogenesis in man. Whilst some effects of EDs may be receptor-mediated, there is growing evidence that these compounds can exert potent effects in vivo by directly interacting with cellular enzyme targets. Here we report on, and review, the effects of alkylphenols and other EDs on two such enzymes: (1) sulfotransferases, which convert active estrogenic steroids to inactive steroid sulfates; and (2) Ca(2+)-ATPases, which are responsible for maintaining low, physiological, intracellular Ca(2+) concentrations. These enzymes are potently inhibited by EDs in both fish and mammalian species. The increased concentrations of active estrogens and the likely cytotoxic effects of elevated concentrations of intracellular Ca(2+) arising from these effects may underlie some of the endocrine disrupting potential of these widespread industrial pollutants.     

The concept of endocrine disruption and human health

In Europe, endocrine disruptors (EDs) have been defined as substances foreign to the body that have deleterious effects on the individuals or their descendants, due to changes in endocrine function. In the United States, EDs have been described as exogenous agents that interfere with the production, release, transport, metabolism, binding, action or elimination of the natural ligands responsible for maintaining homeostasis and regulating body development. These two definitions are complementary, but both indicate that the effects induced by EDs probably involve mechanisms relating in some way to hormonal homeostasis and action. EDs are generally described as substances with anti-oestrogenic, oestrogenic, anti-androgenic or androgenic effects. More recently, other targets have been evidenced such as the thyroid and immune system. Many different EDs are present in the various compartments of the environment (air, water and land) and in foods (of plant and animal origin). They may originate from food packaging, combustion products, plant health treatments, detergents and the chemical industry in general. In addition to the potential effects of these compounds on adults, the sensitivity of embryos and fetuses to many of the xenobiotic compounds likely to cross the placenta has raised considerable concern and led to major research efforts. With the exception of the clearly established links between diethylstilbestrol, reproductive health abnormalities and cancers, very little is known for certain about the effects of EDs on human health. Given the lack of available data, current concerns about the possible involvement of EDs in the increase in the incidence of breast cancer, and possibly of endometriosis and early puberty in girls, remain hypothetical. Conversely, the deterioration in male reproductive health is at the heart of preoccupations and progress in analyses of the relationship between EDs and human health. This literature review aims to describe the current state of knowledge about endocrine disruption, focusing in particular on the problem of food contaminants.     

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.     

The Antiestrogens Tamoxifen and Fulvestrant Abolish Estrogenic Impacts of 17α-ethinylestradiol on Male Calling Behavior of Xenopus laevis

Various synthetic chemicals released to the environment can interfere with the endocrine system of vertebrates. Many of these endocrine disrupting compounds (EDCs) exhibit estrogenic activity and can interfere with sexual development and reproductive physiology. More recently, also chemicals with different modes of action (MOAs), such as antiestrogenic, androgenic and antiandrogenic EDCs, have been shown to be present in the environment. However, to date EDC-research primarily focuses on exposure to EDCs with just one MOA, while studies examining the effects of simultaneous exposure to EDCs with different MOAs are rare, although they would reflect more real, natural exposure situations. In the present study the combined effects of estrogenic and antiestrogenic EDCs were assessed by analyzing the calling behavior of short-term exposed male Xenopus laevis. The estrogenic 17α-ethinylestradiol (EE2), and the antiestrogenic EDCs tamoxifen (TAM) and fulvestrant (ICI) were used as model substances. As previously demonstrated, sole EE2 exposure (10−10 M) resulted in significant alterations of the male calling behavior, including altered temporal and spectral parameters of the advertisement calls. Sole TAM (10−7 M, 10−8 M, 10−10 M) or ICI (10−7 M) exposure, on the other hand, did not affect any of the measured parameters. If frogs were co-exposed to EE2 (10−10 M) and TAM (10−7 M) the effects of EE2 on some parameters were abolished, but co-exposure to EE2 and ICI (10−7 M) neutralized all estrogenic effects. Thus, although EDCs with antiestrogenic MOA might not exhibit any effects per se, they can alter the estrogenic effects of EE2. Our observations demonstrate that there is need to further investigate the combined effects of EDCs with various, not only opposing, MOAs as this would reflect realistic wildlife situations.     

Effects of 4-n-nonylphenol and 17beta-oestradiol on early development of the barnacle Elminius modestus

Pollutants that are present in the aquatic environment and cause abnormal endocrine function in wildlife populations have been termed endocrine disrupting chemicals (EDCs). The impacts of these chemicals on the reproduction and development of vertebrates has been shown to be significant in both field studies and laboratory experiments. Over the past decade the number of investigations into the impacts of EDCs that affect reproductive and sexual characteristics (reproductive EDCs) has increased and evidence of their potency is evident in numerous wildlife species and through data from in vitro tests. However, little information is available on whether chemicals which act as EDCs in vertebrate species affect aquatic invertebrates. The case of imposex in archeogastropods following exposure to tributyltin (TBT) is a notable exception. Moreover, a number of studies have shown that development, fecundity and reproductive output of some aquatic invertebrates are affected significantly by exposure to pollutants. In order to determine whether external signs of exposure to vertebrate EDCs can be observed and monitored in invertebrate species, we exposed larvae of the barnacle Elminius modestus to environmentally realistic concentrations of the xeno-oestrogen, 4-n-nonylphenol (NP), and the natural oestrogen, 17beta-oestradiol (E(2)). Early life stages (nauplii and cyprids) were also exposed in the laboratory to determine whether there were effects on the timing of larval development and settlement. Ovary development and size of juveniles was measured following chronic exposure. Exposure to NP in the concentration range 0.01-10 μg l(-1) resulted in disruption of the timing of larval development. Similar results were obtained with E(2). Pulse exposures showed that the timing of exposure is critical and exposures for a period of 12 months caused long-term effects. A linear, concentration-dependent response was not evident.     

Intersex in the clam Scrobicularia plana: a sign of endocrine disruption in estuaries?

The phenomenon of endocrine disruption is currently a source of growing concern. Feminization of male fish in UK rivers has been shown to occur extensively and has been linked with exposure to endocrine-disrupting compounds present in the environment. Much less is known of the extent and scale of endocrine disruption in estuarine and marine ecosystems, particularly in invertebrates. We present evidence that intersex, in the form of ovotestis, is occurring in the common estuarine bivalve Scrobicularia plana, which is considered to be inherently gonochoristic. We report varying degrees in the severity of ovotestis in male S. plana, and have adopted and developed a grading method to assess the extent of this intersex condition. These findings indicate that S. plana offers potential for widespread screening and investigation of endocrine disruption, helping to focus remediatory strategy.     

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.     

内分泌干扰物的生物学检测和评价方法

内分泌干扰物(endocrine disrupters,EDs)是近年来环境科学研究的热点之一。EDs不仅对陆生动物和人类具有潜在的危害,还可以通过不同途径到达水环境中,影响水生动物的生长和繁殖等。因此,建立EDs快速、灵敏的检测和评价方法非常必要。此外,生物检测方法还能在一定程度上反映化学污染物对生物体的毒害效应。本文就研究EDs的离体和在体实验方法进行了介绍,并从个体、组织细胞、生化与分子(蛋白质、酶、RNA、激素)等不同水平上,重点综述了鱼类生物标志物在检测和评价EDs方面的研究进展。     

Mammalian development in a changing environment: exposure to endocrine disruptors reveals the developmental plasticity of steroid-hormone target organs

Recent findings in the field of environmental endocrine disruption have revealed that developmental exposure to estrogenic chemicals induces morphological, functional, and behavioral anomalies associated with reproduction. The aim of the present study was to determine the effects of in utero exposure to low doses of the estrogenic chemical bisphenol A (BPA) on the development of the female reproductive tissues and mammary glands in CD-1 mice. Humans are exposed to BPA, which leaches from dental materials and plastic food and beverage containers. Here we report that prenatal exposure to BPA induces alterations in tissue organization within the ovaries and mammary glands and disrupts estrous cyclicity in adulthood. Because estrogen receptors are expressed developmentally in these estrogen-target organs, we propose that BPA may directly affect the expression of genes involved in their morphogenesis. In addition, alterations in the sexual differentiation of the brain, and thus the hypothalamic-pituitary-gonadal axis, may further contribute to the observed phenotype. The emerging field of endocrine disruptors promises to provide new insights into the mechanisms underlying the development of hormone-target organs and demonstrates that the environment plays important roles in the making of phenotypes.