非洲爪蟾在生态毒理学研究中的应用（Ⅲ）——生殖内分泌干扰作用的评价

两栖动物生殖内分泌系统(如性别分化、性腺发育、第二性特征)对性激素的敏感性,使得该类动物可用于研究内分泌干扰物(Endocrine Disrupting Chemicals,EDCs)对生殖内分泌系统的干扰作用.两栖动物非洲爪蟾是发育生物学研究的经典模型动物,多年来积累的有关其生殖内分泌的资料可以为生殖内分泌干扰的研究提供参考.论文分析了非洲爪蟾作为评价生殖内分泌干扰作用模型动物的理论基础,总结了评价生殖内分泌干扰作用目前使用的几个指标(性别分化、性腺形态、性腺组织学结构、性激素水平、卵黄蛋白原表达等生物标记),综述了非洲爪蟾在评价酚类化合物、阿特拉津、多氯联苯等几种EDCs的生殖内分泌干扰作用中的应用,最后讨论了非洲爪蟾作为一种评价EDCs生殖内分泌干扰作用的模型动物目前存在的问题.毫无疑问,非洲爪蟾是生殖内分泌干扰研究的良好模型动物,为促进该模型动物更好的应用,需要加强非洲爪蟾生殖生物学和内分泌学的研究.     

浑河流域野生鲫鱼卵黄蛋白原基因表达

为揭示浑河流域水环境内分泌干扰物对水生态的潜在风险,利用兼并引物扩增获得鲫鱼卵黄蛋白原(Vtg)和核糖体蛋白L-7(RPL-7)基因部分碱基序列(分别为825和450bp),建立以RPL-7为内参基因、定量鲫鱼Vtg基因表达的实时定量逆转录聚合酶链反应(qRT-PCR)方法,并将该方法用于定量浑河野生鲫鱼肝组织Vtg基因表达分析。结果显示:与上游对照点(S1)相比,7月下游各点(S4～S8)雄鱼、S4和S6点雌鱼肝组织的VtgmRNA表达水平皆显著升高(P<0.05);11月在雄鱼中未检出VtgmRNA的有效表达,雌鱼也仅在S2和S3点的表达水平升高(P<0.05)。研究表明,浑河流域野生鲫鱼尤其是在7月明显受到了环境雌激素类物质的影响。另外,qRT-PCR方法能够灵敏检测出鲫鱼Vtg基因表达的时空差异。     

全氟辛磺酸（PFOS）对非洲爪蟾（Xenopus laevis）生长发育、甲状腺和性腺组织学的影响

为了揭示全氟辛磺酸(PFOS)的甲状腺激素和性激素干扰效应,并探索运用爪蟾动物模型同时检测多种环境内分泌干扰效应的可能性,将NF48阶段非洲爪蟾(Xenopus laevis)蝌蚪暴露于0.01、0.1和1mg·L-1PFOS中6个月,检测PFOS对爪蟾生长、变态、甲状腺和性腺的影响.结果表明,在各取样时间,PFOS组爪蟾体长、体重和蝌蚪尾长与对照组均无显著差异(p>0.05);2个月后,PFOS组比对照组平均慢1个发育阶段,4和6个月后,0.01mg·L-1PFOS组反而比对照组分别快1和2个发育阶段.6个月后,PFOS组甲状腺出现滤泡上皮细胞增生、胶质减少甚至空泡化等现象,且随着PFOS浓度的增加而加重;各组幼蛙性腺出现间性、睾丸珍珠状和先天性萎缩等畸形现象,外观为间性的性腺主要表现为睾丸组织中产生类似雌性个体的卵巢腔.对照组幼蛙的雌雄比为0.5:1,而暴露组分别为2.3:1(0.01mg·L-1组)、4.5:1(0.1mg·L-1组)和5:1(1mg·L-1组).上述结果表明,PFOS对爪蟾的变态过程具有小剂量刺激效应,能引起甲状腺组织结构的损伤,导致睾丸组织的雌性化和雌雄性比的异常升高,表现出明显的甲状腺激素和性激素干扰效应,可以认定为一种环境内分泌干扰物.实验同时表明非洲爪蟾可以用于多种环境内分泌干扰效应的同时检测.     

Investigating potential for effects of environmental endocrine disrupters on wild populations of amphibians in UK and Japan: status of historical databases and review of methods.

Concern over global declines among amphibians has resulted in increased interest in the effects of environmental contaminants on amphibian populations, and more recently, this has stimulated research on the potential adverse effects of environmental endocrine disrupters in amphibians. Laboratory studies of the effects of single chemicals on endocrine-relevant endpoints in amphibian, mainly anuran, models are valuable in characterizing sensitivity at the individual level and may yield useful bioassays for screening chemicals for endocrine toxicity (for example, thyroid disrupting activity). Nevertheless, in the UK and Japan as in many other countries, it has yet to be demonstrated unequivocally that the exposure of native amphibians to endocrine disrupting environmental contaminants results in adverse effects at the population level. Assessing the potential of such effects is likely to require an ecoepidemiological approach to investigate associations between predicted or actual exposure of amphibians to (endocrine disrupting) environmental contaminants and biologically meaningful responses at the population level. In turn, this demands recent but relatively long-term population trend data. We review two potential sources of such data for widespread UK anurans that could be used in such investigations: records for common frogs and common toads in several databases maintained by the Biological Records Centre (UK Government Centre for Ecology and Hydrology), and adult toad count data from 'Toads on Roads' schemes registered with the UK wildlife charity 'Froglife'. There were little abundance data in the BRC databases that could be used for this purpose, while count data from the Toads on Roads schemes is potentially confounded by the effects of local topology on the detection probabilities and operation of nonchemical anthropogenic stressors. For Japan, local and regional surveys of amphibians and national ecological censuses gathering amphibian data were reviewed to compile survey methodologies and these were compared with methods used in the UK and other countries. Substantial consensus exists in amphibian survey methodologies and this should be exploited in the initiation of coordinated monitoring programs for widespread and common anuran amphibians in Japan and the UK to generate long-term robust and standardized population trend data. Such data would support comparative ecoepidemiological assessments of the impact of environmental endocrine disrupters in these two cooperating countries.     

黑斑蛙与非洲爪蟾蝌蚪急性毒性试验方法敏感性的比较

两栖动物蝌蚪急性毒性试验是评价化学品急性毒性的一种方法。以毒死蜱、乙草胺、重铬酸钾和全氟辛烷磺酸盐(PFOS)为测试物,比较了我国本土黑斑蛙(Rana nigromaculata)与国际通用种非洲爪蟾(Xenopus laevis)在蝌蚪急性毒性试验中的敏感性。结果发现：2类蝌蚪分别进行的11次试验中,空白对照组黑斑蛙蝌蚪死亡率(0.9%)远低于非洲爪蟾蝌蚪的死亡率(5.8%);重铬酸钾和PFOS对黑斑蛙蝌蚪的96 h-LC₅₀分别为34.0 mg·L^-1和81.0 mg·L^-1,而对非洲爪蟾蝌蚪的96 h-LC₅₀分别为51.6 mg·L^-1和92.1 mg·L^-1,显示黑斑蛙蝌蚪对这2种化学品的敏感性略高于非洲爪蟾蝌蚪;毒死蜱和乙草胺对黑斑蛙蝌蚪的96 h-LC₅₀分别为0.41 mg·L^-1和4.1 mg·L^-1,而对非洲爪蟾蝌蚪的96 h-LC₅₀分别为0.12 mg·L^-1和3.1 mg·L^-1,显示黑斑蛙蝌蚪对这2种化学品的敏感性略低于非洲爪蟾。鉴于2类蝌蚪对化学品的敏感性存在差异,且黑斑蛙蝌蚪的自然死亡率低,材料更易获得,笔者认为黑斑蛙蝌蚪比非洲爪蟾更适合作为蝌蚪急性毒性试验的材料,用于我国化学品环境管理中的毒性评价。     

In vitro Screening von Pflanzenschutzmittel-Wirkstoffen auf östrogene Aktivität

Over the last years, the obviously increasing, hormone-dependent impairments observed in humans and animals, as well as the increased occurrence of hormone-dependent types of cancer, are sometimes associated with environmental chemicals which are suspected to imitate or block the effects of natural hormones. For a variety of environmental chemicals an endocrine efficacy could already be demonstrated. Little is known, however, about a possible hormonal activity of plant protection agents which are at present certificated in the Federal Republic of Germany. The aim of the present study was the in vitro testing of at least 57 active ingredients of pesticides and growth regulators certificated in the Federal Republic of Germany, for their possible estrogenic activity. The E-Screen-Assay based on the human breast cancer cell line MCF-7 was used as the suitable test system. For at least 8 of the tested substances, an receptor-mediated estrogenic activity could be shown in vitro. With the exception of one substance, 7 active ingredients displayed a very weak affinity for the human estrogen receptor. Their ability to displace 17β-estradiol from the receptor was low. Only the herbicide Pendimethalin is to be classified as a full estrogen receptor agonist; it is able to displace 17β-estradiol almost completely from the receptor. Howeverm the estrogenic potency of Pendimethalin, in comparison to 17β-estradiol, was seen to be as small as the estrogenic potency of the other 7 substances tested positively in the E-Screen-Assay. Among this 7 substances, two could not be classified as estrogenically active anymore, because their affinity to the human estrogen receptor was too weak. The relevance of the available results for livng organisms should be clarified in further in vivo investigations.     

构建有机化合物斑马鱼雌激素干扰效应的二元分类模型

计算毒理学方法已成为辅助内分泌干扰物(EDCs)管理的决策支持工具。因此,发展内分泌干扰效应指标的(定量)结构活性关系((Q) SAR)等预测模型对于实现EDCs环境管理具有重要的意义。在雌激素受体(Q) SAR模型研究方面,目前主要针对人、牛、大鼠和小鼠等物种的雌激素受体干扰效应进行了研究,而对鱼等水生生物雌激素受体干扰效应等指标的(Q)SAR模型研究还较少。本研究采用基于欧几里德距离的K最近邻(k NN)分类算法,构建了斑马鱼雌激素受体干扰效应的二元分类模型。结果表明,2个最优模型训练集和验证集的预测准确度(Q)、敏感性(Sn)和特异性(Sp)参数均大于0.93,说明模型具有较好的预测能力。因此,能够用所建模型填补模型应用域内其他化合物缺失的斑马鱼雌激素受体干扰效应定性数据。     

基于环境雌激素评估的卵黄蛋白原研究进展

卵黄蛋白原(Vitellogenin,Vtg)是卵黄蛋白(Vitellins,Vn)的前体.无脊椎动物的Vtg主要在肝胰腺、卵巢或脂肪体合成,脊椎动物主要在肝脏合成.在总结国内外相关研究基础上,对基于环境雌激素评估的Vtg研究进展进行了综述.目前的研究已经证明,Vtg的N末端氨基酸序列在动物进化中既具有高度的保守性,又具有一定的特异性.Vtg作为检测环境雌激素效应的生物标志物之一,在环境雌激素类化合物污染的评价中被广泛采用.Vtg的检测方法包括蛋白直接定量检测和mRNA定量分析,选择检测技术要依据实验设计,对于长时间的暴露实验,可通过检测蛋白来完成,短时间的暴露实验则适合半定量RT-PCR检测VtgmRNA的表达.在Vtg的检测中应考虑动物种属的特异性.     

受体报告基因实验及其在维甲酸和维甲酸X受体干扰物监测中的应用

受体报告基因实验具有快速、经济、灵敏、方便等诸多优势,在高通量筛选类或抗雌雄激素等通过核受体起作用的环境内分泌干扰物方面得到了广泛的应用。环境中的维甲酸和维甲酸X受体干扰物如有机锡等有着类似的作用机制,研究者也开始采用受体报告基因实验的方法对该类污染物进行筛选与监测。本文综述了受体报告基因实验的技术方法,包括报告基因和宿主细胞的选择,并介绍了该方法在人工合成的维甲酸和维甲酸X受体干扰物筛选以及环境样品中该类污染监测中的应用。综述总结了应用受体报告基因实验检测环境内分泌干扰物研究中的不足并对该方法的未来发展进行了展望,希望为该方法在环境监测和评估中的应用提供新的思路。     

Interactions of Endocrine-active environmental chemicals with the nuclear receptor PXR

There is growing concern about the human-health impact of environmental chemicals that have the potential to disrupt normal endocrine function. Endocrine disrupting chemicals (EDCs) include structurally diverse organochlorine pesticides, polychlorinated biphenyls (PCBs), plasticizers, fungicides, herbicides and pharmaceutical compounds, and can have a profound impact on development, and on reproductive, neurological and immune system functions. While many studies have focused on the role of androgen receptor, estrogen receptor and aryl hydrocarbon receptor in mediating the effects of EDCs, other nuclear receptors that regulate steroid hormone action and metabolism may also serve as targets of EDC action. This review focuses on two classes of EDCs, PCBs and phthalate monoesters, both of which have been shown to interact with pregnane X receptor (PXR), a member of the nuclear receptor superfamily that regulates a large number of target genes, many of which have important roles in steroid metabolism and transport. Recent findings on the ability of PCBs and phthalate monoesters to activate PXR are discussed and the potential role of PXR and other intracellular receptor proteins in mediating toxicities associated with EDC exposure is considered. Finally, we discuss several gaps in our knowledge regarding the actions of EDCs and the difficulties associated with the evaluation of risks associated with exposure to these endocrine active environmental chemicals.     

唐鱼卵黄蛋白原的诱导、纯化与鉴定

为探讨利用雄性唐鱼(Tanichthys albonubes)卵黄蛋白原(Vitellogenin,Vtg)作为生物标志物检测环境雌激素的可行性,采用浸浴法用50μg·L-117β-雌二醇(E2)对雄性唐鱼进行染毒,30d后将鱼体整体匀浆进行常规聚丙烯酰胺凝胶电泳(Native-PAGE)以分析Vtg的产生;并采用Mg2+-EDTA选择性沉淀和Q Sepharose阴离子交换层析对Vtg进行分离纯化.结果表明,E2诱导下,雄性唐鱼产生了雌性特异蛋白Vtg,并且可在体内积累;利用Mg2+-EDTA选择性沉淀和Q Sepharose阴离子交换层析的两步纯化方法可分离纯化唐鱼整体匀浆Vtg;经Native-PAGE鉴定,确定唐鱼Vtg的分子量为440kDa.以上结果提示,雄性唐鱼Vtg可以作为环境雌激素监测的有效生物学标记物。     

多氯联苯对孔雀鱼卵黄蛋白原的诱导及检测

为探讨利用孔雀鱼(Poecilia reticulata)卵黄蛋白原(Vitellogenin,Vtg)作为生物标志物检测环境雌激素的可行性,采用浸浴法分别使用17β-雌二醇(E2)和多氯联苯(PCBs)对雄性孔雀鱼成鱼进行染毒,30d后测定其性腺系数(GSI)和肝指数(LSI),并将鱼体整体匀浆进行常规聚丙烯酰胺凝胶电泳(Native-PAGE)及磷、脂和糖蛋白特异染色分析.结果表明,PCBs对雄性孔雀鱼具有雌激素效应,和E2均可以诱导雄性孔雀鱼体内产生Vtg,但诱导组雄鱼GSI及LSI与对照组比较均无显著性差异(p>0.05).Native-PAGE及磷、脂、糖蛋白特异性分析表明,孔雀鱼Vtg是一种富含磷、脂、糖的蛋白,具有VtgⅠ、VtgⅡ和VtgⅢ等3种形式,其分子量分别为642kDa、541kDa和441kDa.雄性孔雀鱼Vtg可作为环境雌激素监测的有效生物标记物.     

环境内分泌干扰物低剂量-效应研究进展

环境内分泌干扰物(EDCs)的低剂量-效应已成为生态毒理学界的研究热点.环境内分泌干扰物在接近或低于无可见不良效应浓度水平(NOAEL)时仍可诱发生物效应并存在非单调剂量-效应关系,这对现行的动物繁殖和发育毒理学检测规程以及环境内分泌干扰物风险评价的理论和方法提出了挑战.此外,由于环境中内分泌干扰物呈低剂量长期暴露的特征,研究低剂量-效应对正确进行生态风险评价具有重大的科学意义.在总结国内外相关研究的基础上,对当前环境内分泌干扰物低剂量-效应的研究进展进行了综述.     

农药的内分泌干扰研究

环境污染物引起的内分泌干扰成为全球性环境问题之一.本文介绍了内分泌干扰物的检测方法.并从除草剂、杀虫剂、杀菌剂等三个方面介绍了农药所引起的内分泌干扰现象.在此基础上,简介手性农药内分泌干扰问题的重要性和研究现状,并进行展望.     

苯并（a）芘对雄性罗非鱼肝脏Vtg mRNA表达的影响

卵黄蛋白原（Vitellogenin,Vtg）是检测环境雌激素的重要生物标志物,雄性鱼类VtgmRNA是检测环境雌激素的新兴生物标志物。为研究BaP是否具有环境雌激素效应,本试验对罗非鱼Oreochromis niloticus Vtg mRNA进行研究,用总RNA提取试剂盒提取得到雌鱼肝脏总RNA,以转录得到的cDNA为模板,以Primer设计得到的引物,得到422bp的罗非鱼卵黄蛋白原特异性eDNA扩增片段;采用荧光定量PCR法检测10、50Hg-L。的BaP暴露对罗非鱼肝脏Vtg基因表达的影响,结果发现4d、17d时雄性罗非鱼肝脏内VtgmRNA表达量与对照组无差异（P〉0．05）,10d时10、50ug·L-1 2个剂量组VtgmRNA表达量显著高于对照组（P〈0．01）,分别是对照组的113．18倍和121．79倍。结果意味着BaP可能具有环境雌激素效应。     

Hormonaktive Xenobiotika

Developmental and reproductive processes are most sensitive to the toxic effects of environmental chemicals. Especially alarming is the fact that many groups of industrial chemicals and pesticides are capable of disrupting the endocrine system in man and animals. Of great concern is the reduction in the sperm count in Europe, but also the worldwide increase in a great number of reproductive and developmental disturbances observed in both man and wildlife. A correlation to a high chemical burden has been demonstrated for many different animal species as well as for women. An important consideration in assessing the potential effects of environmental chemicals on the endocrine system is the time at which such an exposure occurs. In the adult organism, hormones initiate functional processes; these effects are usually transient. In early life, hormones control developmental processes and are capable of producing permanent changes in organ structure and function (organizational effects). Therefore, chemical disruption of hormonal functions during development can result in permanent functional changes of the afflicted organ.     

Exposure to metals (Ca,K, Mn) and road salt (NaCl) differentially affect development and survival in two model amphibians

ABSTRACT

Human activity has led to widespread chemical alteration of natural environments. Aquatic ecosystems are especially susceptible to chemical changes, including those caused by runoff and invasive species. Here, we examined the effects of water chemistry, specifically sodium chloride as well as three metals (Ca, K, and Mn) known to differ between native and invasive wetland plant species’ leaf tissues, on the development of two model amphibians: the native Northern leopard frog, Lithobates pipiens, and the non-native African clawed frog, Xenopus laevis. We exposed frog eggs to metal treatment solutions and measured time to hatching (TTH), and following hatching, we exposed tadpoles to a lethal concentration of sodium chloride and recorded time to death (TTD). We found that increasing metal concentrations generally resulted in acceleration of TTH for Xenopus tadpoles, but had no effect on leopard frogs. However, increasing metal concentrations (Ca, Mn) increased leopard frog tadpole susceptibility to NaCl (decreased TTD), while increasing metal concentrations (Ca, K) generally resulted in decreased Xenopus tadpole susceptibility to NaCl. Overall, our data suggest that invasive amphibians may be more tolerant to chemical changes than native amphibians, including those driven by the introduction of invasive plant species.     

近江牡蛎4种类型GST基因cDNA全序列的克隆与分析

为从分子水平上研究不同类型谷胱甘肽S-转移酶(Glutathione S-transferase,GST)基因在近江牡蛎(Crassostrea ariakensis)等贝类体内代谢去毒过程中的作用,采用RT-PCR及RACE法,分离、克隆得到近江牡蛎肝脏4种类型GST基因cDNA全序列.序列分析结果表明,mu、pi、omega、sigma4种类型GST基因cDNA全长分别为970bp、773bp、999bp、1277bp,分别编码215、207、242、203个氨基酸.构建系统进化树发现,除太平洋牡蛎(Crassostrea gigas)pi型GST外,所有类型GSTs序列都能各自聚集成一簇.     

Parasitismus und ‘Endokrine Disruption’ in Fischen

Background

Since the 1960s the scientific community has become increasingly aware of the need for integrative and holistic approaches to solve environmental issues. It has not only been recognized that changing environmental conditions can alter toxicological properties of pollutants but also that susceptibility of organisms to man-made chemicals strongly depends on environmental factors. This is especially true with respect to the endocrine control of reproductive processes in many aquatic species including fish, which are mainly influenced by changes in the surrounding environment such as day length, temperature, water flow, etc. Thus, it is astounding that the use of integrative approaches to address the question of the ecotoxicological relevance of the disruption of endocrine and related reproductive functions is rate. As a consequence of this shortcoming, there is increasing demand both among scientists and politicians regarding more holistic approaches in the endocrine disruptor research.

Aim

This contribution aims to critically discuss current strategies and approaches as well as to identify alternative strategies in context with the environmental risk assessment of endocrine disruptors. This will be exemplified using an ecotoxicological study with bream (Abramis brama) that was conducted along a major European river, the Elbe.

Results and Discussion

Local bream (Abramis brama) populations collected along the river Elbe were characterized by different magnitudes and/or types of endocrine alterations. Although there is a relatively extensive data set available regarding its contamination with wide range of chemicals in the Elbe, it has not been possible to conclusively relate the observed endocrine effects to the exposure to individual compounds or mixtures of chemicals. However, during the gross morphological evaluations of the fish it was observed that at some locations a great number of the individuals caught were parasitized with the tapeworm Ligula intestinalis. Furthermore, statistical analysis revealed that a relatively great percentage of the variation of the biological endpoints observed both among individuals and sampling sites could be explained by the occurrence of this parasite. Overall, the findings of these studies indicate that there is a complex interaction not only among the exposure to chemicals and parasitism but also of both of these aspects with the occurrence of biological effects in fish.

Conclusions

The gaps in our current knowledge as well as the findings from the few studies available indicate, the there is a need to revise our current approaches to characterize and evaluate exposure scenarios regarding their potential to cause endocrine disruption in wild organisms. Given the complexity of most environmental exposure scenarios, involving multiple factors including anthropogenic and natural influences and their interactions, I therefore see the urgent need to initiate a discussion on more integrative approaches in this field in favor of true ‘ecotoxicological studies’ that integrate both ecology and toxicology.     

Arzneimittelwirkstoffe in der Umwelt

During the last decade, the impact of chemical pollution and environmental chemistry has focused almost exclusively on the conventional ‘priority’ pollutants which are regulated in different environmental laws. This spectrum of chemicals, however, is only one piece in the larger puzzle of ‘holistic’ risk assessment. The pharmacentical drugs are a diverse group of bioactive chemicals receiving comparatively little attention as potential environmental pollutants. The aim of the literature review is to describe the most important exposition scenarios for human and veterinary pharmacenticals in different environmental compartments (soil, surface, waste water, ground water, table water, etc.)