两栖动物急性毒性和发育神经毒性评价方法及应用
文献类型：学位论文
| 作者 | 苏红巧 |
| 学位类别 | 硕士 |
| 答辩日期 | 2013 |
| 授予单位 | 中国科学院研究生院 |
| 授予地点 | 北京 |
| 导师 | 秦占芬 |
| 关键词 | 黑斑蛙 Rana nigromaculata 非洲爪蟾 Xenopus laevis 蝌蚪急性毒性 tadpole acute toxicity 胚胎发育毒性 embryo developmental toxicity 发育神经毒性 developmental neurotoxicity 全氟烷基磺酸盐 perfluoroalkyl substances |
| 其他题名 | Establishment and application of amphibian acute toxicity test and developmental neurotoxicity assay |
| 中文摘要 | 近几十年来全球范围内两栖动物种群数量的急剧减少已受到广泛关注。同时，两栖动物的一些独有的生物学特征和对化学污染物尤其是内分泌干扰物的敏感性，使两栖动物成为生态毒理学研究关注的一个热点。另外，两栖动物非洲爪蟾作为实验动物的优点和生物学研究已获得的丰富的数据及积累的成熟的研究方法，为两栖动物用于毒理学研究提供了巨大空间。以上三点共同促进了两栖动物毒理学的发展。近年来，不断有新的基于两栖动物的毒性研究方法出现，其中一些成熟的方法形成标准或导则用于化学品的毒性评价，直接为化学品的环境管理服务。在此背景下，本论文开展了以下研究： 1. 我国本土两栖动物黑斑蛙蝌蚪急性毒性试验方法与应用。建立了黑斑蛙蝌蚪急性毒性试验方法。以毒死蜱、乙草胺、重铬酸钾和全氟辛烷磺酸盐（PFOS）为测试物，比较了黑斑蛙和国际通用两栖种非洲爪蟾在蝌蚪急性毒性试验中的敏感性，并用重铬酸钾为测试物比较了二者的重复性，以及Gosner 26阶段和30阶段黑斑蛙蝌蚪的敏感性。结果发现，两种蝌蚪对化学品的敏感性存在差异，且黑斑蛙蝌蚪具有自然死亡率低、试验重复性好的优点。因此，黑斑蛙蝌蚪比非洲爪蟾蝌蚪更适合作为蝌蚪急性毒性试验的材料。黑斑蛙Gosner 26阶段的蝌蚪对重铬酸钾的敏感性远高于Gosner 30阶段的蝌蚪，所以推荐使用26阶段黑斑蛙蝌蚪进行急性毒性测试。在此基础上，研究了PFOS及其4种替代品、高效氯氟氰菊酯、莎稗磷、1,3-二氯丙烯、丙草胺等几种农药以及废水样品对两种蝌蚪的急性毒性，验证了所建方法的可应用性；另外从结果来看，黑斑蛙蝌蚪急性 毒性试验也适用于废水的毒性评价。 2. 黑斑蛙胚胎发育毒性试验方法与应用。参考非洲爪蟾胚胎致畸试验（FETAX），建立了黑斑蛙胚胎发育毒性试验方法。以视磺酸、五氯酚、重铬酸钾和氯化镉为测试物，比较了黑斑蛙胚胎和非洲爪蟾胚胎的敏感性。结果发现，两种胚胎对化学品的敏感性存在差异，另外黑斑蛙的胚胎颜色更深，容易鉴别胚胎质量；个体更大，易于操作。鉴于黑斑蛙在我国比非洲爪蟾更易获得和以上的原因，黑斑蛙胚胎比非洲爪蟾胚胎更适合作为胚胎毒性试验的材料，用于化学品和废水的毒性评价，为我国相应的环境管理服务。 3. 非洲爪蟾发育神经毒性评价方法和应用。在FETAX试验的基础上，以已知促进神经发育的甲状腺激素T3和抑制神经发育的甲基汞为模式化合物，从神经突形态、神经发育早期分子标记和神经元分化标记三方面，筛选对神经毒物敏感的指标，建立简便快捷的神经毒性评价方法。整体免疫荧光研究发现，T3明显促进爪蟾胚胎神经突的生长，而甲基汞明显抑制神经突的生长。Western blot结果显示T3促进神经突生长相关蛋白GAP-43在头部的表达，而甲基汞抑制GAP-43的表达；同时T3抑制未分化神经元标记NeuroD的表达，而甲基汞促进NeuroD的表达。实时定量PCR研究结果显示，T3明显促进爪蟾神经发育早期关键分子基本转录元件结合蛋白BTEB和甲状腺受体TR mRNA的表达，而甲基汞抑制转录因子神经生长因子诱导的因子NGFI-1A的表达。基于以上，选择这些指标作为评价发育神经毒性的终点指标。另外，鉴于N-tubulin和NCAM是成熟神经元的分子标记，也列为评价发育神经毒性的终点指标。 在此基础上，研究了PFOS、全氟己烷磺酸盐（PFHS）和全氟丁烷磺酸盐（PFBS）的发育神经毒性，结果发现： PFOS所有测试浓度下（0.01-10 μM）对胚胎神经突的生长都没有作用，仅在最高浓度时对GAP43蛋白水平的表达有抑制作用，在所有浓度下对BTEB和TR mRNA表达有促进作用，低浓度时抑制tubulin mRNA表达，高浓度时促进tubulin mRNA表达，对其它指标没有明显作用；PFHS和PFBS对各指标作用的整体趋势与PFOS基本一致，显示三种全氟烷基磺酸对非洲爪蟾早期神经发育的分子事件具有一定的干扰作用，这些干扰作用是否会产生某些明显的生物学效应值得进一步研究。 综上所述，本文建立了我国本土黑斑蛙蝌蚪急性毒性试验方法和胚胎发育毒性试验方法，可为我国化学品环境管理中毒性评价提供技术参考；探索了非洲爪蟾胚胎发育神经毒性评价的方法，并用于评价PFOS、PFHS和PFBS的发育神经毒性，发现三种全氟烷基磺酸盐对早期神经发育的分子事件具有一定的影响。 |
| 英文摘要 | In recent decades, amphibian populations are declining globally at an alarming rate, which has aroused much attention. Meanwhile, amphibians have become one of research focuses in ecotoxicology due to unique biological characteristics and their sensitivity to chemical contaminants especially endocrine disrupting chemicals. In addition, distinct advantages of Xenopus laevis as an amphibian model species, rich available biological data and mature research methods provide a large space for the use of amphibians in toxicology. The above three points have promoted the development of amphibian toxicology in recent years. New methods for studying toxicity based on amphibians are increasing, and some mature methods in the form of standards or guidelines have been used for evaluating toxicity of chemicals. Based the background, the following research work was conducted in this dissertation: 1. Establishment and application of native species Rana nigromaculata tadpole acute toxicity test. R. nigromaculata tadpole acute toxicity test were established. Using dursban, acetochlor, potassium dichromate and perfluorooctane sulfonate (PFOS) as test chemicals, we compared the sensitivity of R. nigromaculata with general species X. laevis in tadpole acute toxicity test. Also, we compared their repeatability in tadpole acute toxicity test and sensitivity between Gosner 26 and Gosner 30 R. nigromaculata tadpoles using potassium dichromate as a test chemical. There is a difference in the sensitivity between R. nigromaculata and X. laevis tadpoles to chemicals. R. nigromaculata tadpoles have lower mortality and higher repeatability. So, R. nigromaculata is more suitable than X. laevis as test species in tadpole acute toxicity test. For R. nigromaculata, the sensitivity of Gosner 26 tadpole to potassium dichromate is much higher than that of Gosner 30 tadpole, showing that R. nigromaculata tadpoles at Gosner 26 are more suitable for acute toxicity test. Then, we investigated acute toxicity of PFOS and its four substitutes, four pesticides (L-cyhalothrin, anilofos, 1,3-dichloro propylene and pretilachor) and two waste water samples using R. nigromaculata and X. laevis tadpoles. The results validated the applicability of the tadpole acute toxicity test. All in all, our study shows that R. nigromaculata is more suitable than X. laevis as test species in tadpole acute toxicity test for Chinese environmental management of chemicals, and that R. nigromaculata tadpole acute toxicity test is also suitable for evaluating toxicity of waste water. 2. Establishment and application of frog embryo developmental toxicity assay-Rana. nigromaculata. Referring to frog embryo teratogenesis assay-Xenopus, we established frog embryo developmental toxicity assay-Rana nigromaculata. Using retinoic acid, pentachlorophenol, potassium dichromate and cadmium chloride as test chemicals, we compared the sensitivity of R. nigromaculata with Xenopus laevis in embryos developmental toxicity assay. Different sensitivity to chemicals was found between R. nigromaculata and X. laevis embryos. The bigger size of R. nigromaculata embryos than X. laevis embryos makes the manipulation easier, and the darker color of R. nigromaculata embryos than X. laevis embryos makes quality identification of embryos more accurately. Considering the availability and reasons mentioned above, R. nigromaculata embryos are more suitable than X. laevis embryos for frog embryo developmental toxicity assay for Chinese environmental management. 3. Establishment and application of X. laevis developmental neurotoxicity test. To establish simple and quick method to evaluate chemical neurotoxicity, based on FETAX, we screened sensitive endpoints for assaying neurotoxic substances from three aspects, including neurite morphology, molecular markers of early neural development and markers of neuronal differentiation, using thyroid hormones T3 and methylmercury (MeHgCl) as model chemicals. Whole-mount immunofluorescence results demonstrated that T3 promoted significantly neurite growth, while MeHgCl inhibited obviously. Correspondingly, T3 was found to promote neurite growth associated protein 43 (GAP43) and inhibit undifferentiation neuron marker NeuroD in western blot assay, while MeHgCl had adverse effects. Using real-time quantification PCR, we found that T3 obviously up-regulated mRNA expression of early neurodevelopmental marker basic transcription element binding protein (BTEB) and thyroid hormone receptor (TR). MeHgCl down-regulated mRNA expression of transcription factor nerve growth factor-inducible factor A (NGFI-A). Thus, these endpoints were selected to evaluate developmental neurotoxicity of chemicals. Two molecular markers of mature neurons, N-tubulin and NCAM, were also selected as endpoint for evaluating developmental neurotoxicity. Then, we examined developmental neurotoxicity of PFOS, perfluorohexanesulfonate (PFHS) and perfluorobutanesulfonate (PFBS). All test concentrations of PFOS (0.01-10μM) had no effect on neurite growth, and 10μM PFOS inhibited GAP43 protein expression. All test concentrations of PFOS up-regulated BTEB and TR mRNA. Low concentrations of PFOS down-regulated N-tubulin mRNA, but high concentrations had adverse effects. PFOS had no effect on other markers. As a whole, PFHS and PFBS had similar effects on these endpoints with PFOS. In short, the three perfluoroalkyl sulfonates (PFASs) had some influences on early neurodevelopment molecule events. Biological effects of these changes are worth further research. To sum up, tadpole acute toxicity test and embryo developmental toxicity assay using Chinese native species R. nigromaculata were established, they could provide technical reference for Chinese environmental management of chemicals. A method for assaying developmental neurotoxicity of chemicals was also established in this dissertation. Developmental neurotoxicity of PFOS, PFHS and PFBS was assayed, and the results show that they had some influences on early neurodevelopmental molecule event. |
| 源URL | [http://ir.rcees.ac.cn/handle/311016/35181]  |
| 专题 | 生态环境研究中心_环境化学与生态毒理学国家重点实验室 |
| 推荐引用方式 GB/T 7714 | 苏红巧. 两栖动物急性毒性和发育神经毒性评价方法及应用[D]. 北京. 中国科学院研究生院. 2013. |
入库方式： OAI收割
来源：生态环境研究中心
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