中国科学院生态环境研究中心机构知识库
Advanced  
RCEES OpenIR  > 中国科学院环境生物技术重点实验室  > 学位论文
题名: 两栖动物用于化学品甲状腺干扰作用研究的方法学及应用
作者: 张银凤
学位类别: 博士
答辩日期: 2014-11
授予单位: 中国科学院研究生院
授予地点: 北京
导师: 李建中
关键词: 两栖动物 ; 非洲爪蟾 ; 黑斑蛙 ; 甲状腺干扰 ; 四溴双酚A,Amphibian ; Xenopus laevis ; Pelophylax nigromaculata ; Thyroid disruption ; Tetrabromobisphenol A
其他题名: Methods for evaluating thyroid disruption by chemicals using amphibians and their application
学位专业: 环境科学
中文摘要:
      越来越多的证据显示,一些化学污染物具有甲状腺干扰作用,建立简便快捷、灵敏可靠的研究方法识别潜在的甲状腺干扰物具有重要意义。两栖动物变态发育是研究 甲状腺干扰作用的良好模型,但总体来看,目前已有的方法存在敏感性的问题。另外,各国使用自己的本土两栖物种发展包括甲状腺干扰作用在内的毒性研究方法已 成一种国际趋势。围绕以上问题,本论文开展以下研究:
      1. OECD非洲爪蟾变态试验的优化及验证:2009年经济合作与发展组织(OECD)颁布了《两栖动物变态试验》导则,用以测试化学品对脊椎动物的甲状腺干 扰作用。本论文以典型的甲状腺抑制剂甲巯咪唑为模式化合物,通过提前暴露起始的发育期和延长暴露时间对方法进行了优化,结果发现优化后的方法检测出的甲巯 咪唑的最低效能浓度比OECD方法更低。论文进一步用另一典型的甲状腺干扰物高氯酸钠对调整后的方法进行验证,结果显示调整后的方法比原方法有更高的敏感 性。
      2. TH诱导的非洲爪蟾变态试验方法探索:一些化学品因化学结构与TH相似,而产生TH信号干扰作用。因此本文以TH诱导的非洲爪蟾变态模型为基础,建立了以 48期蝌蚪肠中TH响应基因表达为基础的2天筛查试验方法和以形态学为基础的6天效应试验方法。在此基础上,以已知在转录水平上具有TH信号干扰作用的四 溴双酚A(TBBPA)为模式化合物,对2天筛查试验进行了验证,结果发现TBBPA表现出对TH信号通路的双向剂量-效应关系。然后应用建立的方法检测 出了低剂量的全氟辛基磺酸 (Perfluorooctanesulfonate, PFOS)和全氟丁酸(Perfluorobutyric acid,PFBA)对TH信号的激活作用。
      3. TBBPA的TH信号干扰作用及对TH依赖的发育的影响:虽然研究显示TBBPA可在体外通过转录水平干扰TH信号通路,但其对TH依赖的脊椎动物发育的 影响还不清楚。本论文利用建立的6天效应验证试验和自然变态试验,对这一问题进行了研究。结果显示,即便低浓度的TBBPA也抑制了T3诱导的TH响应基 因表达和形态学变化,并在10–1000 nM 的浓度范围内呈现明显的剂量-效应关系;单独暴露较高剂量的TBBPA则对变态呈现激活作用。在自然变态过程中对该结果进行了验证,发现当内源性TH处于 高水平(57–66期)时TBBPA表现为TH拮抗剂,反之则为TH激活剂(51–56期)。这一研究提示TBBPA对TH依赖的脊椎动物发育的影响值得 关注。
      4. 基于黑斑蛙的TH信号干扰测试方法的探索及应用:在使用本土生物种开发毒性测试方法的国际背景下,本论文选择已初步实现实验动物化的我国本土两栖种黑斑蛙 为试验种,参考非洲爪蟾TH信号干扰2天筛查试验,建立了基于黑斑蛙的2天筛查试验。并用具有TH信号干扰作用的模式化合物TBBPA对此方法的可靠性和 灵敏度进行了验证。利用建立的方法结合体外TR蛋白结合试验,检测出双酚A的替代品BPF和BPS同BPA一样具有TH信号干扰作用,因此其潜在的健康风 险需要进一步研究。
      总之,本论文以国际通用物种非洲爪蟾和我国本土两栖种黑斑蛙为试验动物,优化并建立了测试甲状腺干扰试验方法,为研究甲状腺干扰作用提供了技术支持;首次 发现TBBPA通过TH信号对脊椎动物的发育的干扰作用呈现阶段依赖性,这一发现对于TH信号通路以及外源化合物的TH信号干扰作用的机制研究具有重要的 意义;首次发现PFBA、BPS、BPF等几种新型污染物具有一定的TH信号干扰作用,其对脊椎动物TH依赖的生长发育的影响值得关注。
英文摘要:
      Increasing evidence has shown that some chemicals have the potential to disrupt the thyroid system. It is very important to establish simple, rapid, sensitive and reliable methods for identifying thyroid disruptors. Amphibian metamorphosis is an ideal model to study thyroid disruption; however, the sensitivity of current methods needs improved. In addition, the use of native species in toxicity assays has become an international trend. In this dissertation, I was focused on these problems and conducted research as follows:
      1. Optimization and validation of amphibian metamorphosis assay (AMA)
      In 2009, Organization for Economic Cooperation and Development (OECD) proposed AMA to screen thyroid disruptors of vertebrate species. This paper aimed to enhance the assay by exposing earlier stage animals and extending exposure time using a typical thyroid inhibitor methimazole as a model compound. The study found that the enhanced assay could identify lower concentrations of effective methimazole than AMA. Further, the paper validated the enhanced assay used another thyroid disruptor perchlorate, and the result showed a higher sensitivity.
      2. Exploration of T3-induced xenopus laevis metamorphosis assay.
      With similar chemical structure to thyroid hormone (TH), some chemicals could disrupt TH signaling by binding to TR. Using T3-induced X.laevis metamorphosis model, a 2-day screening test and a 6-day assay were established by assessing intestinal TH response genes expression and on the basis of the morphology changes, respectively. Then tetrabromobisphenol A (TBBPA) which could bind to TR and affect transcriptional level was tested in the 2-day assay, and it showed double dose-effect on T3-induced gene expression. Then, low concentrations of perfluorooctanesulfonate (PFOS) and perfluorobutyric acid (PFBA) were tested to exhibite agonistic activities via TH signaling pathway in the assay.
      3. The effects of TBBPA on TH signaling pathway and vertebrate development
      TBBPA was found to affect gene transcription level in vitro, however, it’s not clear that the effects of TBBPA on vertebrate development. This paper assayed this in the T3-induced and spontaneous Xenopus laevis metamorphosis. The result showed TBBPA even at low concentrations could exhibite inhibitory effects on T3-induced expression of TH-response genes and morphological changes, and the inhibition was in a concentration-dependent manner (10–1000 nM). And higher concentrations of TBBPA alone promoted the development. In the spontaneous metamorphosis assay, we further found that TBBPA exhibited an antagonistic activity at the developmental stages when animals have high endogenous TH levels (stages 57–66); on the contrary, it acts as an agonist (stages 51–56). This study highlights the adverse influences of TBBPA on TH-dependent development in vertebrates.
      4.Exploration and application of a screening assay to study TH signaling disruption using Pelophylax nigromaculata.
      Using native amphibians to develop toxicity testing assay has become a trend internationally. Referring to 2-day assay of X. laevis, a 2-day screening assay was established using P. nigromaculata which has preliminarily become experimental animals in our country. TBBPA as a model compound was used to validate the assay, and it was found that it’s effective to detect TH signaling disruption of TBBPA. Further, using this assay and TR binding assay, BPF and BPS, the substitutes of BPA were found to affect TH signaling pathway like BPA and should be further studied.
      In a word, this paper optimized and established thyroid disruption assay using X.laeivs and native amphibian P. nigromaculata in our country, providing technical support for thyroid distuption study. It was first time to find that TBBPA disrupts development in a TH-dependent manner, and this is very important for TH signaling and mechanism research of chemicals interference via TH signaling. It was the first time to find that BPS and BPF, as BPS alternatives, can disrupt TH signaling. The effects of these pollutans on TH-dependent development should receive attention.
 
内容类型: 学位论文
URI标识: http://ir.rcees.ac.cn/handle/311016/34542
Appears in Collections:中科院环境生物技术重点实验室_学位论文

Files in This Item:
File Name/ File Size Content Type Version Access License
张银凤--两栖动物用于化学品甲状腺干扰作用研究的方法学及应用.pdf(7301KB)学位论文--限制开放 联系获取全文

Recommended Citation:
张银凤. 两栖动物用于化学品甲状腺干扰作用研究的方法学及应用[D]. 北京. 中国科学院研究生院. 2014.
Service
Recommend this item
Sava as my favorate item
Show this item's statistics
Export Endnote File
Google Scholar
Similar articles in Google Scholar
[张银凤]'s Articles
CSDL cross search
Similar articles in CSDL Cross Search
[张银凤]‘s Articles
Related Copyright Policies
Null
Social Bookmarking
Add to CiteULike Add to Connotea Add to Del.icio.us Add to Digg Add to Reddit
所有评论 (0)
暂无评论
 
评注功能仅针对注册用户开放,请您登录
您对该条目有什么异议,请填写以下表单,管理员会尽快联系您。
内 容:
Email:  *
单位:
验证码:   刷新
您在IR的使用过程中有什么好的想法或者建议可以反馈给我们。
标 题:
 *
内 容:
Email:  *
验证码:   刷新

Items in IR are protected by copyright, with all rights reserved, unless otherwise indicated.

 

 

Valid XHTML 1.0!
Copyright © 2007-2018  中国科学院生态环境研究中心 - Feedback
Powered by CSpace