2,4-二氯酚对斑马鱼性别分化的毒性效应及机制研究

Chlorophenols are common pollutants with estrogenic effects in the aquatic environment and can lead to various toxic effects on aquatic animals. However, the toxic effects and mechanism of chlorophenols on sex differentiation in fish remain unknown. In this project, larval zebrafish (Danio rerio) will be exposed to 2,4-dichlorophenol under constant temperature condition. The techniques or methods including tissue anatomy, ELISA, qPCR, transcriptome analysis and DNA methylation sequence will be used for: (1) the quantitative assessments of gonad structure, sex ratio, and male/female-related gene expression, to explore the toxic effects of 2,4-dichlorophenol on sex differentiation in zebrafish; (2) the analysis on levels of sex hormones (estradiol and testosterone) and their receptors, activities of aromatase, expression of sex differentiation-related genes, degree of DNA methylation, and correlations between these indexes and chlorophenol concentrations, to explore whether or not the hormone synthesis pathway and the alteration of gene expression are involved in 2,4-dichlorophenol toxicity; (3) the screening of differentially expressed genes related to sex differentiation and regulated by DNA methylation, and the identification of specific site for methylation, to explore the role of DNA methylation in regulation of gene expression. The results of this project will uncover the toxicity effects and mechanism of 2,4-dichlorophenol on sex differentiation in fish, and provide basic information for further ecotoxicological studies on chlorophenols.

氯酚是水体中常见的具雌激素效应的环境污染物，可对水生动物产生多种毒性影响。然而目前尚未见关于氯酚影响鱼类性别分化的研究报道。本项目拟在实验室恒温条件下以不同浓度2,4-二氯酚对斑马鱼（Danio rerio）幼鱼染毒，通过性别鉴定与统计、组织解剖、酶联免疫、荧光定量PCR、转录组和DNA甲基化测序等技术方法，分析：（1）斑马鱼的性腺结构特征、性别比例、雌或雄性相关基因表达水平，明确氯酚对斑马鱼性别分化的毒性效应；（2）斑马鱼性激素含量及其受体水平、芳香化酶活性、性别分化相关基因表达水平、DNA甲基化程度等与2,4-二氯酚浓度的效应关系，从激素合成及基因表达两方面探讨氯酚的毒性机制；（3）有差异性表达且受DNA甲基化调控的性别分化相关基因及其甲基化位点，探讨甲基化对基因表达的调控作用。本项目旨在阐明2,4-二氯酚对鱼类性别分化的毒性效应及机制，为深入开展氯酚的生态毒理学研究提供依据。

水环境中常见的氯酚类污染物具有雌激素效应，但目前有关氯酚对鱼类性别分化的影响研究很少。本项目通过室内染毒的方式，以组织解剖、切片观察、酶联免疫、荧光定量PCR、转录组和DNA甲基化分析等技术手段，探讨了氯酚影响斑马鱼性别分化的浓度/时间效应，从激素合成和基因表达两方面揭示了氯酚导致斑马鱼雌性化的分子机制，从DNA甲基化方面证实了DNA的表观遗传修饰在氯酚致斑马鱼雌性化过程中发挥重要作用。本项目圆满完成了原计划的研究任务，达到了预期目标。参与本项目的成员按原计划的分工内容积极开展工作，其资料查阅能力、科研动手能力、数据处理和分析能力、学术报告和论文写作能力都得到了很好的锻炼和提升。依托本项目，共培养青年教师2名，锻炼了研究生9人和本科生4人，已发表SCI期刊论文4篇，国内核心期刊论文1篇，学位论文4篇，还有2篇论文在投稿之中。项目组成员参加全国性学术会议15次，进行学术报告5次，提交摘要4篇。.本项目研究结果发现2,4-二氯酚暴露导致斑马鱼的雌性比例明显升高；受精后20-40天是氯酚影响斑马鱼性别分化的关键期；氯酚暴露明显改变斑马鱼体内性别分化相关基因（如amh、ar、cyp11b、cyp19a、dmrt1、esr1、foxl2、gata4、11β-hsd、17β-hsd、nr5a1a、nr5a2、sox9a、sox9b等）的表达，干扰性激素的合成，影响雌雄激素（如雌酮、雌二醇、雌三醇、睾酮、11-酮基睾酮、雄烯二酮等）的含量；2,4-二氯酚可改变amh, cyp19a1a, sox9a和nr5a2等基因启动子区域的甲基化水平。通过研究，我们筛选出了这些基因启动子中受DNA甲基化影响的位点，探讨了这些位点的甲基化调控基因表达的机制。本项目研究结果为我们科学认识氯酚对水生动物个体及种群的危害，进一步开展氯酚的生态毒理学研究提供了理论依据。