Using Zebrafish to Model Developmental and Multigenerational Volatile Organic Compound Toxicity

项目摘要： 三氯乙烯（TCE）和四氯乙烯（Perchloroethylene; PERC）是两种挥发性有机化合物 在20世纪中期工业使用的遗留问题之后， 世纪。TCE和PERC是已知或疑似致癌物质，与先天性缺陷有关， 神经退行性疾病美国环保署规定饮用水中的TCE和PERC的最大 污染物水平为5 ppb（十亿分之一; µg/L）;然而，地下水水平可能大于300，000 超级基金网站的ppb。拟议中的研究旨在解决有关发展， TCE和PERC的延迟、多代和跨代毒性，尽管使用 斑马鱼（Danio rerio）模型。第二个目标是确定斑马鱼如何代谢这些VOC， 代谢的物种差异是在实验室中模拟人类健康结果的障碍 动物最重要的假设是，发育过程中暴露于TCE和PERC等VOC会导致急性， 延迟和表观遗传毒性，影响整个过程中的身体系统发育和行为结果。 斑马鱼的生命历程和后代。进一步假设TCE和PERC的代谢 是某些毒性作用所必需的，产生的代谢产物依赖于内在代谢产物。 斑马鱼模型的轮廓。该研究的第一个目的是检验挥发性有机化合物TCE 和PERC在发育暴露后引起幼鱼的急性毒性和成年斑马鱼的延迟毒性。 将通过急性毒性试验、行为试验和 形态学测量，而延迟毒性将通过生殖，行为和 形态学终点。第二个目标是检验TCE和PERC引起多和 通过测量F1和F2代后代中的相似终点， 试图通过评估DNA甲基转移酶的表达来确定表观遗传毒性的机制 和TCE暴露的鱼类及其后代的整体DNA甲基化状态。最后，第三个目标将测试 假设TCE和PERC的代谢物是毒性的部分原因， TCE在斑马鱼体内的代谢。将通过GC-MS和HPLC分析监测暴露水， 确定TCE浓度随时间的变化以及斑马鱼排泄的主要代谢物。 将确定TCE和PERC主要代谢物的发育毒性。此外，该基因 将评估常见代谢酶的表达以确定这些酶的表达如何 随着时间的推移和胚胎TCE暴露后的变化。拟议的研究将大大解决关键问题， VOC文献中的空白，并为毒性建模和监管工作提供信息。此外，拟议的 实验的目的是平易近人和直观的本科生，研究生和专业 学生，根据R15 REAP赠款计划的目标。

Project Summary: Trichloroethylene (TCE) and tetrachloroethylene (perchloroethylene; PERC) are two volatile organic compounds (VOCs) that significantly contaminate the environment after a legacy of industrial use in the mid-twentieth century. TCE and PERC are known or suspected carcinogens and are linked to congenital defects and neurodegenerative disease. The US EPA regulates TCE and PERC in drinking water with a maximum contaminant level of 5 ppb (parts per billion; µg/L); however, ground water levels can be greater than 300,000 ppb at Superfund sites. The proposed research seeks to address gaps in knowledge about the developmental, delayed, multigenerational, and transgenerational toxicity of TCE and PERC though focused experiments using the zebrafish (Danio rerio) model. A second goal is to determine how zebrafish metabolize these VOCs, as species differences in metabolism represent an obstacle in modeling human health outcomes in laboratory animals. The overarching hypothesis is that developmental exposure to VOCs like TCE and PERC causes acute, delayed, and epigenetic toxicity that affects body system development and behavioral outcomes throughout the zebrafish life-course and subsequent generations. It is further hypothesized that metabolism of TCE and PERC is necessary for some of the toxic effects and the metabolites produced are dependent on the intrinsic metabolic profile of the zebrafish model. The first aim of the proposed research will test the hypothesis that the VOCs TCE and PERC cause acute toxicity in larvae and delayed toxicity in adult zebrafish after developmental exposure. Developmental toxicity will be evaluated in larvae through acute toxicity assays, tests of behavior, and morphologic measurements, while delayed toxicity will be assessed through reproductive, behavioral, and morphologic endpoints. The second aim will test the hypothesis that TCE and PERC cause multi- and transgenerational toxicity through the measuring of similar endpoints in F1 and F2 generation progeny and also seeks to identify a mechanism of epigenetic toxicity through the evaluation of DNA methyltransferase expression and global DNA methylation status in TCE exposed fish and their progeny. Finally, the third aim will test the hypothesis that metabolites of TCE and PERC are partially responsible for toxicity and will characterize the metabolism of TCE in zebrafish. The exposure water will be monitored via GC-MS and HPLC analysis to determine the change in TCE concentration over time and the predominant metabolites excreted by zebrafish. The developmental toxicity of major metabolites of TCE and PERC will be determined. Additionally, the gene expression of common metabolic enzymes will be evaluated to determine how the expression of these enzymes changes over time and after embryonic TCE exposure. The proposed studies would significantly address key gaps in the VOC literature and inform toxicity modeling and regulatory efforts. Additionally, the proposed experiments are designed to be approachable and intuitive to undergraduate, graduate, and professional students, according to the goal of the R15 REAP grant program.