Investigating the hepatic toxicity of 3,3â-dichlorobiphenyl (PCB-11) in zebrafish (Danio rerio)

! 项目总结/摘要 非酒精性脂肪性肝病（NAFLD）影响30-40%的美国人口，估计美国 每年1030亿美元的经济负担。NAFLD在儿童和青少年中也很普遍， 34.2%的肥胖者和7.6%的总人口。高氯多氯联苯 多氯联苯（PCBs）与NAFLD的病因学有关，然而，对PCBs的肝毒性潜力知之甚少。 低氯多氯联苯，包括3，3 '-二氯联苯（PCB-11），广泛存在于环境和 人体样本，包括孕妇。初步研究表明，急性发育 单独暴露于PCB-11不会导致斑马鱼（Danio rerio）明显的毒理学结果，但是， 错误调节肝脏相关基因，阻碍肝脏发育，并增加肝脏组织空泡化。 此外，与芳香烃受体（Ahr）激动剂组合，PCB-11可以抑制细胞色素C（CYP 2C 19）， 模型PAH诱导的p4501 a（Cyp 1a）酶活性，加重毒性，或预防模型PAH诱导的毒性 PCB-126，强调使用混合物方法的重要性。这项拟议研究的目的是 研究PCB-11代谢物的肝毒性，这些代谢物在啮齿动物体内的半衰期较长， 低剂量接触的初步数据显示，15天鱼的肝脏脂质沉积增加。中央 本研究假设PCB-11诱导的肝毒性归因于其代谢产物 并且在幼年发育期间与NAFLD肝脏表型相关。目标1将决定 酚类和硫酸盐PCB-11代谢物对4日龄鱼毒性贡献。目标2将评估PCB-11 和PCB-11-硫酸盐毒性在30天的幼年期鱼类低剂量慢性单一和混合接触。 执行这些目标的拟议培训计划包括学习和/或执行EROD等方法 测定Cyp 1a活性的试验，评估肝脏发育的荧光显微镜检查，组织病理学，油- 红-O染色以评估脂质沉积，以及流式细胞术（FACS）以评估脂肪酸 肝细胞的组成分析。此外，将使用OECD Fish进行慢性暴露， 早期生命阶段（FELS）毒性试验（OECD TG 210），以获得监管毒性方面的实践经验 方法，并增加作为政府科学家工作的职业目标，以评估 污染物暴露对公众健康的影响。该项目将产生以下方面的体内机制毒性数据： PCB-11及其代谢物在几个发展阶段，可用于公共卫生监管机构。 发起人Alicia Timme-Laragy博士拥有NIH资助的记录，最近是NIH F32的导师。 博士后研究员（现为圣地亚哥州立大学教员），拥有众多设备， 荧光显微镜，分子生物学和生物化学工作，并能够提供指导 以及实现拟议项目目标所需的指导。此外，马萨诸塞大学阿默斯特分校是一个大型R1 它是一个研究机构，拥有多个资源，包括应用生命科学研究所（IALS）。 !

! PROJECT SUMMARY/ABSTRACT Non-alcoholic fatty liver disease (NAFLD) affects 30-40% of the U.S. population and has an estimated U.S. economic burden of $103 billion per year. NAFLD is also prevalent in children and adolescents, affecting 34.2% of obese individuals and 7.6% of this population overall. Higher-chlorinated polychlorinated biphenyls (PCBs) are implicated in the etiology of NAFLD, however, little is known about the hepatic toxicity potential of lower-chlorinated PCBs, including 3,3’-dichlorobiphenyl (PCB-11), widely detected in environmental and human samples, including in pregnant women. Preliminary research shows that acute developmental exposures to PCB-11 alone do not result in overt toxicological outcomes in zebrafish (Danio rerio), however, it misregulates hepatic-associated genes, stunts liver development, and increases vacuolization in liver tissue. Further, in combination with Aryl hydrocarbon receptor (Ahr) agonists, PCB-11 can either inhibit Cytochrome p4501a (Cyp1a) enzyme activity induced by a model PAH, exacerbating toxicity, or prevent toxicity induced by PCB-126, highlighting the importance of using a mixtures approach. The objective of this proposed study is to investigate the hepatic toxicity of PCB-11 metabolites, which have longer half-lives in vivo for rodents, and in preliminary data of low-dose exposures show increased hepatic lipid deposition in 15-day fish. The central hypothesis of this proposed study is that PCB-11 induced hepatic toxicity is attributed to its metabolites and is associated with a NAFLD liver phenotype during juvenile development. Aim 1 will determine the toxicity contribution of phenolic and sulfate PCB-11 metabolites in 4-day old fish. Aim 2 will assess PCB-11 and PCB-11-Sulfate toxicity in 30-day juvenile stage fish after low-dose chronic single and mixture exposures. The proposed training plan to execute these aims includes learning and/or performing methods such as EROD assays to measure Cyp1a activity, fluorescence microscopy to assess liver development, histopathology, Oil- Red-O staining to assess lipid deposition, and Fluorescence-Activated Cell Sorting (FACS) for fatty acid composition analysis of hepatocytes. In addition, chronic exposures will be carried out using the OECD Fish, Early-Life Stage (FELS) toxicity test (OECD TG 210) to gain practical experience in regulatory toxicity approaches and to increase preparation for the career goal of working as a government scientist to assess the public health impacts of contaminant exposures. This project will yield in vivo mechanistic toxicity data on PCB-11 and its metabolites at several developmental stages to be of use for public health regulators. The sponsor, Dr. Alicia Timme-Laragy, has a record of NIH funding, is a recent mentor of an NIH F32 Postdoctoral Fellow (now a faculty member at San Diego State University), has numerous equipment for fluorescence microscopy, molecular biology and biochemistry work, and is capable of providing the mentorship and guidance needed to accomplish the goals of the proposed project. Further, UMass Amherst is a large R1 research institution and houses several resources, including the Institute for Applied Life Sciences (IALS). !