全氟烷基化合物替代品影响斑马鱼鱼鳔发育的分子机制研究

Worldwide attention has been drawn to long-chain PFASs due to their persistence, bioaccumulation, and toxicity. Driven by increasingly stringent restrictions on long-chain PFASs, novel fluorinated compounds have emerged and replaced legacy PFASs in many industrial applications. However, some of these novel PFAS alternatives may still be bioaccumulative and toxic to wildlife and humans..In this proposed project, we hypothesized that PFASs alternatives would act as thyroid disruptors, which may lead to a delay/absence in swim bladder inflation and a decrease in expression of surfactant proteins. Zebrafish (Danio rerio) will be used as model animals, while various approaches including whole-mount in situ hybridization, gene knock-down, morpholino, microinjection, and high-throughput toxicogenomics would be used to investigate the developmental toxicity of PFAS alternatives. .Our proposed project is (1) to investigate the thyroid disrupting effects of PFAS alternatives, by evaluating the expression of critical genes in the regulation of hypothalamic-pituitary-thyroid (HPT) axis, thyroid hormone (TH) biosynthesis, transport, metabolism, and utilization in zebrafish lifecycle (embryo, juveniles and adults). Impact of exposure to PFASs alternatives on plasma and whole body T3/T4 levels of zebrafish will also be analyzed to provide further insight into the effects of alternatives. (2) to assess the link between plasma and whole body T3/T4 levels and swim bladder inflation failure in zebrafish. In the meantime, critical toxic data for PFAS alternatives such as the lowest observed effect concentration (LOEC) and no observed effect concentration (NOEC) which induces swim bladder inflation failure will be investigated. (3) to elucidate the mechanism of PFASs-induced swim bladder inflate failure, and to explore the roles of wnt and hedgehog pathways in the progress of swim bladder inflation; to explore sensitive biomarkers at early stages of PFAS-induced swim bladder damage, and subsequently to build a dose-response relationship between the markers and PFAS exposure..Our project is expected to yield new insight into the toxic responses to PFAS alternatives and to delineate the cross-talk within various pathways. The results may provide important information for assessing the health risks posed to humans and ecosystems exposed to PFAS alternatives.

传统长链PFASs因环境危害而被管控，并将被新型PFASs替代品取代。但部分当前使用的替代品仍具有生物累积性和多种毒性。本项目拟以斑马鱼为研究对象，典型PFASs替代品为目标化合物，采用原位杂交、基因敲降、微注射等技术手段，研究（1）PFASs替代品对斑马鱼不同发育阶段甲状腺轴的干扰效应，明确其对斑马鱼幼鱼不同发育阶段甲状腺激素干扰过程的关键位点。（2）甲状腺激素水平同前、后鱼鳔发育缺陷这一关键分子事件的关系；获得这些PFASs替代品导致鱼鳔缺失的最小可观察效应浓度等效应参数。（3）PFASs替代品影响鱼鳔发育中的关键毒性通路，揭示替代品导致鱼鳔发育异常的分子机制，寻找敏感的早期暴露标志物并建立替代品内暴露水平和敏感标志物之间的浓度（时间）－效应关系模型。研究结果将为新型PFASs替代品的生态毒理及安全性评价提供科学依据，为制定PFASs替代品的管理策略提供科学支撑。

全氟和多氟烷基类化合物（PFASs）是一类新型持久性有机污染物。作为一类内分泌干扰物，PFASs及其替代品暴露干扰鱼类甲状腺激素（THs）代谢，包括合成、分泌、运输和代谢等过程的机制尚不清楚。本项目以斑马鱼为研究对象，从基因和蛋白质水平上研究了几种新型PFASs替代品暴露的发育毒性效应及机制。获得以下创新性结果：（1）同PFOA类似，全氟烷基聚醚羧酸（PFECAs，如PFO3OA，PFO4DA和PFO5DoDA）亚急性暴露能够引起斑马鱼发育毒性，其中鱼鳔未充气是最明显的畸形指标。其毒性大小依次是PFO5DoDA > PFO4DA > PFOA > PFO3OA；发现PFECAs的毒性随着主链上OCF2数目的增多而增强。（2）三种全氟烷基聚醚羧酸（PFO3OA，PFO4DA和PFO5DoDA）存在对甲状腺激素的干扰效应以及外源添加甲状腺素水平可拯救鱼鳔缺失性状。发现PFOA和新型替代品PFECAs亚急性暴露会使斑马鱼体内T3和T4的含量随着化合物浓度的升高显著降低。在基因表达水平上，PFOA以及新型替代品暴露导致甲状腺激素合成相关基因ugt1ab，st1和st5表达显著上调，从而增加甲状腺激素的糖基化和磺基化水平，促进甲状腺激素的降解从而使斑马鱼体内甲状腺激素水平降低。外源添加T3和T4（与四种化合物共暴露），发现畸形率（鱼鳔未充气）显著下降，进一步证明了PFECAs导致鱼鳔未充气是甲状腺激素水平下降所致。（3）研究了6:2 Cl-PFESA慢性暴露对多代斑马鱼甲状腺内分泌系统的干扰作用。发现斑马鱼暴露6:2 Cl-PFESA 180天后，在F1代幼鱼中，发现鱼鳔畸形类型有：鱼鳔未充气，鱼鳔变小和两个鱼鳔等三种类型，即使在F2代幼鱼中，其鱼鳔未充气的畸形率仍然达到8.33%。尽管F2代斑马鱼胚胎及幼鱼体内甲状腺激素水平无明显变化，但HPT轴中关键基因tpo、dio1 和trα的表达量显著下降。.研究结果将为研发新的低毒性替代品提供支撑，为制定PFASs替代品的管理策略提供科学依据。课题组在项目执行期间已公开发表SCI论文5篇，培养博士3名，硕士研究生1名。