CYP4介导前驱物转化的菲律宾蛤仔高累积全氟辛酸机制研究

Perfluorooctanoic acid (PFOA), a kind of emerging persistent organic pollutant, has been found distributed worldwide with extreme stability, high accumulation ability and multi-toxicity. Many researches have certified that PFOA poses serious threat to ecosystem and human, and more and more researchers have paid many attentions on the potential adverse effects of PFOA on organisms and human health. The aquatic animal food is recognized as the primary dietary exposure to PFOA in humans. In China, PFOA had been detected in almost all the aquatic animal food，and the highest levels were mainly found in shellfish, especially Ruditapes philippinarum. It has been demonstrated that PFOA-precursors were the major indirect source of PFOA contamination. Precursor compounds represent chemicals that can ultimately degrade to form PFOA through reactions such as hydrolysis, oxidation, defluorination and other phase I metabolism. There have some evidences that cytochrome CYP4 gene might be involved in the phase I metabolism of the PFOA-precursors path, so understanding the knowledge of the biotransformation of the PFOA-precursors may provide important information for explaining the high level of PFOA in the shellfish. However, the biotransformation behavior and mechanism of PFOA-precursors is still poorly documented in shellfish. In this research, the Ruditapes philippinarum is selected as the research organism for its high residue risk of PFOA. The accumulation, distribution and biotransformation of the predominant precursor (8:2 polyfluoroalkyl phosphate diester, 8:2 diPAP) in clam tissues will be conducted by exposure experiments, followed by the analysis of metabolic pathways and multivariate data using metabolomics technology to screen differential metabolites, which will identify the “precursor-metabolic intermediate-metabolic product” metabolic process and realize the critical role of 8:2 diPAP biotransformation in phase I metabolism process mediated by CYP4 gene. The level of gene expression and the variation of enzyme activities are analyzed. This will illustrated the correlation between CYP4 gene expression and the metabolism of 8:2 diPAP, in order to reveal the high yield of PFOA and endogenous formation process within clams. By this project, we will further understand the metabolic mechanism of PFOA-precursors in Ruditapes philippinarum，and provide theoretical basis for the establishment of maximum residue limits (MRLs) and construction of monitoring program in shellfish in China.

全氟辛酸（Perfluorooctanoic acid, PFOA）为国际社会重点管控的新型POPs，具广布性、持久性及多毒性特点。水产品是PFOA危及消费者健康的主要途径，尤以海洋贝类高累积作用带来的风险最为严峻。已证实前驱物在I相代谢过程中的生物转化是贝类中PFOA风险的重要来源。但是，由于缺乏对贝类中前驱物的代谢过程及关键路径，尤其是CYP4基因参与的核心代谢通路的科学认知，我国仍无法准确评价PFOA的安全风险。项目以我国PFOA风险最高的菲律宾蛤仔为研究对象，利用代谢组学技术解析首要前驱物8:2diPAP的代谢轮廓，通过代谢通路富集分析，明确CYP4基因对前驱物代谢的主导作用，结合解析CYP4基因差异表达-酶活变化-代谢产物的相关性，最终阐释CYP4基因介导前驱物转化的PFOA形成机制。项目有助于深层次了解贝类高累积PFOA的内源机制，为我国科学制定限量标准及构建防控措施提供依据。

为了查明全氟辛酸的前驱物8:2 diPAP在菲律宾蛤仔体内的特征代谢通路，本项目采用转录组-代谢组学联合技术，结合酶活等生化指标，通过代谢轮廓分析，明确8:2 diPAP代谢为PFOA的生物转化路径，再结合解析基因差异表达-酶活变化-代谢产物的相关性，最终阐释CYP4基因参与的8:2 diPAP的代谢过程与机制。研究结果表明，8:2 diPAP可在菲律宾蛤仔体内转化生成7:3 氟调聚羧酸（7:3 fluorotelomer carboxylic acid，7:3 FTCA）、全氟辛酸（Perfluorooctanoic acid，PFOA）、全氟壬酸（Perfluorononanoic acid，PFNA）、全氟庚酸（Perfluoroheptanoic acid，PFHpA）和全氟己酸（Perfluorohexanoic acid，PFHxA）5种I相代谢产物，生物转化率为3.54 mol%。8:2 diPAP诱导菲律宾蛤仔体内抗氧化酶应激，并引起众多基因呈差异表达，酶活与差异表达基因呈显著正/负相关。肝胰腺中8:2 diPAP和代谢产物含量最高，是8:2 diPAP进行富集和代谢转化的靶器官，且8:2 diPAP倾向于随粪便排出。代谢酶-代谢组-转录组的联动分析锁定重要的Ⅰ相和Ⅱ相代谢通路，基因CYP4f6和CYP4f4差异表达，参与细胞色素P450通路，主导菲律宾蛤仔机体的Ⅰ相代谢；谷胱甘肽代谢通路显著富集，是研究发现的主要的Ⅱ相代谢途径；能量代谢调控贯穿氧化应激、Ⅰ相与Ⅱ相代谢过程。研究结果有助于了解菲律宾蛤仔体内由前驱物8:2 diPAP转化为PFOA的内源机制。