PFOS/PFOA通过抑制糖异生途径干扰肝脏中营养物质代谢的分子机制研究

As typical poly-and perfluorinated alkyl substances (PFASs), perfluorooctane sulfonate (PFOS) and perfluorooctanoate (PFOA) can be bio-concentrated through the food chain and finally occur in human bodies, this might pose potential hazards to human health. In the previous studies, we have found that PFOS/PFOA exposure caused abnormal lipid accumulation in livers, atrophied adipose tissues and declined serum glucose level in rodents. Basing on those observations, we hypothesize that PFOS/PFOA may affect lipid and glucose homeostasis by disturbing gluconeogenesis in the liver, and finally this would result in enlarged livers and atrophied adipose tissues of the exposed animals. To test this hypothesis, cultured cells and animal model such as HepG2 cells and BALB/c mice will be used in this study. Following exposure to different doses of the toxicants for certain time, intermediate metabolites such as glucose, pyruvic acid and acetyl-CoA in HepG2 cells, as well as blood, liver and adipose tissues of animals will be measured. In addition, Expression of rate-limiting enzyme involved in the key metabolic pathways of gluconeogenesis, glycolysis and TCA cycle will be examined. Dose-effect curves will be established, aiming to reveal the target molecules of PFOS/PFOA-caused glucose/lipid metabolic disorders. Hormone or receptor antagonist will be used to activate or block certain metabolic pathways in these models. That will give an insight into how PFOS/PFOA affects the gluconeogenesis of HepG2 cells and lipid transportations in exposed animals. Furthermore, molecular mechanism will be explained for PFOS/PFOA-caused glucose/lipid metabolic disorders. Collectively, this study will provide scientific basis for PFOS/PFOA-caused health effects, as well as rationale for substitute products for PFOS.

全氟辛基磺酸（PFOS）和全氟辛基羧酸（PFOA）作为典型的全氟类化合物，可通过食物链传递放大并最终进入人体，对人类健康造成潜在危害。基于实验结果和文献分析，我们提出一个新的科学假设：PFOS/PFOA可通过干扰肝脏的糖异生，影响动物体内的糖/脂代谢平衡，最终造成受试动物肝脏肿大和脂肪组织萎缩。本项目将针对该科学假设，拟结合使用HepG2肝癌细胞株和BALB/c小鼠等离体、活体实验模型，通过测定不同浓度毒物暴露不同时间后，细胞和组织中葡萄糖、丙酮酸、乙酰辅酶A等主要中间代谢物的变化，定量检测细胞内糖异生、糖酵解、三羧酸循环等关键代谢途径中各限速酶的表达水平，建立剂量-效应曲线，以揭示PFOS/PFOA干扰肝脏糖/脂代谢的具体靶分子，再结合利用激素或受体抑制剂激活或阻断实验模型中特定代谢途径，深入研究并阐明PFOS/PFOA干扰肝细胞中糖异生和受试动物体内脂质代谢的作用途径和分子致毒机制。

全氟辛基磺酸（PFOS）和全氟辛基羧酸（PFOA）作为典型的全氟类化合物，因具有和生物体内脂肪酸相似的结构，可干扰生物体脂类代谢，并通过食物链传递进入人体，对人类健康造成潜在危害。我们结合化学分析和离体及活体暴露实验、计算毒理研究手段，揭示了PFOS和PFOA干扰生物体糖脂代谢的分子作用机制及途径。研究结果表明：PFOS和PFOA可以造成小鼠肝脏中脂类物质的累积和脂肪组织的萎缩，血脂和血糖水平降低，同时血液中糖皮质激素水平显著升高；PFOS和PFOA可通过抑制肝脏及脂肪组织中糖异生/甘油异生的关键限速酶磷酸烯醇式丙酮酸羧化酶（PEPCK）的表达，同时抑制关键调控因子糖皮质激素受体（GR）的表达水平，影响糖脂代谢过程中特殊的甘油异生途径，最终造成小鼠脂肪组织的萎缩及肝脏中脂质的异常累积，同时抑制了小鼠血糖水平的升高。我们采用计算模拟手段，分析不同结构的全氟化合物与PEPCK和GR蛋白的分子作用模式，发现PFOS/PFOA等全氟化合物可作为拮抗剂竞争结合于PEPCK和GR的活性口袋，其结合能力与分子结构相关；离体细胞实验也进一步验证了PFOS和PFOA等不同结构（碳链长度不同、末端基团不同）的全氟化合物暴露均可引起HepG2肝细胞中PEPCK基因表达的下调，并存在显著的剂量-效应关系和结构-效应关系。在此基础上，我们进一步研究了PFOS的替代产品全氟丁基磺酸（PFBS）对秀丽隐杆线虫的毒性效应，对比PFOS和PFBS暴露后对线虫的毒性大小和内暴露/外暴露剂量，发现在同等内暴露剂量下，PFBS可产生与PFOS相当的毒性。综上所述，我们系统研究并阐明了PFOS/PFOA干扰生物体中糖脂代谢的分子致毒机制及结构-效应关系，发现全氟化合物的内暴露剂量是决定其毒性大小的关键作用因素之一，这为后续从生物累积性及生物安全性角度科学评估及开发PFOS替代产品奠定了扎实的工作基础。