Sirtuins参与TCEP致细胞损伤的线粒体调控机制研究

Tris(2-chloroethyl) phosphate is widely used as a new organophosphate ester flame retardant and plasticizer.TCEP is recently considered to be an emerging envrionmental pollutant because it can be detected in various environments, including in indoor and outdoor enviornments, water bodies as well as drinking water and in various food products.The in vitro and in vivo test results indicated that TCEP has teratogenic and has liver and renal toxicity, reproductive toxicity, neurotoxicity as well as carcinogenic potential.Therefore,the potential health risks from TCEP exposure has recently attracted special attention in many countries..The mitochodrial is important in cells. The sirtuin family (new tumor-suppressor proteins) affect the mitochodrial-mediated cell cycle, apoptosis. differentiation, and other critical cellular processes (such as balance between the oxidative stress and antioxidant defense and cellular metabolism).. In this project, the cell lines (L02 cells and HepG2 cells) as well as primary rat liver cells were treated with TCEP and TCEP extraction from the water samples, respectively, at the designed concentrations for the indicated time periods. TCEP-induced oxidative stress will be an entry point of the study, impacts of TCEP-induced oxidative stress-dependent cell damage on the mitochondrial functions will be investigated; in addition, TCEP-induced dysfunction in the cell cycle and p53-mediated mitochondrial apoptosis will be investigated after inhibiting the functions of Sirtuins.The project will focus on the roles of Sirtuins in the mitochondrial regulations of TCEP-induec cell damage, using the gene silencing method and the recombinant luciferase reporter plasmid-based cell bioassay. The findings will imply the regulation mechanisms of the TCEP-induced liver cell damage, and provide scientific foundation for the risk assessment of TECP-induced human health.

三(2-氯乙基)磷酸酯(TCEP)是一种广泛使用的新型阻燃增塑剂，并广泛存在于各种环境中。实验室研究已证实TCEP有肝肾毒性、生殖毒性、神经毒性和致癌性。因此，各国政府及科学界已高度关注该物质对人群健康的潜在影响。线粒体是决定细胞命运的关键因素。Sirtuin蛋白作为新的抑癌因子家族参与线粒体对细胞能量代谢、氧化应激、细胞增殖和凋亡的调控。本研究拟用TCEP纯品和饮用水样TCEP提取物处理多种肝细胞(L02和HepG2细胞)和原代大鼠肝细胞，以细胞氧化应激为切入点，研究TCEP引起细胞氧化应激对线粒体功能的影响；通过用Sirtuins的抑制剂来研究p53介导的受试细胞线粒体凋亡机制和/或周期调控机制；结合用基因沉默技术和转染荧光质粒重组技术重点研究Sirtuins在TCEP致肝细胞损伤的线粒体调控机制中的作用。为揭示TCEP致肝细胞损伤机制及评价TCEP的潜在健康风险提供科学依据

本研究主要内容包括：用固相萃取-气质联用技术分析了武汉市C（长江为水源)和H（汉江为水源水厂四季源水、出厂水和末梢水中三（2-氯乙基）磷酸酯（Tris（2-chloroetyl）phosphate, TCEP）含量。评价了水样有机提取物的细胞毒性。分析了TCEP(3.12mg/L、12.50mg/L、50.00mg/L和200.00mg/L)处理L02、HepG2和Chang细胞24h和48h时线粒体功能及细胞周期。用SIRT1干扰剂（EX-527）和siRNA技术探索了PI3K/AKT/mTOR通路在TCEP致细胞生长抑制的调控机制以及p53非依赖p21Waf1/Cip1/Rb信号通路在TCEP诱导细胞衰老中的作用。.本研究重要结果包括：①除两水厂秋季源水及H水厂秋季出厂水外，其余水样均检出TCEP。两水厂冬季源水TCEP检出浓度均最高（C水厂：676.23ng/L；H水厂：504.60ng/L），秋季源水均最低（低于8.80ng/L的检出限）；②C水厂秋季出厂水TCEP检出浓度最高（77.79ng/L），春季最低（33.42ng/L）；H水厂夏季出厂水TCEP浓度最高（104.13ng/L），秋季出厂水低于8.80ng/L的检出限；③两水厂夏秋季末梢水中位TCEP浓度均高于冬春季，但同季两水厂末梢水的TCEP浓度无差异性；④相对其他季节：C水厂夏季源水和H水厂冬季源水的有机提取物对细胞活力影响最大，两水厂四季源水最高有机提取物浓度均诱导了细胞凋亡；⑤TCEP可诱导Chang细胞线粒体功能紊乱，并致细胞周期阻滞；⑥PI3K/AKT/mTOR信号通路参与调控TCEP致肝细胞（L02和HepG2细胞）生长抑制；⑦p53非依赖性p21Waf1/Cip1-Rb信号通路参与调控TCEP致L02细胞的衰老。.本研究揭示了武汉市两个主要水厂的源水、出厂水和末梢水TCEP浓度的季节变化特点及水样有机提取物的细胞毒性。另提示TCEP可致线粒体功能紊乱及肝细胞周期阻滞，并通过PI3K/AKT/mTOR信号通路调控细胞的生长抑制，p53非依赖性p21Waf1/Cip1-Rb信号通路参与了细胞衰老的调控。本研究为水质防控及深入研究TCEP暴露人群的健康损伤提供了科学依据。