磷酸三(1,3-二氯-2-丙基)酯对L-02肝细胞氧化应激及凋亡损伤机制

Oxidative Stress and Apoptosis Mechanism on Damage of L-02 Liver Cells by Tris(1,3-dicholor-2-propyl) phosphate(TDCPP)

在各类环境介质及生物体甚至人体中都检测到有机磷阻燃剂磷酸三(1,3-二氯-2-丙基)酯(tris(1,3-dichloro-2-propyl)phosphate,TDCPP)的存在,为探究TDCPP对人体健康的毒性作用,选取人体正常肝细胞L-02细胞作为模型,考察在体外暴露条件下TDCPP对L-02细胞的细胞存活率、凋亡、氧化应激以及p53通路相关基因方面的影响。MTT结果显示,24 h、48 h、72h的半数致死浓度(LC50)分别为:116.56μmol·L^(-1)、81.89μmol·L^(-1)、65.11μmol·L^(-1)。TDCPP暴露24 h条件下,随着TDCPP暴露浓度的增加,细胞的凋亡率和活性氧(reactive oxygen species,ROS)水平也逐渐增加,100μmol·L^(-1)浓度条件下凋亡高达25.58%±1.61%,ROS水平是对照组的2.07±0.07倍。实时荧光定量PCR法检测线粒体凋亡通路相关基因的表达情况,在TDCPP刺激下,Bax、caspase-3、caspase-9、p53和Apaf-1相对表达量增加,Bcl-2相对表达量减少。蛋白免疫印迹法检测发现Bax/Bcl-2和caspase-3均随浓度增加而递增。本研究为综合评估TDCPP的生物和环境健康毒理效应提供了实验数据及理论支持。

Tris（1,3-dicholor-2-propyl） phosphate（TDCPP）,as one of organophosphorus chemicals widely appliedin fire retardants,whose residual contents has been detected in environmental media,various living organisms and even in human body.In order to investigate the toxicological effects of TDCPP on human health,human normal hepatocytes L-02,treated by in vitro exposure to TDCPP,were detected through the following indice such as cell viability,apoptosis,oxidative stress as well as mRNA expression of p53 pathway-related gene.MTT results indicate that values of LC50 of 24 h,48 h and 72 h were 116.56 μmol·L^-1,81.89 μmol·L^-1 and 65.11 μmol·L^-1 respectively.After cells being exposed to TPCPP for 24 h,it has been found that apoptosis rate and reactive oxygen species（ROS） level both increased as the exposure concentration of TDCPP increased.As compared with the control test（0 μmol·L^-1）,exposure concentration of 100 μmol·L^-1 results in apoptosis rate of 25.58%±1.61% and ROS level up to 2.07±0.07 times greater than that in control.Real-time fluorescence quantitative PCR was applied to detect mitochondrial apoptosis pathway-related gene.Under the stress of TDCPP,the relative expression of Bax,caspase-3,caspase-9,p53 and Apaf-1 increased while the expression of Bcl-2 decreased.Western blot results show that protein expression of Bax/Bcl-2 and caspase-3 increased as TDCPP concentration increased.This research provides experimental data as theoretical support for the comprehensive assessment of toxicological effects of TDCPP in terms of environment and biology.