滴滴涕与纳米二氧化钛对人胚肝细胞DNA损伤作用的协同效应

Synergistic DNA Damage Caused by Nano-TiO_2 and DDT in Human Embryo Liver Cells

[目的]观察滴滴涕(DDT)及纳米二氧化钛(nano-TiO2)单独及联合体外染毒对人胚肝细胞株(L-02)内活性氧(ROS)含量及DNA损伤的影响。[方法]生长良好的L-02细胞,分别加入0.001、0.01、0.1μmol/L的DDT,0.01、0.1、1μg/mL的nano-TiO2,以及上述各浓度的DDT和nano-TiO2联合剂量,染毒时间均为24 h;运用DCFH-DA作为荧光探针,采用流式细胞检测技术检测DDT与nano-TiO2联合作用下L-02细胞内ROS含量,运用碱性和中性两种彗星试验检测各组DNA断裂损伤程度。[结果]0.01μmol/L以上浓度组DDT单独作用24 h引起ROS升高(P<0.05)和DNA损伤增加(P<0.05);各浓度nano-TiO2单独染毒均不能引起DNA损伤增加(P>0.05),但1μg/mL单独染毒可引起ROS升高(P<0.05)。各剂量组单独染毒24 h后均无明显的DNA双链损伤(P>0.05),但可导致DNA单链断裂损伤。析因分析结合反应曲面模型显示两种受试物联合染毒可协同增加ROS水平与DNA损伤作用(P<0.05)。[结论]DDT和nano-TiO2联合作用可协同增加各自对DNA单链断裂水平的影响,诱导产生ROS导致DNA损伤是DDT和nano-TiO2引起机体损伤的主要机制之一。

[Objective] To investigate the effects of DDT and nano-TiO2 on ROS level and DNA damage in human embryo liver L-02 cells.[Methods] L-02 cells were exposed separately to different doses of DDT（0.001,0.01,0.1 μmol/L）,nano-TiO2（0.01,0.1,1 μg/mL）,and different concentration of DDT mixed with nano-TiO2 respectively for 24 h.Reactive oxygen species（ROS） level was determined by flow cytometry and DNA damage was measured by alkali and neutral comet assay.[Results] It was observed that exposure to DDT greater than 0.01 μmol/L or 1 μg/mL nano-TiO2 alone could significantly increase ROS production compared with control cells（P〈 0.05）.In alkaline comet assay,no significant effects on DNA damage were observed in single nano-TiO2 groups（P 〉0.05）,but 0.01 μmol/L concentration of DDT or higher could induce DNA damage（P 〈0.05）.However,there was no statistically significant increase of OTM in all groups compared with control group in neutral comet assay（P 〉0.05）.Mixture of DDT and nano-TiO2 was synergistic to increase ROS generation and DNA single strand breaks（P 〈0.05）[Conclusion] Combination of DDT and nano-TiO2 enhanced their capability synergistically in DNA single strand breaks of L-02 cells.The primary mechanism of toxicity may be that DDT and nano-TiO2 can induce the free radical generation,followed by DNA damage.