Nanoparticles are widely used in cosmetic, pharmaceutical, and food industries, but their safety and genetic toxicity are still unclear. In this study, the genotoxicity of silver nanoparticles (AgNPs) and titanium dioxide nanoparticles (titanium dioxide nanoparticles) were evaluated by in vitro comet assay and PIG-A assay in TK6 cells. We exposed TK6 cells to two types of nanoparticles at the highest concentration of 200 μmol/L for 4 h and conducted the in vitro comet assay. We examined the mutation results of PIG-A gene in vitro after 4 h, 24 ho and 10 days of exposure, respectively. We also examined the endocytosis of nanoparticles in TK6 cells exposed to nanoparticles for 24 h. In the endocytosis assay, with the increase of nano-material concentration, the side scatter (SSC) of TK6 cells in flow cytometry showed a concentration-dependent and time-dependent increase, indicating that TK6 cells could uptake both types of nanoparticles. In the comet assay, AgNPs could induce a concentration-dependent increase in DNA tail intensity. However, titanium dioxide NPs could not induce the concentration-dependent increase of DNA fluorescence intensity of comet tail. In the PIG-A assay, both AgNPs and TiO2NPs did not induce PIG-A gene mutation frequency in TK6 cells. The results showed that AgNPs could induce DNA damage in TK6 cells, but could not induce increase of PIG-A gene mutation frequency. TiO2NPs neither induce DNA damage in TK6 cells nor increase PIG-A mutation frequency. Further tests are needed to determine whether TiO2NPs are genotoxic.
纳米物质以其独特的物理性质广泛用于化妆品、医药和食品工业,但它们的安全性和遗传毒性仍然存在争议。本研究拟采用基于人成淋巴TK6细胞的体外彗星实验整合体外PIG-A基因突变实验对纳米银颗粒(AgNPs)和纳米二氧化钛颗粒(TiO2NPs)的致DNA断裂作用和致突变性进行初步研究。本研究探究了最高至200 μmol/L浓度时,TK6细胞暴露于两种纳米物质4 h时的彗星实验和经过暴露24 h以及10 d基因突变表达期后的PIG-A基因突变结果。同时,本研究还检测了TK6细胞暴露于纳米颗粒4 h及24 h对纳米物质的摄取能力。在纳米物质摄取实验中,随着纳米物质浓度的升高,TK6细胞在流式细胞仪检测图中的侧向散射角(side scatter, SSC)呈现浓度与时间依赖性升高,表明TK6细胞可摄取两种纳米颗粒。在4 h彗星实验中,AgNPs可导致彗星尾DNA荧光%强度呈现浓度依赖性显著升高,为阳性结果。而TiO2NPs则不能诱导彗星尾DNA荧光%强度呈现浓度依赖性显著升高,但最高浓度组尾DNA荧光%超过本实验室阴性历史对照数据,为可疑结果。而在体外PIG-A基因突变实验结果中,AgNPs和TiO2NPs均不能诱导TK6细胞的PIG-A基因突变率出现阳性升高。AgNPs可导致TK6细胞出现DNA损伤,但不能诱导PIG-A基因突变率升高。TiO2NPs既不能诱导TK6细胞出现DNA损伤,也不能诱导PIG-A基因突变率升高,TiO2NPs的遗传毒性有待进一步验证。.
Keywords: comet assay; genotoxicity; in vitro PIG-A assay; silver nanoparticles; titanium dioxide nanoparticles.