适宜纳米材料的体外彗星试验方法研究  

Study on in vitro Comet assay suitable for nanomaterials

作　　者：吴辉 汪祺 杨颖[1] 耿兴超 文海若[3] WU Hui;WANG Qi;YANG Ying;GENG Xingchao;WEN Hairuo(China Pharmaceutical University School,Nanjing 210009,China;Institute of Chinese Traditional Medicine and Ethnic Medicine,National Institutes for Food and Drug Control,Beijing 100050,China;Beijing Key Laboratory of Non-Clinical Drug Safety Evaluation,National Center for Safety Evaluation of Drugs,National Institutes for Food and Drug Control,Beijing 100176,China)

机构地区：[1]中国药科大学,江苏南京210009 [2]中国食品药品检定研究院中药民族药检定所,北京100050 [3]中国食品药品检定研究院国家药物安全评价监测中心,药物非临床安全评价研究北京市重点实验室,北京100176

出　　处：《药物评价研究》2024年第8期1695-1701,共7页Drug Evaluation Research

基　　金：国家重点研发计划(2022YFC2409702)。

摘　　要：目的通过比较体外彗星常规试验方法和添加DNA修复酶——甲酰胺嘧啶DNA糖基化酶(FPG)的改良试验方法,探究适宜评价纳米材料DNA损伤的遗传毒性试验方法。方法使用TK6和CHL细胞,以聚苯乙烯微球为纳米级阴性对照,设置低、中、高浓度(5、10、20μg·mL^(-1))的纳米标准物质Ag40,分别使用体外彗星常规试验方法和FPG酶改良试验方法,在非代谢活化条件下与细胞暴露4、24、72 h(以甲基磺酸甲酯为阳性对照),代谢活化条件下添加2种配方的S9mix(配方A中S9质量分数为10%,配方B中S9质量分数为3.36%,另添加钙离子)与细胞暴露4 h(以环磷酰胺为阳性对照),检测%tail DNA和Olive尾距。结果聚苯乙烯纳米微球与溶媒对照组比较未见统计学差异,体外彗星试验结果为阴性,阳性对照组结果均为阳性。非代谢活化条件下Ag40与TK6、CHL细胞作用4、24、72 h,高、中、低浓度组的%tail DNA、Olive尾距与溶媒对照组相比均明显升高,体外彗星试验结果为阳性。改良FPG酶处理法与常规方法在各条件下相比,Ag40的%tail DNA、Olive尾距均有一定程度的升高。代谢活化条件下,S9 mix配方A和配方B的体外彗星试验阳性结果基本一致。结论添加FPG酶及调整S9含量(质量分数3.36%)后的改良体外彗星试验方法适用于纳米材料的DNA损伤风险评价。Objective To explore the genetic toxicity test method suitable for evaluating the DNA damage of nanomaterials,by comparing the conventional comet test method in vitro with the modified formamide pyrimidine DNA glycosylation enzyme(FPG)enzyme test method.Methods TK6 and CHL cells were used,polystyrene microspheres were used as nanoscale negative controls,and the nanoscale reference material Ag40 in low,medium,and high concentrations(5,10,and 20μg·mL^(-1))were exposed to the cells under non-metabolic activation conditions for 4,24 and 72 h(using methyl methanesulfonate as a positive control)using two different test methods,respectively.Under metabolic activation conditions,two different formulations of S9 mix were added(10%S9 content in formula A,3.36%S9 content in formula B,plus calcium ions)and cells were exposed for 4 h(using cyclophosphamide as a positive control),and%tail DNA and Olive Tail Moment(OTM)were detected.Results Ag40 was treated with TK6 and CHL cells for 4 h,24 h and 72 h under non-metabolic activation conditions,and the%tail DNA of high,medium and low dose groups was significantly different from that of the solvent control group,and the results of comet test in vitro were positive.There was no statistical difference between polystyrene nanospheres and solvent control group,and the results of comet test in vitro were negative.Compared with the conventional method,the OTM of Ag40 was increased to a certain extent by modified FPG enzyme treatment.Under the condition of metabolic activation,the positive results of in vitro comet test of formula A and formula B of S9 mix were basically the same.Conclusion The modified in vitro comet assay method after adding FPG enzyme and adjusting S9 content(3.36%)is suitable for DNA damage risk assessment of nanomaterials.

关 键 词：彗星试验 纳米材料 甲酰胺嘧啶DNA糖基化酶(FPG) DNA损伤 遗传毒性 

分 类 号：R965.1[医药卫生—药理学]