目的 通過比較體外彗星常規(guī)試驗方法和添加DNA修復(fù)酶——甲酰胺嘧啶DNA糖基化酶（FPG）的改良試驗方法，探究適宜評價納米材料DNA損傷的遺傳毒性試驗方法。方法 使用TK6和CHL細(xì)胞，以聚苯乙烯微球為納米級陰性對照，設(shè)置低、中、高濃度（5、10、20 μg·mL^-1）的納米標(biāo)準(zhǔn)物質(zhì)Ag40，分別使用體外彗星常規(guī)試驗方法和FPG酶改良試驗方法，在非代謝活化條件下與細(xì)胞暴露4、24、72 h（以甲基磺酸甲酯為陽性對照），代謝活化條件下添加2種配方的S9mix（配方A中S9質(zhì)量分?jǐn)?shù)為10%，配方B中S9質(zhì)量分?jǐn)?shù)為3.36%，另添加鈣離子）與細(xì)胞暴露4 h（以環(huán)磷酰胺為陽性對照），檢測% tail DNA和Olive尾距。結(jié)果 聚苯乙烯納米微球與溶媒對照組比較未見統(tǒng)計學(xué)差異，體外彗星試驗結(jié)果為陰性，陽性對照組結(jié)果均為陽性。非代謝活化條件下Ag40與TK6、CHL細(xì)胞作用4、24、72 h，高、中、低濃度組的% tailDNA、Olive尾距與溶媒對照組相比均明顯升高，體外彗星試驗結(jié)果為陽性。改良FPG酶處理法與常規(guī)方法在各條件下相比，Ag40的% tail DNA、Olive尾距均有一定程度的升高。代謝活化條件下，S9 mix配方A和配方B的體外彗星試驗陽性結(jié)果基本一致。結(jié)論 添加FPG酶及調(diào)整S9含量（質(zhì)量分?jǐn)?shù)3.36%）后的改良體外彗星試驗方法適用于納米材料的DNA損傷風(fēng)險評價。

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 20 μg·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.

R965.1

國家重點研發(fā)計劃（2022YFC2409702）