Study on in vitro Comet assay suitable for nanomaterials
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.
Comet testnanomaterialsformamide pyrimidine DNA glycosylation enzyme(FPG)DNA damagegenetic toxicity
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