目的 使用人肝癌HepG2细胞筛选亚硝胺的体外彗星试验的S9 mix配方，并对17种常见的亚硝胺化合物开展彗星试验，研究其DNA亲和力和碱基嵌入风险。方法 在非S9活化和S9活化条件下对HepG2细胞进行N-二甲基亚硝胺（NDMA）、N-二乙基亚硝胺（NDEA）、N-亚硝基二丁胺（NDBA）、N-亚硝基二异丙胺（NDIPA）、N-亚硝基-N-甲基-4-氨基丁酸（NMBA）、N-亚硝基甲乙胺（NMEA）、N-亚硝基-N-乙基异丙胺（NEIPA）、N-亚硝基二丙胺（NDPA）、N-甲基-N-亚硝基苯胺（NMPA）、亚硝基二苯胺（NDPh）、二乙醇亚硝胺（NDELA）、亚硝基吗啉（NMOR）、亚硝基-N-甲基-N-（2-苯基）乙胺（NMPEA）、亚硝基吡咯烷（NPYR）、亚硝基哌啶（NPIP）、4-甲基亚硝胺基-1-3-吡啶基-1-丁酮（NNK）、N-亚硝基降烟碱（NNN）给药处理，2种条件均设置溶媒对照（0.5% DMSO）、3个浓度梯度的给药组和阳性对照组，在非S9活化条件下以甲基磺酸甲酯（MMS）为阳性对照，S9活化条件下以环磷酰胺（CP）为阳性对照。以NDMA和NDEA为例比较3种S9 mix配方对亚硝胺化合物体外DNA亲和力和DNA损伤风险，选择效果最优者开展剩余化合物在S9条件下的彗星试验，计算各组尾DNA含量百分率（% tail DNA）的平均值和中位数。结果 在非S9代谢活化条件下17种常见亚硝胺化合物均未导致HepG2细胞核DNA明显损伤。S9 mix配方C中S9体积分数仅为3.36%，但对亚硝胺化合物的代谢活化效果最佳。在该条件下，除NDPh外，其余亚硝胺化合物均对HepG2细胞存在DNA的损伤作用。烷基类亚硝胺化合物对DNA损伤作用强弱顺序依次为NDMA>NEIPA>NDPA>NMEA>NDEA>NDBA>NDIPA，与化合物α氢的数目基本呈正相关。含苯基的亚硝胺化合物对DNA损伤作用强弱顺序依次为NMPEA>NMPA>NDPh，而环状亚硝胺化合物对DNA损伤作用强弱顺序为NMOR>NPIP≈NPYR。结论 提供最新的亚硝胺化合物体外DNA损伤风险数据，并提出适宜亚硝胺化合物的体外彗星试验S9 mix配方，为亚硝胺化合物的毒性评价提供手段。

Objective The S9 mix formula of in vitro comet assay for nitrosamines was evaluated by human hepatoma HepG2 cells, and 17 common nitrosamines were tested by comet assay to study their DNA affinity and base embedding risk. Method HepG2 cells were treated with N-dimethylnitrosamine (NDMA), N-diethylnitrosamine (NDEA), N-nitrosodibutylamine (NDBA), Nnitrosodiisopropylamine (NDIPA), N-nitroson-N-methyl-4-aminobutyric acid (NMBA), N-nitrosomethylethylamine (NMEA), Nnitroson-ethylisopropylamine (NEIPA), N-nitrosodipropyleneamine (NDPA), N-methyl-n-nitroaniline (NMPA), nitrosodiphenylamine (NDPh), diethanol nitrosamine (NDELA), nitrosomorpholine (NMOR), nitroso-N-methyl-N-(2-phenyl) ethylamine (NMPEA), nitrosopyrrolidine (NPYR), nitroso piperidine (NPIP), 4-methylnitrosamine-1-3-pyridyl-1-butanone (NNK), N-nitrosonornicotine (NNN) under non-S9-activated and S9-activated conditions, solvent control (0.5% DMSO), administration group with three concentration gradients and positive control group were set up, and methyl methylate (MMS) was used as the positive control under non-S9-activated conditions meanwhile cyclophosphamide (CP) as the positive control under the S9-activated conditions. Before the comet assay under S9-activated conditions, taking NDMA and NDEA as examples, the DNA affinity and DNA damage risk of three S9 mix formulations to nitrosamines in vitro were compared, then the average and median of%tail DNA in each group were calculated. Results Seventeen common nitrosamines didn't introduce significant nuclear DNA damage in HepG2 under the condition without S9 metabolic activation. The content of S9 in S9 mix formula C is only 3.36%, whereas demonstrated the best metabolic activation effect on nitrosamines, and all nitrosamine compounds had DNA damage effect on HepG2 cells under this condition except NDPh. The order of the strength of alkyl nitrosamines on DNA damage was as follows:NDMA > NEIPA > NDPA > NDIPA > NMEA > NDEA > NDBA, which had a positive correlation with the number of α-hydrogen of compound. The order of DNA damage caused by nitrosamines containing phenyl groups was as follows:NMPEA > NMPA > NDPh, and the order of DNA damage caused by cyclic nitrosamines was as follows:NMOR > NPIP ≈ NPYR. Conclusion This study provides the latest DNA damage risk data in vitro for nitrosamines, and a suitable comet assay S9 mix formula for nitrosamines in vitro was proposed, which provides a powerful method for the toxicity evaluation of nitrosamines.

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国家十三五"重大新药创制"专项（2018ZX09201017）；国家自然科学基金资助项目（81503347）#共同