目的 使用人肝癌HepG2細(xì)胞篩選亞硝胺的體外彗星試驗(yàn)的S9 mix配方，并對17種常見的亞硝胺化合物開展彗星試驗(yàn)，研究其DNA親和力和堿基嵌入風(fēng)險(xiǎn)。方法 在非S9活化和S9活化條件下對HepG2細(xì)胞進(jìn)行N-二甲基亞硝胺（NDMA）、N-二乙基亞硝胺（NDEA）、N-亞硝基二丁胺（NDBA）、N-亞硝基二異丙胺（NDIPA）、N-亞硝基-N-甲基-4-氨基丁酸（NMBA）、N-亞硝基甲乙胺（NMEA）、N-亞硝基-N-乙基異丙胺（NEIPA）、N-亞硝基二丙胺（NDPA）、N-甲基-N-亞硝基苯胺（NMPA）、亞硝基二苯胺（NDPh）、二乙醇亞硝胺（NDELA）、亞硝基嗎啉（NMOR）、亞硝基-N-甲基-N-（2-苯基）乙胺（NMPEA）、亞硝基吡咯烷（NPYR）、亞硝基哌啶（NPIP）、4-甲基亞硝胺基-1-3-吡啶基-1-丁酮（NNK）、N-亞硝基降煙堿（NNN）給藥處理，2種條件均設(shè)置溶媒對照（0.5% DMSO）、3個(gè)濃度梯度的給藥組和陽性對照組，在非S9活化條件下以甲基磺酸甲酯（MMS）為陽性對照，S9活化條件下以環(huán)磷酰胺（CP）為陽性對照。以NDMA和NDEA為例比較3種S9 mix配方對亞硝胺化合物體外DNA親和力和DNA損傷風(fēng)險(xiǎn)，選擇效果最優(yōu)者開展剩余化合物在S9條件下的彗星試驗(yàn)，計(jì)算各組尾DNA含量百分率（% tail DNA）的平均值和中位數(shù)。結(jié)果 在非S9代謝活化條件下17種常見亞硝胺化合物均未導(dǎo)致HepG2細(xì)胞核DNA明顯損傷。S9 mix配方C中S9體積分?jǐn)?shù)僅為3.36%，但對亞硝胺化合物的代謝活化效果最佳。在該條件下，除NDPh外，其余亞硝胺化合物均對HepG2細(xì)胞存在DNA的損傷作用。烷基類亞硝胺化合物對DNA損傷作用強(qiáng)弱順序依次為NDMA>NEIPA>NDPA>NMEA>NDEA>NDBA>NDIPA，與化合物α氫的數(shù)目基本呈正相關(guān)。含苯基的亞硝胺化合物對DNA損傷作用強(qiáng)弱順序依次為NMPEA>NMPA>NDPh，而環(huán)狀亞硝胺化合物對DNA損傷作用強(qiáng)弱順序?yàn)镹MOR>NPIP≈NPYR。結(jié)論 提供最新的亞硝胺化合物體外DNA損傷風(fēng)險(xiǎn)數(shù)據(jù)，并提出適宜亞硝胺化合物的體外彗星試驗(yàn)S9 mix配方，為亞硝胺化合物的毒性評(píng)價(jià)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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國家十三五"重大新藥創(chuàng)制"專項(xiàng)（2018ZX09201017）；國家自然科學(xué)基金資助項(xiàng)目（81503347）#共同