基于γ-H2AX检测的两种基因毒性体外测试方法的比较研究  

Comparison of Two in Vitro Genotoxicity Testing Methods Based on γ-H2AX Analysis

作　　者：瞿敏敏 陈佳 张雅姣 徐斌 徐华 谢剑炜 QU Min-Min;CHEN Jia;ZHANG Ya-Jiao;XU Bin;XU Hua;XIE Jian-Wei(State Key Laboratory of Toxicology and Medical Countermeasures,Institute of Pharmacology and Toxicology,Academy of Military Medical Sciences,Beijing 100850,China)

机构地区：[1]军事科学院军事医学研究院毒物药物研究所,抗毒药物与毒理学国家重点实验室,北京100850

出　　处：《分析化学》2021年第12期2039-2047,共9页Chinese Journal of Analytical Chemistry

基　　金：国家重点研发计划项目(No.2018YFC1602600);国家自然科学基金项目(No.21974151)资助。

摘　　要：以7种基因毒性化合物(GCs)及7种非DNA损伤作用化合物(Non-DCs)为模型,分别采用质谱技术与高内涵技术在两种人源细胞株即肝癌细胞HepG2和宫颈癌细胞HeLa中检测磷酸化组蛋白γ-H2AX,对化合物孵育细胞后的基因毒性进行分析检测,进而对基于γ-H2AX检测的两种基因毒性体外测试方法进行比较。结果表明,两种技术均具有高通量检测的优点,均可在2天内完成实验流程。对于本研究所考察的5个GCs,两种技术测定的细胞γ-H2AX水平显著变化的最低起效浓度(MEC)值相同;针对其余2个GCs,质谱测定的MEC值比高内涵技术低。其中,质谱技术在浓度低至10μmol/L苯丁酸氮芥及0.1μmol/L丝裂霉素C时,即可检测到γ-H2AX水平发生显著变化;而高内涵技术需在100μmol/L苯丁酸氮芥及1μmol/L丝裂霉素C浓度下才能检测到γ-H2AX水平发生显著变化,表明质谱技术具有更高的灵敏度。本研究还表明,质谱技术可特异性检测GCs所诱导的γ-H2AX水平变化;而高内涵技术无法区分检测GCs与Non-DCs导致的γ-H2AX焦点,即高内涵技术对于Non-DCs易呈现假阳性结果。本研究通过比较基于γ-H2AX检测的两种基因毒性体外测试方法发现,基于质谱技术的γ-H2AX检测方法不仅能更灵敏、特异性检测化合物基因毒性,而且可动态监测GCs诱导的DNA损伤修复过程,在化合物的基因毒性测试中显示了可行性及优越性。Taking 7 genotoxic compounds(GCs)and 7 non-DNA damaging compounds(Non-DCs)as examples,mass spectrometry(MS)and high-content analysis(HCA)were used for detecting phosphorylated histoneγ-H2AX in two human cell lines(HepG2 and HeLa)to assess the genotoxicity of compound.The results showed that the both techniques could complete the experimental process within 2 days and have high-throughput characteristics.For the selected 5 GCs,the minimum effective concentration(MEC)value of compound that give rise significant phosphorylation of H2AX determined by MS and HCA was same;for the remaining 2 GCs,the MEC value determined by MS was lower than that by HCA.MS could detect the significant changes inγ-H2AX levels at as low as 10μmol/L chlorambucil and 0.1μmol/L mitomycin C,while HCA detected significant changes ofγ-H2AX levels at the concentration of 100μmol/L chlorambucil and 1μmol/L mitomycin C,indicating that MS had higher sensitivity.We also found that MS could specifically detect the changes ofγ-H2AX levels induced by GCs.However,HCA could not distinguishγ-H2AX focus between GCs and Non-DCs.That is,HCA was prone to false positive results for Non-DCs.Taken together,these results suggested that MS analysis ofγ-H2AX could not only detect compound genotoxicity more sensitively and specifically,but also could dynamically monitor the detail profiles of DNA damage and repair processes,which showed feasibility and superiority in the genotoxicity testing of the compounds.

关 键 词：质谱 高内涵技术 基因毒性 组蛋白H2AX 磷酸化组蛋白H2AX 

分 类 号：R99[医药卫生—毒理学] O657.63[医药卫生—药学]