Citation: QU Min-Min, CHEN Jia, ZHANG Ya-Jiao, XU Bin, XU Hua, XIE Jian-Wei. Comparison of Two in Vitro Genotoxicity Testing Methods Based on γ-H2AX Analysis[J]. Chinese Journal of Analytical Chemistry, ;2021, 49(12): 2039-2047. doi: 10.19756/j.issn.0253-3820.210737 shu

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

State Key Laboratory of Toxicology and Medical Countermeasures, Institute of Pharmacology and Toxicology, Academy of Military Medical Sciences, Beijing 100850, China

Corresponding author: XU Hua, huarxu@163.com
Received Date: 9 September 2021
Revised Date: 3 November 2021

Fund Project: Supported by the National Key R&D Program of China (No.2018YFC1602600) and the National Natural Science Foundation of China(No.21974151).

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 histone γ-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 in γ-H2AX levels at as low as 10 μmol/L chlorambucil and 0.1 μmol/L mitomycin C, while HCA detected significant changes of γ-H2AX levels at the concentration of 100 μmol/L chlorambucil and 1 μmol/L mitomycin C, indicating that MS had higher sensitivity. We also found that MS could specifically detect the changes of γ-H2AX levels induced by GCs. However, HCA could not distinguish γ-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 of γ-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.
- Mass spectrometry,
- High-content analysis,
- Genotoxicity,
- Histone H2AX,
- Phosphorylated histone H2AX
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