Nikolova Teodora, Dvorak Mirek, Jung Fabian, Adam Isabell, Krämer Elisabeth, Gerhold-Ay Aslihan, Kaina Bernd
Institute of Toxicology, University Medical Center, Obere Zahlbacher Str. 67, D-55131 Mainz, Germany.
Institute of Medical Biometry, Epidemiology and Informatics, University Medical Center, Obere Zahlbacher Str. 67, D-55131 Mainz, Germany.
Toxicol Sci. 2014 Jul;140(1):103-17. doi: 10.1093/toxsci/kfu066. Epub 2014 Apr 17.
DNA double-strand breaks (DSBs) and blocked replication forks resulting from bulky adducts and inhibitors of replication activate the DNA damage response (DDR), a signaling pathway marked by phosphorylation of histone 2AX (H2AX). The phosphorylated form, γH2AX, accumulates at the site of the damage and can be visualized as foci by immunocytochemistry. The objective of this study was to assess if γH2AX is a reliable biomarker for genotoxic exposures. To this end, we selected 14 well-known genotoxic compounds and compared them with 10 nongenotoxic chemicals, using CHO-9 cells because they are well characterized as to DNA repair and DDR. We quantified γH2AX foci manually and automatically. In addition, total γH2AX activation was determined by flow cytometry. For all chemicals the cytotoxic dose response was assayed by a metabolic cytotoxicity assay. We show that (1) all genotoxic agents induced γH2AX dose-dependently whereas nongenotoxic agents do not; (2) γH2AX was observed for genotoxicants in the cytotoxic dose range, revealing a correlation between cytotoxicity and γH2AX for genotoxic agents; for nongenotoxic agents cytotoxicity was not related to γH2AX; (3) manual scoring of γH2AX and automated scoring provided comparable results, the automated scoring was faster and investigator independent; (4) data obtained by foci counting and flow cytometry showed a high correlation, suggesting that γH2AX scoring by flow cytometry has the potential for high-throughput analysis. However, the microscopic evaluation can provide additional information as to foci size, distribution, colocalization and background staining; (5) γH2AX foci were colocalized with 53BP1 and Rad51, supporting the notion that they represent true DSBs. Collectively, the automated analysis of γH2AX foci allows for rapid determination of genetic damage in mammalian cells. The data revealed that the induction of γH2AX by genotoxicants is related to loss of viability and support γH2AX as a reliable bio-indicator for pretoxic DNA damage.
由大分子加合物和复制抑制剂导致的DNA双链断裂（DSB）及受阻的复制叉会激活DNA损伤反应（DDR），这是一条以组蛋白2AX（H2AX）磷酸化为特征的信号通路。磷酸化形式的γH2AX会在损伤部位积累，通过免疫细胞化学可将其可视化为焦点。本研究的目的是评估γH2AX是否为遗传毒性暴露的可靠生物标志物。为此，我们选择了14种知名的遗传毒性化合物，并将它们与10种非遗传毒性化学物质进行比较，使用CHO-9细胞，因为它们在DNA修复和DDR方面具有良好的特征。我们手动和自动定量γH2AX焦点。此外，通过流式细胞术测定总γH2AX激活情况。对于所有化学物质，通过代谢细胞毒性测定法测定细胞毒性剂量反应。我们发现：（1）所有遗传毒性剂均剂量依赖性地诱导γH2AX，而非遗传毒性剂则不会；（2）在细胞毒性剂量范围内观察到遗传毒性剂诱导γH2AX，这揭示了遗传毒性剂的细胞毒性与γH2AX之间的相关性；对于非遗传毒性剂，细胞毒性与γH2AX无关；（3）γH2AX的手动评分和自动评分提供了可比的结果，自动评分更快且与研究者无关；（4）通过焦点计数和流式细胞术获得的数据显示出高度相关性，这表明通过流式细胞术对γH2AX进行评分具有高通量分析的潜力。然而，显微镜评估可以提供有关焦点大小、分布、共定位和背景染色的额外信息；（5）γH2AX焦点与53BP1和Rad51共定位，支持它们代表真正DSB的观点。总体而言，对γH2AX焦点的自动分析能够快速确定哺乳动物细胞中的遗传损伤。数据表明，遗传毒性剂诱导γH2AX与活力丧失有关，并支持γH2AX作为中毒前DNA损伤的可靠生物指标。
