Sánchez-Flores María, Pásaro Eduardo, Bonassi Stefano, Laffon Blanca, Valdiglesias Vanessa
*DICOMOSA Group, Department of Psychology, Area of Psychobiology, Universidade da Coruña, Edificio de Servicios Centrales de Investigación, Campus Elviña s/n, 15071 Coruña, Spain and Unit of Clinical and Molecular Epidemiology, IRCCS San Raffaele Pisana, Via di Val Cannuta, 247, 00166 Rome, Italy.
*DICOMOSA Group, Department of Psychology, Area of Psychobiology, Universidade da Coruña, Edificio de Servicios Centrales de Investigación, Campus Elviña s/n, 15071 Coruña, Spain and Unit of Clinical and Molecular Epidemiology, IRCCS San Raffaele Pisana, Via di Val Cannuta, 247, 00166 Rome, Italy
Toxicol Sci. 2015 Apr;144(2):406-13. doi: 10.1093/toxsci/kfv011. Epub 2015 Jan 22.
H2AX histone phosphorylation represents an early event in the cellular response against DNA double-strand breaks (DSBs), and plays a central role in sensing and repairing DNA damage. Therefore, the analysis of H2AX phosphorylated (γH2AX) may be possibly used as biomarker of genotoxicity and genomic instability with a number of applications in human epidemiology. However, the lack of an experimental standard leads to a wide heterogeneity in the results obtained and their interpretation, affecting the reliability of the assay. To address the most critical issues limiting the use of the γH2AX assay in human population studies, a flow cytometry analysis was performed to establish differences in γH2AX levels between fresh or cryopreserved peripheral blood lymphocytes, and to assess the influence of phytohemagglutinin (PHA) stimulation. To this purpose, cells were treated with 4 known genotoxic chemicals with different mechanisms of DSB induction, ie, bleomycin, methyl methanesulfonate, camptothecin, and actinomycin. According to our results, both unstimulated and stimulated fresh lymphocytes can be efficiently employed to evaluate γH2AX levels, but the sensitivity of the assay is depending upon the kind of damage observed. On the other hand, cryopreserved lymphocytes require PHA stimulation since unstimulated cells showed too high basal damage. Consequently, the protocol conditions will depend on the expected mechanism of production of DSB and the characteristics of the study design (sample collection and storage conditions, type of epidemiological study). Further studies are required to standardize the protocol of γH2AX assay to be employed as biomarker of genotoxicity or genomic instability in human population studies.
H2AX组蛋白磷酸化是细胞对DNA双链断裂（DSB）作出反应的早期事件，在感知和修复DNA损伤中起核心作用。因此，对磷酸化H2AX（γH2AX）的分析可能用作遗传毒性和基因组不稳定性的生物标志物，在人类流行病学中有许多应用。然而，缺乏实验标准导致所获得的结果及其解释存在很大异质性，影响了该检测方法的可靠性。为了解决限制γH2AX检测方法在人群研究中应用的最关键问题，进行了流式细胞术分析，以确定新鲜或冷冻保存的外周血淋巴细胞之间γH2AX水平的差异，并评估植物血凝素（PHA）刺激的影响。为此，用4种已知的具有不同DSB诱导机制的遗传毒性化学物质处理细胞，即博来霉素、甲基磺酸甲酯、喜树碱和放线菌素。根据我们的结果，未刺激和刺激的新鲜淋巴细胞均可有效用于评估γH2AX水平，但检测方法的敏感性取决于所观察到的损伤类型。另一方面，冷冻保存的淋巴细胞需要PHA刺激，因为未刺激的细胞显示出过高的基础损伤。因此，方案条件将取决于预期的DSB产生机制和研究设计的特点（样本采集和储存条件、流行病学研究类型）。需要进一步研究以标准化γH2AX检测方法的方案，以便在人群研究中用作遗传毒性或基因组不稳定性的生物标志物。
