Factors to Consider for the Correct Use of γH2AX in the Evaluation of DNA Double-Strand Breaks Damage Caused by Ionizing Radiation

SIMPLE SUMMARY: The increase of exposure to ionizing radiation (IR) from medical procedures has prompted research into improving methodologies for the detection of DNA double-strand breaks (DSBs). The measurement of γH2AX by immunofluorescence has become the gold standard for this analysis. However, for the correct use of γH2AX as a biomarker for the assessment of IR-induced DNA DSBs, several exogenous and endogenous conditions that can influence γH2AX levels must be taken into consideration. Here, we describe the conditions leading to H2AX phosphorylation, the most widely used methods for its detection, the principal applications, and the related problems of γH2AX scoring, with particular regard to clinical studies. ABSTRACT: People exposed to ionizing radiation (IR) both for diagnostic and therapeutic purposes is constantly increasing. Since the use of IR involves a risk of harmful effects, such as the DNA DSB induction, an accurate determination of this induced DNA damage and a correct evaluation of the risk–benefit ratio in the clinical field are of key relevance. γH2AX (the phosphorylated form of the histone variant H2AX) is a very early marker of DSBs that can be induced both in physiological conditions, such as in the absence of specific external agents, and by external factors such as smoking, heat, background environmental radiation, and drugs. All these internal and external conditions result in a basal level of γH2AX which must be considered for the correct assessment of the DSBs after IR exposure. In this review we analyze the most common conditions that induce H2AX phosphorylation, including specific exogenous stimuli, cellular states, basic environmental factors, and lifestyles. Moreover, we discuss the most widely used methods for γH2AX determination and describe the principal applications of γH2AX scoring, paying particular attention to clinical studies. This knowledge will help us optimize the use of available methods in order to discern the specific γH2AX following IR-induced DSBs from the basal level of γH2AX in the cells.