High Throughput Measurement of γH2AX DSB Repair Kinetics in a Healthy Human Population

Abstract

The Columbia University RABiT (Rapid Automated Biodosimetry Tool) quantifies DNA damage using fingerstick volumes of blood. One RABiT protocol quantifies the total γ-H2AX fluorescence per nucleus, a measure of DNA double strand breaks (DSB) by an immunofluorescent assay at a single time point. Using the recently extended RABiT system, that assays the γ-H2AX repair kinetics at multiple time points, the present small scale study followed its kinetics post irradiation at 0.5 h, 2 h, 4 h, 7 h and 24 h in lymphocytes from 94 healthy adults. The lymphocytes were irradiated ex vivo with 4 Gy γ rays using an external Cs-137 source. The effect of age, gender, race, ethnicity, alcohol use on the endogenous and post irradiation total γ-H2AX protein yields at various time points were statistically analyzed. The endogenous γ-H2AX levels were influenced by age, race and alcohol use within Hispanics. In response to radiation, induction of γ-H2AX yields at 0.5 h and peak formation at 2 h were independent of age, gender, ethnicity except for race and alcohol use that delayed the peak to 4 h time point. Despite the shift in the peak observed, the γ-H2AX yields reached close to baseline at 24 h for all groups. Age and race affected the rate of progression of the DSB repair soon after the yields reached maximum. Finally we show a positive correlation between endogenous γ-H2AX levels with radiation induced γ-H2AX yields (RIY) (r=0.257, P=0.02) and a negative correlation with residuals (r=-0.521, P=<0.0001). A positive correlation was also observed between RIY and DNA repair rate (r=0.634, P<0.0001). Our findings suggest age, race, ethnicity and alcohol use influence DSB γ-H2AX repair kinetics as measured by RABiT immunofluorescent assay.

Persistent γH2AX: A promising molecular marker of DNA damage and aging

“…The majority of DSBs ($80%) are repaired during the first phase of the repair process and the remaining portion ($20%) repair at a slower pace during the slower phase [55,56]. The slow gH2AX repair kinetics reported in lymphocytes from healthy donors following exposure to IR is consistent with the findings that showed $25% of residual gH2AX foci at 7 h after exposure to 4 Gy of IR in lymphocytes [57,58]. Evidence from several studies suggests that 60% of initial IR-induced DSBs are transient and repair in a relatively fast manner, often with half-lives of approximately 1-18 min [59,60].…”

“…The rate of gH2AX foci loss and the presence of residual foci has also been correlated with cellular radiosensitivity and absorbed radiation dose [47,56,[67][68][69][70][71]. Estimation of DSB repair rate from the decline kinetics of gH2AX foci was reported as a useful parameter to evaluate cellular radiosensitivity [58]. The persistent gH2AX foci may be present in the form of large foci.…”

“…Clinical studies have demonstrated that the stochastic gH2AX foci induction and loss after external and internal radiation exposure in different types of cell depend on (i) the amount or type of IR (e.g. high dose (radiotherapy), low dose X-ray examination, or computed tomography (CT) scan), chemotherapeutic drug and genotoxic compound used; (ii) type of sample or part of body exposed to IR; (iii) duration or fractionation of exposure; (iv) inter individual radiosensitivity or damage response; (v) methods to measure gH2AX immunoreactivity; (vi) time-points for the kinetics of gH2AX foci formation and loss; (vii) time elapsed between the exposure and the gH2AX analysis, particularly if genotoxic exposure is acute rather than chronic [58,75,76].…”

“…Therefore, persistence of gH2AX following the exposure to IR in human lymphocytes could be used as a maker to identify the radiosensitivity and the ability of individuals to recover from IR-related damage. The effect of age, gender, race, ethnicity, and alcohol use was investigated on IR-induced persistent gH2AX (24 h) in lymphocytes from healthy adults [58]. Of these demographic variables, there was a decline of persistent gH2AX…”

THE DECADE OF THE RABiT (2005–15)

“…An assay for quantifying DNA repair kinetics (28,29) was also developed (under NIEHS funding) by modifying the gamma-H2AX RABiT assay. In the modified assay, an irradiated sample is split and analyzed at multiple time points post irradiation, generating a curve as seen in Figure 4.…”