Induction and Repair of DNA DSB as Revealed by H2AX Phosphorylation Foci in Human Fibroblasts Exposed to Low- and High-LET Radiation: Relationship with Early and Delayed Reproductive Cell Death

Abstract

The spatial distribution of radiation-induced DNA breaks within the cell nucleus depends on radiation quality in terms of energy deposition pattern. It is generally assumed that the higher the radiation linear energy transfer (LET), the greater the DNA damage complexity. Using a combined experimental and theoretical approach, we examined the phosphorylation-dephosphorylation kinetics of radiation-induced γ-H2AX foci, size distribution and 3D focus morphology, and the relationship between DNA damage and cellular end points (i.e., cell killing and lethal mutations) after exposure to gamma rays, protons, carbon ions and alpha particles. Our results showed that the maximum number of foci are reached 30 min postirradiation for all radiation types. However, the number of foci after 0.5 Gy of each radiation type was different with gamma rays, protons, carbon ions and alpha particles inducing 12.64 ± 0.25, 10.11 ± 0.40, 8.84 ± 0.56 and 4.80 ± 0.35 foci, respectively, which indicated a clear influence of the track structure and fluence on the numbers of foci induced after a dose of 0.5 Gy for each radiation type. The γ-H2AX foci persistence was also dependent on radiation quality, i.e., the higher the LET, the longer the foci persisted in the cell nucleus. The γ-H2AX time course was compared with cell killing and lethal mutation and the results highlighted a correlation between cellular end points and the duration of γ-H2AX foci persistence. A model was developed to evaluate the probability that multiple DSBs reside in the same gamma-ray focus and such probability was found to be negligible for doses lower than 1 Gy. Our model provides evidence that the DSBs inside complex foci, such as those induced by alpha particles, are not processed independently or with the same time constant. The combination of experimental, theoretical and simulation data supports the hypothesis of an interdependent processing of closely associated DSBs, possibly associated with a diminished correct repair capability, which affects cell killing and lethal mutation.

Measurement of complex DNA damage induction and repair in human cellular systems after exposure to ionizing radiations of varying linear energy transfer (LET)

“…The existence of more than one DSB under a focus can be an issue for underestimation and the correct assignment of focus size can improve this situation but up to a point. The yields we found for low-LET (Figure 2) are compatible with previous findings of 10-20 DSB foci/Gy/cell [37,42,43] and the same stands also for a-particles [35,44,45] and Ar ions [46], although in the specific study 40 Ar ions of a much lower LET (125 keV/lm) were used. Previous studies using cold lysis PFGE protocols in order to minimize conversion of heat-labile sites to DSBs suggest relatively increased numbers of $25 DSBs/Gy/cell for normal human skin fibroblasts (GM5758) for low-LET radiations [47].…”

“…Therefore in the case of a c-H2AX focus which can spread to a chromosome region up to 10-20 Mbp [19], one can expect roughly $0.2-0.4 DSB and 2-4 TSB per focus (low-LET and 1 Gy). DSB clusters are expected for higher doses or high-LET particles where there is a non-homogenous density of breaks along the particle track and one expects even 8 DSBs per focus for a-particles based also on PARTRAC simulations [35]. The electron energies studied cover the low-medium LET range (1-20 keV/lm), so the results can be used to explain some of the characteristics associated with the transition from low-to high-LET radiation.…”

“…In all cases, a variation can be found in literature due to the different cell lines, time points and other technical factors. Comparison of DSB yields for radiations of different LETs shows an unexpected lower yield for DSBs for higher LET, which is probably explained by the DSB clustering and the underestimation of the actual true DSB number under one focus [35]. In order to improve the statistics of foci measurements due to the inherent heterogeneity of cell sub-populations (G0-G1, S-G2), we applied a specific statistical analysis methodology in the case of HepG2 cells as described in the Supplementary data section.…”

Analysis of the miRNA–mRNA networks in malignant transformation BEAS-2B cells induced by alpha-particles

“…Data showed that exposure to alpha particles in vitro produced induced DNA and chromosomal damage, consequently decreasing cell survival rate (Antonelli et al, 2015;Sumption et al, 2015). In our study, cell survival rate was also reduced in a dose-dependent manner.…”

Visualization of complex DNA damage along accelerated ions tracks

“…It should be noted that the efficiency of the DNA DSBs repair induced by radiation of different quality based on the γH2AX foci formation and elimination kinetics was also shown in [4]. However in this study only a qualitative analysis of the complexity of the induced lesions was performed.…”