Induction of Clustered DNA Damage in Human Cells and Ex Vivo Epidermis by Low Doses of Low Dose Rate Simulated Solar Particle Event Protons

Dose rate and radiation quality are key issues in predicting and mitigating cancer risk from space radiation. Solar particle events (SPEs) present a major radiation-related risk for manned exploratory missions in deep space. Astronauts may be exposed to significant radiation doses from exposure to high fluences of low energy, high linear energy transfer (LET) protons within a short time frame. While these acute exposures can be mitigated with shielding, space travelers still have the possible risk of receiving significant radiation exposures during SPEs. Dose equivalents from these exposures have been estimated as being between 5–40 cSv through 10g•cm of aluminum shielding [1]. Such exposures have the potential to increase risk of radiation-induced carcinogenesis in human space travelers. We recently reported results on our studies of clonogenic survival and induction of neoplastic transformation (i.e., acquisition of anchorage-independent growth in soft agar) in human cells irradiated with monoenergetic protons having similar energy spectra to the 1989 SPE delivered at Brookhaven National Laboratory’s NASA Space Radiation Laboratory (NSRL). Low doses (0–30 cGy) of protons (50, 100, and 1000 MeV) were delivered to apparently normal human fibroblasts at a SPE-like dose-rate of 1.65 cGy•min with high dose-rate (33.3 cGy•min) sets run in parallel. In the low dose range (≤ 30cGy), sparing effects of the low dose-rate exposures were observed both for cell survival and transformation [2]. Importantly, low dose-rate transformation frequencies increased above high dose-rate transformation frequencies as doses >30 cGy were accumulated. Transformation potential also increased with increasing LET and decreasing particle energy. These results indicate that both dose-rate effects and radiation quality are of critical importance in evaluating the biological effects and potential carcinogenic risks of space radiation exposure. The induction of clustered DNA damage and its subsequent processing may play a significant mechanistic role in the acquisition of the carcinogenic mutations and genomic instability in cells surviving proton irradiation which is likely related to the increased transformation frequencies we observe with protons of increasing LET. Clustered DNA damage is bistranded and consists of two or more oxidized bases, abasic sites or strand breaks on opposing strands within a few helical turns – these complex damage sites are considered critical lesions underlying the observed refractory repair and increased biological effectiveness of high LET-induced DNA damage. Studies performed in our laboratory on megabase pair-scale genomic DNA irradiated with different HZE particles or protons of different energies in radioquenching conditions (representative of the cellular microenvironment) showed that the spectrum of clustered DNA damages likewise changes with radiation quality and increases with LET. However, the induction and processing of clustered DNA damages in human cells and whole tissues irradiated with protons of different energies at low dose-rates typical of the space radiation environment is currently not well understood. Companion experiments were conducted in which apparently normal human fibroblasts and ex vivo human epidermis samples were irradiated with both high and 1989 SPE-like low dose-rate proton exposures and clustered DNA damage levels and DNA double strand breaks were measured electrophoretically and by immunocytochemical staining of DSB-associated nuclear foci (γ-H2AX pS139, pATM pS1981). We report here on the induction and processing of clustered damage and DSBs induced in these samples by low dose, low dose-rate proton exposures. We show clustered DNA damage yields increase with increasing LET and are mitigated by low dose-rate exposures but are readily detectable at doses as low as 2–5 cGy of low dose-rate protons.