Purpose: Free OH radicals are considered to be the common mediator of DNA damage after ionizing radiation and oxidative stress. In particular, double-strand breaks (dsb) have a major impact on cell killing after irradiation, while the mechanism of cell killing is less clear for oxidative injury. The latter not only affects DNA, but also equally other cell compartments, such as membranes and mitochondria, which may trigger cell death. This study intended to clarify the relationship between DNA damage induction, repair and cell inactivation for hydrogen peroxide and ionizing radiation.
Materials and methods: Chinese hamster ovary (CHO) cells were treated with H2O2 in serum-free medium in combination with/ without X-irradiation. DNA damage was measured using the alkaline unwinding method or neutral constant-field gel electrophoresis. Cell survival was recorded using the colony-formation assay.
Results: Hydrogen peroxide induced a large number of single-strand breaks (ssb>36000/cell) without impairing cell survival. This number reached a maximum (36 Gy-equiv. at 3 x 10(-4) mol/dm3) without further increase after higher concentrations. Repair kinetics of ssb were similar to those after irradiation. Dsb were found only after very high concentrations of H2O2 (>3 x 10(-2) mol/dm3), which is different from irradiation which generated ssb and dsb in the same dose range. A linear-quadratic increase of dsb was found with increasing concentrations of H2O2 suggesting a single or a pairwise action of OH radicals to form a dsb. After either irradiation or peroxide treatment cell killing was observed only after doses which also allowed dsb detection. The number of dsb calculated per lethal event was in the same range but slightly higher after irradiation (1.7-fold) than after H2O2 treatment.
Conclusions: Cell killing after irradiation or hydrogen peroxide appears to be due to dsb, whereas cells withstand large numbers of single-strand lesions and other types of non-DNA damage occurring at lower concentrations of hydrogen peroxide. The number of ssb saturates at intermediate concentrations of H2O2 suggesting that a limited amount of chromatin-bound metal ions is available for OH radical generation.