Ascorbate as a “redox sensor” and protector against irradiation-induced oxidative stress in 32D CL 3 hematopoietic cells and subclones overexpressing human manganese superoxide dismutase

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PurposeTo determine whether increased expression of manganese superoxide dismutase (MnSOD) protects cells from irradiation by preventing the production of reactive oxygen species (ROS), a new approach to detecting free radical intermediates using ascorbate as an endogenous spin trap was used. Materials and methodsCells from the 32D cl 3 hematopoietic cell line or a subclone overexpressing MnSOD (2C6) were incubated with dehydroascorbate for 30 min and irradiated to doses from 0 to 50 Gy. Radical intermediates reacting with spin traps or ascorbate were measured by electron spin resonance spectroscopy. Results were compared to irradiation-induced changes in thiol levels, irradiation survival curves, and accumulation of 8-OHdG as a measurement of DNA oxidative damage. ResultsManganese superoxide dismutase-overexpressing 2C6 cells maintained higher levels of ascorbate (5.4 ± 0.5 and 2.6 ± 0.5 nmol/106 cells, respectively) and thiols (14.0 ± 0.1 and 11.1 ± 0.2 nmol/106 cells) compared to 32D cl 3 parent cells. Cells overexpressing MnSOD produced lower levels of ROS than did the parental 32D cl 3 cells, as evidenced by lower expenditure of ascorbate and GSH after irradiation. Increased ascorbate levels protected both 32D cl 3 and 2C6 cells from irradiation killing, as demonstrated by an increased shoulder on survival curves and decreased DNA 8-OHdG accumulation. ConclusionsManganese superoxide dismutase overexpression protects 2C6 cells from irradiation damage by scavenging ROS that readily interact with major endogenous antioxidants—ascorbate and GSH—in nontransfected hematopoietic 32D cl 3 cells.