Ascorbate and H 2O 2 induced oxidative DNA damage in Jurkat cells

Abstract

The effect of vitamin C (ascorbate) on oxidative DNA damage was examined by first incubating cells with dehydroascorbate, which boosts the intracellular concentration of ascorbate, and then exposing cells to H 2O 2. Oxidative DNA damage was estimated by the analysis of 5-hydroxy-2′-deoxycytidine (oh 5dCyd) and 8-oxo-7,8-dihydro-2′-deoxyguanosine (oxo 8dGuo). The presence of a high concentration of ascorbate (30 mM), compared to the absence of ascorbate in cells, when exposed to H 2O 2 (200 μM), resulted in a remarkable sensitization of oh 5dCyd from 2.7 ± 0.6 to 40.8 ± 6.1 lesions /10 6 dCyd (15-fold). In contrast, the level of oxo 8dGuo increased from 8.4 ± 0.4 to 12.1 ± 0.5 lesions/10 6 dGuo (50%). The formation of oh 5dCyd was also observed at lower concentrations of intracellular ascorbate and exogenous H 2O 2. Additional studies showed that replacement of H 2O 2 with tert-butyl hydroperoxide completely abolished damage, and that preincubation with iron and desferroxamine increased and decreased this damage, respectively. The latter studies suggest that a Fenton reaction is involved in the mechanism of damage. In conclusion, we report a novel model system in which ascorbate sensitizes H 2O 2-induced oxidative DNA damage in cells, leading to elevated levels of oh 5dCyd and oxo 8dGuo, with a strong bias toward the formation of oh 5dCyd.