Ascorbate and H2O2 induced oxidative DNA damage in Jurkat cells

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 H2O2. Oxidative DNA damage was estimated by the analysis of 5-hydroxy-2'-deoxycytidine (oh(5)dCyd) and 8-oxo-7,8-dihydro-2'-deoxyguanosine (oxo(8)dGuo). The presence of a high concentration of ascorbate (30 mM), compared to the absence of ascorbate in cells, when exposed to H2O2 (200 mu M), resulted in a remarkable sensitization of oh(5)dCyd from 2.7 +/- 0.6 to 40.8 +/- 6.1 lesions/10(6) dCyd (15-fold). In contrast, the level of oxo(8)dGuo increased from 8.4 +/- 0.4 to 12.1 +/- 0.5 lesions/10(6) dGuo (50%). The formation of oh(5)dCyd was also observed at lower concentrations of intracellular ascorbate and exogenous H2O2. Additional studies showed that replacement of H2O2 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 H2O2-induced oxidative DNA damage in cells, leading to elevated levels of oh(5)dCyd and oxo(8)dGuo, with a strong bias toward the formation of oh(5)dCyd. (c) 2006 Elsevier Inc. All rights reserved.