Hydrogen peroxide is responsible for UVA-induced DNA damage measured by alkaline comet assay in HaCaT keratinocytes

We investigated the role of different reactive oxygen species (ROS) in ultraviolet A (WA)-induced DNA damage in a human keratinocyte cell line, HaCaT. UVA irradiation increased the intracellular levels of hydrogen peroxide (H2O2), detected by a fluorescent probe carboxydichlorodihydrofluorescein, and caused oxidative DNA damage, single strand-breaks and alkali-labile sites, measured by alkaline single cell gel electrophoresis (comet assay). Superoxide anion (O-2(.-)) was a likely substrate for H2O2 production since diethyldithiocarbamate (DDC), a superoxide dismutase blocker, decreased the level of intracellular H2O2. Hydrogen peroxide was shown to play a central role in DNA damage. Increasing the intracellular levels of H2O2 with aminotriazole (AT) (a catalase blocker) and buthionine sulfoximine (BSO) (an inhibitor of glutathione synthesis) potentiated the UVA-induced DNA damage. Exogenous H2O2 was also able to induce DNA damage. Since H2O2 alone is not able to damage DNA directly, we investigated the significance of the H2O2-derived hydroxyl radical ((OH)-O-.). Addition of FeSO4, that stimulates (OH)-O-. formation from H2O2 (Fenton reaction) resulted in a twofold increase of DNA-damage. Desferrioxamine, an iron chelator that blocks the Fenton reaction, prevented UVA-induced DNA damage. We also employed a panel of less specific antioxidants and enzyme modulators. Sodium selenite (Na-Se) present in glutathione peroxidase and thioredoxin reductase and addition of glutathione (GSH) prevented DNA-damage. Tocopherol potently prevented UVA- and H2O2-induced DNA damage and reduced intracellular H2O2-levels. Ascorbic acid reduced H2O2 production, but only partly prevented DNA damage. Singlet oxygen (O-1(2)) did not seem to play an important role in the UVA-induced DNA-damage since the specific O-1(2) scavenger sodium azide (NaN3) and the less specific O-1(2) scavenger beta -carotene did not markedly prevent either DNA-damage or H2O2 production. In conclusion the conversion of H2O2 to (OH)-O-. appears to be the most important step in UVA-induced generation of strand breaks and alkali-labile sites and the bulk H2O2 appears to originate from O-2(.-) generated by UVA irradiation. (C) 2000 Elsevier Science B.V. All rights reserved.