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

Abstract

We investigated the role of different reactive oxygen species (ROS) in ultraviolet A (UVA)-induced DNA damage in a human keratinocyte cell line, HaCaT. UVA irradiation increased the intracellular levels of hydrogen peroxide (H 2O 2), 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 H 2O 2 production since diethyldithiocarbamate (DDC), a superoxide dismutase blocker, decreased the level of intracellular H 2O 2. Hydrogen peroxide was shown to play a central role in DNA damage. Increasing the intracellular levels of H 2O 2 with aminotriazole (AT) (a catalase blocker) and buthionine sulfoximine (BSO) (an inhibitor of glutathione synthesis) potentiated the UVA-induced DNA damage. Exogenous H 2O 2 was also able to induce DNA damage. Since H 2O 2 alone is not able to damage DNA directly, we investigated the significance of the H 2O 2-derived hydroxyl radical ( ·OH). Addition of FeSO 4, that stimulates ·OH formation from H 2O 2 (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 H 2O 2-induced DNA damage and reduced intracellular H 2O 2-levels. Ascorbic acid reduced H 2O 2 production, but only partly prevented DNA damage. Singlet oxygen ( 1O 2) did not seem to play an important role in the UVA-induced DNA-damage since the specific 1O 2 scavenger sodium azide (NaN 3) and the less specific 1O 2 scavenger β-carotene did not markedly prevent either DNA-damage or H 2O 2 production. In conclusion the conversion of H 2O 2 to ·OH appears to be the most important step in UVA-induced generation of strand breaks and alkali-labile sites and the bulk H 2O 2 appears to originate from O 2 ·− generated by UVA irradiation.