Petersen A B, Gniadecki R, Vicanova J, Thorn T, Wulf H C
Department of Dermatology, Bispebjerg Hospital, Copenhagen, Denmark.
J Photochem Photobiol B. 2000 Dec;59(1-3):123-31. doi: 10.1016/s1011-1344(00)00149-4.
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 (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 (O2*-) 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). Addition of FeSO4, that stimulates *OH 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 (1O2) did not seem to play an important role in the UVA-induced DNA-damage since the specific 1O2 scavenger sodium azide (NaN3) and the less specific 1O2 scavenger beta-carotene did not markedly prevent either DNA-damage or H2O2 production. In conclusion the conversion of H2O2 to OH 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 O2- generated by UVA irradiation.
我们研究了不同活性氧（ROS）在人角质形成细胞系HaCaT中紫外线A（UVA）诱导的DNA损伤中的作用。用荧光探针羧基二氯二氢荧光素检测发现，UVA照射会增加细胞内过氧化氢（H₂O₂）的水平，并通过碱性单细胞凝胶电泳（彗星试验）检测到其会导致氧化性DNA损伤、单链断裂和碱不稳定位点。超氧阴离子（O₂⁻·）可能是H₂O₂产生的底物，因为超氧化物歧化酶阻滞剂二乙基二硫代氨基甲酸盐（DDC）会降低细胞内H₂O₂的水平。过氧化氢在DNA损伤中起核心作用。用氨基三唑（AT）（一种过氧化氢酶阻滞剂）和丁硫氨酸亚砜胺（BSO）（一种谷胱甘肽合成抑制剂）提高细胞内H₂O₂水平会增强UVA诱导的DNA损伤。外源性H₂O₂也能够诱导DNA损伤。由于单独的H₂O₂不能直接损伤DNA，我们研究了H₂O₂衍生的羟基自由基（·OH）的重要性。添加硫酸亚铁（FeSO₄）可刺激H₂O₂形成·OH（芬顿反应），导致DNA损伤增加两倍。去铁胺是一种阻断芬顿反应的铁螯合剂，可预防UVA诱导的DNA损伤。我们还使用了一组特异性较低的抗氧化剂和酶调节剂。谷胱甘肽过氧化物酶和硫氧还蛋白还原酶中的亚硒酸钠（Na-Se）以及添加谷胱甘肽（GSH）可预防DNA损伤。生育酚能有效预防UVA和H₂O₂诱导的DNA损伤并降低细胞内H₂O₂水平。抗坏血酸可减少H₂O₂的产生，但只能部分预防DNA损伤。单线态氧（¹O₂）似乎在UVA诱导的DNA损伤中不起重要作用，因为特异性的¹O₂清除剂叠氮化钠（NaN₃）和非特异性的¹O₂清除剂β-胡萝卜素均未显著预防DNA损伤或H₂O₂的产生。总之，H₂O₂向·OH的转化似乎是UVA诱导产生链断裂和碱不稳定位点的最重要步骤，而大部分H₂O₂似乎源自UVA照射产生的O₂⁻·。
