Chignell Colin F, Sik Robert H
Laboratory of Pharmacology and Chemistry, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709-2233, USA.
Free Radic Biol Med. 2003 Apr 15;34(8):1029-34. doi: 10.1016/s0891-5849(03)00022-4.
There have been several attempts to implicate reactive oxygen species in UVA-induced damage by loading cells with 2',7'-dichlorofluorescin (DCFH) and following the appearance of 2',7'-dichlorofluorescein (DCF), its highly fluorescent oxidation product. However, both DCF and DCFH have significant absorption in the 300-400 nm range so it is possible that photochemical reactions will occur in cells containing these dyes when they are irradiated with UVA. HaCaT keratinocytes loaded with DCFH were irradiated with 0, 1, 2, or 4 J/cm(2) UVA and DCF fluorescence was measured. A dose-dependent increase in DCF fluorescence was observed, with the cells exposed to 4 J/cm(2) UVA exhibiting an almost 10-fold increase over dark controls. However, there was no difference in cell viability, as measured by the MTS assay or LDH release, between the dark and the 4 J/cm(2) UVA-exposed groups. Furthermore, a large increase in DCF fluorescence was observed when a cell-free system containing DCF, DCFH, and horseradish peroxidase was UVA irradiated. As a control, keratinocytes loaded with DCFH were incubated in the dark with either exogenously added H(2)O(2) or 5-hydroxy-1,4-naphthoquinone (juglone), which redox cycles to generate superoxide (and H(2)O(2)). In both cases, the cells showed a concentration-dependent increase in DCF fluorescence and a concomitant decrease in viability. Our findings suggest that DCFH can not be used to detect the UVA-induced generation of reactive oxygen species in cells when the dye is present during exposure.
已有多项研究试图通过用2',7'-二氯荧光素（DCFH）加载细胞并跟踪其高荧光氧化产物2',7'-二氯荧光素（DCF）的出现，来证明活性氧在UVA诱导的损伤中的作用。然而，DCF和DCFH在300 - 400 nm范围内都有显著吸收，因此当含有这些染料的细胞受到UVA照射时，可能会发生光化学反应。用0、1、2或4 J/cm²的UVA照射加载了DCFH的HaCaT角质形成细胞，并测量DCF荧光。观察到DCF荧光呈剂量依赖性增加，暴露于4 J/cm² UVA的细胞与黑暗对照组相比，荧光增加了近10倍。然而，通过MTS测定法或LDH释放测量，黑暗组和暴露于4 J/cm² UVA的组之间的细胞活力没有差异。此外，当含有DCF、DCFH和辣根过氧化物酶的无细胞系统受到UVA照射时，观察到DCF荧光大幅增加。作为对照，加载了DCFH的角质形成细胞在黑暗中与外源添加的H₂O₂或5-羟基-1,4-萘醌（胡桃醌）一起孵育，后者通过氧化还原循环产生超氧化物（和H₂O₂）。在这两种情况下，细胞都显示出DCF荧光的浓度依赖性增加和活力的相应下降。我们的研究结果表明，当在暴露过程中存在染料时，DCFH不能用于检测细胞中UVA诱导的活性氧生成。
