A photochemical study of cells loaded with 2′,7′-dichlorofluorescin: implications for the detection of reactive oxygen species generated during UVA irradiation

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/cm2 UVA and DCF fluorescence was measured. A dose-dependent increase in DCF fluorescence was observed, with the cells exposed to 4 J/cm2 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/cm2 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 H2O2 or 5-hydroxy-1,4-naphthoquinone (juglone), which redox cycles to generate superoxide (and H2O2). 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.