KINETICS OF DNA STRAND BREAKS AND PROTECTION BY ANTIOXIDANTS IN UVA-IRRADIATED OR UVB-IRRADIATED HACAT KERATINOCYTES USING THE SINGLE-CELL GEL-ELECTROPHORESIS ASSAY

The aim of this study was to characterize the genotoxic action of UVA and UVB in human keratinocytes by application of the single cell gel e lectrophoresis assay (SCGE assay). Dose dependence of DNA damage, the time course of its repair, and the influence of cellular antioxidant s tatus were assessed. Irradiation with UVA or UVB both resulted in a do se-dependent increase in the level of DNA damage. A time course study to evaluate the repair kinetics in keratinocytes irradiated with 5 J/c m(2) UVA revealed an immediate occurrence of DNA effects which subsequ ently disappeared within about 1 h, indicating removal of DNA lesions. This rapid repair of DNA damage is consistent with the observation th at 5 J/cm(2) UVA did not impair cellular viability. In contrast, expos ure to 15 mJ/cm(2) UVB resulted in a prolonged repair of DNA damage wh ich lasted about 25 h, Thus, the repair kinetics of UVA-and UVB-induce d DNA damage clearly differed from each other, implicating the inducti on of different types of DNA lesions by UVA and UVB. Neither a pretrea tment with Mg-ascorbyl phosphate or D,L-alpha-tocopherol, nor depletio n of endogenous glutathione altered cellular sensitivity to UVB. In co ntrast, the DNA damaging effects of WA could be counteracted by a pret reatment with these antioxidants. These observations confirm that the UVA-induced effects on DNA are related to radical mediated strand brea ks and DNA lesions forming alkali-labile sites. The UVB-induced effect s mainly occur as a consequence of excision repair-related strand brea ks. The observed repair kinetics of DNA lesions and the influence of c ellular antioxidant status may help to elucidate protective mechanisms against the carcinogenic effects of UV radiation present in sunlight. (C) 1998 Elsevier Science B.V.