The skin as largest organ of the human body and main barrier to the environment is permanently exposed to different damaging factors, especially ultraviolet light. It is well-known that UV radiation is involved in the generation of free radicals and reactive oxygen species in epidermal tissue. A protection against cell damages could be achieved due to a topical application of free radical scavengers. In the present study, various substances (melatonin, N-acetylserotonin, quercetin, kaempferol, rutin) with described antioxidant capacities were investigated mainly regarding to their cytotoxic and cytoprotective properties on human keratinocytes. Low concentrations of quercetin (5, 10 and 15 µM) and kaempferol (3, 6 and 12 µM) led to a dose dependent decrease of the proliferation rate and the living cell number already after 24 h whereas rutin, melatonin and the model substance N-acetylserotonin were well tolerated in the in vitro cell system. Furthermore it could be shown that a preincubation (2 h) with rutin (50, 100 and 200 µM), melatonin (0,1 and 1 mM) or N-acetylserotonin (1 mM) respectively together with an incubation during the UV-B irradiation (100 mJ/cm²) led to a cytoprotection which is partly associated with the UV-B absorption abilities. By way of contrast, a simultaneous incubation with the highest melatonin concentration of 1 mM during the UV-A irradiation (15 J/cm²) caused an additional decrease of the proliferation rate and the living cell number compared to the irradiated control. In addition to this finding an induction of apoptosis could be detected with the APO2.7-test and the Annexin-V/PI test using flow cytometry. Moreover, a strong increase of intracellular peroxides (DHR test) was measured after melatonin supplementation and UV-A irradiation. On the other hand, a supplementation with rutin during the UV-A irradiation showed photoprotective effects. Furthermore, a UV-A and UV-B induced generation of intracellular peroxides was reduced due to a pretreatment with rutin. Finally it can be concluded, that a topical application of rutin seems to be useful, while an external use of melatonin appears questionable because of the photoinstability and phototoxicity on human keratinocytes. |