DNA repair efficacy of photolyase (Photosome^(®)) in human keratinocytes after UVB irradiation using the comet assay

L Decome PhD^a, MP De Méo PhD^a, C Robert PhD^b, O Doucet PhD^b, A Botta Pr^a

^aLaboratoire de Biogénotoxicologie et Mutagenèse Environnementale, Faculté de Médecine, Marseille, France, ^bCell Pharmacology department, Coty Beauty Research and development Center, Monaco

Aim. The aim of the present study was to evaluate the DNA repair efficacy of photolyase on human keratinocytes after a single dose of UVB irradiation using the comet assay. Methods. Keratinocytes primary cultures were incubated with photolyase (Photosome^®, 0.5%) and L-ergothioneine (4 mM) then cells were harvested and comet slides were prepared. Then, the slides were irradiated with 0.06 J/cm² UVB. A second UVA irradiation (1.2 J/cm²) was performed to activate the photolyase enzyme and the slides were incubated for 60 min at 37 °C in humidified chamber. Finally, the slides were immersed in cold lysing solution. After lysis, slides were incubated with SDS-Proteinase K (0.5%:0.1mg/ml) for 60 min at 37°C then were washed with enzymatic buffer before treatment with T4 endonuclease V (T4NV, 75 ng) for 30 min at 37 °C in humidified chamber. Slides were allowed to unwind for 20 min and electrophoresis was performed with an additional period of 20 min. DNA single strand breaks were quantified using an image analysis system. The Tail moment χ² values were compared with Dunnet's test. Results. In order to photoactive photolyase (Photosome^®), cells were irradiated with 1.2 J/cm² UVA after UVB irradiation. However, UVA induced DNA single strand breaks immediately after irradiation via the production of reactive oxygen species. Preliminary experiments showed that L-ergothioneine decreased UVA-induced single strand breaks in dose-dependent manner. For an optimal antioxidant protection against UVA irradiation, L-ergothioneine was used at a concentration of 4mM in culture medium for 30 min of incubation at 37°C prior UVB irradiation. If pyrimidine dimers were not repaired by photolyase, damaged sites were blocked and T4 endonuclease V could not repair the pyrimidine dimers sites. A SDS-Proteinase K treatment step was included to allow T4NV action by the release of unrepaired pyrimidine dimers sites. With this additional treatment, the number of DNA single strand breaks was increased after T4NV treatment. With this modified protocol of the comet assay, the photolyase treatment significantly increase DNA repair efficacy in normal human keratinocytes in vitro. Percentage of increase was 52%. Conclusions. L-ergothioneine effectively protects cells of DNA single strand breaks due to reactive oxygen species produced by UVA irradiation. Photolyase (Photosome ^®) increases DNA repair efficacy in normal human keratinocytes and could be used to improve sunscreen protection against UVB irradiation.

Paper presented at the International Symposium on Predictive Oncology and Intervention Strategies; Nice, France; February 7 - 10, 2004; in poster session 898 (Oncogenesis).