Evaluation of photolyase (Photosome) repair activity in human keratinocytes after a single dose of ultraviolet B irradiation using the comet assay.
Photosome is constituted of photolyases included in liposomes. Photolyase is a bacterial enzyme that can repair ultraviolet B (UVB)-induced cyclobutane pyrimidine dimers (CPD) in eukaryotic cells. A modified version of the alkaline comet assay has been set up to evaluate the repair activity of this enzyme after a single dose of UVB (312 nm, 0.06 J/cm2) in human keratinocytes. The formation of single strand breaks (SSB) induced by the UVA photoactivation of the enzyme (1.2 J/cm2) was inhibited by the pretreatment of the cells with 4 mM L-ergothioneine (ERT) during 30 min at 37 degrees C. To increase the sensitivity of the comet assay, an additional lysis was used with a buffer containing sodium dodecyl sulfate (0.5%) and proteinase K (0.1 mg/ml) for 60 min at 37 degrees C. Unrepaired CPD by photolyase were revealed by a second enzymatic treatment with T4 endonuclease V, a CPD specific glycosylase. UVB irradiation increased the SSB level in keratinocytes and additional T4NV treatment enhanced this SSB level by 1.5-2.0-fold confirming that CPD were the major base modifications generated by UVB irradiation. UVA-photoactivated Photosome repaired CPD lesions and decreased the SSB levels by 2.6-3.3-fold. Photosome could be an additional component of sunscreens to reduce the development of skin cancer.
Decome
Laetitia
L
Laboratoire de Biogénotoxicologie et Mutagenèse Environnementale (EA 1784), Facultés de Pharmacie et de Médecine, Université de la Méditerranée, 27 Bd jean Moulin, Marseille Cedex 5, France.
De Méo
Michel
M
Geffard
Anne
A
Doucet
Olivier
O
Duménil
Gérard
G
Botta
Alain
A
D002478:Cells, Cultured; D020552:Comet Assay; D004255:Deoxyribodipyrimidine Photo-Lyase / Q000378:metabolism*; D043224:Deoxyribonuclease (Pyrimidine Dimer) / Q000378:metabolism; D019286:Endopeptidase K / Q000378:metabolism; D004880:Ergothioneine / Q000494:pharmacology; D006801:Humans; D015603:Keratinocytes / Q000166:cytology / Q000187:drug effects / Q000201:enzymology* / Q000528:radiation effects*; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology; D014466:Ultraviolet Rays; D014764:Viral Proteins / Q000378:metabolism
D014764:Viral Proteins; D012967:Sodium Dodecyl Sulfate; D004880:Ergothioneine; C470698:endonuclease V, phage T4; D043224:Deoxyribonuclease (Pyrimidine Dimer); D019286:Endopeptidase K; D004255:Deoxyribodipyrimidine Photo-Lyase