Evaluation of photolyase (Photosome®) repair activity in human keratinocytes after a single dose of ultraviolet B irradiation using the comet assay

被引:28
作者
Decome, L
De Méo, M
Geffard, A
Doucet, O
Duménil, G
Botta, A
机构
[1] Univ Mediterranee, Fac Pharm & Med, Lab Biogenotoxicol & Mutagenese Environm, Marseille 5, France
[2] Skin Res Dept, Coty Monaco, Monaco
关键词
photolyase; photosome (R); L-ergothioneine; T4 endonuclease V; comet assay; cyclobutane pyrimidine dimers;
D O I
10.1016/j.jphotobiol.2004.11.022
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Photosome((R)) 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/cm(2)) in human keratinocytes. The formation of single strand breaks (SSB) induced by the UVA photoactivation of the enzyme (1.2 J/cm(2)) 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((R)) repaired CPD lesions and decreased the SSB levels by 2.6-3.3-fold. Photosome((R)) could be an additional component of sunscreens to reduce the development of skin cancer. (c) 2005 Elsevier B.V. All rights reserved.
引用
收藏
页码:101 / 108
页数:8
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