In vitro tools for photobiological testing: molecular responses to simulated solar UV of keratinocytes growing as monolayers or as part of reconstructed skin.
Epidermal keratinocytes are critical targets for UV-induced genotoxicity as their transformation by sunlight overexposure can lead to skin cancer such as basal cell carcinoma (BCC) and squamous cell carcinoma (SCC). Therefore, assessment of photoprotection should involve early markers associated with DNA photodamage. Here, the same normal human keratinocytes either in monoculture (KC) or in full thickness reconstructed skin (RS) were compared with respect to their response to simulated solar UV (SSUV) exposure. Irradiation conditions (spectral power distribution and doses) were designed to mimic environmental zenithal UV from sunlight. At doses where survival was higher than 80%, comet assay showed more single strand breaks (SSB) and cyclobutane pyrimidine dimers (CPD) in keratinocytes in RS than in KC one hour post-exposure. The transcription factor p53 was activated in both models. While in KC p53 accumulation displayed a linear dose-dependency up to 24 h post-exposure, in RS it followed a bell-shaped profile and reverted to its basal rate. QRT-PCR demonstrated that among genes controlled by p53, P21 and MDM2 were clearly induced by SSUV in KC, whereas GADD45 expression was strongly and almost exclusively up-regulated in RS. Nrf2-dependent antioxidant genes (Ferritin light chain, NQO1) were only induced in RS, yet at low doses for NQO1. In vitro models such as KC or RS allowing the development of quantitative methodologies should be used as surrogates for in vivo tests assessing photogenotoxicity.
Marrot
Laurent
L
L'OREAL, International Department of Safety Research, Aulnay sous bois, France.
Planel
Emilie
E
Ginestet
Anne-Claire
AC
Belaïdi
Jean-Philippe
JP
Jones
Christophe
C
Meunier
Jean-Roch
JR
D016475:3T3 Cells; D000328:Adult; D000818:Animals; D018929:Cell Culture Techniques; D002454:Cell Differentiation; D049109:Cell Proliferation / Q000528:radiation effects; D053960:DNA Breaks / Q000528:radiation effects; D019281:Dimerization; D004307:Dose-Response Relationship, Radiation; D005260:Female; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism / Q000528:radiation effects; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation / Q000528:radiation effects; D006801:Humans; D015603:Keratinocytes / Q000166:cytology* / Q000378:metabolism / Q000528:radiation effects*; D007700:Kinetics; D051379:Mice; D018384:Oxidative Stress / Q000528:radiation effects; D018462:Photobiology / Q000379:methods*; D012867:Skin / Q000166:cytology* / Q000254:growth & development* / Q000378:metabolism / Q000528:radiation effects; D013941:Thymine / Q000378:metabolism; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism; D014466:Ultraviolet Rays / Q000009:adverse effects*
D016159:Tumor Suppressor Protein p53; D013941:Thymine