Validation of a predictive method for sunscreen formula evaluation using gene expression analysis in a Chinese reconstructed full-thickness skin model.

OBJECTIVE: This study aimed to establish a predictive in vitro method for assessing the photoprotective properties of sunscreens using a reconstructed full-thickness skin model. MATERIALS AND METHODS: A full-thickness skin model reconstructed with human fibroblasts and keratinocytes isolated from Chinese skin was exposed to daily UV radiation (DUVR). We examined the transcriptomic response, identifying genes for which expression was modulated by DUVR in a dose-dependent manner. We then validated the methodology for efficacy evaluation of different sunscreens formulas. RESULTS: The reconstructed skin model was histologically consistent with human skin, and upon DUVR exposure, the constituent fibroblasts and keratinocytes exhibited transcriptomic alterations in pathways associated with oxidative stress, inflammation and extracellular matrix remodelling. When used to evaluate sunscreen protection on the model, the observed level of protection from UV-induced gene expression was consistent with the corresponding protection factors determined clinically and allowed for statistical ranking of sunscreen efficacy. CONCLUSIONS: Within this study we show that quantification of gene modulation within the reconstructed skin model is a biologically relevant approach with sensitivity and predictability to evaluate photoprotection products.

OBJECTIF: Cette étude visait à établir une méthode prédictive in vitro permettant d'évaluer les propriétés photoprotectrices des écrans solaires à l'aide d'un modèle de peau reconstruite sur toute son épaisseur. MATÉRIELS ET MÉTHODES: Un modèle de peau reconstruite sur toute son épaisseur avec des fibroblastes et des kératinocytes humains isolés à partir de peaux chinoises a été exposé au rayonnement UV quotidien (DUVR). Nous avons examiné la réponse transcriptomique en identifiant les gènes dont l'expression était modulée par le DUVR de façon dépendante à la dose. Nous avons ensuite validé la méthodologie d'évaluation de l'efficacité des formules des différents écrans solaires. RÉSULTATS: Le modèle de peau reconstruite correspondait histologiquement à de la peau humaine, et lors de l'exposition à des DUVR, les fibroblastes et les kératinocytes qui la composaient présentaient des altérations transcriptomiques des voies associées au stress oxydatif, à l'inflammation et au remodelage de la matrice extracellulaire. Lorsque ce modèle a été utilisé pour évaluer la protection solaire, le niveau de protection observé de l'expression génique induite par les UV correspondait aux facteurs de protection cliniques déterminés correspondants et permettait un classement statistique de l'efficacité de la protection solaire. CONCLUSIONS: Dans cette étude, nous montrons que la quantification de la modulation génique dans le modèle de peau reconstruite est une approche biologiquement pertinente offrant une sensibilité et une prédictibilité pour évaluer les produits de photoprotection.

Differential behaviors toward ultraviolet A and B radiation of fibroblasts and keratinocytes from normal and DNA-repair-deficient patients.

Xeroderma pigmentosum (XP) and trichothiodystrophy (TTD) are rare genodermatoses transmitted as recessive and autosomal traits that result in reduced capacity to repair UV-induced DNA lesions. Although XP, but not TTD, patients are prone to basal and squamous cell carcinomas, to date no comparative studies of the XP and TTD phenotypes have included epidermal keratinocytes. We compared the DNA repair capacity (by unscheduled DNA synthesis) and cell survival (by clonal analysis) of epidermal keratinocytes and dermal fibroblasts grown from normal individuals and patients with xeroderma pigmentosum and trichothiodystrophy following UVA and UVB irradiation. The same dose of UVB (1000 J/m2) induced twice as many DNA lesions in normal fibroblasts as in normal keratinocytes. UV survival rates were always higher in keratinocytes than in fibroblasts. Normal and TTD keratinocytes survived better following UVA and UVB irradiation than XP-C and XP-D keratinocytes. XP-C keratinocytes exhibited exacerbated sensitivity toward UVA radiation. Unscheduled DNA synthesis at UV doses leading to 50% cell survival indicated that the ratio of DNA repair capacity to cell survival is higher in keratinocytes than in fibroblasts. In addition, UVA and UVB irradiation induced a transition from proliferative to abortive keratinocyte colonies. This transition varied between donors and was in part correlated with their cancer susceptibility. Altogether these data provide the first evidence of the differential behaviors of normal, XP, and TTD keratinocytes toward UV radiation.

Cyclobutane pyrimidine dimers are the main mutagenic DNA photoproducts in DNA repair-deficient trichothiodystrophy cells.

We have used the replicating shuttle vector pR2 to determine the role of ultraviolet C (UVC)-induced cyclobutane pyrimidine dimers (CPDs) and nondimer photoproducts in mutagenesis in human trichothiodystrophy (TTD) cells and in their repair-proficient counterparts obtained after complementation with the wild-type XPD/ERCC2 repair gene (TTD + ERCC2 cells). Before transfection in human cells, the UVC-irradiated vector DNA was treated with Anacystis nidulans photolyase [photoreactivation (PR) procedure] that selectively removed CPDs, leaving nondimer photoproducts intact. The mutant frequency of the UV-irradiated pR2 plasmid treated by PR was similar after replication in TTD or in TTD + ERCC2 cells. This result indicates that TTD cells were able to repair nondimer photoproducts as efficiently as TTD cells complemented with the wild-type repair gene and that in TTD cells, CPDs were the major photoproducts generating an increased mutant frequency after UVC irradiation. Sequence analysis of > 300 mutant plasmids indicated that PR of the DNA increased the relative level of tandem mutations and decreased the relative level of multiple mutations in TTD cells. In both cell lines, we observed that CPDs mostly led to GC-AT transitions; whereas only nondimer photoproducts were responsible for the induction of GC-TA transversions in TTD and TTD + ERCC2 cells.

Recovery of normal DNA repair and mutagenesis in trichothiodystrophy cells after transduction of the XPD human gene.

To determine whether expression of the XPD/ERCC2 repair gene in trichothiodystrophy (TTD) group D cells could restore mutagenesis characteristics of repair-proficient cells, we compared the UV mutagenesis of normal cells, TTD group D cells, and TTD group D cells retrovirally transduced by the wild-type XPD/ERCC2 gene (TTD + ERCC2 cells). We first verified the expression of the XPD protein, correction of UV cell survival, and DNA repair ability of TTD + ERCC2 cells. UV-induced mutations were studied using the pR2 shuttle vector. The addition of the XPD/ERCC2 gene in TTD cells led to a significant but partial decrease of mutation frequency compared with the parental TTD cells. Types of mutations of TTD + ERCC2 cells get closer to those observed in normal cells (ie., a reduction of multiple mutations). New hotspots appeared and some disappeared in the complemented line, suggesting that hotspot distribution is particular to each cell line and cannot be correlated with the repair status of the cells. In conclusion, the expression of the XPD/ERCC2 repair gene completely corrected UV hypersensitivity and almost all types of mutations of TTD group D cells, whereas hypermutagenesis was partially corrected.

Characteristics of UV-induced mutation spectra in human XP-D/ERCC2 gene-mutated xeroderma pigmentosum and trichothiodystrophy cells.

To study the relationships between mutagenesis and carcinogenesis, we compared the mutations and their frequency induced by ultraviolet irradiation at 254 nm (UV-C) in XP-D (GM-08207B/XP6BE), TTD/XP-D (TTD1VI-LAS-KMT11) and wild-type (MRC-5V1) human cells. XP-D and TTD/XP-D cells, mutated in the same XP-D/ERCC2 gene, are deficient in nucleotide excision repair. Whereas XP-D patients develop early skin tumors, TTD patients do not exhibit abnormal levels of cancers. After verification of UV hypersensitivity and DNA repair defect of the immortalized cell lines XP-D and TTD compared with a wild-type cell line, UV-induced mutagenesis was studied with a new shuttle vector pR2, carrying the target lacZ' gene. The UV-mutation frequencies in XP-D and TTD cells were similar and significantly increased compared with normal cells. Sequence analysis of 312 independent mutant plasmids revealed that more rearrangements were induced in TTD cells (16%) than in XP-D (5%) and normal cells (1%), while XP-D cells exhibited a twofold higher rate of tandem mutations compared with TTD and normal cells. In the three cell lines, a predominance of G:C to A:T transitions was found, especially in chiefly on the cytosine at 5'-TC-3' sites. The types of UV-induced point mutations in TTD cells were, however, more similar to those in normal cells than those found in XP-D cells. XP-D mutations were preferentially located in 5'-TCPur-3' sites, while mutations in normal and TTD cells were mostly at 5'-TCC-3' sites. Analysis of mutation spectra revealed differences in the location of the mutational hotspots between the three lines. Although the mutation frequency of the UV-irradiated pR2 vector is much higher in TTD and XP-D cells than in normal cells, the mutation spectrum is closer between TTD and normal cells as compared with XP-D cells. These dissimilarities could contribute to an explanation of some of the differences between the two syndromes.