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Notes: The skin provides a physical barrier between the host and the environment, in particular against solar radiations. Ultra-violet (UV) radiation exposure is associated with an increased risk of skin aging and carcinogenesis. The UV light that reaches the surface of the earth is divided into UVA (320–400 nm) and UVB (290–320 nm) components that have distinct biological effects. UVA is the predominant component of solar UV radiation. Although it has been considered to be only weakly carcinogenic, it causes aging and wrinkling of the skin and penetrates more deeply into the skin to reach the dermis. Hence fibroblasts are the main cell targets for UVA radiation. UVA affects cellular DNA integrity by the activation of photosensitizers that consequently generate reactive oxygen species (ROS). Among them superoxide anion (O2°–), singlet oxygen (1O2), hydrogen peroxide (H2O2) and hydroxyl radical (OH°) can react with DNA bases or sugar and induce oxidative DNA damage and strand breaks.The comet assay (known also as the single cell gel electrophoresis assay) is a popular method that allows the microscopic detection of strand breaks and oxidative damage in DNA [1]. It is based on the analysis of migration in alkaline conditions of the DNA of individual cells lysed in an agarose gel spread onto a microscope slide. Denatured DNA comes unwound at the sites of strand breaks and migrates faster than the supercoiled fraction. It gives the DNA supercoiled structure the appearance of a comet. The numbers of strand breaks (SB) and alkali-labile sites (ALS) are evaluated by the measurement of the percentage of DNA that migrates into the tail of the comet (Tail DNA). The specificity of the method can be extended to the detection of oxidated purines (mainly 8-oxodGuo) by the addition of a step in which DNA is digested by a repair enzyme (Fpg: formamido pyrimidine DNA-N-glycosylase) that specifically excises the last oxidative damage [2]. The comet assay is also a convenient method to follow the repair of damaged DNA. Non-repaired lesions and transient strand breaks may be monitored during incubation after application of a stress.We have used this very sensitive and specific method to compare the sensitivity to oxidative stress (H2O2 exposition)of human dermal fibroblasts established from sun-exposed (SE) and sun-protected (SP) female Japanese skin. We evaluated the amount of initial damage and the repair capacities (SSB, ALS, and Fpg sites) of these cells.〈section xml:id="abs1-2"〉〈title type="main"〉Materials and methods〈section xml:id="abs1-3"〉〈title type="main"〉SubjectsVolunteer recruitment and biopsy removal were performed at Laboratoire DermExpert (Paris, France) in accordance with ethical procedures. The group consisted of 10 healthy Japanese women living in France with age ranging from 30 to 45 years (mean: 36 years). Clinical evaluations and biopsies were performed by a dermatologist on each subject.〈section xml:id="abs1-4"〉〈title type="main"〉Cell cultureThe cultures of human dermal fibroblasts were established by outgrowth of 3 mm punches taken from sun-exposed (SE) and sun-protected (SP) parts of the forearm of the 10 volunteers. The cells were cryopreserved at the second passage and subsequently cultured for the experiments before passage 5.〈section xml:id="abs1-5"〉〈title type="main"〉Comet assayThe comet experiments were performed as described in [3] using sub-confluent cells. The cells were submitted to H2O2 exposition (20 mM) for 5 min at 4°C in PBS. They were then re-fed with the reserved culture medium and transferred to the CO2 incubator at 37°C to allow DNA repair. The cells were processed for the comet experiments at different times after the stress over an 8-h period.The viability of the cells was monitored by the MTT test 24 h after the H2O2 treatment. Approximately 85% of cells remained viable.We used the Komet 3.0 software from Kinetic Imaging to analyse the slides. Fifty nuclei were scored per slide and duplicate slides were processed for each experimental point. The amount of damage was evaluated by the Tail DNA (percentage of DNA in the tail of the comet). Four repair curves (Tail DNA as a function of repair time) were obtained for each donor, for SSB + ALS and for Fpg sites in both SE and SP cells.〈section xml:id="abs1-6"〉〈title type="main"〉StatisticsLinear regression curves were established for the correlation between the amount of residual damage and age. Significant differences are noted by * on the graph. anova analyses were conducted to assess the correlation between the % of residual damage and life habits.〈section xml:id="abs1-7"〉〈title type="main"〉ResultsTypical images of comets are shown in 〈link href="#f1-18"〉Fig. 1.〈figure xml:id="f1-18"〉1〈mediaResource alt="image" href="urn:x-wiley:01425463:ICS254_18_18:ICS_254_f1-18"/〉Fluorescent microscope imaging of non-damaged and damaged DNA after the electrophoresis in alkaline medium and ethidium bromide staining.For the 10 subjects, comet assay results revealed that the initial amount of damage (SSB + ALS, Fpg sites) and the damage induction by the oxidative stress were equivalent in cells from SE and SP area.Repair of SSB + ALS was significantly faster in SP cells compared with SE cells at 1 h after stress. Moreover, SP cells totally repaired the damage in 2 h while it took 4 h for the SE cells (〈link href="#f2-18"〉Fig. 2). For repair of Fpg sites, no difference was observed between SP and SE cells. It is important to stress that 8 h after the stress, return to the initial level of damage was not observed, either in the SE cells or in the SP cells.〈figure xml:id="f2-18"〉2〈mediaResource alt="image" href="urn:x-wiley:01425463:ICS254_18_18:ICS_254_f2-18"/〉Repair of SSB and ALS for SE and SP fibroblasts (mean of results obtained for each cell line). Statistically significant difference (*P 〈 0.05) between SE and SP cells.Using the clinical evaluations made by a dermatologist, we looked at the correlations that could be statistically established between the damage repair kinetics and age, skin features and life habits for each donor.Striking features appeared concerning the SP fibroblasts: the repair kinetics of the damage (SSB + ALS and Fpg sites) was strongly inversely correlated to the age of the donors. Hence, the fibroblasts from older donors had the highest levels of damage after 4 h repair (〈link href="#f3-18"〉Fig. 3a and b; r = 0.85 and 0.71 respectively). On the contrary, no correlation existed in SE cells.〈figure xml:id="f3-18"〉3〈mediaResource alt="image" href="urn:x-wiley:01425463:ICS254_18_18:ICS_254_f3-18"/〉(a) Residual (SSB + ALS) as a function of age after 4 h repair, for SP cells. (b) Residual Fpg sites as a function of age after 4 h repair, for SP cells.Repair was slower and residual damage higher for smokers or former smokers (n = 3) than for donors who had never smoked (n = 7). On the contrary, no correlation was established in SE cells.The level of overall sun exposure of the donors influenced the amount of oxidative residual damage (Fpg sites). In SE cells, residual Fpg sites were higher in cells from donors with heavy sun exposure habits. SP cells coming from donors with little sun exposure behaviour (assimilated to never exposed cells) tended to have higher Fpg-sensitive residual sites than SP fibroblasts from heavy exposed donors (assimilated to moderately exposed cells).There was no correlation between DNA repair and heliodermy, phototype, skin tanning index. Correlation with life habits questionnaire showed no influence of food consuming (rice, tea, alcohol, etc.) on DNA repair.〈section xml:id="abs1-8"〉〈title type="main"〉Discussion and conclusionAs expected, the response of the normal dermal fibroblasts, taken from sun-exposed and sun-protected skin of Japanese women, to H2O2 treatment showed inter-individual variations. Fibroblasts from SE and SP area did not display any difference in their sensitivity to the action of H2O2 stress. Although the in vitro subculture could era