Influence of UV exposure on DNA damage in Chinese skin
Authors: Bonnet Duquennoy M. , Lachmann N. , Noblesse E. , Pincemail J. , Kurfürst R. , Bonté F. 
Published: September 2010

Introduction

Sunlight is essential for human life but we need to avoid overexposure to the sun. Chronic exposure of the skin to UV radiation leads to photoageing (sunburn, wrinkles, spots, freckles, skin texture changes, and dilated blood vessels), immunosuppression, and sometime more serious lesions. UV radiation also causes DNA damage, which is a critical event in skin photoageing and photocarcinogenesis [1]. Exposure to UV light induces wide range of DNA lesions on skin cells by direct absorption (UVB), or via oxidative stress (UVA and UVB). We choose to work on healthy Chinese women skin because it is demonstrated that Asian people developed less skin cancer than Caucasian, and few studies have been done on DNA damage in the skin of Asian subjects [2, 3].

The formation of 8‐hydroxy‐2'deoxyguanosine (8‐oxo‐dG) is a major type of DNA lesion resulting from oxidative stress. It is a biological marker of oxidative stress on DNA. It causes the transversion of G : C to T : A during DNA replication, which occurs frequently in some genes in human skin lesions on areas exposed to sunlight [4]. Skin cells have evolved several DNA repair mechanisms to counteract immediately the deleterious effects of such lesions that can lead to genomic instability. These repair pathways are present in all living cells and are extremely well conserved. P53 is a phosphoprotein that is activated by stress, up‐regulates DNA repair enzymes, participates directly in DNA repair, blocks cell cycles during DNA repair, and induces apoptosis in critically damaged cells. After exposure to UV, basal keratinocytes repair damaged DNA whereas differentiating keratinocytes undergo cell death, both processes are regulated by p53 [5].

Repeated exposure of the skin to UV light induces pigmentation and thickening of the epidermis, both of which help to increase tolerance of sunlight. Some studies have shown that this photo‐adaptation can help preserve epidermal DNA from UV injury [2, 6–8]. However, as most of these studies were performed by exposing the skin to a single or several acute bursts of radiation, they do not necessarily reflect the effect of chronic exposure to sunlight. The first aim of this study on 15 healthy Chinese women was to determine the effects of chronic exposure to sunlight on DNA damage in the skin. We compared, on sun‐protected and sun‐exposed skin of the same patient, the amounts of p53 protein and 8‐oxo‐dG determined by immunohistochemistry method. Blood samples from these women were used to measure their anti‐oxidant stress status and hence their intrinsic defence capacities.

We also evaluated how changes in the skin induced by chronic exposure to sunlight helped preserve DNA from an acute radiation. The two areas (chronically exposed to and protected from sunlight) were exposed to a relatively low dose of 1 minimal erythema dose (MED). The amounts of the markers (p53 protein and 8‐oxo‐dG) and the responses of the two areas were compared 24 h after exposure.

Methods

Volunteers

Volunteers were recruited and biopsies removed at the Laboratoires Dermexpert (Paris, France), in accordance with international ethical procedures.

Volunteers completed a questionnaire that allowed us to estimate their skin sensitivity, phototype, life styles and eating habits. The skin of patients was clinically evaluated by a dermatologist. Blood and urine were taken and analysed. Skin punch biopsies (3 mm) were taken from the anterior surface of the upper arm (protected area) and the posterior surface of the forearm (exposed area). MED was determined. Two zones (one exposed and one protected) on the other arm were irradiated at 1 MED. Punch biopsies (3 mm) were taken from the two irradiated zones 24 h after irradiation.

MED determination

The MED values were determined using a multiport solar light (601 model). The anterior forearm test site was exposed to six increasing doses (13.44; 16.8; 21.0; 26.46; 32.76 and 40.95 mJ cm–²). The skin reaction was evaluated visually 24 h later. The lowest dose of UV energy that caused a perceptible demarcated erythema was considered to be 1 MED. Those subjects that had a very mild erythema after the maximum dose (40.95 mJ cm–²) were to be irradiated at 51.24 mJ cm–².

Oxidative stress status

Samples of blood and morning urine were carried out from fasting subjects. The blood samples were used to measure the following parameters: antioxidants (vitamin C, vitamin E, carotenoids, glutathione, thiol proteins, uric acid, total antioxidant capacity), trace metals (selenium, copper, zinc), markers of oxidative stress (lipid peroxidation), and iron status (free iron, ferritin, transferrin). The urine samples were used to assay 8‐oxo‐dG. Assays were performed by Probiox SA (Liege, Belgium) [9, 10].

Immunohistochemistry

The punch biopsies were immediately placed in 10% neutral buffered formalin (4% formaldehyde) and fixed for 24 h at ambient temperature. They were then embedded in fresh wax and p53 and 8‐oxo‐dG detected immunohistochemically using BP53‐12‐1 and N45.1 monoclonal antibodies respectively [11]. Two hundred epidermal cells were counted per sample and the numbers of p53 or 8‐oxo‐dG positive and negative cells were determined.

The statistical methods used were analysis of variance (anova) and linear regression, and all data were assessed for statistical significance (P < 0.05).

Results

The 15 healthy Chinese women living in France with a age range of 31‐‐43 years (mean: 36 years). The clinical evaluation of the skin performed by a dermatologist and based on wrinkles, heliodermy and pigmentation disorders, showed that all 15 women had similar degree of cutaneous photoageing. Similarly, analysis of global anti‐oxidant stress status showed that all 15 women had the same antioxidant potential, providing same intrinsic defence capacities.

The amounts of 8‐oxo‐dG in protected (six to 64 positive cells per 200 epidermal cells; mean: 18.8) and exposed areas (two to 40 positive cells per 200 epidermal cells; mean: 17.1) varied greatly from one individual to another. No difference in the 8‐oxo‐dG contents was observed between the two areas (Fig. 1). And there was no statistical correlation between the 8‐oxo‐dG in the skin and in the urine. The large differences in 8‐oxo‐dG in the skin between individuals were not correlated with any of the life style parameters in the questionnaire or with any of the blood antioxidant parameters.

Epidermal detection of p53 protein and 8‐oxo‐dG in sun‐protected and chronically sun‐exposed skin. Data are the mean of 15 biopsies. These was no statistical difference in the p53 or 8‐oxo‐dG in the protected and exposed areas.

image

Immunochemical staining for p53 in biopsies of protected skin from all individuals showed one to seven (mean 1.8) stained cells per 200 cells in the epidermis. The skin chronically exposed to sunlight had the same number of p53‐stained cells (mean: 1.7) for all women (Fig. 1). P53‐positive nuclei were found in the basal and suprabasal cells of the epidermis. Thus, the sun‐protected and sun‐exposed areas of skin contained the same amounts of both 8‐oxodG and p53.

The second part of the study compared the responses of protected and chronically exposed samples of skin from the same patient to a single acute UV radiation. The skin areas of all 15 subjects were exposed to 1 MED and the removal of DNA lesions (8‐oxo‐dG) and p53 were quantified 24 h later. The MED were similar for 15 volunteers (40.95 or 51.24 mJ cm–²), suggesting that they were all similarly sensitive to sunlight. There was erythema in both areas 24 h after radiation, but it was more pronounced in the protected areas. For p53, weak increase was observed in sun‐protected area. In sun‐exposed skin, a significant increase of p53 level was measured (Fig. 2). The increase in p53 after UV radiation was significantly greater in chronically exposed skin than in protected skin. Large inter‐individual variations in the epidermal p53 response were observed. The increase in p53 expression was not correlated with any of the life style parameters or skin parameters described in the questionnaires, or with any of the blood antioxidant status parameters. There was no significant increase in 8‐oxo‐dG in either the protected or chronically exposed areas of skin after the single irradiation (Fig. 2).

p53 protein expression (a) and 8‐oxo‐dG amount (b) in protected and chronically exposed areas of skin before and 24 h after UV radiation. Data are the means of 15 biopsies. Statistically significant difference (*P < 0.05) between skin before and after acute radiation.

image

Discussion

We have studied the influence of exposure to UV light on DNA damage and p53 in the skin of Chinese women. All 15 women were similar in terms of age, life style, cutaneous photoageing determined by dermatological evaluation, MED, and blood antioxidant potential, indicating the same intrinsic defence capacities.

The first part shows that chronically exposure to sunlight of skin from all the 15 women (mean age 36 y) has no influence on basal DNA damage (8‐oxo‐dG) and p53, the ‘genome guardian’ protein, production. Thus there is no accumulation of DNA damage in healthy, chronically exposed skin suggesting a good repair capacity.

We then compared epidermal responses to UV light from protected and chronically exposed areas of human skin. Twenty‐four hours after the acute radiation of 1 MED, level of 8‐oxodG, was small compared with published data for a Caucasian population [6]. Our results suggest that DNA damage (8‐oxo‐dG) on Chinese skin is already repaired 24 h after stress, regardless of the exposure of the skin to sunlight.

We confirmed that the amount of p53 in the epidermis is increased in response to acute exposure [3, 6–8], but the increase varied greatly for one individual to another, in spite of the great similarity of the volunteers studied [8]. Moreover, significantly more p53 was detected in chronically exposed skin than in protected skin. Similarly, deWinter et al. [7] showed that there was more p53 in skin with a high MED (dark skin type) than in skin with a lower MED (light skin type) 24 h after an acute exposure. The reasons for the differences in skin responses to UV light are unknown. Epidermal cells chronically exposure to sunlight could become more reactive to a stress like UV irradiation. p53 is known activated by stress, and it up‐regulates DNA repair enzymes, participates directly in DNA repair, blocks cell cycles during DNA repair, and induces apoptosis of critically damaged cells. A stronger p53 response could result in better DNA repair. Our findings suggest that areas of skin chronically exposed to sunlight are more adapted to respond to DNA injury, as in Caucasian skin [7, 8]. The results thus confirm that is essential to better understand the influence of chronic exposure to sunlight on DNA protection.

Acknowledgements

The authors thank A. Bernois and Delphine Pelle de Queral for the statistical analyses Dr Adhoute for biopsies, and Jean Baptiste Grieu, Catherine Heusèle and Virginie Nollent for their support.

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