Novel screening method for ultraviolet protection: Combination of a human skin‐equivalent model and 8‐hydroxy‐2′‐deoxyguanosine

To the Editor: Ultraviolet (UV) radiation is a threat to human skin not only because of photoaging but also because of skin cancer. Therefore, attenuation of UV-induced damage at the epidermal surface is important, and various chemicals have thus far been shown to be beneficial. Previously, these kinds of experiments were performed using laboratory animals. However, current ethical concerns for the use of laboratory animals made it sometimes unacceptable to use animals for massive screening purposes in certain countries. 8-Hydroxy-2′-deoxyguanosine (8-OHdG), an oxidatively modified DNA base, is induced either by a hydroxyl radical, singlet oxygen, a photodynamic reaction or peroxynitrite, and is mutagenic when present during DNA replication. We reported increase in the amount of epidermal 8-OHdG not only after acute UV exposure but also after chronic repeated low-dose exposure in mice. Furthermore, ogg1 knockout mice, lacking the repair enzyme for 8-OHdG present in the genome, were shown to be susceptible to UV-induced skin cancer. Here we propose that a combination of human skinequivalent model (HSEM) and 8-OHdG immunohistochemistry presents an ideal screening strategy for protection against UV-induced skin damage. This model (EpiDerm Skin Model, EPI-200; MatTek Corporation, Ashland, MA, USA) is distinct in that: (i) cultured human epidermal cells are 3-D, mimicking normal skin structure; and (ii) the cells are fed via supporting mesh attached to basal cell-equivalent bottom cells, with the top surface keratinized and dry under air. Chemicals for screening purposes were applied to the surface of the HSEM followed by UV irradiation for a variety of periods. After UVB irradiation at various doses (27.0–135 mJ/cm via 12–60 min exposure, corresponding to 1.0–5.0 minimal erythema dose, respectively) using FL20SE lamps (Toshiba Medical Supply, Tokyo, Japan), the cultured cells were fixed with phosphatebuffered 10% formalin for 1 h. A center portion of 5 mm in diameter was punched out with Trepan (Kai Medical, Tokyo, Japan) followed by immersion in 95% ethanol and routine processes for paraffin embedding. The specimens were cut at 3 mm and put on MAS-coated glass slides (Matsunami, Tokyo, Japan). HE staining and immunohistochemical staining for 8-OHdG (clone N45.1, Japan Institute for the Control of Aging, Fukuroi, Shizuoka, Japan) were performed as previously described except for minor modifications. Antigen retrieval with a microwave (100°C, 5 min) was performed in 10 mmol/L glycine buffer at pH 3.0, which gave optimal results. No major histological changes were observed with HE staining immediately after the UVB exposure (data not shown). In the unexposed control skin samples, 8-OHdG immunostaining was positive in the nuclei of basal cell areas mimicking results of normal mouse skin. Notably, nuclei of the epidermal cells in upper layer corresponding to the spinous and granular layers were intensely immunostained in proportion to the UV dose up to 36 min (Fig. 1). We chose 18 min for the evaluation of UV protective ability of various chemicals (10 mL topical application: 10 mmol/L ascorbate, 10 mmol/L b-carotene, Tokyo Kasei, Tokyo, Japan; 100 mmol/L Cu,Zn-superoxide dismutase; Sigma, St Louis, MO, USA) that were associated with the metabolism of reactive oxygen species. The results showed a significant decrease in the immunohistochemical positivity microscopically as well as by quantification with NIH image freeware (8-OHdG index;Fig. 2a–c). This was confirmed by [3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay performed as described after 60 min UVB exposure (Fig. 2d), demonstrating that prior evaluation with 8-OHdG immunohistochemistry was indeed associated with the following epidermal cell degeneration and death. In a recent publication we showed that real-time chemiluminescent imaging to detect reactive oxygen species is also possible in HSEM after UVB exposure. The present results show that a combination of HSEM and 8-OHdG immunohistochemistry is a distinct screening strategy for agents preventing UV-induced skin damage with the following characteristics: (i) it is an alternative to animal experiments; (ii) expensive instruments are not required; and (iii) it is a more sensitive method as compared with the animal models. Currently, there is an emerging concept that there are fragile genomic sites to oxidative stress as well as UV-specific DNA base modifications. This 3-D model is further applicable to the analysis of each epidermal layer with laser microdissection.