Akira Date

Visualization and characterization of UVB-induced reactive oxygen species in a human skin equivalent model

Reactive oxygen species (ROS) play important roles in the process of ultraviolet-induced skin damage or photoaging. Although many enzymatic and chemical methods have been developed for evaluating ROS, evaluation methods for ROS generation in living systems are quite limited. Here we propose a unique system to visualize UVB-induced ROS and investigate the biological impact of ROS. In brief, a human skin equivalent model (HSEM) was exposed to UVB. Emitted luminescence from the HSEM was visualized and semi-quantified by using a chemiluminescent probe (CLA) and an ultra low-light imaging apparatus. The effects of anti-oxidative compounds such as ascorbate, β-carotene, superoxide dismutase (SOD), and yeast ferment filtrate (YFF) on the HSEM were evaluated by semi-quantification of emitted chemiluminescence (CL) intensities, MTT assay and 8-hydroxy-2′-deoxyguanosine (8-OHdG) staining. Visualization of time- and space-dependent dynamics of ROS generation in the HSEM was successfully achieved by utilizing a sensitive two-dimensional ultra-low light luminograph. Treatments with β-carotene and SOD effectively suppressed CL intensity, indicating the generation of 1O2 and O2·− in the HSEM under UVB exposure. Tested anti-oxidative compounds also attenuated UVB-induced CL and ameliorated the induced skin damages in terms of 8-OHdG formation and cell death. As a conclusion, this model is useful for not only visualizing the production of UVB-induced ROS in real-time but also evaluating the efficacy of topically applied anti-oxidative compounds to suppress ROS generation and attenuate sequential chemical and biological responses.

Comparison of cathepsin L activity in cheek and forearm stratum corneum in young female adults.

Purpose: The objective is to determine the usefulness of cathepsin L (Cat L) activity derived from the stratum corneum as an index of skin conditions by comparing the cheek and the forearm, in 20 female young adults.

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.

An analytical technique for measuring protein carbonyl in the stratum corneum using surface plasmon resonance.

Protein carbonyl groups in the stratum corneum may be used as a biomarker for skin photo‐stress. To evaluate the relationship between the protein carbonyl to total protein (TP) ratio (carbonylation ratio) and skin photo‐stress, the authors established a methodology by which protein carbonyl can be easily and highly sensitively analysed using an optical technique based on surface plasmon resonance (SPR). To collect the stratum corneum, tape stripping was employed. Firstly, the protein carbonyl was reacted with 2,4‐dinitrobenzenesulfonic acid dihydrate, and the quantity of dinitrophenylated (DNP)‐protein carbonyl was determined using an anti‐dinitrophenyl (anti‐DNP) antibody. The mass of DNP‐protein carbonyl was measured using SPR. A truncated sampling‐reporting cycle of <5 min allowed speedy reporting of DNP‐protein carbonyl levels. A significant difference was observed in the protein carbonyl/TP ratio (carbonylation ratio) between a sun‐protected area (mid‐ventral arm) and a sun‐exposed area (upper cheek, P < 0.05). Additionally, the carbonylation ratio of the sun‐exposed area showed a higher value than that of the sun‐protected area. It was suggested that the carbonylation ratio might be a useful index of skin photo‐stress.

Noninvasive biosensor for cathepsin L in the stratum corneum

The objective is to propose an on‐site testing biosensor of cathepsin L (CatL) activity in the stratum corneum, which can be used for the evaluation of skin conditions noninvasively and easily.

A novel analytical technique of skin photo-stress biomarker using surface plasmon resonance

It is possible that protein carbonylation in the stratum corneum might be used as an index of skin photo-stress. In order to evaluate the relationship between the protein carbonyl / total protein ratio (carbonylation ratio) and skin photo-stress, the authors established a methodology by which protein carbonyl can be quantitatively analyzed using an optical technique based on surface plasmon resonance (SPR). Firstly, the protein carbonyl was reacted with 2,4-Dinitrophenylhydrazine (DNP), and the quantity of DNP-protein carbonyl was determined using an anti-DNP antibody. The mass of DNP-protein carbonyl was measured using SPR. A significant difference was observed in the carbonylation ratio between a sun-protected area (mid-ventral arm) and a sun-exposed area (upper cheek, p &#60; 0.05). Moreover, the carbonylation ratio of the sun-exposed area showed a higher value than that of the sun-protected area. It was suggested that the carbonylation ratio might be a useful index of skin photo-stress.