VISUALIZATION AND CHARACTERIZATION OF UVB-INDUCED REACTIVE OXYGEN SPECIES IN A HUMAN SKIN EQUIVALENT MODEL.

This study by researchers at Procter and Gamble, Kyoto University and Kyoto Pharmaceutical University demonstrated that MatTek’s EpiDerm in vitro 3-D human skin tissue equivalent is useful for visualizing the production of UVB-induced Reactive Oxygen Species (ROS) in real-time, and for evaluating the efficacy of topically applied anti-oxidative compounds to suppress ROS generation and attenuate sequential chemical and biological responses. 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 human skin equivalent model 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, beta-carotene, superoxide dismutase (SOD), and yeast ferment filtrate (YFF) on the human skin equivalent model 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 human skin equivalent model was successfully achieved by utilizing a sensitive two-dimensional ultra-low light luminograph. Treatments with beta-carotene and SOD effectively suppressed CL intensity, indicating the generation of 1O2 and O2- in the human skin equivalent model 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.

8-hydroxy-2'-deoxyguanosine (8-OHdG), Anti-oxidative compounds, Chemiluminescence (CL), EPI-200, EpiDerm, Human skin equivalent model, Immunostaining, ROS, Reactive oxygen species, Visualization

Beta-carotene, Sodium ascorbate, Superoxide dismutase (SOD), UVB, Yeast ferment filtrate (YFF)