Arch Dermatol Res (2008) 300 (Suppl 1):S51–S56 DOI 10.1007/s00403-007-0804-3 123 ORIGINAL PAPER Visualization and characterization of UVB-induced reactive oxygen species in a human skin equivalent model Tomohiro Hakozaki · Akira Date · Takashi Yoshii · Shinya Toyokuni · Hiroyuki Yasui · Hiromu Sakurai Published online: 30 October 2007  Springer-Verlag 2007 Abstract Reactive oxygen species (ROS) play important roles in the process of ultraviolet-induced skin damage or photoaging. Although many enzymatic and chemical meth- ods 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-quantiWed by using a chemilumi- nescent probe (CLA) and an ultra low-light imaging appa- ratus. The eVects of anti-oxidative compounds such as ascorbate, -carotene, superoxide dismutase (SOD), and yeast ferment Wltrate (YFF) on the HSEM were evaluated by semi-quantiWcation 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-dimen- sional ultra-low light luminograph. Treatments with -carotene and SOD eVectively suppressed CL intensity, indicating the generation of 1 O 2 and O 2 ·¡ in the HSEM under UVB expo- sure. Tested anti-oxidative compounds also attenuated UVB-induced CL and ameliorated the induced skin dam- ages 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 eval- uating the eYcacy of topically applied anti-oxidative com- pounds to suppress ROS generation and attenuate sequential chemical and biological responses. Keywords Reactive oxygen species · Visualization · Human skin equivalent model · Chemiluminescence · 8-OHdG Introduction Skin is the only organ directly exposed to UV-light from the sun. Oxidative cellular stress and DNA damage caused by UV exposure have been recognized to participate in var- ious pathogeneses of the skin [11, 13]. Among several oxi- dative stressors, ROS have been associated with the process of UV-induced skin damage including photoaging, immu- nomodulation, melanogenesis, and ultimately photocarci- nogenesis (skin cancer) [1, 14]. It has already been shown that ROS level increases in the skin under UV exposure [15, 25, 26] and the cell injury in the epidermis is caused by ROS [2, 3, 7]. Antioxidant enzymes such as SOD, catalase, and glutathione peroxidase play important roles in protect- ing the skin against these degenerative changes [10, 19, 20]. To examine the impact of UV-induced ROS and pro- tective eYcacy of anti-oxidative compounds in the skin, it is essential to observe the ROS changes quantitatively in real-time. However, because ROS are extremely short-lived and essentially non-emissive, ROS are diYcult to detect directly. For this purpose, several evaluation methods such as chemiluminescence (CL) [17], photoemission [21], and T. Hakozaki (&) · A. Date · T. Yoshii Kobe Technical Center 7F, Procter & Gamble Japan K.K., Naka 1-17, Koyo-cho, Higashinada-ku, Kobe 658-0032, Japan e-mail: hakozaki.t.1@pg.com S. Toyokuni Department of Pathology and Biology of Diseases, Graduate School of Medicine, Kyoto University, Kyoto, Japan H. Yasui · H. Sakurai Department of Analytical and Bioinorganic Chemistry, Kyoto Pharmaceutical University, Kyoto, Japan