49 In vitro Method to Visualize UV-induced Reactive Oxygen Species in a Skin Equivalent Model

When the skin is exposed to ultraviolet (UV) light consisting of UVA (320–400 nm) and UVB (290–320 nm), reactive oxygen species (ROS) such as superoxide anion radical ( O2 ), hydrogen peroxide ( H2O2), hydroxyl radical (OH), singlet oxygen (O2), as well as lipid peroxides, and their radicals (LOOH and LOO ) are formed [1, 2]. It is well documented that these free radicals and ROS cause oxidative cellular stress, cell injury, and DNA damage in the epidermis [3, 4], and eventually induce inflammation, skin photoaging, phototoxicity, or malignant tumors [5–8]. To protect skin from these radical species, there are multiple natural defense mechanisms in the skin. For instance, antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) play important roles in protecting the skin against degenerative changes by free radicals or ROS [9, 10]. In order to examine the impact and role of UVinduced free radicals or ROS in the skin, it is essential to detect and visualize them in a real time. However, due to extremely short-lived and essentially nonemissive nature, free radials and ROS are difficult to detect directly. Several evaluation methods such as chemiluminescence (CL) probe [1, 11], photon emission detection [12–15], fluorescence detection [16, 17], and electron spin resonance (ESR) spectroscopy using spin probes [18–21] have been developed to detect ROS and investigate their species and behaviors. Meanwhile, in the past 2 decades, human skin equivalent models have been developed for multiple purposes, such as a replacement of animal models for compound safety evaluation. Now several models have become commercially available, and are used not only for safety assessment, but also to investigate biological functions of skin or responses against various stimulations such as compound treatment or UV exposure.