UV-induced free radicals in the skin detected by ESR spectroscopy and imaging using nitroxides

Reactive free radicals and reactive oxygen species (ROS) induced by ultraviolet irradiation in human skin are strongly involved in the occurrence of skin damages like aging and cancer. In the present work an ex vivo method for the detection of free radicals/ROS in human skin biopsies during UV irradiation is presented. This method is based on the Electron Spin Resonance (ESR) spectroscopy and imaging and uses the radical trapping properties of nitroxides. The nitroxides 2,2,6,6-Tetramethylpiperidine-1-oxyl (TEMPO), 3-Carbamoyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl (PCM), and 3-Carboxy-2,2,5,5-tetramethylpyrrolidine-1-oxyl (PCA), were investigated for their applicability of trapping reactive free radicals and reactive oxygen species in skin during UV irradiation. As a result of the trapping process the nitroxides were reduced to the EPR silent hydroxylamins. The reduction rate of TEMPO was due to both the UV radiation and the enzymatic activity of the skin. The nitroxides PCM and PCA are sufficiently stable in the skin and are solely reduced by UV-generated free radicals/ROS. The nitroxide PCA was used for imaging the spatial distribution of UV-generated free radicals/ROS. As a result of the homogeneous distribution of PCA in the skin, it was possible to estimate the penetration of UVA and UVB irradiation: The UV irradiation decreased the PCA intensity corresponding to its irradiance and penetration into the skin. This reduction was shown to be caused mainly by UVA radiation (320-400 nm).