This study was designed to investigate the generation of free radicals (including reactive oxygen species) during exposure to ultraviolet light (UV) in intact mouse skin by an in vivo EPR (electron paramagnetic resonance) spectrometer. Two kinds of nitroxyl probes, TEMPONE (4-oxo-2,2,6,6-tetramethyl-piperidine-d16-1-oxyl) and CMP (3-carbamoyl- 2,2,5,5-tetramethylpyrrolidine-N-oxyl), were injected intraperitoneal and/or directly supplicated to skin. Signal decays of nitroxyl probes were monitored with the in vivo EPR spectrometer equipped with a surface-coil-resonator after UV (UVA＋B) irradiation for 3 minutes. In the direct skin supplication study, there was no significant difference between the control and UV-irradiation groups in half-lives (sec.) of TEMPONE. However, half-lives of CMP were significantly prolonged in the UV-irradiation group compared with the control group, and the difference was substantially eliminated with a spin-trapping agent (PBN, N-tert-butyl-α-phenylnitrone) before UV-irradiation. In the intraperitoneal injection study, half-lives of CMP measured in the skin were significantly prolonged in the UVirradiation group compared to that of the control group. Our results suggest that direct supplicating the nitroxyl probe to the skin is useful as an extremely noninvasive method to evaluate free radical generation after UV-irradiation.
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