In Vivo Measurement of Reactive Oxygen Species Generated in Skin under Ultraviolet Light Irradiation

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Distinct electron spin resonance(ESR) signal of 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) adduct of hydroxyl radical (OH) was observed in uroporphyrin or hematoporphyrin photodynamic reactions in the presence of reducing reagents, although no adduct of OH was observed using 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide (DEPMPO) as a spin-trapping agent. Both DMPO and DEPMPO adducts of OH appeared, as the spin-trapping was performed with DEPMPO in the presence of DMPO. The results indicate that the DMPO-mediated generation of OH occurs during the spin-trapping in photodynamic reactions. Thus, in vivo ESR spectroscopy with a nitroxyl spin probe was employed to detect reactive oxygen species generated in skin.An aqueous solution of carbamoyl-PROXYL was injected intravenously to an anesthetized hair-removed ddY mouse or hairless mouse, and L-band ESR spectra of the probe were recorded at the dorsal region of the mice. A surface-coil-type resonator was used to detect carbamoyl-PROXYL in skin. The decay rate of the ESR signal increased by irradiation with UV light (UVA+B). The enhancement of signal decay was suppressed by pre-administration of a spin-trapping reagent, N-t-butyl-α-phenylnitrone (PBN), while PBN did not change the decay rate for non-irradiated mouse. Peroxyl radical caused the decay of carbamoyl-PROXYL signal in vitro, and the decay was inhibited by PBN. These observations suggest that peroxyl radical was generated in skin of mouse under ultraviolet light irradiation.