An In Vivo Study of Free Radicals Generated in Murine Skin by Protoporphyrin IX and Visible Light

Abstract

Lipids extracted from the skin of C57BL/6J mice injected subcutaneously with alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (POBN) and exposed to topical protoporphyrin IX (PPIX) and visible light had significantly higher levels of POBN spin adducts compared with dark PPIX exposed or vehicle-treated controls. Computer analysis of the POBN adduct electron paramagnetic (spin) resonance (EPR) spectra indicated that two radical species were present in each extract, one of which was a lipid-derived carbon-centered adduct (1, a(N) = 14.8 G and a(H) = 2.6 G), whereas the other (2, a(N) = 13.8 G and a(H) = 1.8 G) was probably oxygen centered. Adduct 2 was present in greater proportion in lipids extracted from PPIX/light-exposed mice compared with dark or vehicle-treated controls. These findings suggest that PPIX/light generates free radicals in mouse skin, thus providing a radical mechanism for PPIX-induced photosensitivity. Our approach may be useful for the detection of free radicals generated by other skin photosensitizers and may also provide a means for testing putative skin-protecting agents.