Free Radical Reduction Mechanisms in Mouse Epidermis Skin Homogenates

Abstract

Scavenging mechanisms for persistent free radicals were investigated using nitroxide-type radicals as model compounds. The free radical reducing activity of a) isolated thioredoxin reductase, a flavin containing oxidoreductase, b) skin homogenates, and c) the epidermis of hairless mice was studied by electron spin resonance spectroscopy. In all three systems, reduction rates of different classes of nitroxide free radicals exhibited the following order: oxazolidinoxy greater than piperidinoxy greater than dihydropyrroloxy. The main reductant for piperidinoxy radicals in mouse skin homogenate is ascorbic acid. Other reducing activities were stimulated by NAD(P)H and could be inhibited by N-ethyl maleimide, suggesting involvement of thiol-dependent processes. Mammalian thioredoxin, a competitive inhibitor of nitroxide reduction by thioredoxin reductase, significantly stimulates nitroxide scavenging in skin homogenate. Thioredoxin reductase did not significantly participate in nitroxide reduction in skin homogenates. At the surface of mouse epidermis a cationic dihydropyrroloxy nitroxide, which was stable in the presence of mammalian thioredoxin reductase was readily reduced. The epidermal reduction was inhibited by zinc, N-ethyl maleimide, and by heat (70 degrees C, 5 min). At least for mouse epidermis, reduction of a variety of nitroxides is a complex phenomenon involving enzymatic and nonenzymatic mechanisms and cannot be used as a specific assay for an enzyme, e.g., thioredoxin reductase. The study indicates the epidermis contains an effective antioxidant system that scavenges ascorbate-sensitive piperidinoxy nitroxides as well as more reducing radicals exemplified by dihydropyrroloxy nitroxides.