Electron paramagnetic resonance studies on nitroxide radical 2,2,5,5-tetramethyl-4-piperidin-1-oxyl (TEMPO) redox reactions in human skin.

Electron paramagnetic resonance (EPR) is currently being explored for the study of living biological systems. Among biophysical and biochemical applications, the study of nitroxide radical interactions with tissue antioxidants and oxidants is of growing interest. Skin is a target organ of the EPR methodology and is frequently exposed to oxidative stress. We investigated the piperidine-type nitroxide 2,2,5,5-tetramethyl-4-piperidin-1-oxyl (TEMPO) because it is skin permeable and readily accepts electrons in biological systems. TEMPO is readily scavenged on the surface of cultured human skin. Pretreatment of skin cultures with butylhydroperoxide, which decreases intracellular ascorbate and glutathione, causes inhibition of nitroxide scavenging. Exposure of skin cultures to dehydroascorbate, which is internalized and converted to ascorbate, leads to stimulation of nitroxide scavenging. In human keratinocytes and fibroblasts, the TEMPO radical is reversibly reduced to the hydroxylamine depending on the oxygen concentration and the availability of intracellular glutathione and ascorbate. Cell exposure to the glutathione synthetase inhibitor buthionine-sulfoximine depleted intracellular glutathione and inhibited nitroxide reduction; exposure to dehydroascorbate or glutathione-monoethylester increased intracellular ascorbate or glutathione concentration and stimulated nitroxide reduction. Quantitative considerations indicate that the major reduction site of TEMPO in skin and skin cells is the cytosol ascorbate/glutathione redox cycle. We suggest that analysis of TEMPO radical scavenging by the EPR technique is a convenient method for measuring skin ascorbate and thiol-dependent antioxidant activity in vitro and in vivo.