Studies on the Role of Reactive Oxygen Species in Mediating Lipid Peroxide Formation in Epidermal Microsomes of Rat Skin

Abstract

The role of superoxide, hydrogen peroxide, and singlet oxygen in mediating nonenzymic and NADPH-supported enzymic lipid peroxidation in skin microsomes was investigated. Incubation of skin microsomes with NADPH and/or Fe+3-ADP or ascorbate resulted in the formation of lipid peroxides. The epidermis was the major target site for microsomal lipid peroxide formation in skin. Enzymic peroxidation of epidermal microsomes required NADPH and was oxygen-dependent. Addition of the nonenzymic catalysts, Fe+3 and ADP, to the enzymic peroxidation system had an additive effect on the generation of lipid peroxide in epidermal microsomes. Epidermal microsomal lipid peroxidation was inhibited by singlet oxygen quenchers such as dimethylfuran, histidine, and beta-carotene. Hydroxyl ion scavengers such as mannitol, benzoate, or ethyl alcohol and the enzymic scavenger of superoxide, superoxide dismutase, were all ineffective in this respect. Addition of EDTA, Mn+2, cytochrome c+3, and catalase to the NADPH-supported enzymic peroxidation system resulted in strong inhibition of lipid peroxide formation in skin. Glutathione or epidermal cytosol added alone or in combination to the NADPH-supported incubation system enhanced peroxidation of microsomal lipids. Vitamin E (alpha-tocopherol) inhibited lipid peroxidation. These results indicate that singlet oxygen may mediate lipid peroxide formation in epidermal microsomes.