Search

This study was directed to examine the role of type II (photodynamic) reactions involving the production of reactive oxygen species (singlet oxygen, superoxide anion, and hydroxy radicals) in in vitro and in vivo photosensitization reactions induced by skin photosensitizing chemicals. Several porphyrins and psoralens, as model compounds representing examples of endogenous and exogenous photosensitizers, were examined for their ability to (a) produce singlet oxygen and superoxide anions, (b) induce damage to membranes and associated microsomal P-450, (c) promote lipid peroxidation of microsomal lipids of liver and epidermal cells, and (d) induce skin photosensitization reactions in vivo. Dose-response study in vitro of singlet oxygen production in H2O and D2O and inhibition studies involving the production of singlet oxygen and superoxide anion by specific quenchers indicated significant production of singlet oxygen by porphyrins, about 5-20 times higher than psoralen at 10(-5) M and 10(-6) M concentration and irradiation dose of 1-5 J/cm2 of UVA (greater than 320-400 nm radiation). The comparative studies on aerobic photodegradation of microsomal P-450 of guinea pig epidermis and liver indicated a significantly greater destruction of P-450 with porphyrins than with psoralens. A membrane-lipid peroxidation study involving malondialdehyde production, using liver and epidermal microsomal fractions with and without porphyrins, psoralens, and UVA radiation, indicated 10-20 times increased production of malondialdehyde with UVA and porphyrins than with psoralens.

The role of reactive oxygen (1O2 and O2-.) in skin photosensitization and tanning reaction has been examined. Riboflavin (RF), hematoporphyrin (HP), 3-carbethoxypsoralen (3-CP), and 8-methoxypsoralen (8-MOP), upon photoexcitation under aerobic conditions, produced singlet O2 (1O2). RF, 3-CP, and 8-MOP also produced superoxide anion (O2-.). Reactive O2 produced by photosensitized RF, 3-CP, and 8-MOP was found to oxidize tyrosine and dopa to dopachrome and subsequently their conversion to melanin. HP did not oxidize tyrosine to dopachrome, and 3-CP and RF revealed substantial oxidation of tyrosine. Dopa was oxidized to dopachrome and subsequently to melanin by all photosensitizers tested at a variable rate as follows: RF greater than 3-CP greater than HPD greater than 8-MOP. UVA alone and to a lesser extent UVB also produced 1O2 which induced the oxidation of tyrosine and dopa to dopachrome and subsequently to melanin. The production of dopachrome was higher with dopa compared to tyrosine under all irradiation conditions. These observations appear to have relevance to the O2-requiring immediate tanning reaction of the skin stimulated by solar radiation and in the induction of skin photosensitization.

Superoxide dismutase provides a protective defense mechanism in cellular compartments against the potential cytotoxicity of superoxide anion generated by ultraviolet radiation. Little information is available about the nature of superoxide dismutase in mammalian skin. We report the isolation and characterization of superoxide dismutase from human, guinea pig, and mouse epidermis. Copper-zinc superoxide dismutase was detected in all the mammalian skin specimens examined. Manganese superoxide dismutase was detected in human and guinea pig epidermis but not in the newborn or adult albino CD1 mouse epidermis. Electrophoresis studies of the extracted and partially purified skin superoxide dismutase on polyacrylamide gel slabs in the presence of sodium dodecylsulfate showed the characteristic molecular weights for subunits of 16,500 for copper-zinc superoxide dismutase, and 23,500 for mangano superoxide dismutase. Studies under nondenaturing conditions revealed significant differences in the mobility of the enzymes, depending on the sources of superoxide dismutase. The mouse epidermal copper-zinc superoxide dismutase was found similar to the bovine liver copper-zinc superoxide dismutase used as an internal standard. The copper-zinc superoxide dismutase of human skin and guinea pig skin showed activity-stained bands characterized by a higher mobility than the same enzyme from mouse or bovine liver. Quantitative data using the beta-NADH oxidation method indicated a 5-10-fold lower content of superoxide dismutase in mammalian epidermis in comparison with other tissues examined during this study, or compared with reported values in the literature.