Protective role of copper, zinc superoxide dismutase against UVB-induced injury of the human keratinocyte cell line HaCaT

On the basis of our recent observation that copper, zinc-superoxide dismuta se and manganese-superoxide dismutase change differently following a single exposure to ultraviolet-B irradiation in the human keratinocyte cell line HaCaT, we have examined the possible role of endogenous copper,zinc-superox ide dismutase or manganese-superoxide dismutase against ultraviolet-B-induc ed reactive-oxygen- species-mediated keratinocyte injury in vitro. To evalu ate the individual defensive roles of copper, zinc-superoxide dismutase and manganese-super-oxide dismutase, we treated HaCaT cells with diethyldithio carbamate, a chelating agent of ionic copper that inactivates copper,zinc-s uperoxide dismutase activities, tumor necrosis factor alpha, which enhances manganese-superoxide dismutase levels, or transforming growth factor beta( 1), which inhibits manganese-superoxide dismutase levels. After the treatme nt with each reagent, HaCaT cells in the three different conditions were ex posed to a single dose of ultraviolet-B irradiation. We assessed ultraviole t-B-induced cytotoxicity by measuring both lactate dehydrogenase leakage an d cell viability using trypan blue dye exclusion assay. The lactate dehydro genase leakage in the supernatant from damaged HaCaT cells whose copper,zin c-superoxide dismutase levels were inactivated by diethyldithiocarbamate wa s significantly increased and the cell viability was significantly decrease d in comparison with untreated groups at 8 and 24 h after ultraviolet-B irr adiation. On the other hand, the lactate dehydrogenase release and cell via bility for HaCaT cells whose manganese-superoxide dismutase levels were enh anced by tumor necrosis factor alpha or inhibited by transforming growth fa ctor beta(1) showed no significant difference from untreated groups. Furthe rmore, increased production of intracellular peroxides in HaCaT cells treat ed with diethyldithiocarbamate was observed by flow cytometric analysis at 8 h after ultraviolet-B irradiation. These results suggest that copper,zinc -superoxide dismutase may play a primary protective role against ultraviole t-B-induced injury of the human keratinocyte cell line HaCaT.