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 dismutase 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- superoxide dismutase or manganese-superoxide dismutase against ultraviolet-B- induced reactive-oxygen-species-mediated keratinocyte injury in vitro. To evaluate the individual defensive roles of copper, zinc-superoxide dismutase and manganese-superoxide dismutase, we treated HaCaT cells with diethyldithiocarbamate, a chelating agent of ionic copper that inactivates copper, zinc-superoxide dismutase activities, tumor necrosis factor α, which enhances manganese-superoxide dismutase levels, or transforming growth factor β₁, which inhibits manganese-superoxide dismutase levels. After the treatment with each reagent, HaCaT cells in the three different conditions were exposed to a single dose of ultraviolet-B irradiation. We assessed ultraviolet-B-induced cytotoxicity by measuring both lactate dehydrogenase leakage and cell viability using trypan blue dye exclusion assay. The lactate dehydrogenase leakage in the supernatant from damaged HaCaT cells whose copper, zinc-superoxide dismutase levels were inactivated by diethyldithiocarbamate was significantly increased and the cell viability was significantly decreased in comparison with untreated groups at 8 and 24h after ultraviolet-B irradiation. On the other hand, the lactate dehydrogenase release and cell viability for HaCaT cells whose manganese-superoxide dismutase levels were enhanced by tumor necrosis factor α or inhibited by transforming growth factor β₁ showed no significant difference from untreated groups. Furthermore, increased production of intracellular peroxides in HaCaT cells treated with diethyldithiocarbamate was observed by flow cytometric analysis at 8h after ultraviolet-B irradiation. These results suggest that copper,zinc-superoxide dismutase may play a primary protective role against ultraviolet-B-induced injury of the human keratinocyte cell line HaCaT.