Effects of calcium concentration on the SOD activity and UVB-induced cytotoxicity in cultured human keratinocytes

Background/Purpose: Cellular differentiation due to the extracellular calcium (Ca²⁺) concentration affects the level of several antioxidant enzymes in cultured human keratinocytes. Because the epidermis includes well- and un-differentiated keratinocytes, we expected that keratinocytes possess different antioxidant capacity and sensitivity to damaging effects of ultraviolet-B (UVB) depending on the differentiation. We examined the effects of Ca²⁺ concentration of culture medium (DMEM (Dulbecco's modified Eagle's medium)) on the superoxide dismutase (SOD) activity and UVB-induced cytotoxicity in cultured human keratinocytes in order to investigate the relationship between cell differentiation and antioxidant defense. Methods: Human keratinocytes (HaCaT cells) were incubated in high Ca²⁺ (> 1mM) or low Ca²⁺ (< 0.1 mM) concentration DMEM for 24 h at 37°C in 5%) CO₂. Then, we measured total SOD activity and also individual Cu,Zn- and Mn-SOD activities in keratinocytes. Furthermore, after incubation in high or low Ca²⁺ concentration DMEM, human keratinocytes were irradiated with 10, 20 or 30 mJ/cm² UVB. ne quantity of lactate dehydrogenase (LDH) leaked in the supernatant from damaged keratinocytes, cell viability and TdT-mediated dUTP nick end labelings (TUNEL) positive keratinocytes imae numsmed at 24 h aRer UVB irradiation. Results: Total SOD activity and Cu,Zn-SOD activity in human keratinocytes cultured in low Ca²⁺ were significantly lower than in keratinocytes cultured in high Ca²⁺ concentration DMEM. In contrast, MnSOD activity was not affected. LDH leakage in the supernatant from keratinocytes cultured in low Ca²⁺ concentration was significantly higher than that from keratinocytes cultured in high Ca²⁺ concentration DMEM after UVB irradiation. The cell viability of keratinocytes cultured in low Ca²⁺ concentration DMEM was significantly decreased compared to that of keratinocytes cultured in high Ca²⁺ concentration DMEM after UVB irradiation. Furthermore, UVB-induced apoptosis was increased in keratinocytes cultured in low Ca²⁺ concentration DMEM by the TUNEL method. Conclusions: These results suggest that cellular differentiation due to the change of Ca²⁺ concentration of culture medium affects the Cu,Zn-SOD activity and UVB-induced cytotoxicity in cultured human keratinocytes.