Premium
Effects of calcium concentration on the SOD activity and UVB‐induced cytotoxicity in cultured human keratinocytes
Author(s) -
Sasaki Hiroko,
Itoh Taketo,
Akamatsu Hirohiko,
Okamoto Hiroyuki,
Horio Takeshi
Publication year - 2005
Publication title -
photodermatology, photoimmunology and photomedicine
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.736
H-Index - 60
eISSN - 1600-0781
pISSN - 0905-4383
DOI - 10.1111/j.1600-0781.2005.00117.x
Subject(s) - hacat , keratinocyte , lactate dehydrogenase , superoxide dismutase , microbiology and biotechnology , cytotoxicity , chemistry , calcium , viability assay , extracellular , antioxidant , cell culture , biochemistry , apoptosis , biology , enzyme , in vitro , genetics , organic chemistry
Background/Purpose: Cellular differentiation due to the extracellular calcium (Ca 2+ ) 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 Ca 2+ 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 Ca 2+ (>1 mM) or low Ca 2+ (<0.1 mM) concentration DMEM for 24 h at 37°C in 5% CO 2 . Then, we measured total SOD activity and also individual Cu,Zn‐ and Mn‐SOD activities in keratinocytes. Furthermore, after incubation in high or low Ca 2+ concentration DMEM, human keratinocytes were irradiated with 10, 20 or 30 mJ/cm 2 UVB. The quantity of lactate dehydrogenase (LDH) leaked in the supernatant from damaged keratinocytes, cell viability and TdT‐mediated dUTP nick end labelings (TUNEL) positive keratinocytes were measured at 24 h after UVB irradiation. Results: Total SOD activity and Cu,Zn‐SOD activity in human keratinocytes cultured in low Ca 2+ were significantly lower than in keratinocytes cultured in high Ca 2+ concentration DMEM. In contrast, Mn‐SOD activity was not affected. LDH leakage in the supernatant from keratinocytes cultured in low Ca 2+ concentration was significantly higher than that from keratinocytes cultured in high Ca 2+ concentration DMEM after UVB irradiation. The cell viability of keratinocytes cultured in low Ca 2+ concentration DMEM was significantly decreased compared to that of keratinocytes cultured in high Ca 2+ concentration DMEM after UVB irradiation. Furthermore, UVB‐induced apoptosis was increased in keratinocytes cultured in low Ca 2+ concentration DMEM by the TUNEL method. Conclusions: These results suggest that cellular differentiation due to the change of Ca 2+ concentration of culture medium affects the Cu,Zn‐SOD activity and UVB‐induced cytotoxicity in cultured human keratinocytes.