Effects of calcium concentration on the SOD activity and UVB-induced cytotoxicity in cultured human keratinocytes.
OBJECTIVE
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 degrees 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.
Sasaki
Hiroko
H
Department of Dermatology, Kansai Medical University, 10-15 Fumizono-cho, Moriguchi, Osaka 570-8507, Japan. sasakih@takii.kmu.ac.jp
Itoh
Taketo
T
Akamatsu
Hirohiko
H
Okamoto
Hiroyuki
H
Horio
Takeshi
T
D017209:Apoptosis / Q000528:radiation effects*; D002118:Calcium / Q000378:metabolism*; D002461:Cell Line, Transformed; D006801:Humans; D015603:Keratinocytes / Q000201:enzymology / Q000378:metabolism* / Q000528:radiation effects*; D013482:Superoxide Dismutase / Q000378:metabolism*; D014466:Ultraviolet Rays
D013482:Superoxide Dismutase; D002118:Calcium