SIN-1 cytotoxicity to PC12 cells is mediated by thiol-sensitive short-lived substances generated through SIN-1 decomposition in culture medium.
As a generator of peroxynitrite (ONOO(-)), 3-morpholinosydnonimine (SIN-1) is widely used in the study of oxidative/nitrosative stress in cultured cells, although controversy exists regarding active species responsible for cytotoxicity. In this study, we report that unstable thiol-sensitive substances, generated from the reaction of SIN-1 with components in culture medium, play a crucial role in SIN-1 cytotoxicity in PC12 cells. Exposure of cells to culture medium obtained after almost complete SIN-1 decomposition at 37 degrees C for 2h demonstrated almost the same degree of cytotoxicity as did fresh SIN-1. The cytotoxicity of SIN-1-decomposed medium largely depended on serum, decayed with time, and could be completely abolished by the addition of thiols. Degradation of synthetic ONOO(-) in the culture medium did not reproduce the unstable cytotoxicity. The presence of superoxide dismutase (SOD) during SIN-1 decomposition prevented the formation of the cytotoxic substances, whereas SOD had no protection against the cytotoxicity itself, suggesting a crucial role of simultaneously generated superoxide and nitric oxide in the formation of the toxicants, but not in their cytotoxic action. The cytotoxicity of fresh SIN-1 is dramatically suppressed in a basal medium (Hanks balanced salt), suggesting that the cytotoxicity of fresh SIN-1 also requires components of culture medium. These results suggest that SIN-1 cytotoxicity in PC12 cells is mediated via the generation of cytotoxic substances in the medium during its decomposition.
Konishi
Kanako
K
Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba, Japan.
Watanabe
Nobuo
N
Arai
Takao
T
D000818:Animals; D053148:Caspase 3 / Q000378:metabolism; D016923:Cell Death; D002478:Cells, Cultured; D004791:Enzyme Inhibitors / Q000633:toxicity*; D008981:Molsidomine / Q000031:analogs & derivatives* / Q000633:toxicity; D018384:Oxidative Stress; D016716:PC12 Cells; D030421:Peroxynitrous Acid / Q000378:metabolism; D051381:Rats; D013438:Sulfhydryl Compounds / Q000378:metabolism*; D013482:Superoxide Dismutase / Q000037:antagonists & inhibitors / Q000378:metabolism
D004791:Enzyme Inhibitors; D013438:Sulfhydryl Compounds; D030421:Peroxynitrous Acid; C002385:linsidomine; D008981:Molsidomine; D013482:Superoxide Dismutase; D053148:Caspase 3