Bicarbonate Plays a Critical Role in the Generation of Cytotoxicity during SIN-1 Decomposition in Culture Medium
Figure 3
SIN-1 cytotoxicity to PC12 cells after its complete decomposition in bicarbonate. Serially diluted SIN-1 in 10 mM NaHCO3, either freshly prepared or allowed to decompose in advance for 4 h, was supplemented with glucose and applied to cells in 96-well plates (3000 cells/well) for 2 h. The cells were then incubated with fresh FCS/RPMI for an additional 24 h, and viability was assessed. (a) Comparison of the cytotoxicity of fresh SIN-1 and 4-h-decomposed SIN-1 in 10 mM NaHCO3 at 50 μL/well or 100 μL/well. (b) Evaluation of the cytotoxic potency of decomposed SIN-1 by MTT assay. Cells were treated with fresh SIN-1 or 4-h decomposed SIN-1 in 10 mM NaHCO3 as in (a), and cell viability was assessed by MTT reduction activity. (c) Decline in cytotoxicity of decomposed SIN-1. Fresh or 4-h- or 24-h-decomposed SIN-1 (1 mM)-dependent LDH leakage measured in (a) was normalized to fresh SIN-1-dependent leakage as 100%. Values are means ± inter-assay deviations, expressed as SD, from six (control and 4-h) or three (24-h) independent experiments. (d) Comparison of the cytotoxicity of 4-h-decomposed SIN-1 prepared in 10 mM NaHCO3 and in 10 mM NaOH. (e) Concentration dependence of NaHCO3 on decomposed SIN-1 cytotoxicity. SIN-1 (1 mM) was decomposed in the indicated concentrations of NaHCO3 or NaOH for 4 h, and cytotoxicity was measured as in (a). Unless otherwise specified, all the values are means ± intra-assay deviations, expressed as SD, from four or five wells in a representative experiment. *P < 0.05, **P < 0.01 and ***P < 0.001 versus respective control. In (a) ##P < 0.01 versus respective 50 μL/well, and in (c) ###P < 0.001 versus 4-h-decomposed SIN-1.
