Bicarbonate Plays a Critical Role in the Generation of Cytotoxicity during SIN-1 Decomposition in Culture Medium
Figure 5
Effects of thiols on the cytotoxicity of 4-h-decomposed SIN-1 in bicarbonate. (a) Effects of amino acids, antioxidants, metal chelators, and air saturation on the cytotoxicity of decomposed SIN-1. Cells were treated with 4-h-decomposed SIN-1 (1 mM) prepared in NaHCO3 in conjunction with the indicated compound. The concentration for each compound was 100 M unless otherwise specified. The far-right column represents 4-h-decomposed SIN-1 (1 mM) saturated with oxygen by voltex mixing aerobically for 5 min. The values are means ± intra-assay deviations, expressed as SD, from four or five wells in a representative experiment. versus decomposed SIN-1 alone. (b) HPLC profile of GSH after incubation with SIN-1-decomposed bicarbonate. GSH (50 M) was added to 4-h-decomposed SIN-1 (1 mM) in NaHCO3 or to the control NaHCO3 and incubated in a CO2 incubator for 2 h. Glu-Glu was added to the samples as an internal standard. The samples were derivatized and separated by HPLC on an NH2 column. GSH eluted at 22 min, GSSG at 27 min, and Glu-Glu at 12 min. The inset graph shows the GSH levels normalized to the area for Glu-Glu. The values are means ± inter-assay deviations, expressed as SD, from four independent assays. Note that the GSH level is consistently lower in the decomposed SIN-1 medium than in the control medium.
