Rapid sodium cyanide depletion in cell culture media: outgassing of hydrogen cyanide at physiological pH

During the course of in vitro studies on cyanide exposure with SH-SY5Y human neuroblastoma cells, we found that sodium cyanide (NaCN) up to a concentration of 10 mM had no significant toxic effect under our culture conditions. Further investigation of this apparent cyanide resistance revealed that the sodium cyanide was being rapidly depleted from the cell culture medium. Cyanide was interacting with constituents of the cell culture medium and was somehow being detoxified or removed from solution. The reaction of cyanide with cell culture media in 96-well culture plates reduced cyanide concentrations rapidly (80-90% in 2 h at 37 degrees C). Running the same reaction in capped tubes significantly reduced cyanide loss from solution. Incubation of cyanide with individual constituents of the cell culture medium in solution showed that glucose, phenol red, and amino acids all acted to detoxify or remove cyanide from solution. When amino acids or buffers were incubated with sodium cyanide in aqueous solution at pH 7.4, hydrogen cyanide (HCN) was found to degas from the solutions. We compared HCN outgassing over a range of pH values. As expected, HCN remained very soluble at high pH, but as the pH was reduced to 7.0, the rate of HCN formation and outgassing increased dramatically. Acid-base reactions involving cyanide and proton donors, such as amino acids and other cell culture media constituents, at physiological pH result in rapid HCN outgassing from solution at 37 degrees C. These results indicate that previous in vitro cyanide toxicity studies done in standard culture media with prolonged incubation times using gas-exchanging culture containers might have to be reevaluated in light of the fact that the effective cyanide concentrations in the culture media were significantly lower than reported.