A nitrogen dioxide delivery system for biological media

Nitrogen dioxide is formed endogenously via the oxidation of NO by O2 or O2− and from NO2− via peroxidases, among other pathways. This radical has many potential biological targets and its concentration, like that of NO and other reactive nitrogen species, is thought to be elevated at sites of inflammation. To investigate the specific cytotoxic or mutagenic effects of NO2, it is desirable to be able to maintain its concentration at constant, predictable, and physiological levels in cell cultures, in the absence of NO. To do this, a delivery system was constructed in which NO2-containing gas mixtures contact a liquid within a small (110 ml) stirred reactor. In such gas mixtures NO2 is present in equilibrium with its dimer, N2O4. The uptake of NO2 and N2O4 was characterized by measuring the accumulation rates of NO2− and NO3−, the stable products of N2O4 hydrolysis, in buffered aqueous solutions. In some experiments NO2-reactive 2,2′-azino-bis(3-ethyl-benzothiazoline-6-sulfonate) (ABTS) was included and formation of the stable ABTS radical was measured. A reaction–diffusion model was developed that predicts the accumulation rates of all three products to within 15% for gas-phase concentrations of NO2 spanning 3 orders of magnitude. The model also provides estimates for the NO2 concentration in the liquid. This system should be useful for exposing cells to NO2 concentrations similar to those in vivo.

► We demonstrate quantitative delivery of nitrogen dioxide by gas–liquid contact.
► We provide a detailed model of mass transfer and chemical kinetics.
► We also give guidelines for nitrogen dioxide delivery to cell culture.