Diffusion and reaction of nitric oxide in cell cultures

Abstract

Nitric oxide (NO) is synthesized throughout the body and its intercellular signalling and other functions are necessary for health. However, its mutagenic effects make it a potential cause of cancer, if it is produced at a high enough rate for a sufficient time. Sustained overproduction of NO may occur, for example, in macrophages as part of the immune response to certain chronic infections. The mutagenic effects of NO are thought to be due largely to trace compounds such as nitrous anhydride (N2O3) and peroxynitrite (ONOO-), formed during its reaction with molecular oxygen or superoxide anion (O2-)- It is not technically feasible to monitor the concentrations of these species under most conditions of interest, yet assessing the amounts of these species reaching target cells is essential to correlate levels of toxicity and to extrapolate to pathological situations in the body. Accordingly, mathematical models are needed to predict the concentrations of NO and related compounds in cell cultures. A reaction-diffusion model was developed to predict the fate of NO released by activated macrophages attached to microcarrier beads suspended in a stirred vessel, where experimental data were obtained previously. In the analysis the reactor was divided into a "stagnant film" with position-dependent concentrations and a well-mixed bulk solution. Model predictions based on independently determined kinetics and mass transfer parameters were found to be in good agreement with the experimental results for the concentration of NO in bulk solution, nitrite and nitrate accumulation rate, as well as the NO loss to the head space. The coupling between reaction and diffusion was found to greatly influence the spatial distributions of NO, O2-, as well as N2O3 and peroxynitrite, the potential mediators of cytotoxicity and mutagenicity.

(cont.) Superoxide and peroxynitrite were predicted to be present only within very localized regions, whereas NO and N203 were found to distribute everywhere, more or less uniformly. The CO2 pathway was shown to be the most important pathway for peroxynitrite decomposition and the reaction involving HCO3- was suggested to appreciably reduce the level of N203 and therefore the formation of N-nitroso compounds. Reaction-diffusion models were employed also to describe the spatiotemporal behavior of NO and related species in "plate cultures", a configuration that is employed frequently to examine the toxicity and/or mutagenicity of NO. In such experiments macrophages are maintained on the bottom of culture dishes in submonolayer amounts, typically with 1-4 mm of medium separating the cells from a head space. The reaction-diffusion analysis revealed that there exists in the culture medium a distinctive spatial segregation among the key reactions, which leads to important simplifications in the kinetics and suggests relatively simple approximations to the concentration fields on both the macroscopic (culture medium depth) and microscopic (cell diameter or cell spacing) length scales. Transient effects were found to be unimportant. In contrast to the usual assumption made by those who have used this type of culture system, the calculations showed that NO loss to the head space is not negligible ...