Prediction of concentrations of reactive nitrogen species in aqueous solutions and cells

Abstract

Reactive nitrogen species (RNS) derived from nitric oxide (NO) have been implicated in cancer and other diseases, but their intracellular concentrations are largely unknown. To estimate them under steady-state conditions representative of inflamed tissues, a kinetic model was developed that included the effects of cellular antioxidants, amino acids, proteins, and lipids. For an NO concentration of 1 [mu]M, total peroxynitrite (Per, the sum of ONOO- and ONOOH), nitrogen dioxide (NO2), and nitrous anhydride (N20 3) were calculated to have concentrations in the nM, pM, and fM ranges, respectively. The concentrations of NO2 and N20 3 were predicted to decrease markedly with increases in glutathione (GSH) levels, due to the scavenging of each by GSH. Although lipids accelerate the oxidation of NO by 02 (because of the high solubility of each in hydrophobic media), lipid-phase reactions were calculated to have little effect on NO2 or N20 3 concentrations. The major sources of intracellular NO2 were found to be the reaction of Per with metals and with CO 2, whereas the major sinks were its reactions with GSH and ascorbate (AH-). The radical-scavenging ability of GSH and AH- caused 3-nitrotyrosine to be the only tyrosine derivative predicted to be formed at a significant rate. The major GSH reaction product was S-nitrosoglutathione. Analytical (algebraic) expressions were derived for the concentrations of the key reactive intermediates, allowing the calculations to be extended readily. To investigate the mutagenic and toxic effects of NO on cells, methods are needed to expose them to constant, physiological levels of NO for hours to days. One way to do this is to co-culture target cells with activated macrophages, which can synthesize NO at constant rates for long periods. A novel method, developed in the laboratory of Professor G. N. Wogan at MIT, involves the use of TranswellTM permeable supports (Coming), in which a porous membrane separates two chambers in a culture dish. Target cells and macrophages are placed on the top and bottom of the insert, respectively. Although the two cell types are in close diffusional contact, the target cells can be recovered separately for viability and mutation assays. To infer the NO concentration at the level of the cells from measured rates of formation of nitrite (N02-), a reaction-diffusion model was developed to calculate NO and 02 concentrations as a function of height in the medium. In this system the oxidation of NO to NO2 competes with the diffusional loss of NO to the incubator gas. It was shown that a one-dimensional, steady-state formulation is justified. The key factors affecting NO and 02 concentrations are the total rate of respiratory 02 consumption by the cells and their net rate of NO generation. Because the overall rate of the multi-step NO oxidation is second order in NO, the fractional loss of NO from the system by diffusion increases as the NO concentration is reduced. Also, the fractional loss of NO is increased if cellular 02 consumption is elevated. The cellular NO concentration was predicted to be nearly proportional to the square root of the NO2 formation rate. Thus, in experiments in the Wogan laboratory in which NMA (an inhibitor of NO synthase) was added to the culture medium, reducing NO2 formation by 90%, the cellular NO concentration was calculated to decrease only by about two-thirds (from 1.1 [mu]M to 0.36 [mu]M). To facilitate the use of the reaction-diffusion model by other laboratories, a graphical method was developed to allow cellular NO concentrations to be estimated from measured rates of NO2 accumulation. The controlled delivery of NO2 into aqueous solutions, in the absence of NO, would be useful in investigating its rates of reaction with biological molecules and in isolating its effects on cells from those of other RNS. Two possible NO2 delivery methods were investigated theoretically. One was the direct contact of NO2 gas mixtures with stirred aqueous solutions, and the other was diffusion of NO2 through gas-permeable tubing (such as polydimethylsiloxane, PDMS) into such solutions. In gases and in water, NO2 dimerizes reversibly to form dinitrogen tetroxide (N204), which reacts rapidly with water to produce nitrite and nitrate. Thus, it was necessary to describe the coupled reaction and diffusion of NO2 and N20 4 in each kind of system. Microscopic models were developed to describe spatial variations in concentrations near the gasliquid interface, or within the tubing wall and immediately adjacent liquid. These were used to predict parameter values (such as mass transfer coefficients) in macroscopic models designed to describe bulk aqueous concentrations. Because the direct measurement of NO2 and N20 4 concentrations at the low levels desired for biological experiments is impractical, the combined models are needed to estimate bulk NO2 and N20 4 concentrations from measurable quantities such as rates of N0 2- accumulation. For direct gas-liquid contacting, the utility of a quasiequilibrium approximation (QEA) was examined. This assumes that the NO2 and N20 4 concentrations are related as for dimerization equilibrium. At relatively high NO2 concentrations in the delivery gas, the results from the QEA and exact equations were in excellent agreement. As the NO2 level was reduced, the QEA eventually fails, because NO2 increasingly resembles an unreactive species as its concentration approaches zero. However, the QEA was found to be quite accurate throughout the practical range of concentrations (0.001% to 1% NO2 gas), the relative error in total fluxes not exceeding 6%. The results show that it is desirable to use as low an NO2 concentration as is analytically feasible (such as 0.001% NO2 gas). This minimizes both the concentration of N20 4 and the effects of concentration nonuniformities in the aqueous boundary layer. For NO2 delivery through gas-permeable tubing such as PDMS, the modeling was more complicated and the results more uncertain. The main complication was due to the presence of a concentration boundary layer within the membrane next to the liquid, which required that the governing equations be rescaled for that region. The major source of uncertainty is the unknown solubility of N20 4 in PDMS. However, as the gas concentration was lowered, the results became insensitive to this parameter. For 1% NO2 gas, the estimated bulk NO2 concentrations were 7.1 pM for the direct gas contact and 0.35 pM for the gas-permeable tubing. For 0.001% NO2 gas, the estimated NO2 concentrations were 0.45 [mu]M for the direct gas contact and 0.14 [mu]M for the gas-permeable tubing. For both methods, the times to reach steady state were predicted to be quite fast, at most 10 seconds.