Nitrosation of amines by stimulated macrophages

Rats and mice treated in vivo with Escherichia coli lipopolysaccharide (LPS) synthesize and excrete large quantities of nitrate. Murine peritoneal macrophages, elicited in vivo with thioglycolate and stimulated in vitro with LPS and/or gamma-interferon (IFN), produce copious amounts of nitrate and nitrite. We report here experiments showing N-nitrosamine formation by macrophages immunostimulated in vitro. Macrophage cell lines J774.1, PU5-1.8, WEHI-3 and RAW 264 and freshly isolated macrophages from C3H/He mice were used. Macrophages were cultured in Dulbecco's modified Eagle's medium (pH 7.5) supplemented with calf serum (10%). Supernatant NO2- and NO3- were measured. N-Nitrosamines were extracted with dichloromethane and the extracts analyzed by a gas chromatography--thermal energy analyzer. Cells (1.5 X 10(6)/ml) were incubated with LPS (10 micrograms/ml) and morpholine (15 mM) for 72 h at 37 degrees C. Under these conditions, all of the cell types listed above produced nitrite (40-70 microM) and N-nitrosomorpholine (NMOR; 114-940 nM). LPS was required for both processes, and this effect was enhanced by IFN. Nitrite (150 microM) incubated with morpholine in cell-free medium did not form NMOR nor did cells plus morpholine and NO2-. The rate of NMOR formation in the J774.1 cell line was highest in the middle incubation period (24-36 h) although [NO2-] was highest in the final incubation period (48-72 h). Thus, the cells do not catalyze nitrosamine formation per se, rather the amine traps out a reactive nitrosating species prior to the formation of NO2- and NO3-. These results suggest that immunostimulated macrophages may be capable of nitrosamine formation under physiological conditions.