In vivo nitrosation of morpholine in mice by inhaled NO2

Groups of 3-5 mice were gavaged with aqueous solutions of 2 mg morpholine and immediately exposed to 2-50 ppm levels of NO2 in inhalation chambers for periods of up to 3 hours. After exposure, mice were frozen and pulverized in liquid nitrogen, and aliquots of frozen powder (congruent to 8 g) were extracted with cold 35% aqueous methanol and methylene chloride. Concentrated methylene chloride solutions were analyzed for N-nitrosomorpholine (NMOR), using gas chromatography with a Thermal Energy analyzer detector. NMOR levels in controls (exposed to NO2 alone, or dosed with morpholine without subsequent NO2 exposure) were less than 5 ng/mouse. NMOR yields in test mice were time-dependent with respect to duration of NO2 exposure and dose-dependent with respect to NO2 levels. These preliminary studies demonstrate the in vivo nitrosating potential of nitrogen oxides.