A STUDY OF CHEMICAL CARCINOGENESIS .66. A SENSITIVE METHOD FOR DETECTING INVIVO FORMATION OF N-NITROSOMORPHOLINE AND ITS APPLICATION TO RATS GIVEN LOW-DOSES OF MORPHOLINE AND SODIUM-NITRITE

Abstract

A method was developed to monitor the in vivo formation of N-nitrosomorpholine. N-Nitroso(2-hydroxyethyl)glycine, a major urinary metabolite of N-nitrosomorpholine, was quantified as its methyl ester-trimethylsilyl ether derivative, using gas chromatography. When the method was applied to rats, the in vivo formation of, or exposure to, as little as 0.6 .mu.g of N-nitrosomorpholine could be quantified. The method was also applicable to human urine, with a detection limit of .apprx. 0.5 .mu.g of N-nitroso(2-hydroxyethyl)glycine/100-ml urine sample. The formation of N-nitrosomorpholine was measured in rats treated by gavage with a wide range of doses of morpholine and NaNO2. Depending on the dose, 0.5-12% of the morpholine was nitrosated. N-Nitrosomorpholine formation showed a high degree of variability among rats treated with a given dose of morpholine and NaNO2, but the levels of N-nitrosomorpholine formed were generally in agreement with expectations based on in vitro studies in which dependence on morpholine concentration multiplied by nitrite concentration squared had been established. The formation of N-nitrosomorpholine was also measured in rats administered a diet containing 50 ppm of morpholine and 1000 ppm of NaNO2, a regimen which induces liver cell tumors. The mean daily formation of N-nitrosomorpholine under these conditions was estimated to be 0.88 .+-. 0.59 .mu.mol/rat (SD), which is high enough to account for the observed tumor incidence. The results of this study provide quantitative support for the assumption that in vivo formation of N-nitrosomorpholine leads to tumor development.