Evidence for endogenous formation of the hepatocarcinogen N-nitrosodihydrouracil in rats treated with dihydrouracil and sodium nitrite: A potential source of human hepatic DNA carboxyethylation

An earlier study demonstrated that hydrolysates of all human liver DNA samples analyzed contain the DNA adduct 7-(2′-carboxyethyl)guanine (7-CEGua) with an average level of 74.6 adducts per 10⁹ nucleotides. One possible source of this DNA adduct would be endogenous nitrosation of the normal pyrimidine metabolites dihydrouracil (DHU) and β-ureidopropionic acid (β-UPA), yielding the corresponding nitroso compounds N-nitrosodihydrouracil, a potent hepatocarcinogen, and N-nitroso-β-ureidopropionic acid. Another potential source would be reaction of endogenously formed acrylic acid with DNA. We tested these hypotheses in a study in which rats were treated with NaNO₂ in the drinking water, alone, or in combination with dietary DHU or β-UPA, or with acrylic acid in the drinking water, for either 2 or 4 weeks. Hepatic DNA from these rats was analyzed for 7-CEGua, using liquid chromatography-tandem mass spectrometry-selected reaction monitoring with confirmation by high resolution mass spectrometry. The results demonstrated consistent statistically significant increases of 7-CEGua in hepatic DNA of the rats treated with the combination of NaNO₂ and DHU compared to the corresponding controls, while the other treatments gave variable results. These results support the hypothesis that endogenous nitrosation of DHU could be a major source of 7-CEGua in human hepatic DNA. Development of methodology for analysis of 7-CEGua in human leukocyte DNA is also reported, which will allow testing of this hypothesis in epidemiologic and clinical studies.