Identification and quantification of DNA adducts formed by carcinogenic nitrosamines

Nitrosamines are an important class of potentially carcinogenic and mutagenic compounds. Nitrosamines have been shown to induce tumors of the liver, lung, oral cavity, esophagus, kidney, nasal, and respiratory tract in wide variety of experimental animals. N-nitrosomorpholine (NMOR), N-nitrosopyrrolidine (NPYR), and N-nitrosopiperidine (NPIP) are potent carcinogens. The metabolism of these cyclic nitrosamines occurs predominantly through cytochrome P-450 mediated &agr;-hydroxylation. The resulting &agr;-hydroxy-nitrosamine decomposes to a higly reactive diazonium ion which can react with DNA. Thus, the electrophilic diazonium ion formed from NMOR, NPYR, and NPIP react with DNA, producing 2-(2-ethoxyacetaldehyde)-(EA), 4-butanal, and 5-pentanal adducts, respectively.;To date, only purine adducts are reported resulting from NMOR exposure. There are no reports for pyrimidine adducts formed from the diazonium ion resulting from NMOR. The first major aim of the research was to investigate the formation of pyrimidine adducts resulting from the reaction with &agr;-hydroxy-N-nitrosomorpholine (HONMOR), the key metabolite of the metabolism of NMOR. This was accomplished by the reaction of a stable precursor &agr;-hydroperoxy-N-nitrosomorpholine (HOONMOR) with nucleosides and DNA. The unstable aldehyde (EA) adducts were reduced to the more stable reduced alcohols (rEA). Identification and quantification was done with the use of synthetic standards. In reactions of nucleosides, the yields of 2-(2-ethoxyacetaldehye) are in the order of O2C > O4T > O2T > N3T. The N4-dC adduct was not detected in the nucleoside reaction. In the reaction of DNA, yields of pyrimidine adducts are in the order of O2T > O 2C > O4T. Adducts N3T and N4-dC were not observed in the reaction of HONMOR with DNA. Another goal of this research was to examine the aqueous stability of pyrimidine adducts formed in the reaction of nucleosides and DNA. The results for the decay of EA adducts in nucleoside and DNA shows the overall order of O2T > N3T > O4T > O2C and O4T > O2T > O 2C, respectively. The results of the stability study indicate an enhancement in the rate of decay for the transition from nucleoside to DNA for all adducts.;The second major aim of this research was the examination of the N3-Ade, N7-Gua, and O6-Gua adducts formed by the diazonium ions derived from the carcinogenic nitrosamines NMOR, NPYR, and NPIP. To date, only two adducts are reported for NPYR resulting from the direct reaction of diazonium ion with DNA. There are no reports for adducts formed from the diazonium ion generated from NPIP metabolism. For the investigation of diazonium ion derived adducts, stable precursors to the reactive carcinogens HOONMOR, &agr;-hydroperoxy-N-nitrosopyrrolidine (HOONPYR), and &agr;-hydroperoxy-N-nitrosopiperidine (HOONPIP), and synthetic standards were used. All three adducts under investigation were observed in the reaction of nucleosides and DNA with the cyclic nitrosamines in the overall order of N7-Gua > O6-Gua > N3-Ade. The results obtained from the nucleoside reactions indicate that NMOR and NPYR show similar yields of alkylation with adduct yields 50-80% higher than NPIP.