Reactions of alpha-acetoxy-N-nitrosopyrrolidine with deoxyguanosine and DNA

We investigated the reactions of alpha-acetoxy-N-nitrosopyrrolidine (alpha-acetoxyNPYR) with dGuo and DNA. Alpha-acetoxyNPYR is a stable precursor to the major proximate carcinogen of NPYR, alpha-hydroxyNPYR (3). Our goal was to develop appropriate conditions for the analysis of DNA adducts of NPYR formed in vivo. Products of the alpha-acetoxyNPYR-dGuo reactions were analyzed directly by HPLC or after treatment of the reaction mixtures with NaBH3CN. Products of the alpha-acetoxyNPYR-DNA reactions were released by enzymatic or neutral thermal hydrolysis of the DNA, then analyzed by HPLC. Alternatively, the DNA was treated with NaBH3CN prior to hydrolysis and HPLC analysis. The reactions of alpha-acetoxyNPYR with dGuo and DNA were complex. We have identified 13 products of the dGuo reaction-6 of these were characterized in this reaction for the first time. They were four diastereomers of N2-(3-hydroxybutylidene)dGuo (20, 21), 7-(N-nitrosopyrrolidin-2-yl)Gua (2), and 2-(2-hydroxypyrrolidin-1-yl)deoxyinosine (12). Adducts 20 and 21 were identified by comparison to standards produced in the reaction of 3-hydroxybutanal with dGuo. Adduct 2 was identified by its spectral properties while adduct 12 was characterized by comparison to an independently synthesized standard. With the exception of adduct 2, all products of the dGuo reactions were also observed in the DNA reactions. The major product in both the dGuo and DNA reactions was N2-(tetrahydrofuran-2-yl)dGuo (10), consistent with previous studies. Several other previously identified adducts were also observed in this study. HPLC analysis of reaction mixtures treated with NaBH3CN provided improved conditions for adduct identification, which should be useful for in vivo studies of DNA adduct formation by NPYR.