Identification of Adducts Formed in the Reaction of α-Acetoxy-N-nitrosopyrrolidine with Deoxyribonucleosides and DNA

Abstract

N-Nitrosopyrrolidine (NPYR) is a well-established hepatocarcinogen in the rat. NPYR requires metabolic activation by cytochrome P450-catalyzed α-hydroxylation to express its carcinogenic activity. This produces α-hydroxyNPYR (2) which spontaneously ring opens to 4-oxobutanediazohydroxide (4), a highly reactive intermediate which may itself modify DNA or yield a cascade of electrophiles which react with DNA to produce adducts. Multiple dGuo adducts formed in this reaction have been previously characterized, but there are no examples of adducts formed with other DNA nucleobases. In this study, we used α-acetoxyNPYR (3) as a stable precursor to α-hydroxyNPYR (2) and 4. α-AcetoxyNPYR (3) was allowed to react with DNA. The DNA was enzymatically hydrolyzed to deoxyribonucleosides and the products were analyzed by LC-ESI-MS and LC-ESI-MS/MS. Reactions of α-acetoxyNPYR with individual deoxyribonucleosides were also carried out. The products were identified by their MS, UV, and NMR spectra as N 6 -(tetrahydrofuran-2-yl)dAdo (16) and N 4 -(tetrahydrofuran-2-yl)dCyd (17) in addition to the previously characterized N 2 -(tetrahydrofuran-2-yl)dGuo (13). Unstable dThd adducts were also formed. Further characterization of the adducts was achieved by NaBH 3 CN reduction of the reaction mixtures of α-acetoxyNPYR with deoxyribonucleosides or DNA. This produced N 6 -(4-hydroxybut-1-yl)dAdo (21), N 4 -(4-hydroxybut-1-yl)dCyd (22), O 2 -(4-hydroxybut-1-yl)dThd (23), O 4 -(4-hydroxybut-1-yl)dThd (24), and 3-(4-hydroxybut-1-yl)dThd (25). Adducts 21 and 22 were characterized by their spectral properties while the dThd adducts 23-25 were identified by comparison to synthetic standards. The results of this study demonstrate that α-acetoxyNPYR forms adducts with dAdo, dCyd, and dThd in DNA, in addition to the previously characterized dGuo adducts. These newly characterized standards can be used to investigate DNA adduct formation in rats treated with NPYR.

Analysis of Adducts in Hepatic DNA of Rats Treated with N-Nitrosopyrrolidine

“…The adduct levels observed in our study (Table 1) therefore probably represent a steady state between adduct formation and repair under conditions of chronic NPYR treatment, but this requires further study. While dThd adducts 15 and 16 are stable in DNA, they are quite unstable at the deoxyribonucleoside level (12), which prevents their quantitation in the absence of NaBH 3 CN treatment of the DNA.…”

Mass spectrometry for the assessment of the occurrence and biological consequences of DNA adducts

“…Alternatively, if the isotope-labeled standards are not available, a putative DNA adduct standard can be synthesized and analyzed in parallel with the analyte to establish whether the analyte exhibits identical retention time as well as similar MS and tandem MS as the synthetic standard. 6,100 However, special attention needs to be paid in this case to avoid misidentification of analytes.…”

Versatility of Y-family Sulfolobus solfataricus DNA Polymerase Dpo4 in Translesion Synthesis Past Bulky N2-Alkylguanine Adducts

“…The bulkiness of DNA adducts may be a differentiating factor in the misincorporation and blockage of DNA polymerases. The N2 atom of guanine is susceptible to modification by various potential carcinogens, including formaldehyde (4), acetaldehyde (5)(6)(7)(8), styrene oxide (9), N-nitrosopyrrolidine (10), oxidation products of heterocyclic aromatic amines (e.g. N-hydroxy-2-amino-3-methylimidazo [4,5-f]quinoline and N-hydroxy-2-amino -3,8-dimethylimidazo [4,5-f]quinoxaline (11)), and the oxidation products of various polycyclic aromatic hydrocarbons (e.g.…”