Identification of adducts formed in the reaction of alpha-acetoxy-N-nitrosopyrrolidine with deoxyribonucleosides and DNA.
N-Nitrosopyrrolidine (NPYR) is a well-established hepatocarcinogen in the rat. NPYR requires metabolic activation by cytochrome P450-catalyzed alpha-hydroxylation to express its carcinogenic activity. This produces alpha-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 that 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 alpha-acetoxyNPYR (3) as a stable precursor to 2 and 4. Compound 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 3 with individual deoxyribonucleosides were also carried out. The products were identified by their MS, UV, and NMR spectra as N6-(tetrahydrofuran-2-yl)dAdo (16) and N4-(tetrahydrofuran-2-yl)dCyd (17) in addition to the previously characterized N2-(tetrahydrofuran-2-yl)dGuo (13). Unstable dThd adducts were also formed. Further characterization of the adducts was achieved by NaBH3CN reduction of the reaction mixtures of 3 with deoxyribonucleosides or DNA. This produced N6-(4-hydroxybut-1-yl)dAdo (21), N4-(4-hydroxybut-1-yl)dCyd (22), O2-(4-hydroxybut-1-yl)dThd (23), O4-(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 3 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.
Wang
Mingyao
M
The Cancer Center, University of Minnesota, Mayo Mail Code 806, 420 Delaware Street Southeast, Minneapolis, Minnesota 55455, USA.
Lao
Yanbin
Y
Cheng
Guang
G
Shi
Yongli
Y
Villalta
Peter W
PW
Hecht
Stephen S
SS
D002851:Chromatography, High Pressure Liquid; D018736:DNA Adducts / Q000737:chemistry*; D003853:Deoxyribonucleosides / Q000737:chemistry*; D015394:Molecular Structure; D009242:N-Nitrosopyrrolidine / Q000031:analogs & derivatives* / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization
D018736:DNA Adducts; D003853:Deoxyribonucleosides; C036669:alpha-acetoxy-N-nitrosopyrrolidine; D009242:N-Nitrosopyrrolidine