title: Analysis of adducts in hepatic DNA of rats treated with N-nitrosopyrrolidine.
abstract: N-Nitrosopyrrolidine (NPYR) is a hepatocarcinogen in rats. It is metabolically activated by cytochrome P450 enzymes in the liver leading to the formation of 4-oxobutanediazohydroxide (4) and related intermediates that react with DNA to form adducts. Because DNA adducts are thought to be critical in carcinogenesis by NPYR, we analyzed hepatic DNA of NPYR-treated rats for several adducts: N2-(tetrahydrofuran-1-yl)dGuo (N2-THF-dGuo, 13), N6-THF-dAdo (14), N4-THF-dCyd (17), and dThd adducts 15 and 16. The rats were treated with NPYR in the drinking water, 600 ppm for 1 week, or 200 ppm for 4 or 13 weeks. Hepatic DNA was isolated, enzymatically hydrolyzed, and analyzed by capillary LC-ESI-MS-SIM, which indicated the presence of adducts 13, 14, and 17. Because these adducts can be unstable at the deoxyribonucleoside level, further analyses were carried out using DNA treated with NaBH3CN, which converts adducts 13-17 to N2-(4-hydroxybut-1-yl)dGuo [N2-(4-HOB)dGuo, 18], N6-(4-HOB)dAdo (19), O2-(4-HOB)dThd (20), O4-(4-HOB)dThd (21), and N4-(4-HOB)dCyd (22). [15N]-Labeled analogues of adducts 18-20 and 22 were synthesized and used in this analysis, which was performed by capillary LC-ESI-MS/MS-SRM. Convincing evidence for the presence of adducts 18-22 was obtained. Levels of 18, 19, 20, and 21 were (mumol/mol dGuo): 3.41-5.39, 0.02-0.04, 2.56-3.87, and 2.28-5.05, respectively. Compound 22 was not quantified due to interfering peaks. These results provide the first evidence for tetrahydrofuranyl-substituted DNA adducts in the livers of rats treated with NPYR. The finding of dAdo and dThd adducts is of particular interest since previous studies have shown that NPYR causes mutations at AT base pairs in DNA of rat liver.
authors: The Cancer Center, University of Minnesota, Mayo Mail Code 806, 420 Delaware Street Southeast, Minneapolis, Minnesota 55455, USA.
authors: Mingyao
authors: M
authors: Wang
authors: Yanbin
authors: Y
authors: Lao
authors: Guang
authors: G
authors: Cheng
authors: Yongli
authors: Y
authors: Shi
authors: Peter W
authors: PW
authors: Villalta
authors: Akiyoshi
authors: A
authors: Nishikawa
authors: Stephen S
authors: SS
authors: Hecht
mesh_terms: D000818:Animals; D020029:Base Pairing; D018736:DNA Adducts / Q000032:analysis* / Q000737:chemistry* / Q000378:metabolism; D008099:Liver / Q000737:chemistry* / Q000187:drug effects* / Q000378:metabolism; D015394:Molecular Structure; D009242:N-Nitrosopyrrolidine / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology*; D051381:Rats; D011916:Rats, Inbred F344; D021241:Spectrometry, Mass, Electrospray Ionization
chemical_list: D018736:DNA Adducts; D009242:N-Nitrosopyrrolidine