Investigation of 2′-Deoxyadenosine-Derived Adducts Specifically Formed in Rat Liver and Lung DNA by N′-Nitrosonornicotine Metabolism

Yupeng Li, Erik S. Carlson, Adam T. Zarth, Pramod Upadhyaya, Stephen S. Hecht

Research output: Contribution to journalArticlepeer-review

6 Scopus citations


The International Agency for Research on Cancer has classified the tobacco-specific nitrosamines N′-nitrosonornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) as "carcinogenic to humans"(Group 1). To exert its carcinogenicity, NNN requires metabolic activation to form reactive intermediates which alkylate DNA. Previous studies have identified cytochrome P450-catalyzed 2′-hydroxylation and 5′-hydroxylation of NNN as major metabolic pathways, with preferential activation through the 5′-hydroxylation pathway in some cultured human tissues and patas monkeys. So far, the only DNA adducts identified from NNN 5′-hydroxylation in rat tissues are 2-[2-(3-pyridyl)-N-pyrrolidinyl]-2′-deoxyinosine (Py-Py-dI), 6-[2-(3-pyridyl)-N-pyrrolidinyl]-2′-deoxynebularine (Py-Py-dN), and N6-[4-hydroxy-1-(pyridine-3-yl)butyl]-2′-deoxyadenosine (N6-HPB-dAdo) after reduction. To expand the DNA adduct panel formed by NNN 5′-hydroxylation and identify possible activation biomarkers of NNN metabolism, we investigated the formation of dAdo-derived adducts using a new highly sensitive and specific liquid chromatography-nanoelectrospray ionization-high-resolution tandem mass spectrometry method. Two types of NNN-specific dAdo-derived adducts, N6-[5-(3-pyridyl)tetrahydrofuran-2-yl]-2′-deoxyadenosine (N6-Py-THF-dAdo) and 6-[2-(3-pyridyl)-N-pyrrolidinyl-5-hydroxy]-2′-deoxynebularine (Py-Py(OH)-dN), were observed for the first time in calf thymus DNA incubated with 5′-acetoxyNNN. More importantly, Py-Py(OH)-dN was also observed in relatively high abundance in the liver and lung DNA of rats treated with racemic NNN in the drinking water for 3 weeks. These new adducts were characterized using authentic synthesized standards. Both NMR and MS data agreed well with the proposed structures of N6-Py-THF-dAdo and Py-Py(OH)-dN. Reduction of Py-Py(OH)-dN by NaBH3CN led to the formation of Py-Py-dN both in vitro and in vivo, which was confirmed by its isotopically labeled internal standard [pyridine-d4]Py-Py-dN. The NNN-specific dAdo adducts Py-THF-dAdo and Py-Py(OH)-dN formed by NNN 5′-hydroxylation provide a more comprehensive understanding of the mechanism of DNA adduct formation by NNN.

Original languageEnglish (US)
Pages (from-to)1004-1015
Number of pages12
JournalChemical research in toxicology
Issue number4
StatePublished - Apr 19 2021

Bibliographical note

Funding Information:
This study was supported by grant CA-81301 from the National Cancer Institute. Mass spectrometry was carried out in the Analytical Biochemistry Shared Resource of the Masonic Cancer Center, University of Minnesota, supported in part by Cancer Center Support grant CA-077598.

Publisher Copyright:
© 2021 American Chemical Society. All rights reserved.

PubMed: MeSH publication types

  • Journal Article
  • Research Support, N.I.H., Extramural


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