Development of a quantitative liquid chromatography/electrospray mass spectrometric assay for a mutagenic tobacco specific nitrosamine-derived DNA adduct, O6-[4-oxo-4-(3-pyridyl)butyl]-2′-deoxyguanosine

Nicole M. Thomson, Renée S. Mijal, Rebecca Ziegel, Nancy L. Fleischer, Anthony E. Pegg, Natalia Y. Tretyakova, Lisa A. Peterson

Research output: Contribution to journalArticle

21 Scopus citations

Abstract

Liquid chromatography with electrospray tandem mass spectrometry (LC/ESI-MS/MS) was employed to quantify O6-[4-oxo-4-(3-pyridyl)butyl] -2′-deoxyguanosine (O6-pobdG), a mutagenic adduct formed by pyridyloxobutylating nitrosamines. Selected reaction monitoring (SRM) of the neutral loss of the sugar from protonated molecules of the adduct, [M + H - 116]+, was utilized for detection of O6-pobdG in pyridyloxobutylated DNA from both in vitro and in vivo sources. Quantitation was based on isotope dilution with synthetic O6-[1,2,2- 2H3-4-oxo-4-(3-pyridyl)butyl]-2′-deoxyguanosine. The detection limits in this study were less than 5 fmol of pure standard and 50 fmol in 1.5 mg of DNA. This method was validated by comparing adduct levels measured with the LC/ESI-MS/MS method to those obtained with radiochemical methods in DNA alkylated with the model pyridyloxobutylating agent, [5- 3H]4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone ([5- 3H]NNKOAc). The pyridyloxobutyl 2′-deoxyguanosine adduct coeluting with the deuterated standard disappeared when NNKOAc-treated DNA had been reacted with the repair protein, O6-alkylguanine-DNA alkyltransferase. This result confirms that the coeluting peak is solely O 6-pobdG. Preliminary studies with liver DNA isolated from NNKOAc-treated mice demonstrated that this method can be used to quantify O 6-pobG in DNA from in vivo sources. The improved sensitivity and specificity of adduct detection afforded by this LC/ESI-MS/MS method will allow us to explore the role of O6-pobdG in the toxicological properties of pyridyloxobutylating nitrosamines.

Original languageEnglish (US)
Pages (from-to)1600-1606
Number of pages7
JournalChemical research in toxicology
Volume17
Issue number12
DOIs
StatePublished - Dec 1 2004

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