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.
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 O6-pobdG. Preliminary studies with liver DNA isolated from NNKOAc-treated mice demonstrated that this method can be used to quantify O6-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.
Thomson
Nicole M
NM
Division of Environmental Health Sciences, Department of Medicinal Chemistry, and The Cancer Center, University of Minnesota, Minneapolis, Minnesota 55455, USA.
Mijal
Renée S
RS
Ziegel
Rebecca
R
Fleischer
Nancy L
NL
Pegg
Anthony E
AE
Tretyakova
Natalia Y
NY
Peterson
Lisa A
LA
D000818:Animals; D002853:Chromatography, Liquid / Q000379:methods*; D018736:DNA Adducts / Q000032:analysis* / Q000737:chemistry; D003849:Deoxyguanosine / Q000031:analogs & derivatives* / Q000032:analysis* / Q000737:chemistry; D005260:Female; D006147:Guanine / Q000031:analogs & derivatives; D051379:Mice; D009153:Mutagens / Q000032:analysis* / Q000737:chemistry; D009602:Nitrosamines / Q000737:chemistry* / Q000633:toxicity; D011725:Pyridines / Q000032:analysis* / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D014026:Nicotiana / Q000633:toxicity*
D018736:DNA Adducts; D009153:Mutagens; D009602:Nitrosamines; C473029:O(6)-(4-oxo-4-(3-pyridyl)butyl)guanine; D011725:Pyridines; D006147:Guanine; D003849:Deoxyguanosine