Comprehensive High-Resolution Mass Spectrometric Analysis of DNA Phosphate Adducts Formed by the Tobacco-Specific Lung Carcinogen 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone

Abstract

The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK, 1) is a potent lung carcinogen in laboratory animals and is believed to play a key role in the development of lung cancer in smokers. Metabolic activation of NNK leads to the formation of pyridyloxobutyl DNA adducts, a critical step in its mechanism of carcinogenesis. In addition to DNA nucleobase adducts, DNA phosphate adducts can be formed by pyridyloxobutylation of the oxygen atoms of the internucleotidic phosphodiester linkages. We report the use of a liquid chromatography–nanoelectrospray ionization–high-resolution tandem mass spectrometry technique to characterize 30 novel pyridyloxobutyl DNA phosphate adducts in calf thymus DNA (CT-DNA) treated with 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone (NNKOAc, 2), a regiochemically activated form of NNK. A 15N3-labeled internal standard was synthesized for one of the most abundant phosphate adducts, dCp[4-oxo-4-(3-pyridyl)butyl]dC (CpopC), and this standard was used to quantify CpopC and to estimate the levels of other adducts in the NNKOAc-treated CT-DNA. Formation of DNA phosphate adducts by NNK in vivo was further investigated in rats treated with NNK acutely (0.1 mmol/kg once daily for 4 days by subcutaneous injection) and chronically (5 ppm in drinking water for 10, 30, 50, and 70 weeks). This study provides the first comprehensive structural identification and quantitation of a panel of DNA phosphate adducts of a structurally complex carcinogen and chemical support for future mechanistic studies of tobacco carcinogenesis in humans.

Methyl DNA Phosphate Adduct Formation in Rats Treated Chronically with 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone and Enantiomers of Its Metabolite 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanol

“…A total of 23, 21, and 22 out of 32 possible methyl DNA phosphate adducts (B 1 pMeB 2 ) were detected and quantified in the lung tissues of rats treated with NNK, ( S )-NNAL, and ( R )-NNAL, respectively. Taken together with our previous studies in which we characterized 30 B 1 p(POB)B 2 adducts 10 and 107 B 1 p(PHB)B 2 adducts, 12 we have collectively identified 160 different structurally unique DNA phosphate adducts in tissues of rats treated with NNK, which establishes NNK as the source of the most structurally diverse panel of DNA adducts identified to date from any carcinogen.…”

“…7 In another study, male Wistar rats were intraperitoneally injected with a single dose of NDMA at 10 mg/kg. TpMeT was detected and quantified in the liver with a half-life of 7 days, significantly longer than the corresponding O 6 -methylguanine with a half-life of 25 h. 24 The persistence of DNA phosphate adducts was also observed in our studies of pyridyloxobutyl 10 and pyridylhydroxybutyl 12,13 phosphate adducts in the NNK- and NNAL-treated rats. The persistence of these DNA phosphate adducts could be partially due to their chemical stability.…”

“…4,10,12,13 The levels of total B 1 p(POB)B 2 phosphate adducts in the lung of NNK-treated rats were reported previously. 10 The levels of total B 1 p(POB)B 2 and B 1 p(PHB)B 2 phosphate adducts in the lung of ( R )- and ( S )-NNAL-treated rats and total B 1 p(PHB)B 2 phosphate adducts in the lung of NNK-treated rats were obtained from other studies. 12,13 The levels of one methyl base adduct, O 6 -methylguanine, three major POB base adducts, O 2 -[4-(3-pyridyl)-4-oxobut-1-yl]thymidine, 7-[4-(3-pyridyl)-4-oxobut-1-yl]guanine, and O 6 -[4-(3-pyridyl)-4-oxobut-1-yl]deoxyguanosine, and two major PHB base adducts, O 2 -[4-(3-pyridyl)-4-hydroxybut-1-yl]thymidine and 7-[4-(3-pyridyl)-4-hydroxybut-1-yl]guanine, were reported previously.…”

“…1,4,8,10,25 To determine the relative proportion of methyl phosphate adducts to the total measured DNA adducts formed in rats treated with NNK or NNAL, the levels of the other five types of adducts were obtained from our previous studies (Table 2). 4,10,12,13 The levels of total B 1 p(POB)B 2 phosphate adducts in the lung of NNK-treated rats were reported previously. 10 The levels of total B 1 p(POB)B 2 and B 1 p(PHB)B 2 phosphate adducts in the lung of ( R )- and ( S )-NNAL-treated rats and total B 1 p(PHB)B 2 phosphate adducts in the lung of NNK-treated rats were obtained from other studies.…”

“…12 The major POB and PHB base adducts and B 1 p(POB)B 2 and B 1 p(PHB)B 2 phosphate adducts have been characterized, and their levels in NNK- and NNAL-treated animals have been quantified in our laboratory. 4,8,10−14 α-Hydroxylation also occurs on the α-methylene carbons of NNK and NNAL, which produces intermediates α-methylenehydroxyNNK ( 4 ) and α-methylenehydroxyNNAL ( 5 ), respectively. Both 4 and 5 decompose to methane diazohydroxide ( 8 ), which reacts with DNA to form methyl DNA base adducts (Figure 1).…”