Mass Spectrometric Quantitation of Pyridyloxobutyl DNA Phosphate Adducts in Rats Chronically Treated with N′-Nitrosonornicotine

Abstract

The tobacco-specific carcinogens N´-nitrosonornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) require metabolic activation to exert their carcinogenicity. NNN and NNK are metabolized to the same reactive diazonium ions, which alkylate DNA forming pyridyloxobutyl (POB) DNA base and phosphate adducts. We have characterized the formation of both POB DNA base and phosphate adducts in NNK-treated rats, and the formation of POB DNA base adducts in NNN-treated rats. However, POB DNA phosphate adducts in NNN-treated rats are still uncharacterized. In this study, we quantified the levels of POB DNA phosphate adducts in tissues of rats chronically treated with (S)-NNN or (R)-NNN for 10, 30, 50, and 70 weeks during a carcinogenicity study. The highest amounts of POB DNA phosphate adducts were observed in the esophagus of the (S)-NNN-treated rats, with a maximum level of 5400 ± 317 fmol/mg DNA at 50 weeks. The abundance of POB DNA phosphate adducts in the esophagus was consistent with the results of the carcinogenicity study showing that the esophagus was the primary site of tumor formation from treatment with (S)-NNN. Compared to the (R)-NNN group, the levels of POB DNA phosphate adducts were higher in the oral mucosa, esophagus and liver, while lower in the nasal mucosa of the (S)-NNN-treated rats. Among ten combinations of all isomers of POB DNA phosphate adducts, Ap(POB)C and combinations with thymidine predominated across all the rat tissues examined. In the primary target tissue, esophageal mucosa, Ap(POB)C accounted for ~20% of total phosphate adducts in the (S)-NNN treatment group throughout the 70 weeks, with levels ranging from 780 ± 194 to 1010 ± 700 fmol/mg DNA. The results of this study showed that POB DNA phosphate adducts were present in high levels and persisted in target tissues of rats chronically treated with (S)- or (R)-NNN. These results improve our understanding of DNA damage during NNN-induced carcinogenesis. The predominant POB DNA phosphate isomers observed, such as Ap(POB)C, may serve as biomarkers for monitoring chronic exposure of tobacco-specific nitrosamines in humans.

Recent Studies on DNA Adducts Resulting from Human Exposure to Tobacco Smoke

“…Since 2012, multiple DNA adducts formed by tobacco-specific carcinogens (i.e., NNK and NNN) have been characterized and quantified in in vitro models and in NNK- or NNN-treated animals [19,20,21,31,34,35,36,123,124]. In addition to DNA base adducts, which have been extensively studied, we have recently characterized and measured a panel of DNA phosphate adducts formed by NNK and NNN [20,34,35,36].…”

“…In order to characterize and measure all of the 32 possible combinations, a specific and powerful analytical method is required. We have developed a series of LC-NSI-HRMS/MS-based methods and characterized four different types of NNK- and NNN-derived DNA phosphate adducts, including pyridyloxobutyl DNA phosphate adducts [B 1 p(POB)B 2 ], pyridylhydroxybutyl DNA phosphate adducts [B 1 p(PHB)B 2 : B 1 p(PHB) s B2 + B 1 p(PHB) b B 2 ], and methyl DNA phosphate adducts [B 1 pMeB 2 ] [20,34,35,36,124].…”

“…In the chronically treated rats, some B 1 p(POB)B 2 adducts are persistent and abundant over 70 weeks, which suggested that those adducts could potentially be detected in smokers and be used as biomarkers to investigate chronic exposure to NNK [20]. The B 1 p(POB)B 2 adducts were also detected in rats treated with NNAL [35] or NNN [124].…”

Replication of Pyridyloxobutyl Phosphotriester Lesions in Cells

“…Many methylating agents, for example, N -methyl- N -nitrosourea, diazomethane, and methylmethanesulfonate, could attack noncarbon-bonded oxygen atoms at the backbone phosphate to yield methyl-PTEs. − Likewise, reactive metabolites of NNN and NNK can attack backbone phosphate groups in DNA to form pyridyloxobutyl phosphotriesters (POB-PTEs) in vitro and in rats (Figure ). , In addition, PTEs constitute the major alkylation products of DNA induced by N -ethyl- N -nitrosourea, and 55–73% of the total POB DNA adducts induced by NNK reside on the phosphate backbone, suggesting that POB-PTEs can serve as biomarkers for some tobacco-related diseases, for example, lung cancer . Furthermore, POB-PTEs appear to be poorly repaired in mammalian tissues, and these lesions remain highly abundant for over 70 weeks in lung tissue of rats treated with drinking water containing 5 ppm of NNN …”

“…21,22 In addition, PTEs constitute the major alkylation products of DNA induced by N-ethyl-N-nitrosourea, 23 and 55−73% of the total POB DNA adducts induced by NNK reside on the phosphate backbone, 21 suggesting that POB-PTEs can serve as biomarkers for some tobacco-related diseases, for example, lung cancer. 21 Furthermore, POB-PTEs appear to be poorly repaired in mammalian tissues, and these lesions remain highly abundant for over 70 weeks in lung tissue of rats treated with drinking water containing 5 ppm of NNN. 22 Since the alkyl group could neutralize the negative charge on the phosphate backbone, PTE lesions were shown to perturb the interactions of DNA with DNA-binding proteins including MutS, 24 MutY, 25 and a RNA polymerase.…”