Metabolic activation of the carcinogenic tobacco-specific nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N′-nitrosonornicotine (NNN) results in formation of reactive electrophiles that modify DNA to produce a variety of products including methyl, 4-(3-pyridyl)-4-oxobutyl (POB)-, and 4-(3-pyridyl)-4-hydroxybutyl adducts. Among these are adducts such as 7-POB-deoxyguanosine (N7POBdG) which can lead to apurinic/apyrimidinic (AP) sites by facile hydrolysis of the base-deoxyribonucleoside bond. In this study, we used a recently developed highly sensitive mass spectrometric method to quantitate AP sites by derivatization with O-(pyridin-3-yl-methyl)hydroxylamine (PMOA) (detection limit, 2 AP sites per 108 nucleotides). AP sites were quantified in DNA isolated from tissues of rats treated with NNN and NNK and from human lung tissue and leukocytes of cigarette smokers and nonsmokers. Rats treated with 5 or 21 mg/kg bw NNK for 4 days by s.c. injection had 2-6 and 2-17 times more AP sites than controls in liver and lung DNA (p < 0.05). Increases in AP sites were also found in liver DNA of rats exposed for 10 and 30 weeks (p < 0.05) but not for 50 and 70 weeks to 5 ppm of NNK in their drinking water. Levels of N7POBG were significantly correlated with AP sites in rats treated with NNK. In rats treated with 14 ppm (S)-NNN in their drinking water for 10 weeks, increased AP site formation compared to controls was observed in oral and nasal respiratory mucosa DNA (p < 0.05). No significant increase in AP sites was found in human lung and leukocyte DNA of cigarette smokers compared to nonsmokers, although AP sites in leukocyte DNA were significantly correlated with urinary levels of the NNK metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL). This is the first study to use mass spectrometry based methods to examine AP site formation by carcinogenic tobacco-specific nitrosamines in laboratory animals and to evaluate AP sites in DNA of smokers and nonsmokers.
Bibliographical noteFunding Information:
This work was supported by the U.S. National Cancer Institute [CA-081301 to S.S.H., P01 CA-160032 to R.J.T]. 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. We thank Sarah Reisinger and Professors Peter G. Shields and Dorothy Hatsukami for providing leukocyte samples from smokers and nonsmokers participating in an observational study supported in part by the U.S. National Cancer Institute [N01 PC-064402]. We thank Laura Maertens for providing leukocyte samples from the Tobacco Research Programs Repository, Bob Carlson for editorial support, Yupeng Li for teaching JG how to isolate the rat tissues, and Steven Carmella and Guang Cheng for help obtaining the human samples. We also thank Nicole Thomson from the Sharon E. Murphy laboratory in the Masonic Cancer Center for measurements of urinary cotinine and NNAL.
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