Mass Spectrometric Quantitation of Apurinic/Apyrimidinic Sites in Tissue DNA of Rats Exposed to Tobacco-Specific Nitrosamines and in Lung and Leukocyte DNA of Cigarette Smokers and Nonsmokers

Abstract

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.

Liquid Chromatography-Nanoelectrospray Ionization-High-Resolution Tandem Mass Spectrometry Analysis of Apurinic/Apyrimidinic Sites in Oral Cell DNA of Cigarette Smokers, e-Cigarette Users, and Nonsmokers

“…Ten percent of the filtrate was taken to measure dGuo by LC–MS/MS. The rest was further purified by solid-phase extraction (SPE) …”

“…Quantitation of PMOA-dR was conducted with a Linear Trap Quadropole (LTQ) Orbitrap Velos instrument (Thermo Scientific, Waltham, MA) interfaced with a Dionex Ultimate 3000 UHPLC system (NCS-3500RS pump and WPS-3000PL autosampler) using NSI. The instrument conditions were summarized previously . The chromatograms obtained from oral cell DNA samples from a blank, a nonsmoker, a smoker, and an e-cigarette user are shown in Figure .…”

“…Spontaneous cleavage of the base-deoxyribonucleoside bond of some DNA adducts (such as 7-methyldeoxyguanosine, 7-Me-dGuo) or mechanisms such as base excision repair can result in an apurinic/apyrimidinic (AP) site, which is a location in DNA that has lost either a purine or pyrimidine base, leaving a deoxyribosyl moiety. , Our previous study found that exposure of rats to the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) resulted in increased formation of AP sites in rat liver and lung DNA both in vivo and in vitro . Increased AP sites were also found in nasal and oral mucosa DNA, target tissues of N ′-nitrosonornicotine (NNN), in ( S )-NNN exposed rats . Although cells have active excision repair mechanisms which restore the normal base sequence in DNA, some unrepaired AP sites can cause genomic instability and result in mutations and cancer. , The quantification of AP sites may provide a general measurement of DNA exposure to mutagens …”

“…Several approaches have been developed to stabilize deoxyribose to quantify AP sites in genomic DNA . Chen et al used O -(pyridin-3-yl-methyl)­hydroxylamine (PMOA) to react with AP sites to form (3 S ,4 R )-3,4,5-trihydroxypentanal,- O -pyridin-3-yl-methyl oxime (PMOA-dR) (Scheme ) before DNA isolation to limit the formation of artifacts during DNA processing .…”

“…Then, they used liquid chromatography tandem mass (LC–MS/MS) to measure PMOA-dR in rat tissues. We have applied their approach to derivatize and measure AP sites from rat and human tissues …”