The tobacco-specific nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N'-nitrosonornicotine (NNN) are considered carcinogenic to humans by the International Agency for Research on Cancer. Metabolic activation of NNK and 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 (PHB)- adducts that have been previously characterized. Among these are adducts such as 7-POB-deoxyguanosine and O2-POB-deoxycytidine that can result in apurinic/apyrimidinic (AP) sites by facile hydrolysis of the base-deoxyribonucleoside bond. The purpose of this study was to quantitatively examine the AP sites induced by NNN and NNK in different tissues of rats using a novel validated mass spectrometric method. Incubation of 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone (NNKOAc), a model compound for methyl hydroxylation of NNK, with rat liver homogenate, produced apurinic sites quantified by derivatization with O-(pyridin-3-ylmethyl)hydroxylamine (PMOA) and analysis by LC-ESI-MS/MS. The amount found in the NNKOAc incubations was 4.54 ± 0.41 mol/107 mol nucleotides (n=4), significantly greater (p&lt;0.001) than in controls (2.00 ± 0.16 mol/107 mol nucleotides, n=4) lacking NNKOAc. No significant difference (p&gt;0.05) was found in the levels of AP sites in the livers of rats treated with 5 ppm NNK in their drinking water for 10 weeks or 70 weeks, compared to control untreated rats in a carcinogenicity study. Further investigation of AP site formation in different tissues of NNK and NNN exposed rats is in progress. This is the first study to examine AP site formation by carcinogenic tobacco-specific nitrosamines.

Mass spectrometric quantitation of apurinic/apyrimidinic sites in tissues of rats treated with 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and N'-nitrosonornicotine

2019 Fall National Meeting

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