Quantitation of pyridyloxobutyl-DNA adducts in tissues of rats treated chronically with (R)- or (S)-N'-nitrosonornicotine (NNN) in a carcinogenicity study - PubMed (original) (raw)

. 2013 Oct 21;26(10):1526-35.

doi: 10.1021/tx400235x. Epub 2013 Sep 18.

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Quantitation of pyridyloxobutyl-DNA adducts in tissues of rats treated chronically with (R)- or (S)-N'-nitrosonornicotine (NNN) in a carcinogenicity study

Lijiao Zhao et al. Chem Res Toxicol. 2013.

Abstract

We quantified DNA adducts resulting from 2'-hydroxylation of enantiomers of the tobacco-specific nitrosamine N'-nitrosonornicotine (NNN) in tissues of male F-344 rats after 10, 30, 50, and 70 weeks of treatment with 14 ppm in the drinking water. These rats were in subgroups of a carcinogenicity study in which (S)-NNN was highly tumorigenic in the oral cavity and esophagus, while (R)-NNN was relatively weakly active. DNA adducts were quantified by liquid chromatography-electrospray ionization-tandem mass spectrometry in six tissues: oral mucosa, esophageal mucosa, nasal respiratory mucosa, nasal olfactory mucosa, liver, and lung. O²-[4-(3-Pyridyl)-4-oxobut-1-yl]thymidine (O²-POB-dThd, 7) and 7-[4-(3-pyridyl)-4-oxobut-1-yl]-2'-deoxyguanosine (7-POB-dGuo, 8), the latter as 7-[4-(3-pyridyl)-4-oxobut-1-yl]guanine (7-POB-Gua, 11), were detected at each time point in each tissue. In the target tissues for carcinogenicity, oral mucosa and esophageal mucosa, levels of 7-POB-Gua (11) and O²-POB-dThd (7) were similar, or 11 predominated, while in all other tissues at all time points for both enantiomers, 7 was clearly present in greater amounts than 11. Total measured DNA adduct levels in esophageal mucosa and oral mucosa were higher in rats treated with (S)-NNN than (R)-NNN. The highest adduct levels were found in the nasal respiratory mucosa. DNA adducts generally persisted in all tissues without any sign of substantial decreases throughout the 70 week time course. The results of this study suggest that inefficient repair of 7-POB-dGuo (8) in the rat oral cavity and esophagus may be important in carcinogenesis by NNN and support the development of these DNA adducts as potential biomarkers of NNN metabolic activation in people who use tobacco products.

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Figures

Figure 1

Figure 1

Structures of (S)-NNN (1) and (R)-NNN (2)

Figure 2

Figure 2

Structures of the POB-DNA adducts discussed in this study.

Figure 3

Figure 3

Typical SRM chromatograms obtained upon analysis of oral mucosa DNA isolated from (A) control rats; (B) (R)-NNN-treated rats; and (C) (S)-NNN-treated rats after 70 weeks of treatment. Individual POB-DNA adducts or internal standards were monitored as indicated on each channel.

Figure 4

Figure 4

Plots of total adduct levels (fmol/mg of DNA) vs time (weeks) in (A) oral mucosa, (B) esophageal mucosa, (C) nasal respiratory mucosa, (D) nasal olfactory mucosa, (E) liver, and (F) lung DNA from (R)- and (S)-NNN-treated rats. Values of the total adduct levels are the sum of the amounts of all POB-DNA adducts measured in oral, esophageal, nasal respiratory, nasal olfactory, liver and lung DNA at each time point ± S. D. Symbol designations are ■, total adduct levels from (R)-NNN treatment and □, total adduct levels from (S)-NNN treatment. All differences were statistically significant (P<0.05) except oral mucosa, 70 weeks (P = 0.07); esophageal mucosa, 70 weeks (P = 0.06), and nasal respiratory mucosa, 50 weeks (P = 0.06); see Table 3, Supporting Information.

Figure 5

Figure 5

Levels of each POB-DNA adduct in the (A) oral mucosa of (R)-NNN-treated rats, (B) oral mucosa of (S)-NNN-treated rats, (C) esophageal mucosa of (R)-NNN-treated rats, (D) esophageal mucosa of (S)-NNN-treated rats, (E) nasal respiratory mucosa of (R)-NNN-treated rats, (F) nasal respiratory mucosa of (S)-NNN-treated rats, (G) nasal olfactory mucosa of (R)-NNN-treated rats, (H) nasal olfactory mucosa of (S)-NNN-treated rats, (I) liver of (R)-NNN-treated rats, (J) liver of (S)-NNN-treated rats, (K) lung of (R)-NNN-treated rats, and (L) lung of (S)-NNN-treated rats. Adduct levels are shown in sequence at each time point, as follows: open bars 7-POB-Gua; striped bars _O_2-POB-dThd. All differences between levels of 7-POB-Gua and _O2_-POB-dThd were significant (P<0.05) in panels E-L. The only significant differences in the other panels were in panel A, 10 and 30 weeks, and panel C, 10 weeks. See Table 4, Supporting Information.

Figure 6

Figure 6

Comparison of total POB-DNA adduct levels (fmol/mg DNA) vs time (weeks) in oral mucosa, esophageal mucosa, nasal respiratory mucosa, nasal olfactory mucosa, liver and lung DNA from (A); (R)-NNN-treated rats and (B); (S)-NNN-treated rats. Symbols and colors are as follows: nasal respiratory, red closed square; liver, magenta open square; lung, green triangle; esophageal mucosa, blue circle; oral mucosa, orange diamond; nasal olfactory mucosa, grey cross. Statistical comparisons are summarized in Table 5 of the Supporting Information.

Figure 7

Figure 7

Chromatograms obtained upon analysis of _O_6-POB-dGuo in nasal respiratory mucosa DNA of rats treated for 70 weeks with (S)-NNN, using (A) the TSQ Quantum Vantage triple quadrupole mass spectrometer and (B) the high resolution LTQ Orbitrap Velos mass spectrometer.

Scheme 1

Scheme 1

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