Analysis of pyridyloxobutyl DNA adducts in F344 rats chronically treated with (R)- and (S)-N'-nitrosonornicotine

Abstract

NNN (1) is an esophageal carcinogen in rats. 2'-Hydroxylation of NNN is believed to be the major bioactivation pathway for NNN tumorigenicity. (S)-NNN is preferentially metabolized by 2'-hydroxylation in cultured rat esophagus, whereas there is no preference for 2'-hydroxylation versus 5'-hydroxylation in the metabolism of (R)-NNN. 2'-Hydroxylation of NNN generates the reactive intermediate 4-oxo-4-(3-pyridyl)butanediazohydroxide (8), resulting in the formation of pyridyloxobutyl (POB)-DNA adducts. On the basis of these observations, we hypothesized that (S)-NNN treatment would produce higher levels of POB-DNA adducts than that by (R)-NNN in the rat esophagus. We tested this hypothesis by treating male F344 rats with 10 ppm of (R)-NNN or (S)-NNN in drinking water. After 1, 2, 5, 10, 16, or 20 weeks of treatment, POB-DNA adducts in esophageal, liver, and lung DNA were quantified by HPLC-ESI-MS/MS. In the rat esophagus, (S)-NNN treatment generated levels of POB-DNA adducts 3-5 times higher than (R)-NNN treatment, which supports our hypothesis. 7-[4-(3-Pyridyl)-4-oxobut-1-yl]guanine (7-POB-Gua, 14) was the major adduct detected, followed by O2-[4-(3-pyridyl)-4-oxobut-1-yl]thymidine (O2-POB-dThd, 11) and O2-[4-(3-pyridyl)-4-oxobut-1-yl]cytosine (POB-Cyt, 15). O6-[4-(3-Pyridyl)-4-oxobut-1-yl]-2'-deoxyguanosine (O6-POB-dGuo, 10) was not detected. The total POB-DNA adduct levels in the esophagus were 3-11 times higher than those in the liver for (R)-NNN and 2-6 times higher than those for (S)-NNN. In contrast to the esophagus and liver, (R)-NNN treatment produced more POB-DNA adducts than (S)-NNN treatment in the rat lung, which suggested an important role for cytochrome P450 2A3 in NNN metabolism in the rat lung. In both the liver and lung, O2-POB-dThd was the predominant adduct and accumulated during the experiment. The results of this study demonstrate that individual POB-DNA adducts form and persist in the esophagi, livers, and lungs of rats chronically treated with NNN enantiomers and demonstrate that (S)-NNN produces higher levels of POB-DNA adducts in the esophagus than (R)-NNN, suggesting that (S)-NNN is more tumorigenic than (R)-NNN to the rat esophagus.

Figures

Figure 1

Water consumption by (A) control rats; (B) (R)-NNN-treated rats; (C) (S) NNN-treated rats; and (D) growth chart of rats in control (▨), (R)-NNN (■), and (S)-NNN (□) groups. Values are mean ± S.D. of measurements of all rats in each group.

Figure 2

Typical SRM chromatograms obtained upon analysis of esophageal DNA isolated from (A) control rats; (B) (R)-NNN-treated rats; and (C) (S)-NNN-treated rats after 16 weeks of treatment. Individual POB-DNA adducts or internal standards were monitored as indicated on each channel.

Figure 3

Levels of POB-DNA adducts in esophageal DNA isolated from rats treated with (A) (R)-NNN and (B) (S)-NNN. Symbol designations are: ▨, O2-POB-dThd; ■, 7-POB-Gua; □, O2-POB-Cyt. Each value is the mean ± S.D. of single analyses of DNA samples from 3 pools of 3 rats per group.

Figure 4

Plots of total adduct levels (fmol/mg DNA) versus time (weeks) in (A) esophageal, (B) liver and (C) lung DNA from (R)- and (S)-NNN-treated rats. Values of the total adduct levels are the sum of amounts of all POB-DNA adducts measured in esophageal, liver and lung DNA at each time point ± S. D. Symbol designations are: ■, total adduct levels from (R)-NNN treatment; □, total adduct levels from (S)-NNN treatment.

Figure 5

Typical SRM chromatograms obtained upon analysis of liver DNA from (A) control rats; (B) (R)-NNN-treated rats; and (C) (S)-NNN-treated rats after 10 weeks of treatment. Individual POB-DNA adducts or internal standards were monitored as indicated on each channel. In Figure 5A, the peak eluting at the position of O2-POB-dThd, the area of which is 0.2 % of [pyridine-D4]O2 -POB-dThd, originates from the internal standard.

Figure 6

Levels of POB-DNA adducts in liver DNA isolated from rats treated with (A) (R)-NNN and (B) (S)-NNN. Symbol designations are: ▨, O2-POB-dThd; □, 7-POB-Gua; ■, O2-POB-Cyt. Each value is the mean ± S.D. of single analyses of DNA samples from three rats per group.

Figure 7

Typical SRM chromatograms obtained upon analysis of lung DNA from (A) control rats; (B) (R)-NNN-treated rats; and (C) (S)-NNN-treated rats after 16 weeks of treatment. Individual POB-DNA adducts or internal standards were monitored as indicated on each channel. In Figure 7A, the peak eluting at the position of O2-POB-dThd, the area of which is 0.2 % of [pyridine-D4]O2-POB-dThd, originates from the internal standard.

Figure 8

Levels of POB-DNA adducts in lung DNA isolated from rats treated with (A) (R)-NNN and (B) (S)-NNN. Symbol designations are: ▨, O2-POB-dThd; ■, 7-POB-Gua; □, O2-POB-Cyt. Each value is the mean ± S.D. of single analyses of DNA samples from three rats per group.

Scheme 1

Overview of NNN metabolism and DNA adduct formation. NTH: neutral thermal hydrolysis.

Chart 1

Structures of (R)-NNN, (S)-NNN and NNK,

Chart 2

Structures of POB-DNA adducts (–15) and DNA adducts derived from 5′-hydroxylation of NNN ( and 17). dR: 2′-deoxyribosyl.