Analysis of O(6)-[4-(3-Pyridyl)-4-oxobut-1-yl]-2'-deoxyguanosine and Other DNA Adducts in Rats Treated with Enantiomeric or Racemic N'-Nitrosonornicotine

Abstract

(S)-N'-Nitrosonornicotine [(S)-NNN] and racemic NNN are powerful oral and esophageal carcinogens in the F344 rat, whereas (R)-NNN has only weak activity. Tumor formation in these tissues of rats treated with racemic NNN was far greater than the sum of the activities of the individual enantiomers. We hypothesized that metabolites of (R)-NNN enhanced levels of DNA adducts produced by (S)-NNN. A test of that hypothesis necessitated the development of a novel liquid chromatography-nanoelectrospray ionization-high resolution tandem mass spectrometry method for the analysis of O(6)-[4-(3-pyridyl)-4-oxobut-1-yl]-2'-deoxyguanosine (O(6)-POB-dGuo), a highly mutagenic DNA adduct not previously quantified in rats treated with NNN. The new method, with a limit of detection of 6.5 amol for diluted standard and 100 amol for DNA samples, was applied in this study. Groups of nine F344 rats were treated with doses as follows: 7 ppm (R)-NNN, 7 ppm (S)-NNN, and 14 ppm racemic NNN; 14 ppm (R)-NNN, 14 ppm (S)-NNN, and 28 ppm racemic NNN; or 28 ppm (R)-NNN, 28 ppm (S)-NNN, and 56 ppm racemic NNN for 5 weeks, and tissues were analyzed for DNA adducts. We found statistically significant, but modest, synergistic enhancement of levels of O(6)-POB-dGuo in the esophagus but not the oral cavity of rats treated with racemic NNN (low and median doses only) compared to the sum of the amounts formed in these tissues of rats treated with (S)-NNN or (R)-NNN. There was no synergy in the formation of other POB-DNA adducts of NNN in oral cavity and esophagus, nor was there any evidence for synergy in nasal respiratory and olfactory epithelium, lung, or liver. Our results provide the first quantitation of O(6)-POB-dGuo in DNA from tissues of rats treated with NNN and evidence for synergy in DNA adduct formation as one possible mechanism by which (R)-NNN enhances the carcinogenicity of (S)-NNN in rats.

Figure 1

Structures of (S)-NNN (1), (R)-NNN (2) and NNK (3)

Figure 2

Structures of the POB-DNA adducts (4 - 9). dR = 2′-deoxyribose.

Figure 3

Method characterization for O⁶-POB-dGuo quantitation by LC-NSI-HRMS/MS. The analyte is O⁶-POB-dGuo and the internal standard is [pyridine-D₄]O⁶-POB-dGuo. (A) Calibration curve for O⁶-POB-dGuo. The amount of [pyridine-D₄]O⁶-POB-dGuo was maintained constant at 7.5 fmol on-column, while the amount of O⁶-POB-dGuo ranged from 6.5 to 422 amol on-column. (B) Relationship between added and measured O⁶-POB-dGuo in 0.3 mg calf thymus DNA. Each data point represents the average of three replicates. The error bar represents the standard deviation.

Figure 4

Extracted ion chromatograms (EIC) for O⁶-POB-dGuo from 7 ppm (R)-NNN-treated rat esophageal mucosa using LC-ESI-MS/MS (A) and LC-NSI-HRMS/MS (C); EIC for O⁶-POB-dGuo from 56 ppm racemic NNN-treated rat esophageal mucosa using LC-ESI-MS/MS (B) and LC-NSI-HRMS/MS (D). For the LC-ESI-MS/MS method, the following ion transitions were monitored: m/z 415 to 148 for O⁶-POB-dGuo, and m/z 419 to 152 for the internal standard [pyridine-D₄]O⁶-POB-dGuo. For the LC-NSI-HRMS/MS method, the following ion transitions were monitored with accurate mass extracted: m/z 415 to 148.0757 and 152.0567 for O⁶-POB-dGuo; m/z 419 to 152.0567 and 152.1008 for [pyridine-D₄]O⁶-POB-dGuo.

Figure 5

Levels of O⁶-POB-dGuo in (A) esophageal mucosa and (B) oral mucosa. Low dosage level: 7 ppm (R)-NNN, 7 ppm (S)-NNN and 14 ppm racemic NNN; Median dosage level: 14 ppm (R)-NNN, 14 ppm (S)-NNN and 28 ppm racemic NNN; High dosage level: 28 ppm (R)-NNN, 28 ppm (S)-NNN and 56 ppm racemic NNN. Open bars represent (R)-NNN treated groups; Grey bars represent (S)-NNN groups; Black bars represent racemic NNN groups; the stacked bars represent the additive sum of the corresponding (R)-NNN and (S)-NNN treated groups. Each column is the average of three replicates and the error bars represent the standard deviations.