Analysis of pyridyloxobutyl and pyridylhydroxybutyl DNA adducts in extrahepatic tissues of F344 rats treated chronically with 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and enantiomers of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol

Abstract

The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and its metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) are potent pulmonary carcinogens in rats. NNK and NNAL require metabolic activation to express their carcinogenicity. Cytochrome P450-catalyzed alpha-hydroxylation at the methyl position of NNK or NNAL generates reactive intermediates, which alkylate DNA to form pyridyloxobutyl (POB)-DNA adducts or pyridylhydroxybutyl (PHB)-DNA adducts. NNK is metabolized to NNAL in a reversible and stereoselective manner, and the tissue-specific retention of (S)-NNAL is believed to be important to the carcinogenicity of NNK. In the present study, we investigated the formation of POB- and PHB-DNA adducts in extrahepatic tissues of F344 rats treated chronically with NNK and (R)- and (S)-NNAL (10 ppm in the drinking water, 1-20 weeks). POB- and PHB-DNA adducts were quantified in nasal olfactory mucosa, nasal respiratory mucosa, oral mucosa, and pancreas of treated rats. Adduct formation in the nasal respiratory mucosa exceeded that in the other tissues. O(2)-[4-(3-Pyridyl)-4-oxobut-1-yl]thymidine (O(2)-POB-dThd) or O(2)-[4-(3-pyridyl)-4-hydroxybut-1-yl]thymidine (O(2)-PHB-dThd) was the major adduct, followed by 7-[4-(3-pyridyl)-4-oxobut-1-yl]guanine (7-POB-Gua) or 7-[4-(3-pyridyl)-4-hydroxybut-1-yl]guanine (7-PHB-Gua). There was a remarkable similarity in adduct formation between the NNK and the (S)-NNAL groups, both of which were distinctively different from that in the (R)-NNAL group. For example, in the nasal olfactory mucosa, POB-DNA adduct levels in the NNK and (S)-NNAL groups were not significantly different from each other, while (R)-NNAL treatment generated 6-33 times lower amounts of POB-DNA adducts than did NNK treatment. In contrast, (R)-NNAL treatment produced significantly higher levels of PHB-DNA adducts than did NNK or (S)-NNAL treatment. Similar trends were observed in the nasal respiratory mucosa, oral mucosa, and pancreas. These results suggest extensive retention of (S)-NNAL in various tissues of NNK-treated rats and support a mechanism in which the preferential metabolism of NNK to (S)-NNAL, followed by sequestration of (S)-NNAL in the target tissues and reoxidation to NNK, is important to NNK tumorigenesis.

Figures

Figure 1

LC-ESI-MS/MS chromatograms obtained upon the analyses of (A) POB-DNA adducts; and (B) PHB-DNA adducts in nasal respiratory mucosa DNA of rats treated with NNK for 16 weeks. Individual POB- and PHB-DNA adducts and their internal standards were monitored as indicated on each channel. SRM transitions are summarized in Table 1.

Figure 2

Total POB-DNA adduct levels (fmol/mg DNA) ± SD vs time (weeks) in the (A) nasal olfactory mucosa; (B) nasal respiratory mucosa; (C) oral mucosa; and (D) pancreas of NNK-, (R)-NNAL-, and (S)-NNAL-treated rats. Symbol designations are: ◆, NNK; ■, (R)-NNAL; and □, (S)-NNAL.

Figure 3

Levels of each POB-DNA adduct in the (A) nasal olfactory mucosa; (B) nasal respiratory mucosa; (C) oral mucosa; and (D) pancreas of NNK treated rats. Adduct levels are shown in sequence at each time point, as follows: □, O2-POB-Cyt; ■, 7-POB-Gua; , O2-POB-dThd; , O6-POB-dGuo.

Figure 4

Total PHB-DNA adduct levels (fmol/mg DNA) ± SD vs time (weeks) in the (A) nasal olfactory mucosa; (B) nasal respiratory mucosa; (C) oral mucosa; and (D) pancreas of NNK-, (R)-NNAL-, and (S)-NNAL-treated rats. Symbol designations are: ◆, NNK; ■, (R)-NNAL; and □, (S)-NNAL.

Figure 5

Levels of each PHB-DNA adduct in the (A) nasal olfactory mucosa; (B) nasal respiratory mucosa; (C) oral mucosa; and (D) pancreas of (R)-NNAL treated rats. Adduct levels are shown in sequence at each time point, as follows: ■, 7-POB-Gua; , O2-POB-dThd; , O6-POB-dGuo.

Figure 6

Comparison of adduct levels in the liver, lung, nasal olfactory mucosa, nasal respiratory mucosa, oral mucosa, and pancreas of treated rats: (A) total POB-DNA adduct levels (fmol/mg DNA) vs time (weeks) in NNK-treated rats; (B) total PHB-DNA adduct levels (fmol/mg DNA) vs time (weeks) in (R)-NNAL-treated rats. Levels of POB- and PHB-DNA adducts in the liver and lung were measured previously (24, 25). Symbol designations are: ◆, liver; ■, lung; ▲, nasal olfactory mucosa; X, nasal respiratory mucosa; □, oral mucosa; and ● pancreas.

Scheme 1

Overview of NNK and NNAL metabolism and DNA adduct formation.

Scheme 2

Proposed mechanisms for the interconversion of NNK and NNAL, and their DNA adduct formation in rats.

Chart 1

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

Chart 2

Structures of POB- and PHB-DNA adducts.