Tumorigenicity and metabolism of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol enantiomers and metabolites in the A/J mouse

Pramod Upadhyaya, Patrick M.J. Kenney, J. Bradley Hochalter, Mingyao Wang and Stephen S. Hecht1

University of Minnesota Cancer Center, Box 806 Mayo, 420 Delaware Street SE, Minneapolis, MN 55455, USA


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), a major metabolite of the tobacco-specific pulmonary carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), has a chiral center but the tumorigenicity of the NNAL enantiomers has not been previously examined. In this study, we assessed the relative tumorigenic activities in the A/J mouse of NNK, racemic NNAL, (R)-NNAL, (S)-NNAL and several NNAL metabolites, including [4-(methylnitrosamino)-1-(3-pyridyl)but-(S)-1-yl] ß-O-D-gluco-siduronic acid [(S)-NNAL-Gluc], 4-(methylnitrosamino)-1-(3-pyridyl N-oxide)-1-butanol, 5-(3-pyridyl)-2-hydroxytetrahydrofuran, 4-(3-pyridyl)butane-1,4-diol and 2-(3-pyridyl) tetrahydrofuran. We also quantified urinary metabolites of racemic NNAL and its enantiomers and investigated their metabolism with A/J mouse liver and lung microsomes. Groups of female A/J mice were given a single i.p. injection of 20 µmol of each compound and killed 16 weeks later. Based on lung tumor multiplicity, (R)-NNAL (25.6 ± 7.5 lung tumors/mouse) was as tumorigenic as NNK (25.3 ± 9.8) and significantly more tumorigenic than racemic NNAL (12.1 ± 5.6) or (S)-NNAL (8.2 ± 3.3) (P < 0.0001). None of the NNAL metabolites was tumorigenic. The major urinary metabolites of racemic NNAL and the NNAL enantiomers were 4-hydroxy-4-(3-pyridyl)butanoic acid (hydroxy acid), NNAL-N-oxide and NNAL-Gluc, in addition to unchanged NNAL. Treatment with (R)-NNAL or (S)-NNAL gave predominantly (R)-hydroxy acid or (S)-hydroxy acid, respectively, as urinary metabolites. While treatment of mice with racemic or (S)-NNAL resulted in urinary excretion of (S)-NNAL-Gluc, treatment with (R)-NNAL gave both (R)-NNAL-Gluc and (S)-NNAL-Gluc in urine, apparently through the metabolic intermediacy of NNK. (S)-NNAL appeared to be a better substrate for glucuronidation than (R)-NNAL in the A/J mouse. Mouse liver and lung microsomes converted NNAL to products of {alpha}-hydroxylation, to NNAL-N-oxide, to adenosine dinucleotide phosphate adducts and to NNK. In lung microsomes, metabolic activation by {alpha}-hydroxylation of (R)-NNAL was significantly greater than that of (S)-NNAL. The results of this study provide a metabolic basis for the higher tumorigenicity of (R)-NNAL than (S)-NNAL in A/J mouse lung, namely preferential metabolic activation of (R)-NNAL in lung and preferential glucuronidation of (S)-NNAL.

Abbreviations: diol, 4-(3-pyridyl)butane-1,4-diol; hydroxy acid, 4-hydroxy-4-(3-pyridyl)butanoic acid; lactol, 5-(3-pyridyl)-2-hydroxytetrahydrofuran; LC-MS/MS, liquid chromatography–tandem mass spectrometry; MMPB, methyl 4-[{alpha}-methylbenzylcarbamoyl]-4-(3-pyridyl)butanoate; NNAL, 4-(methyl-nitrosamino)-1-(3-pyridyl)-1-butanol; NNAL(ADP)+, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol adenosine dinucleotide phosphate; NNAL-Gluc, [4-(methylnitrosamino)-1-(3-pyridyl)but-(S)-1-yl] ß-O-D-glucosiduronic acid; NNAL-N-oxide, 4-(methylnitrosamino)-1-(3-pyridyl N-oxide)-1-butanol; NNK, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone; NNK-N-oxide, 4-(methylnitrosamino)-1-(3-pyridyl N-oxide)-1-butanone; pyridyl-THF, 5-(3-pyridyl)tetrahydrofuran; UDPGA, uridine 5'-diphosphoglucoronic acid


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) (Figure 1Go)is formed from nicotine during the curing and processing of tobacco (1,2). It is present in the tobacco that is used to manufacture cigarettes and snuff, as well as in cigarette smoke. Smokers and snuff users are exposed to substantial amounts of this tobacco-specific carcinogen, which is hypothesized to play an important role in the etiology of cancers associated with the use of tobacco products (19). NNK is a potent pulmonary carcinogen in rodents (14). Metabolically, it is extensively and rapidly converted to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) in rodents and humans (4). NNAL is also a strong pulmonary carcinogen in mice and rats (4).



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Fig. 1. NNK and NNAL metabolism pathways as determined by studies in laboratory animals and humans (see ref. 4 for details).

 
The metabolism of NNK and NNAL is summarized in Figure 1Go. NNAL is converted to its O-glucuronide, [4-(methynitrosamino)-1-(3-pyridyl)but-1-yl] ß-O-D-glucosiduronic acid (NNAL-Gluc), which has been detected along with NNAL in the urine of smokers, snuff users, people exposed to environmental tobacco smoke and newborns of mothers who smoked (1017). Pyridine N-oxidation of NNAL and NNK leads to 4-(methylnitrosamino)-1-(3-pyridyl N-oxide)-1-butanol (NNAL-N-oxide) and 4-(methylnitrosamino)-1-(3-pyridyl N-oxide)-1-butanone (NNK-N-oxide), respectively (4). NNAL-N-oxide has been detected in the urine of smokers and snuff users (18). NNAL and NNK also form adducts with ADP in vitro (19,20). The metabolic activation of NNAL and NNK proceeds by hydroxylation of the carbons {alpha} to the N-nitroso group producing intermediates that bind to DNA and protein (4). {alpha}-Hydroxylation of the NNK methyl group produces 4-(3-pyridyl)-4-oxobutane-1-diazohydroxide (6) which pyridyloxobutylates DNA. {alpha}-Hydroxylation at the methylene group gives methanediazohydroxide (7) which ultimately methylates DNA. Similarly, pyridylhydroxybutylating and methylating intermediates are formed upon {alpha}-hydroxylation of NNAL. Analysis of DNA isolated from tissues of rats treated with NNAL demonstrated the presence of pyridyloxobutylated and methylated DNA, suggesting that reconversion of NNAL to NNK may have been involved in DNA adduct formation by NNAL (21).

A chiral center is introduced into the NNAL molecule when it is formed from NNK (Figure 2Go). Previously, we have determined the absolute configuration of the NNAL enantiomers (22). However, no information is available on their relative tumorigenic activities or metabolism. Therefore, in this study, we evaluated the tumorigenicity of (R)- and (S)-NNAL in the A/J mouse, a model that has been used extensively for tumorigenicity studies of NNK and its metabolites (4). The tumorigenic activities of other products formed in the NNAL metabolic cascade were also examined in this study. We tested (S)-NNAL-Gluc, NNAL-N-oxide, 5-(3-pyridyl)-2-hydroxytetrahydrofuran (lactol), 4-(3-pyridyl)butane-1,4-diol (diol) and 2-(3-pyridyl)tetrahydrofuran (pyridyl-THF) (Figure 1Go). We also investigated the in vitro metabolism of (R)- and (S)-NNAL by A/J mouse liver and lung microsomes and the urinary metabolites of these compounds as well as those of (S)-NNAL-Gluc. The results provide some new insights on metabolic activation and detoxification pathways of NNAL and NNK.



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Fig. 2. Structures of (S)- and (R)-NNAL, (S)- and (R)-hydroxy acid and (S,S)- and (R,S)-MMPB.

 

    Materials and methods
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 Materials and methods
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Chemicals
[5-3H]NNK (3.05 Ci/mmol) was purchased from Chemsyn Science Laboratories (Lenexa, KS) and converted to racemic [5-3H]NNAL by NaBH4 reduction, as described (23). It was purified by reverse phase HPLC system 2. The radiochemical purity was 99%, with no contamination by [5-3H]NNK. NNK (24), racemic NNAL (23), (R)-NNAL and (S)-NNAL (22) were prepared as described; [5-3H](R)-NNAL and [5-3H](S)-NNAL were prepared in the same way and their purities established by HPLC radioflow analysis, as for racemic [5-3H]NNAL. The enantiomeric purities of (R)- and (S)-NNAL were determined by derivatization with Mosher's acid chloride and analysis by HPLC as described (22). There was no detectable cross-contamination of the enantiomers (<0.5%). NNAL-N-oxide (25), lactol (26), diol (23), pyridyl-THF (27), 4-hydroxy-4-(3-pyridyl)butanoic acid (hydroxy acid) (28) and methyl 4(S)- and 4(R)-[(S)-{alpha}-methylbenzylcarbamoyl]-4-(3-pyridyl)butanoate [(S,S)- and (R,S)-MMPB] (29) were synthesized. (S)-NNAL-Gluc was prepared by incubation of NNAL with rat liver microsomes, uridine 5'-diphosphoglucoronic acid (UDPGA) and cofactors as described, except that incubations were carried out overnight (20). (S)-NNAL-Gluc was purified by HPLC system 1. Its purity (>99%) was determined by reinjection on the same system, with photodiode array detection. Hydrolysis of (S)-NNAL-Gluc with ß-glucuronidase gave (S)-NNAL (>99%), as determined by HPLC and, after silylation, by gas chromatography–nitrosamine selective detection. [5-3H](R)-NNAL-Gluc was similarly prepared by incubation of [5-3H]NNAL with patas monkey liver microsomes, UDPGA and cofactors.

All biochemical reagents were purchased from Sigma Chemical Co. (St Louis, MO).

Bioassay and collection of urine
Female A/J mice, aged 5 weeks, were obtained from Jackson Laboratories (Bar Harbor, ME). They were maintained in a Specific Pathogen Free area of the animal facilities. The rooms were temperature controlled (20–22°C) with a 12 h light/dark cycle. The mice were housed in groups of five in plastic 7x11 inch microisolator cages with corn cob bedding (Bed-O'-Cobs; Anderson's Cob Division, Malumee, OH). Water was provided ad libitum. They were maintained on AIN-93G diet until aged 15 weeks, then switched to AIN-93M diet. Food was dispensed from metal feeders (model 30604; Lab Products, Maywood, NJ). At age 7 weeks they were given a single i.p. injection of 20 µmol of the appropriate compound in 0.2 ml saline. Treatment groups are summarized in Table IGo. The (S)-NNAL-Gluc group had 14 mice, due to a limitation in the amount of compound available; all other groups started with 20 mice. The mice were killed 16 weeks after the injections and lung tumors were counted. Statistical comparisons were carried out by one way ANOVA and by {chi}2 analysis.


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Table I. Lung tumorigenicity of NNAL enantiomers and metabolites in A/J micea
 
The mice in groups 2–5 were switched to metabolism cages following the i.p. injections. They were housed five per cage. Urine was collected at 24 h intervals for 72 h. The urine was cooled to 0°C during collection. When collection was complete, the mice were returned to their standard cages.

Analysis of urine
Aliquots of urine (0.5 ml) were centrifuged at 4000 r.p.m. for 10 min. The pH of the supernatant was adjusted to 2.0 and it was extracted with ethyl acetate. The ethyl acetate extracts were discarded. The pH was then readjusted to 7.0 and the resulting mixture was filtered through a 0.45 µmx3 mm Acrodisc (Gelman Sciences, Ann Arbor, MI) and 50 µl aliquots were analyzed for NNAL-N-oxide, NNAL-Gluc and NNAL by HPLC system 2 with UV detection (254 nm). Quantitation of metabolites was based on standard curves. Hydroxy acid and its enantiomers were determined by conversion to (S,S)- and (R,S)-MMPB followed by liquid chromatography–tandem mass spectrometry (LC-MS/MS) as described (30).

In vitro metabolism
Liver and lung microsomes were prepared by the methods of Guengerich (31) and Flammang et al (32). NNAL metabolism was carried out essentially as described previously (20). Reaction mixtures contained 1 µM [5-3H]NNAL, 3 mM MgCl2, 5 mM glucose 6-phosphate, 5 mM NADP+, 3.8 U glucose 6-phosphate dehydrogenase, 0.5 (liver) or 0.3 mg/ml (lung) protein in a total volume of 0.5 ml 100 mM potassium phosphate buffer (pH 7.0). Reactions were carried out at 37°C and were terminated by placing the mixture in an ice bath and adding 0.2 vol each of 0.3 N ZnSO4 and 0.3 N Ba(OH)2. Precipitated protein was pelleted by centrifugation. The mixture was filtered through an Acrodisc and analyzed with HPLC system 3.

HPLC analysis
HPLC was carried out with a Waters Associates system equipped with a Model 440 UV detector, a ß-RAM radioflow detector (IN/US Systems, Fairfield, NJ) and a 3.9x300 mm Phenomenex Bondclone C18 10 µm column (Phenomenex, Torrance, CA). Elution systems were as follows: (1) a linear 60 min gradient of 0–30% MeOH in 20 mM sodium phosphate buffer (pH 7.0) at 1 ml/min; (2) 10 min of 20 mM sodium phosphate buffer, then the same as system 1, followed by a wash with 100% MeOH; (3) a gradient of 0–8% solvent B in solvent A for 15 min, a hold at 8% B for 15 min, 8–35% B over 55 min, 35–50% B over 5 min, then a wash with 50% B. Solvent A was 20 mM sodium phosphate buffer (pH 7.0) and solvent B was 95:5 MeOH:H2O


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The results of the tumorigenicity assay are presented in Table IGo. There were no significant effects of any treatment on body weight or survival. Three mice died during the experiment; two in the NNK group and one in the (S)-NNAL group. Lung tumors had the same appearance as in previous studies which have shown that the adenomas observed at 16 weeks, as in this experiment, later progress to adenocarcinomas (4). Based on lung tumor multiplicity, (R)-NNAL was as tumorigenic as NNK and was significantly more tumorigenic than either racemic NNAL or (S)-NNAL (P < 0.0001). None of the NNAL metabolites was tumorigenic.

Urinary metabolites of racemic NNAL, (R)-NNAL and (S)-NNAL were quantified to determine whether there were major differences in extents of {alpha}-hydroxylation, pyridine N-oxidation, glucuronidation or reconversion to NNK, which would all influence NNAL tumorigenicity. Metabolites of (S)-NNAL-Gluc were analyzed to assess its uptake and conversion to NNAL. Urine was collected at 0–24, 24–48 and 48–72 h. Metabolite peaks were observed by UV mainly in the 0–24 h urine samples; only these were quantified. NNAL-N-oxide, NNAL-Gluc and NNAL were identified by comparison of their retention times with those of standards and, in the case of the NNAL-Gluc diastereomers, by conversion to NNAL with ß-glucuronidase. These metabolites were quantified by HPLC UV. Hydroxy acid was converted to [S,S]- and [R,S]-MMPB (Figure 2Go), which were identified and quantified by LC-MS/MS. The results of these studies are summarized in Table IIGo.


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Table II. Urinary metabolites of racemic NNAL, NNAL enantiomers and (S)-NNAL-Gluca
 
There was no difference in total urinary hydroxy acid, the major product of NNAL {alpha}-hydroxylation, among the groups treated with racemic and enantiomeric NNAL. The enantiomeric composition of hydroxy acid was: 91 ± 0.3% (R), 9.4 ± 0.3% (S) in mice treated with (R)-NNAL; 13 ± 0.8% (R), 87 ± 0.8% (S) in mice treated with (S)-NNAL; 59 ± 2.4% (R), 41 ± 2.4% (S) in mice treated with racemic NNAL (Figure 3Go).



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Fig. 3. LC-MS/MS analysis of hydroxy acid enantiomers in the urine of A/J mice treated with (A) racemic NNAL, (B) (R)-NNAL and (C) (S)-NNAL. Diastereomers of MMPB were produced by converting urinary hydroxy acid to its methyl ester, followed by reaction with (S)-{alpha}-methylbenzyl isocyanate. (S,S)-MMPB is formed from (S)-hydroxy acid and (R,S)-MMPB from (R)-hydroxy acid. The peaks elute at slightly different retention times in each run, as determined by comparison with deuterium labeled internal standards (30).

 
(S)-NNAL-Gluc comprised 16 and 29% of the dose in the mice treated with racemic NNAL or (S)-NNAL, respectively, and 4.6% of the dose in the mice treated with (R)-NNAL. (R)-NNAL-Gluc was not detected in the urine of the mice treated with racemic or (S)-NNAL and comprised 3.0% of the dose in the (R)-NNAL-treated mice.

In mice treated with (S)-NNAL-Gluc, there was little evidence of conversion to NNAL based on recovered urinary metabolites. (S)-NNAL-Gluc in urine accounted for 50% of the dose. Only small amounts of hydroxy acid, NNAL-N-oxide and NNAL were observed.

The microsomal metabolism of racemic and enantiomeric NNAL was investigated to provide further information potentially relevant to their tumorigenic activities. The results of these studies are summarized in Table IIIGo. HPLC traces were similar to those previously illustrated (20). In liver, NNK was the predominant metabolite of racemic, (R)- and (S)-NNAL. For the latter two, its rate of formation was significantly greater than that of all other metabolites (P < 0.01). Lactol, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol adenosine dinucleotide phosphate [NNAL(ADP)+] and NNAL-N-oxide were the other major metabolites. In lung, NNAL-N-oxide was the predominant metabolite of all three compounds and its rate of formation was significantly greater (P < 0.01) than those of all other metabolites in the case of racemic and (R)-NNAL.


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Table III. A/J mouse liver and lung microsomal metabolism of racemic NNAL and NNAL enantiomersa
 
The most striking difference in metabolism of the NNAL enantiomers was observed in lung. (R)-NNAL was clearly a better substrate for oxidative metabolism than was (S)-NNAL. Thus, rates of formation of NNAL-N-oxide, lactol, diol, NNK and pyridyl-THF were all significantly greater (P < 0.01) from (R)-NNAL than from (S)-NNAL. This was also observed in liver for diol and lactol (P < 0.01), but not for the other metabolites.


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The results of the bioassay in A/J mice clearly demonstrate that (R)-NNAL is a more effective lung tumorigen than either (S)-NNAL or racemic NNAL. The tumorigenicity of (R)-NNAL in this model is equivalent to that of NNK. These results are important in furthering our understanding of NNK tumorigenesis. In rodents and humans, NNK is rapidly and extensively converted to NNAL (4). In CD-1 mice, the half-life of NNK is only 12 min, while that of NNAL is 29 min, suggesting that NNAL plays a significant role in the tumorigenicity of NNK (33). Both compounds alkylate DNA upon metabolic activation and therefore both can potentially contribute to tumor induction by NNK (4). Although the enantiomeric composition of metabolically formed NNAL is not yet known, the equivalent tumorigenic activities of (R)-NNAL and NNK, together with the available pharmacokinetic data, suggest that (R)-NNAL is important in NNK tumorigenesis. The preferential metabolic activation of (R)-NNAL in mouse lung is consistent with this hypothesis.

Three previous studies have shown that NNK is a stronger pulmonary tumorigen than racemic NNAL in the A/J mouse (3436). One study used the same single dose protocol as employed here, except that the doses were lower (35). In that bioassay, single doses of 10 µmol of racemic NNAL or NNK produced 3.2 and 7.3 lung tumors/mouse, respectively. Based on those results, we chose a single dose of 20 µmol for the present study because we wanted to be certain that an adequate number of tumors would be produced to assess the relative activities of the NNAL enantiomers and metabolites, some of which were likely to be less tumorigenic than racemic NNAL. Although tumor multiplicity in this study was somewhat higher than expected based on the previous results, the relative activities of racemic NNAL and NNK were about the same as seen earlier.

NNAL-Gluc has been considered as a detoxification product of NNK (4). The lack of tumorigenicity of (S)-NNAL-Gluc, observed for the first time in this study, supports this view. We did not test (R)-NNAL-Gluc because sufficient material was not available. NNAL-Gluc and free NNAL have both been detected in human urine and the ratio of these metabolites is highly variable among individuals (1017). We have suggested that the NNAL-Gluc/NNAL ratio may be an indicator of lung cancer risk (11,12).

NNAL-N-oxide is another NNAL metabolite found in human urine, although its levels are relatively low (18). Based on the lower tumorigenicity of NNK-N-oxide compared with NNK (35), it was reasonable to expect that NNAL-N-oxide would be less tumorigenic than NNAL, but this had not been previously demonstrated. The other three NNAL metabolites tested here (lactol, diol and pyridyl-THF) are end products of NNAL {alpha}-hydroxylation and were not expected to be tumorigenic, but had not been tested. Lactol was of particular interest because it is also a major metabolite of N'-nitrosonornicotine and is expected to react with deoxyguanosine (4,37). These metabolites had no tumorigenic activity.

This is the first study to analyze urinary metabolites of NNAL administered to laboratory animals. The detection of hydroxy acid, NNAL-Gluc, NNAL-N-oxide and NNAL in urine is consistent with expectations based on previous studies of urinary NNK metabolites in A/J mice, although administration of NNK also produced significant amounts of urinary 4-oxo-4-(3-pyridyl)butanoic acid, which was not observed here (38).

The relative proportions of urinary NNAL-Gluc diastereomers after administration of enantiomeric and racemic NNAL were of interest. (S)-NNAL-Gluc was a major urinary metabolite of both (S)-NNAL and racemic NNAL, with no (R)-NNAL-Gluc being detected in either of these experiments. However, administration of (R)-NNAL produced both (R)-NNAL-Gluc and (S)-NNAL-Gluc in urine. Therefore, some of the (R)-NNAL must be metabolically converted to NNK, which is then reduced to both enantiomers of NNAL. (S)-NNAL is apparently a better substrate for mouse UDP-glucuronosyl transferases than is (R)-NNAL and the (S)-NNAL which is produced by reduction of metabolically formed NNK is rapidly glucuronidated. In this experiment, some urinary (R)-NNAL-Gluc was also observed, presumably because (R)-NNAL was originally present in great excess. When racemic NNAL was administered, only (S)-NNAL-Gluc was observed in urine, probably because the two enantiomers were originally present in the same amounts and (S)-NNAL is a better substrate for glucuronidation. Collectively, these results indicate that (S)-NNAL is an excellent substrate for detoxification via glucuronidation in the A/J mouse, while (R)-NNAL is less effectively glucuronidated.

The results of the analyses of hydroxy acid enantiomers in urine support our assignment of the absolute configuration of (R)- and (S)-hydroxy acid. These assignments were previously made based upon in vitro metabolism of (S)-NNAL to (S)-hydroxy acid and by considering the HPLC retention times of the diastereomers formed by reaction of NNAL or hydroxy acid with {alpha}-methylbenzyl isocyanate (29). Detection in urine of predominantly (R)-hydroxy acid in mice treated with (R)-NNAL and (S)-hydroxy acid in mice treated with (S)-NNAL is consistent with our previous assignment. In each case, ~10% of the opposite enantiomer was observed, suggesting that the overall extent of metabolic conversion of NNAL to NNK is ~20% in A/J mice. In rats, in vivo conversion of NNAL to NNK was estimated at ~10–20% (21,39). The conversion of NNAL to NNK is also consistent with the detection of (S)-NNAL-Gluc in the urine of mice treated with (R)-NNAL, as discussed above. Metabolic oxidation of NNAL to NNK could play a role in tumor induction by NNAL. Previously, we observed pyridyloxobutylation of DNA in rats treated with NNAL, indicating that reconversion to NNK was involved in DNA adduct formation (21).

In the mice treated with (S)-NNAL-Gluc, 50% of this material was detected unchanged in the urine. Small amounts of hydroxy acid, NNAL-N-oxide and NNAL were also detected in the urine of these mice, consistent with a previous study which examined the metabolism of (S)-NNAL-Gluc in rats (40). These results suggest that some (S)-NNAL-Gluc is reconverted to NNAL by intestinal ß-glucuronidase following uptake and excretion into bile. However, the small amount of NNAL released in this way is insufficient for tumor induction.

The results of the in vitro studies are consistent with a previous study of NNAL microsomal metabolism in rats (20). The same metabolites were observed in liver and lung, and their rates of formation were generally similar to those in the rat. However, that study examined only racemic NNAL. Here, we have shown that the metabolic activation of (R)-NNAL in the A/J mouse lung is significantly faster than that of (S)-NNAL, based on the relative rates of formation of lactol, diol and pyridyl-THF. These results suggest that DNA alkylation by (R)-NNAL in lung would exceed that of (S)-NNAL, consistent with the tumorigenicity results, as mentioned above. This will be examined in future studies. However, it should be noted that the rate of formation of NNAL-N-oxide, a detoxification product, was also significantly greater from (R)-NNAL than from (S)-NNAL in lung. Metabolic activation of (R)-NNAL was also greater than that of (S)-NNAL in liver, but the differences were smaller. The preferential metabolic activation of (R)-NNAL together with the preferential glucuronidation of (S)-NNAL appear to provide a rationale for the higher tumorigenicity of (R)-NNAL than (S)-NNAL in the A/J mouse.


    Acknowledgments
 
We thank Chap Le for his assistance with the statistical analyses and David Schlagel for assistance in the preparation of (S)-NNAL-Gluc. This study was supported by grants CA-44377 and CA-81301 from the National Cancer Institute.


    Notes
 
1 To whom correspondence should be addressed Email: hecht002{at}tc.umn.edu Back


    References
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 Abstract
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 Materials and methods
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 Discussion
 References
 

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Received December 23, 1998; revised March 12, 1999; accepted March 30, 1999.