Stereoselective metabolism and tissue retention in rats of the individual enantiomers of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), metabolites of the tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)

Cheryl L. Zimmerman1, Zheng Wu, Pramod Upadhyaya and Stephen S. Hecht

College of Pharmacy and Cancer Center, University of Minnesota, Minneapolis, MN 55455, USA

1 To whom correspondence should be addressed Email: zimme005{at}umn.edu


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is reduced to its main metabolite, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) in a reaction that is both stereoselective and reversible. (S)-NNAL has been shown to be equivalent to NNK in carcinogenic potency, and significantly more potent than (R)-NNAL. It was hypothesized that stereoselective differences in metabolism or tissue distribution contributed to the difference in carcinogenicity between the enantiomers. The individual NNAL enantiomers were therefore administered to bile duct-cannulated rats. Male Fisher F344 rats received i.v. doses of either (R)-NNAL (n = 10) or (S)-NNAL (n = 9) and bile, urine, blood and tissue samples were collected over 24 h. (R)/(S)-NNAL and metabolites were quantified by HPLC and radioflow detection. NNAL was also collected from the HPLC and silylated, and the two NNAL enantiomers were separated by chiral GC-TEA. (S)-NNAL had a much larger tissue distribution (Vss = 1792 ± 570 ml) than did (R)-NNAL (Vss = 645 ± 230 ml). Overall, (R)-NNAL tended to enter detoxification pathways, particularly glucuronidation, while reversible metabolism of (S)-NNAL to NNK was favored. For example, after (R)-NNAL administration, ~50% of the dose was excreted as (R)-NNAL-Gluc in bile and urine, and <5% was excreted as NNK or NNK metabolites. In contrast, only 10% of an (S)-NNAL dose was excreted as a glucuronide, while almost 20% of the (S)-NNAL dose was excreted as NNK or NNK metabolites. In tissues, particularly the lung, (S)-NNAL appeared to be stereoselectively retained. These findings suggest that the difference in carcinogenicity between the NNAL enantiomers may be attributed to stereoselective differences in tissue distribution and excretion.

Abbreviations: hydroxy acid, 4-hydroxy-4-(3-pyridyl)butyric acid; keto acid, 4-oxo-4-(3-pyridyl)butyric acid; NNAL, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol; NNAL-Gluc, [4-(methylnitrosamino)-1-(3-pyridyl)but-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


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is the most carcinogenic of the tobacco-specific nitrosamines (1). These compounds are formed from nicotine and related tobacco alkaloids during processing of tobacco leaves. The carcinogenicity of NNK in rodents is remarkably lung-specific and independent of the route of NNK administration (1,2). The mechanism for the lung specificity of NNK is complex and not completely understood (2,3). However, it has been shown that one of the metabolites of NNK, (S)-4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol [(S)-NNAL], is stereoselectively retained in the rat lung after administration of either NNK or racemic NNAL (4).

NNK, a pro-chiral compound, is reduced to its main metabolite, NNAL, in a reversible reaction (Figure 1). NNAL has a chiral center and there are remarkable differences in metabolism, pharmacokinetics and carcinogenic activities between the two enantiomers (5). In the A/J mouse, (S)-NNAL was shown to be as potent as NNK in causing lung tumors; (R)-NNAL was considerably less potent (5). It is possible that the difference in carcinogenicity between NNAL enantiomers may be due to stereoselective differences in metabolism or tissue distribution. The objective of the present investigation was to evaluate the metabolism of the individual NNAL enantiomers as well as to investigate their differential tissue exposure and retention.



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Fig. 1. NNK and NNAL metabolic pathways.

 

    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Chemicals
[5-3H]NNAL enantiomers were synthesized and purified as reported previously (6). ß-Glucuronidase, type IXA and pentobarbital were purchased from Sigma Chemical (St Louis, MO). Picofluor-40 was purchased from Packard Instruments (Meridan, CT). Bis-trimethylsilyltrifluoroacetamide containing 1% trimethylchlorosilane was purchased from Pierce (Rockford, IL). All other chemicals were of analytical grade.

Animal treatment and dosing
Male Fisher 344 rats [281 ± 16 g, n = 10 for the (R)-NNAL treatment group and 293 ± 22 g, n = 9 for the (S)-NNAL treatment group] were obtained from Harlan (Indianapolis, IN). They were housed two per cage under standard conditions (20 ± 2°C, 12 h light/dark cycle), and were given food and water ad libitum.

The study of individual enantiomers was carried out in three separate groups. In brief, the first group of rats was used to investigate the general pharmacokinetics and biliary and urinary excretion of the compounds, while the second and third groups were used to investigate their tissue distribution. In the first group, rats were anesthetized with pentobarbital (50 mg/kg, i.p.), and the right femoral artery, vein and bile duct were cannulated as described previously (4). The rats were placed in metabolic cages and allowed to recover overnight (at least 12 h) before the experiment.

Rats in the first group were given [5-3H](R)-NNAL or [5-3H](S)-NNAL at a dose of 0.65 ± 0.067 mg/kg for (R)-NNAL (n = 4) and 1.07 ± 0.12 mg/kg for (S)-NNAL (n = 4), each with a specific activity of ~560 µCi/mg. The dosing solution was prepared in 0.9% NaCl. The dose was given through the femoral vein as a bolus of ~200 µl. Serial blood, urine and bile samples were collected up to 24 h. The blood samples (200 µl) were collected through the arterial cannula into microcentrifuge tubes, which were immediately centrifuged at 10 000 g for 5 min; the resultant plasma samples were placed on ice, then frozen at –20°C. Urine and bile were collected at intervals over 24 h. A 10 µl aliquot of each sample was removed for liquid scintillation counting (LSC), while the rest was stored at –20°C until analysis. At 24 h, the rats were killed by an overdose of pentobarbital, and lung, liver and kidneys were collected. Lung, liver and kidney tissues were rinsed in 0.9% NaCl solution, blotted dry and weighed, then frozen.

Tissue distribution and metabolic profiles of the NNAL enantiomers were investigated in two additional groups of animals. On the day before dosing, these animals were cannulated in the femoral vein for administration of either tritiated [5-3H](S)- or (R)-NNAL. The rats were allowed to recover overnight (at least 12 h) before the experiment. Rats were killed at either 1 or 4 h after administration of (R)- or (S)-NNAL. The dose for the (R)-NNAL treatment groups was 0.11 ± 0.03 mg/kg (n = 6); the dose for the (S)-NNAL treatment groups was 0.198 ± 0.051 mg/kg (n = 5). Because of shortage of compound, only two animals were dosed for the (S)-NNAL group that was to be killed at 4 h post-dose. The liver, lungs and kidneys were harvested, and handled as described above.

Quantification of NNAL enantiomers and metabolites
The tissues (~1 g for the lung, 2 g each for the liver and kidney) were prepared for analysis as described previously (4) except that tissue solubilization for LSC was carried out with the use of hyamine hydroxide (Sigma). Plasma samples were prepared for HPLC by filtration through Amicon Centrifree Micropartition Filters (Amicon, Beverly, MA). Urine and bile samples were spiked with non-radiolabeled standards and injected directly onto the HPLC. The volume of injection for plasma samples was typically 100 µl; the volumes of injection for bile and urine samples were typically 50–100 µl.

NNAL and its metabolites were separated and quantified by reversed-phase HPLC with radioflow detection as described previously (4). The identities of the glucuronide metabolites were confirmed by treating the samples with glucuronidase and analyzing the amount of NNAL released. Since (R)- and (S)-NNAL could not be separated by HPLC, racemic NNAL from various samples was collected, then silylated and analyzed by chiral GC-TEA as described previously (4).

Pharmacokinetic analysis
The plasma concentration versus time data for NNAL enantiomers were analyzed by non-compartmental analysis (7). The slope of the terminal phase of the urinary excretion rate versus time curve was determined by fitting a mono-exponential function to the log urinary excretion rate versus time data. A least-squares linear regression approach was used (Microsoft ExcelTM, PC version 97, Microsoft, Redmond, WA). The terminal rate constant, {lambda}, was determined from the slope. The area under the plasma concentration–time curve was calculated with the linear trapezoidal rule up to the last measured concentration, with integration of the remaining area to infinity (7). The urinary elimination half-life (t1/2) was calculated as 0.693/{lambda}. The total body clearance (CL), steady state volume of distribution (VSS), renal clearance (CLr) and biliary clearance (CLb) were calculated as described previously (4).

Differences in the parameters were assessed by analysis of variance (ANOVA) or paired Student's t-tests (Sigma StatTM v. 3.06, Jendal Scientific, San Rafael, CA), with P < 0.05 considered to be significant.


    Results
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 Abstract
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 Materials and methods
 Results
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 References
 
The pharmacokinetics of the individual enantiomers of NNAL were studied in chronically bile duct-cannulated rats. After 24 h, roughly 65% of a total dose of (S)-NNAL (14.9 ± 3.0% from bile and 48.0 ± 5.1% from urine) was recovered, while almost 90% of the total dose (43.6 ± 7.0% from bile and 44.6 ± 12.0% from urine) was recovered after (R)-NNAL administration (Table I). The difference in the overall excretion of the enantiomer is accounted for by a much lower percentage of (S)-NNAL being excreted in the bile (Figure 2). The lower percent of the dose appearing in bile after a dose of (S)-NNAL is accounted for by the difference in biliary elimination of (R)-NNAL-Gluc. (R)-NNAL-Gluc made up 39.5 ± 7.3% of the total dose following (R)-NNAL administration but only 9.1 ± 2.1% of the total dose following (S)-NNAL administration (Figure 3). Even after a dose of (S)-NNAL, little (S)-NNAL-Gluc appeared in the bile (0.60 ± 0.17% of the dose), indicating that (S)-NNAL was a poor substrate for glucuronidation, a detoxification pathway. This was an initial indication of the relative retention of (S)-NNAL.


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Table I. Summary of pharmacokinetic parameters after i.v. doses of either (S)-NNAL or (R)-NNALa

 


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Fig. 2. Percent of the total dose excreted as a function of time in bile after administration of (R)-NNAL (open circle, n = 3) or (S)-NNAL (filled circle, n = 4).

 


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Fig. 3. Biliary metabolic profile after administration of (R)-NNAL (open bars, n = 3) or (S)-NNAL (closed bars, n = 4). Each bar represents the percentage of total dose.

 
(R)-NNAL had a large volume of distribution (645 ± 230 ml) and a total body clearance of 2.7 ± 1.1 ml/min (Table I). (S)-NNAL had a total body clearance of 4.0 ± 1.2 ml/min, but its large volume of distribution (1792 ± 570 ml) was significantly greater than that of (R)-NNAL (<0.05). Biliary and renal clearances for the unmetabolized enantiomers were small proportions of their respective total body clearances.

The plasma levels of NNAL and metabolites reached the limit of quantification at short times after administration of either enantiomer (8), so the terminal portion of the urinary excretion rate versus time profile was used for estimation of the elimination half-lives of NNAL enantiomers and metabolites (Table II). Except for the half-life of (R)-NNAL-Gluc after administration of (S)-NNAL, the apparent terminal half-lives of the metabolites were not significantly different from the NNAL enantiomer from which they arose, indicating formation rate-limited elimination. However, in each case the metabolites' half-lives after (R)-NNAL were significantly shorter than after (S)-NNAL (P < 0.05). This was a second indication of the relative retention of (S)-NNAL.


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Table II. Urinary elimination half-lives (h) of NNAL and metabolites following (R)-NNAL or (S)-NNAL administrationa

 
Although similar percentages of the administered doses were excreted in the urine after administration of the enantiomers (Figure 4), their urinary excretion profiles (Figure 5) were markedly different. For the (R)-enantiomer, 44.6 ± 12.6% of the dose was excreted in the urine. The urinary metabolites consisted largely of unchanged NNAL (14.1 ± 6.2% of the dose), 4-hydroxy-4-(3-pyridyl)butyric acid (hydroxy acid) (15.3 ± 6.3% of the dose) and (R)-NNAL-Gluc (9.7 ± 4.7% of the dose). For the (S)-enantiomer, 48.0 ± 5.1% of the dose was excreted in the urine. NNK and its metabolites were a significant proportion of the urinary excretion (NNK, 1.4 ± 1.4% of the (S)-NNAL dose; keto acid, 13.8 ± 4.3% of the dose; NNK-N-oxide, 4.3 ± 0.7% of the dose). It was evident from the urinary excretion profile that (S)-NNAL underwent significant metabolism back to NNK, since keto acid is formed from NNK, and the interconversion of hydroxy acid and keto acid is minimal (9). The excretion of unchanged NNAL in the urine was 7.0 ± 1.1% of total dose following (S)-NNAL administration. It was estimated that of the 7% of the dose excreted as NNAL, ~60% was (S)-NNAL.



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Fig. 4. Percent of total dose excreted as a function of time in urine after administration of (R)-NNAL (open circle) or (S)-NNAL (filled circle), n = 4.

 


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Fig. 5. Urinary metabolic profile after administration of (R)-NNAL (open bars) or (S)-NNAL (closed bars), n = 4. Each bar represents the percentage of total dose.

 
The overall tissue retention of metabolites was evaluated (Table III). On average, more of the dose was retained in liver, lung and kidney 24 h following (S)-NNAL administration as compared with (R)-NNAL administration. The quantification of individual metabolites in the tissues gave additional insights into the overall disposition of these compounds (Tables IV and V). At 1 h following (S)-NNAL administration, NNAL was proportionally the most abundant metabolite in all tissues (Table IV), accounting for at least 40% of all the metabolites quantified in each tissue. The (S)/(R)-NNAL ratio indicates the proportion of the two enantiomers found in each tissue. A significant percentage of the NNAL in tissues 1 h after (S)-NNAL administration was (R)-NNAL. This observation indicates that the metabolism of the (S)-NNAL to NNK and subsequent reduction to (S)- and (R)-NNAL was very fast and extensive.


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Table III. Percent of total dose (% dose/g tissue) remaining in tissues following administration of the individual enantiomers of NNALa

 

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Table IV. Tissue metabolite profiles after administration of (S)-NNAL

 

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Table V. Tissue metabolite profiles after administration of (R)-NNAL

 
In the tissue distribution profiles 1 h after administration of (S)-NNAL, each organ had a unique metabolic profile. Hydroxy acid (36.0 ± 34.2% of total metabolites) was favored in the liver, NNAL-N-oxide (27.0 ± 18.8% of total metabolites) in the lung, and keto acid (32.1 ± 21.5% of total metabolites), an NNK metabolite, in the kidney.

At 24 h following the administration of the (S)-NNAL enantiomer (Table IV), a substantial amount of NNAL was retained in the lung (77.9 ± 36.2% of total metabolites), and this NNAL was largely of the (S) form [with an (S)/(R)-ratio of 4.23]. This shift of (S)/(R) ratio from the 1 h value suggests a relative retention of the (S)-enantiomer. Keto acid was a major metabolite in the liver and kidney; this indicates significant (S)-NNAL reversible metabolism, since keto acid is formed almost entirely from NNK. It should also be noted that at 24 h after a dose of (S)-NNAL, there was no NNK detectable in any organ, including its carcinogenic target, the lung.

The metabolic profile in the tissues 1 h following (R)-NNAL administration was drastically different from those after (S)-NNAL administration. NNAL still made up the greatest proportion of the radioactivity in most tissues (Table V). Most of the NNAL in the tissue, however, was (R)-NNAL. In addition, no NNK or NNK metabolites were observed. In the kidney the predominant metabolite was hydroxy acid (60.4 ± 9.6% of total metabolites), while NNAL-N-oxide (38.1% of total metabolites) was favored in lung at early times.

At 24 h after the (R)-NNAL dose, NNAL was undetectable in any tissue by the HPLC radioflow assay. There were no quantifiable metabolites in the lung. In the liver, keto acid made up ~30% of the metabolites found, indicating that (R)-NNAL did undergo some conversion to NNK. In one of four rats, NNAL enantiomers could be detected by the stereoselective GC-TEA assay in all tissues; (R)-NNAL predominated, although the (S)/(R)-ratio shifted toward (S)-NNAL from the 1-h value.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The metabolism of NNK to NNAL had been shown previously to be stereoselective and reversible. NNK and NNAL metabolism were investigated in vitro in rodent liver and lung microsomes and cytosol and red blood cells, as well as in human liver microsomes and cytosol. (S)-NNAL was the predominant enantiomer formed from NNK in those tissues (10). The incubation of individual NNAL enantiomers with rat liver microsomes showed that the metabolism of (S)-NNAL to NNK was >10-fold faster than the conversion of (R)-NNAL to NNK. In the rat lung, (S)-NNAL was more rapidly metabolized than (R)-NNAL by all oxidative pathways, including those associated with the formation of activated metabolites. The results demonstrated that NNK was preferentially metabolized to (S)-NNAL, and (S)-NNAL was preferentially re-oxidized to NNK (10).

The findings of the in vitro metabolism studies were relevant to tumorigenicity studies carried out in mice. Groups of female A/J mice were given single i.p. injections of 20 mmol of either NNK, racemic NNAL, (R)-NNAL, (S)-NNAL or several other NNAL metabolites. The animals were killed 16 weeks later. (S)-NNAL was shown to be as potent (25.6 ± 7.5 lung tumors/mouse) as NNK (25.3 ± 9.8 lung tumors/mouse); (R)-NNAL was considerably less potent (8.2 ± 3.3 lung tumors/mouse) (5). These studies indicated that stereoselective metabolism had toxicological implications and in vivo metabolism studies were indicated.

Studies of racemic NNAL carried out in chronically bile-duct-cannulated male F344 rats (4) supported the in vitro findings that the enantiomers of NNAL were stereoselectively metabolized and excreted. Of major importance was the finding that (S)-NNAL was stereoselectively retained in the lung. At 24 h after racemic NNAL administration, NNAL comprised an average of 75.4% of total radioactivity in the lung with an (S)/(R) ratio of >20. Overall, (R)-NNAL tended to enter detoxification and excretory pathways, while (S)-NNAL appeared to be retained in the tissues of rats, specifically the lung (4). However, the reversibility of NNK reduction made the interpretation of the data difficult after racemic NNAL was administered. For example, when the NNAL racemate was administered, NNK was formed, but it was not possible to determine whether the source of NNK was mainly (S)-NNAL or (R)-NNAL. One of the main objectives of the present study was to quantify the stereoselectivity of the reversible metabolism between NNK and NNAL in vivo. The pharmacokinetic properties of the individual NNAL enantiomers were characterized, as well as their metabolic profiles and tissue-specific disposition.

The present study showed that the metabolic profiles of the two enantiomers were significantly different. When the (R)-enantiomer was dosed, almost 40% of the dose was excreted in the bile as the (R)-NNAL-Gluc. (R)-NNAL-Gluc excretion in bile was predominant over (S)-NNAL-Gluc even when the (S)-enantiomer was dosed. When (S)-NNAL was administered, only marginal amounts of (S)-NNAL-Gluc were recovered in the bile. Although (R)-NNAL-Gluc was identified in the plasma and urine in addition to the bile samples, the liver tissue concentration of (R)-NNAL-Gluc was minimal (data not shown). This suggests that the glucuronide metabolite was probably transported into the bile by an active process. In fact, (R)-NNAL-Gluc has been shown to be a substrate for MRP2 (11), a transporter expressed on the bile canalicular membrane (12).

Besides differences in the metabolic profile between the two enantiomers, they also appeared to be distributed differently in the body, as indicated by the apparent volume of distribution (Vss). The Vss value for (S)-NNAL was 1792 ± 570 ml and that of (R)-NNAL was 645 ± 230 ml, a significant difference. These large values suggest extensive tissue binding, with the (S)-NNAL volume of distribution indicating a more extensive distribution. Therefore, tissue binding appears to be stereoselective. In addition, (S)-NNAL was retained in the lung 24 h following its own administration. Thus, (S)-NNAL has a more extensive tissue distribution than (R)-NNAL, and is retained for a longer period of time in the target tissue for carcinogenesis.

NNK itself was rapidly metabolized and could not be detected in lung tissue 4 h after it was administered intravenously to chronically bile duct-cannulated male F344 rats (4). In rats receiving multiple doses of NNK over 16 weeks, NNAL was the only metabolite that accumulated in the lung (13). Additionally, hepatic DNA from animals treated with racemic NNAL released 4-hydroxy-1-(3-pyridyl)-butanone, a metabolite of NNK (14), suggesting that the source of DNA adducts in animals dosed with NNAL is NNK. Since NNK is so rapidly eliminated from the lung tissue, (S)-NNAL sequestration may be one mechanism for prolonging exposure of the lung, particularly because (S)-NNAL can be more easily re-converted to NNK than (R)-NNAL.

Significantly, these findings were reflected in human studies in which NNAL and NNAL-Gluc were measured in the urine of people who had stopped smoking (15) or stopped using smokeless tobacco (16). Despite nicotine and nicotine metabolites reaching background levels within 7 days, NNAL and NNAL-Gluc had elimination half-lives of 40–45 days (15). More importantly, the enantiomeric ratios of (S)-NNAL/(R)-NNAL and (S)-NNAL-Gluc/(R)-NNAL-Gluc in urine were significantly (3.1–5.7 times) higher 7 days after cessation than at baseline in both smokeless tobacco users and smokers, indicating stereoselective retention of (S)-NNAL in humans (16). With (S)-NNAL being the more carcinogenic enantiomer, its relative retention may have implications for its carcinogenic activity in humans.

In summary, compared with (R)-NNAL, the (S)-enantiomer had a much larger volume of distribution, indicating a significantly greater tissue distribution. In the bile, the major metabolite after administration of either (R)- or (S)-NNAL was (R)-NNAL-Gluc, indicating a stereoselectivity in the glucuronidation process. In the urine, the metabolic profiles were significantly different; (S)-NNAL favored reversible metabolism through NNK, while (R)-NNAL did not. In the tissues, particularly the lung, (S)-NNAL appeared to be selectively retained after the administration of either compound. These findings suggest that differences in the pharmacokinetics of the enantiomers may contribute to their stereoselective tumorigenicity. Thus, further studies to determine the mechanism of the stereoselective pulmonary retention of (S)-NNAL are warranted, and may ultimately lead to new chemopreventive strategies.


    Acknowledgments
 
The authors wish to thank Steven G.Carmella and Zhihong Li for excellent technical assistance. This study was supported by PHS grant CA-81301 from the National Cancer Institute. S.S.H. is an American Cancer Society Research Professor, supported by ACS grant RP-00-138.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

  1. Hecht,S.S. and Hoffmann,D. (1988) Tobacco-specific nitrosamines, an important group of carcinogens in tobacco and tobacco smoke. Carcinogenesis, 9, 875–884.[Abstract]
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  4. Wu,Z., Upadhyaya,P., Carmella,S.G., Hecht,S.S. and Zimmerman,C.L. (2002) Disposition of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) in bile duct-cannulated rats: stereoselective metabolism and tissue distribution. Carcinogenesis, 23, 171–179.[Abstract/Free Full Text]
  5. Upadhyaya,P., Kenney,P.M.J., Hochalter,J.B., Wang,M. and Hecht,S.S. (1999) Tumorigenicity and metabolism of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) enantiomers and metabolites in the A/J mouse. Carcinogenesis, 20, 1577–1582.[Abstract/Free Full Text]
  6. Hecht,S.S., Spratt,T.E. and Trushin,N. (1997) Absolute configuration of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) formed metabolically from 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Carcinogenesis, 18, 1851–1854.[Abstract]
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  14. Hecht,S.S. and Trushin,N. (1988) DNA and hemoglobin alkylation by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and its major metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol in F344 rats. Carcinogenesis, 9, 1665–1668.[Abstract]
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Received July 30, 2003; revised February 9, 2004; accepted February 10, 2004.





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