Formation and metabolism of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol enantiomers in vitro in mouse, rat and human tissues

Pramod Upadhyaya, Steven G. Carmella, F.Peter Guengerich1 and Stephen S. Hecht2

University of Minnesota Cancer Center, Minneapolis, MN 55455 and
1 Department of Biochemistry, Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, TN, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) is a major metabolite of the tobacco-specific lung carcino- gen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). NNAL has a chiral center at the 1-position, but little is known about the stereochemical aspects of its metabolic formation from NNK or its further metabolism. We investigated the metabolism of NNK to enantiomers of NNAL in microsomes and cytosol from male F-344 rat liver and lung, female A/J mouse liver and lung, and human liver, as well as in red blood cells from rats, mice and humans. In all systems, (S)-NNAL was the predominant enantiomer formed, ranging from 90 to 98% in the rodent tissues and averaging 64, 90 and >95% in human liver microsomes, liver cytosol and red blood cells, respectively. In rat liver microsomes, (R)- and (S)-NNAL were metabolized at similar rates by {alpha}-hydroxylation, considered to be the major metabolic activation pathway of NNAL. Pyridine-N-oxidation and adenosine dinucleotide phosphate adduct formation also occurred at similar rates from both enantiomers, while reoxidation to NNK was favored with (S)-NNAL as substrate. In rat lung microsomes, (S)-NNAL was more rapidly metabolized than (R)-NNAL by all oxidative pathways. In human liver microsomes, there were no significant differences in the rates of {alpha}-hydroxylation, pyridine-N-oxidation and reoxidation to NNK between the two enantiomers. The results of this study demonstrate that (S)-NNAL, the more tumorigenic enantiomer in mice, is preferentially formed from NNK in rodent and human tissues, and is a substrate for oxidative metabolism in rodent and human tissue microsomes.

Abbreviations: diol, 4-(3-pyridyl)butane-1,4-diol; GC-TEA, gas chromatography with nitrosamine selective detection; hydroxy acid, 4-hydroxy-4-(3-pyridyl)butanoic acid; iso-NNAL, 4-(methylnitrosamino)-4-(3-pyridyl)-1-butanol; lactol, 5-(3-pyridyl)-2-hydroxytetrahydrofuran; 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; pyridyl-THF, 5-(3-pyridyl)tetrahydrofuran; RBC, red blood cells.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The tobacco specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is believed to play a significant role as a cause of lung cancer in smokers (1,2). Several lines of evidence support this conclusion. Extensive international data clearly demonstrate the presence of substantial amounts of NNK in unburned tobacco as well as in tobacco smoke (3,4). The uptake of NNK by smokers and people exposed to environmental tobacco smoke has been established by analysis of its metabolites in urine (2,5). The dose of NNK encountered by smokers in a lifetime of smoking is not dissimilar from the lowest total dose of NNK required to produce lung tumors in rats (2). The selective and potent carcinogenicity of NNK for the lung has been repeatedly demonstrated in extensive studies in rats and mice (1,2). NNK also causes lung tumors in hamsters and mink (1,2). Although the lung is the primary site of NNK carcinogenicity in rodents, this compound also induces pancreatic tumors in rats (2). As the only pancreatic carcinogen in tobacco smoke, it may play a role as a cause of pancreatic cancer in smokers. NNK and the related tobacco-specific nitrosamine, N'-nitrosonornicotine, are the most prevalent strong carcinogens present in smokeless tobacco products and are considered as likely causative agents for oral cavity cancer associated with the use of these products (13,6,7).

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) is a major metabolite of NNK in rodents and humans (2). NNAL, like NNK, is a potent pulmonary carcinogen in rats and mice (2). The carcinogenic properties of NNK and NNAL depend on their metabolism (2). Pathways of NNAL metabolism are summarized in Figure 1Go. {alpha}-Hydroxylation is believed to be the major pathway of metabolic activation of NNK and NNAL. {alpha}-Hydroxylation at the methylene group of NNAL produces 5-(3-pyridyl)-2-hydroxytetrahydrofuran (lactol) and methane- diazohydroxide, which methylates DNA. Lactol can be further oxidized to 4-hydroxy-4-(3-pyridyl)butanoic acid (hydroxy acid). {alpha}-Hydroxylation of the methyl group of NNAL gives a potential DNA pyridylhydroxybutylating intermediate and formaldehyde. The former is converted 4-(3-pyridyl)butane-1,4-diol (diol) and 5-(3-pyridyl)tetrahydrofuran (pyridyl-THF). DNA methylation and pyridyloxobutylation are important in carcinogenesis by NNAL and NNK. NNAL is detoxified by glucuronidation yielding [4-(methylnitrosamino)-1-(3-pyridyl)-but-1-yl]-ß-O-D-glucosiduronic acid (NNAL-Gluc), and by pyridine-N-oxidation to 4-(methylnitrosamino)-1-(3-pyridyl-N-oxide)-1-butanol (NNAL-N-oxide). Pyridine-N-oxidation is also a detoxification pathway of NNK. Both NNK and NNAL form adenosine dinucleotide phosphate (ADP) adducts in vitro, but the biological significance of these metabolites is not clear.



View larger version (12K):
[in this window]
[in a new window]
 
Fig. 1. Overview of NNAL formation and metabolism. NNK is converted to NNAL in most systems studied to date. NNAL can be reoxidized to NNK, which undergoes metabolic activation by {alpha}-hydroxylation. {alpha}-Hydroxylation of NNAL produces lactol, hydroxy acid, diol and pyridyl-THF, along with intermediates that bind to DNA. Metabolism of NNAL to NNAL-Gluc and NNAL-N-oxide are detoxification pathways, while the biological significance of NNAL-ADP is unclear at present.

 
NNAL has a chiral center at its carbinol carbon (Figure 2Go), but relatively little is known about the stereochemistry of NNAL formation from NNK, or about the further metabolism of NNAL. (S)-NNAL [the original assignments of NNAL absolute configuration (Carcinogenesis, 18, 1851–1854, 1997) are reversed (Carcinogenesis, 21, p. 850, 2000, Corrigendum); this is the first paper to use the revised assignments] is the predominant enantiomer of NNAL formed in rat liver microsomal metabolism of NNK (8). (R)-NNAL-Gluc predominates in the urine of rats treated with NNK, whereas (S)-NNAL-Gluc is the major diastereomer in patas monkey urine (8). Mice treated with racemic NNAL excrete mainly (R)-NNAL-Gluc (9). (S)-NNAL-Gluc predominates in the urine of smokers, while free NNAL in smokers' urine is an approximately 1:1 mixture of enantiomers (10).



View larger version (17K):
[in this window]
[in a new window]
 
Fig. 2. Comparison of the formation and metabolism of NNAL enantiomers in humans and rats, based on the data presented here for human and rat liver, and on previous studies (8,10,22). Large arrows, favored pathways relative to the other enantiomer. Note that the revised assignments of NNAL enantiomers are used here for the first time.

 
(S)-NNAL is more tumorigenic than (R)-NNAL in the A/J mouse (9). The metabolism of the NNAL enantiomers has been studied in A/J mouse lung and liver (9). The rate of microsomal {alpha}-hydroxylation of (S)-NNAL is greater than that of (R)-NNAL (9). The results of studies on NNAL formation and metabolism clearly indicate the potential for considerable stereoselectivity and species differences. Therefore, in the present study, we systematically examined in vitro the stereochemistry of NNAL formation from NNK as well as the further metabolism of the NNAL enantiomers. We investigated NNAL formation from NNK in rat, mouse and human liver microsomes and cytosol; rat and mouse lung microsomes and cytosol; and rat, mouse and human red blood cells. We also studied the metabolism of the NNAL enantiomers in rat liver and lung microsomes and in human liver microsomes.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Chemicals
Unlabelled NNK and NNAL were synthesized as described elsewhere (11,12). [5-3H]NNK (4.6 Ci/mmol) was purchased from Chemsyn Science Laboratories (Lenexa, KS). It was converted to racemic [5-3H]NNAL by NaBH4 reduction and purification by reverse-phase HPLC as described previously (12). [5-3H](R)-NNAL and [5-3H](S)-NNAL were prepared by reaction of racemic [5-3H]NNAL with (R)-(–)-{alpha}-methoxy-{alpha}-(trifluoromethyl)phenyl- acetic acid chloride and separation of the diastereomers by normal-phase HPLC on a 250x4.6 mm Luna 5 µm silica column (Phenomenex, Torrance, CA) with isocratic elution by 72% hexane (Fisher HPLC grade), 26% CHCl3 (Mallinckrodt, CromAR HPLC grade, ethanol stabilized), 2% methanol (Mallinckrodt ChromAR HPLC grade) at 1 ml/min (8). Both rotamers of (S)-[4-(methylnitrosamino)-1-(3-pyridyl)but-1-yl](S)-{alpha}-methoxy-{alpha}-(trifluoromethyl)phenyl acetate [(S)-NNAL-(S)-MTPA] eluted from 24–26 min and those of (R)-NNAL-(S)-MTPA eluted from 28–30 min. The diastereomers were collected and hydrolyzed with 0.1 N NaOH. (R)- and (S)-NNAL were then purified as described (8). Metabolite standards were prepared by published procedures (1217). All biochemical reagents were purchased from Sigma Chemical Co. (St Louis, MO).

In vitro metabolism
Microsomes and cytosol were prepared as described (18) from 10 human liver samples obtained from organ donors through the Tennessee Donor Services (Nashville, TN) and the Veterans Administration Medical Center (Little Rock, AR). The same methodology was used to prepare microsomes and cytosol from the livers or lungs of male F-344 rats (15–20 weeks old, obtained from Charles River, Wilmington, MA), and the livers or lungs of female A/J mice (7 weeks old, obtained from Jackson Labs, Bar Harbor, ME). Male F-344 rats and female A/J mice were chosen for these experiments because they have been used in most tumorigenesis studies of NNK and NNAL (2). The rats were maintained on Harlan Teklad diet 7001, and the mice on AIN-93G diet. Protein concentrations were determined using the Micro Protein kit, Sigma Diagnostics.

Human blood was collected from five male volunteers. Rat and mouse blood were obtained by cardiac puncture under halothane anesthesia. Blood was collected in EDTA-containing tubes (Vacutainer; Becton and Dickinson, Rutherford, NJ). Red blood cells (RBC) were pelleted at 9000 g as described (19). The supernatant and buffy coat were removed and the RBC pellet was washed three times in 0.9% saline. After the final wash, the pellet was resuspended in a volume of 0.9% saline solution equal to the volume of RBC.

Conversion of NNK to NNAL was studied using 1 µM NNK. Microsomal incubations (total volume 0.5 ml) were carried out in 100 mM potassium phosphate buffer (pH 7.4) containing 3 mM MgCl2, 1 mM EDTA, an NADPH generating system (5 mM glucose-6-phosphate, 1 mM NADP+, and 1.5 U glucose-6-phosphate dehydrogenase) with protein concentrations of 0.5 mg/ml (liver microsomes) or 0.3 mg/ml (lung microsomes). The same conditions were used for cytosolic incubations, except that the protein concentrations were 1.5 mg/ml (rodent) and 2.5 mg/ml (human). Incubation times were 10 min for rodent liver preparations, 15 min for rodent lung preparations and 20 min for human liver preparations. Reactions were performed 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 supernatant was removed and 4-(methylnitrosamino)-4-(3-pyridyl)-1-butanol (iso-NNAL) was added as internal standard. The resulting mixture was extracted three times with ethyl acetate. The extracts were concentrated to dryness and stored at –20°C until analysis by gas chromatography with nitrosamine selective detection (GC-TEA).

For the RBC studies, 0.3 ml of the RBC solution and 0.2 ml of phosphate buffer were incubated with 1 µM NNK for 20 min. The solution was centrifuged at 3000 r.p.m. and iso-NNAL was added to the supernatant which was then worked up as above for analysis by GC-TEA.

Metabolism of racemic [5-3H]NNAL and [5-3H]NNAL enantiomers was investigated using 1 µM [5-3H]NNAL. Each incubation contained 1 µCi. Microsomal metabolism was carried out as described above for conversion of NNK to NNAL, except that the rat liver and lung studies used 5 mM NADP+ and 3.8 U of glucose-6-phosphate dehydrogenase. Incubation times were 10 min for rat liver, 15 min for rat lung, and 20 min for human liver. At the end of the incubations, precipitated protein was pelleted by centrifugation. The supernatant was filtered through a 0.45 µmx3 mm Acrodisc (Gelman Sciences, Ann Arbor, MI) and analyzed by HPLC.

GC-TEA analysis of NNAL enantiomers
The concentrated extracts containing iso-NNAL internal standard were silylated with bis-trimethylsilyltrifluoroacetamide containing 1% trimethylchlorosilane (20), then analyzed by GC-TEA using a chiral capillary column as described (10).

HPLC analysis of NNAL metabolites
HPLC was carried out with a Waters Associates system equipped with a Model 440 UV detector and a ß-RAM radioflow detector (IN/US Systems, Fairfield, NJ). Metabolites were analyzed with a 4.6x250 mm Luna C18 column (Phenomenex). Eluting solvents were 20 mM sodium phosphate buffer (pH 7.0) and methanol. The following gradient was used: 0–8% methanol in 15 min, hold at 8% for 15 min, 8–23% methanol in 30 min, hold at 23% methanol for 20 min, and 23–35% methanol in 15 min. Metabolites were identified by coelution with standards.

Statistical analyses
Statistical analyses were accomplished using Student's t-test.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Formation of NNAL from NNK
Incubations were carried out for 10 min with rodent hepatic preparations, 15 min with rodent pulmonary preparations and 20 min with human hepatic preparations as well as rodent and human RBC. These time points were chosen based on previous studies of NNK and NNAL metabolism (21,22). Investigation of the (R)-NNAL:(S)-NNAL ratio produced when human liver microsomes were incubated with NNK demonstrated that there was no significant change over the course of 1–20 min (data not shown). All incubations were carried out with 1 µM NNK, the same concentration as that used for NNAL metabolism as described below.

Chiral column GC-TEA proved to be a useful method for determining the ratio of NNAL enantiomers formed from NNK. (R)- and (S)-NNAL-TMS were well separated in this system (10). Conversion of NNK to NNAL was assessed with iso-NNAL as internal standard.

The results of the rodent tissue studies are summarized in Table IGo. All preparations produced predominantly (S)-NNAL [range 90–98%; P < 0.0001 compared with (R)-NNAL]. There were no major differences among species, tissues or subcellular fractions.


View this table:
[in this window]
[in a new window]
 
Table I. Formation of NNAL enantiomers from NNK in rodent tissue preparations in vitroa
 
The results of the human liver studies are summarized in Table IIGo. In these 10 samples, there was relatively little variation in the enantiomeric composition of the NNAL formed from NNK. (S)-NNAL accounted for 64 ± 18 and 90 ± 10% of total NNAL in microsomes and cytosol, respectively. These values were significantly greater than those of (R)-NNAL (P = 0.002 in microsomes and P < 0.0001 in cytosol). (S)-NNAL formation in cytosol was significantly greater than in microsomes (P = 0.00025).


View this table:
[in this window]
[in a new window]
 
Table II. Formation of NNAL enantiomers from NNK in human liver microsomes and cytosola
 
We analyzed the conversion of NNK to NNAL in five samples of human RBC, collected from non-smoking laboratory personnel. All samples had >95% (S)-NNAL.

Metabolism of NNAL
Incubation times were based on previous studies and were the same as those used in the NNAL formation experiments: 10 min for rodent liver microsomes, 15 min for rodent lung microsomes and 20 min for human liver microsomes (21,22). Metabolites were analyzed by HPLC. Chromatograms were similar to those published previously (21,22).

The results of the rat liver microsomal experiments are summarized in Table IIIGo. In liver, there were no significant differences between the metabolism of (R)- and (S)-NNAL, except for reconversion to NNK, which was significantly more rapid when (S)-NNAL was the substrate (P < 0.001). In lung, all metabolites except NNAL(ADP)+ were formed at significantly greater rates from (S)-NNAL than from (R)-NNAL. These differences were particularly striking for diol, the product of {alpha}-methyl hydroxylation, and NNAL-N-oxide, from pyridine-N-oxidation (P < 0.001).


View this table:
[in this window]
[in a new window]
 
Table III. Rat liver and lung microsomal metabolism of racemic NNAL and NNAL enantiomersa
 
HPLC chromatograms of NNAL metabolites formed by human liver microsomes were similar to those reported previously (21). There was considerable variation in rates of NNAL metabolism among the 10 samples analyzed. For example, ranges of metabolite formation from racemic NNAL were as follows, in pmol/min/mg protein: lactol (0–0.13); hydroxy acid (0.01–0.16); diol (0–0.14); NNAL-N-oxide (0.01–0.15); NNK (0.15–1.40). Similar ranges were observed in the metabolism of the enantiomers. The results of these experiments are summarized in Table IVGo. As in our previous study, reoxidation to NNK proceeded more rapidly than {alpha}-hydroxylation or pyridine-N-oxidation (21). There were no significant differences among racemic NNAL and the NNAL enantiomers in the rates of formation of any of the metabolites.


View this table:
[in this window]
[in a new window]
 
Table IV. Human liver microsomal metabolism of racemic NNAL and NNAL enantiomersa
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
All human liver cytosol samples and human RBC examined in this study converted NNK mainly to (S)-NNAL. This enantiomer was also the predominant one formed in nine of 10 human liver microsomal samples incubated with NNK, although the percentage of (S)-NNAL was somewhat lower than in cytosol or RBC. NNK is extensively converted to NNAL in human tissues (2). These results indicate that, in humans exposed to NNK, (S)-NNAL will be the major circulating enantiomer. Consistent with this, the total of (S)-NNAL plus (S)-NNAL-Gluc in smokers' urine exceeds that of (R)-NNAL plus (R)-NNAL-Gluc (10). The extensive formation of (S)-NNAL from NNK is potentially significant because (S)-NNAL is the more tumorigenic enantiomer in mice (9). (S)-NNAL is more readily glucuronidated than is (R)-NNAL in monkeys, and probably in humans (8,23). Thus, the urine of NNK-treated monkeys as well as the urine of smokers contains more (S)-NNAL-Gluc than (R)-NNAL-Gluc (8,10,23). In humans, it is therefore likely that the initial amounts of free (S)-NNAL will be diminished by glucuronidation and excretion, although individual humans differ widely in their capacity to glucuronidate NNAL (24).

Rodent subcellular fractions and RBC also converted NNK mainly to (S)-NNAL. In rat liver microsomes, (R)-NNAL is a better substrate for glucuronidation than (S)-NNAL, and (R)-NNAL-Gluc is the major diastereomer found in rat urine upon treatment with NNK (8,22,23,25). Glucuronidation of NNAL is a relatively minor pathway of NNK metabolism in rats (26), probably due to the availability of relatively small amounts of (R)-NNAL. In mice treated with NNAL enantiomers, (R)-NNAL is more readily glucuronidated than is (S)-NNAL. Therefore, in rodents, the ratio (S)-NNAL:(R)-NNAL will be increased somewhat by removal of (R)-NNAL via glucuronidation. Stereochemical aspects of NNAL formation and further metabolism, based on studies in human and rat liver microsomes and in vivo, are summarized in Figure 2Go.

While both rodent and human tissue preparations favored production of (S)-NNAL from NNK, comparison of the further metabolism of NNAL demonstrated some differences between rat and human liver microsomes. In rat liver microsomes, pyridine-N-oxidation and reoxidation to NNK were the most prevalent metabolic pathways and the latter was significantly more rapid from (S)-NNAL. In human liver microsomes, reoxidation to NNK was clearly the dominant transformation; there was no difference between the NNAL enantiomers in this or any other pathway. This is summarized in Figure 2Go. There are some limitations to the comparison of NNAL metabolism by rat and human liver microsomes. The rat microsomes were freshly isolated from young healthy inbred rats, whereas the human liver microsomes were prepared from tissue that had been stored for >10 years and originated from older individuals of unknown health status. It is not known how such factors may affect rates of NNAL metabolism. However, we have observed that storage of rat and mouse liver microsomes for 6 months results in a decrease in the stereoselectivity of (S)-NNAL formation from NNK (data not shown), and there may also be storage effects on NNAL metabolism.

In rat lung, pyridine-N-oxidation of NNAL was the most prevalent pathway, consistent with previous studies of NNAL and NNK metabolism in rodent lung (2). The strong preference for (S)-NNAL as a substrate for {alpha}-hydroxylation, pyridine-N-oxidation, and reoxidation to NNK in rat lung is similar to the results of our previous study in A/J mouse lung, in which rates of {alpha}-hydroxylation and pyridine-N-oxidation were greater starting with (S)-NNAL (9). This indicates similarity in the P450 enzymes that metabolize NNAL in the mouse and rat lung. (S)-NNAL is the more tumorigenic enantiomer of NNAL in mice, and based on the similarities between rat and mouse metabolism, (S)-NNAL would also be expected to be the more tumorigenic enantiomer in rats. We did not examine human lung metabolism of NNAL in this study because our previous work suggests that the conditions used here are not appropriate for human lung microsomal carcinogen metabolism.

The results of this study indicate that there is considerable stereoselectivity for production of (S)-NNAL in NNK metabolism by rodents and humans. Overall, the greatest stereoselectivity was observed in cytosol and RBC. Carbonyl reductase (EC 1.1.1.184), a ubiquitous NADPH-dependent system, is likely to be involved in the reduction of NNK to NNAL in cytosol and RBC (2,27,28). Previous studies have demonstrated stereoselectivity in the reduction of a number of drugs by carbonyl reductase. These include dolasteron (29), haloperidol (30), fenofibrate (31) and several other ketones (32). The stereochemistry of reduction has been predicted based on results in bacteriological systems, but it is not clear to what extent these are generalizable to mammalian enzymes (33). However, the results presented here, in which NNK is reduced mainly to (S)-NNAL, are consistent with these predictions (33). The results are also consistent with those of our previous study showing that a structurally related compound, 4-oxo-4-(3-pyridyl)butanoic acid is converted primarily to (S)-hydroxy acid (see structure in Figure 1Go) in vivo in rats and humans (34,35).

Stereoselectivity in carbonyl reduction also was observed in the microsomal fractions investigated here. Maser and co-workers have demonstrated that 11ß-hydroxysteroid dehydrogenase 1 (EC 1.1.1.146) is involved in the conversion of NNK to NNAL by mouse liver and lung microsomes (36,37). Apparently the stereoselectivity of this and related enzymes is similar to that of the non-microsomal carbonyl reductases.

In summary, the results of this study demonstrate that (S)-NNAL is the predominant enantiomer formed upon metabolic reduction of NNK in rodent liver, lung, and RBC as well as in human liver and RBC. There is little stereoselectivity in the further oxidative metabolism of (R)- versus (S)-NNAL by rat and human liver microsomes, whereas rat lung microsomes preferentially metabolize (S)-NNAL. These results indicate that (S)-NNAL, the more tumorigenic enantiomer in mouse lung, is preferentially formed from NNK in rodents and humans, and is a substrate for oxidative metabolism in rodent and human tissue microsomes.


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


    Acknowledgments
 
This study was supported by grants CA-81301 from the National Cancer Institute and ES-00267 from the National Institute of Environmental Health Sciences. S.S.H. is an American Cancer Society Research Professor.


    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]
  2. Hecht,S.S. (1998) Biochemistry, biology and carcinogenicity of tobacco-specific N-nitrosamines. Chem. Res. Toxicol., 11, 559–603.[ISI][Medline]
  3. Hoffmann,D., Brunnemann,K.D., Prokopczyk,B. and Djordjevic,M.V. (1994) Tobacco-specific N-nitrosamines and areca-derived N-nitrosamines: chemistry, biochemistry, carcinogenicity and relevance to humans. J. Toxicol. Environ. Health, 41, 1–52.[ISI][Medline]
  4. Spiegelhalder,B. and Bartsch,H. (1996) Tobacco-specific nitrosamines. Eur. J. Cancer Prev., 5, 33–38.
  5. Hecht,S.S. (1999) Tobacco smoke carcinogens and lung cancer. J. Natl Cancer Inst., 91, 1194–1210.[Abstract/Free Full Text]
  6. Magee,P.N. (1996) Nitrosamines and human cancer: introduction and overview. Eur. J. Cancer Prev., 5, 7–10.[ISI][Medline]
  7. Bartsch,H. and Spiegelhalder,B. (1996) Environmental exposure to N-nitroso compounds (NNOC) and precursors: an overview. Eur. J. Cancer Prev., 5, 11–18.[ISI][Medline]
  8. Hecht,S.S., Spratt,T.E. and Trushin,N. (1997) Absolute configuration of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) formed metabolic- ally from 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Carcinogenesis, 18, 1851–1854.[Abstract]
  9. 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]
  10. Carmella,S.G., Ye,M., Upadhyaya,P. and Hecht,S.S. (1999) Stereochemistry of metabolites of a tobacco-specific lung carcinogen in smokers' urine. Cancer Res., 59, 3602–3605.[Abstract/Free Full Text]
  11. Hecht,S.S., Lin,D. and Castonguay,A. (1983) Effects of {alpha}-deuterium substitution on the mutagenicity of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Carcinogenesis, 4, 305–310.[ISI][Medline]
  12. Hecht,S.S., Young,R. and Chen,C.B. (1980) Metabolism in the F344 rat of 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone, a tobacco specific carcinogen. Cancer Res., 40, 4144–4150.[Abstract]
  13. Castonguay,A., Tjälve,H., Trushin,N. and Hecht,S.S. (1984) Perinatal metabolism of the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in C57B1 mice. J. Natl Cancer Inst., 72, 1117–1126.[ISI][Medline]
  14. Peterson,L.A., Ng,D.K., Stearns,R.A. and Hecht,S.S. (1994) Formation of NADP(H) analogs of tobacco specific nitrosamines in rat liver and pancreatic microsomes. Chem. Res. Toxicol., 7, 599–608.[ISI][Medline]
  15. Loozen,H.J.J., Godefroi,E.F. and Besters,J.S.M.M. (1975) A novel and efficient route to 5-arylated {delta}-lactones. J. Org. Chem., 40, 892–894.[ISI]
  16. McKennis,H., Schwartz,S.L., Turnbull,L.B., Tamaki,E. and Bowman,E.R. (1964) The metabolic formation of {gamma}-(3-pyridyl)-{gamma}-hydroxybutyric acid and its possible intermediary role in the mammalian metabolism of nicotine. J. Biol. Chem., 239, 3981–3989.[Free Full Text]
  17. Spratt,T.E., Peterson,L.A., Confer,W.L. and Hecht,S.S. (1990) Solvolysis of model compounds for {alpha}-hydroxylation of N'-nitrosonornicotine and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone: evidence for a cyclic oxonium ion intermediate in the alkylation of nucleophiles. Chem. Res. Toxicol., 3, 350–356.[ISI][Medline]
  18. Guengerich,F.P. (1994) Analysis and characterization of enzymes. In Hayes,A.W. (ed.), Principles and Methods of Toxicology. Raven Press, New York, pp. 1259–1313.
  19. Murphy,S.E. and Coletta,K.A. (1993) Two types of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone hemoglobin adducts, from metabolites which migrate into or are formed in red blood cells. Cancer Res., 53, 777–783.[Abstract]
  20. Carmella,S.G., Akerkar,S. and Hecht,S.S. (1993) Metabolites of the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in smokers' urine. Cancer Res., 53, 721–724.[Abstract]
  21. Staretz,M.E., Murphy,S.E., Nunes,M.G., Koehl,W., Amin,S., Koenig,L., Guengerich,F.P. and Hecht,S.S. (1997) Comparative metabolism of the tobacco smoke carcinogens benzo(a)pyrene, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol and N'-nitrosonornicotine in human hepatic microsomes. Drug Metab. Dispos., 25, 154–162.[Abstract/Free Full Text]
  22. Staretz,M.E., Koenig,L. and Hecht,S.S. (1997) Effects of long term phenethyl isothiocyanate treatment on microsomal metabolism of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol in F344 rats. Carcinogenesis, 18, 1715–1722.[Abstract]
  23. Hecht,S.S., Trushin,N., Reid-Quinn,C.A., Burak,E.S., Jones,A.B., Southers,J.L., Gombar,C.T., Carmella,S.G., Anderson,L.M. and Rice,J.M. (1993) Metabolism of the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in the Patas monkey: pharmacokinetics and characterization of glucuronide metabolites. Carcinogenesis, 14, 229–236.[Abstract]
  24. Carmella,S.G., Akerkar,S., Richie,J.P.Jr and Hecht,S.S. (1995) Intraindividual and interindividual differences in metabolites of the tobacco-specific lung carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in smokers' urine. Cancer Epidemiol. Biomarkers Prev., 4, 635–642.[Abstract]
  25. Ren,Q., Murphy,S.E., Dannenberg,A.J., Park,J.Y., Tephly,T.R. and Lazarus,P. (1999) Glucuronidation of the lung carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) by rat UDP-glucuronosyltransferase 2B1. Drug Metab. Disp., 27, 1010–1016.[Abstract/Free Full Text]
  26. Morse,M.A., Eklind,K.I., Toussaint,M., Amin,S.G. and Chung,F.-L. (1990) Characterization of a glucuronide metabolite of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and its dose-dependent excretion in the urine of mice and rats. Carcinogenesis, 11, 1819–1823.[Abstract]
  27. Felsted,R.L. and Bachur,N.R. (1980) Mammalian carbonyl reductases. Drug Metab. Rev., 11, 1–60.[ISI][Medline]
  28. Jez,J.M., Flynn,T.G. and Penning,T.M. (1996) A nomenclature system for the aldo-keto reductase superfamily. In Weiner,H., Lindahl,R., Crabb,D.W. and Flynn,T.G. (eds), Enzymology and Molecular Biology of Carbonyl Metabolism. Plenum Press, New York, pp. 579–589.
  29. Dow,J. and Berg,C. (1995) Stereoselectivity of the carbonyl reduction of dolasetron in rats, dogs and humans. Chirality, 7, 342–348.[ISI][Medline]
  30. Eyles,D.W. and Pond,S.M. (1992) Stereospecific reduction of haloperidol in human tissues. Biochem. Pharmacol., 44, 867–871.[ISI][Medline]
  31. Weil,A., Caldwell,J., Guichard,J.-P. and Picot,G. (1989) Species differences in the chirality of the carbonyl reduction of [14C] fenofibrate in laboratory animals and humans. Chirality, 1, 197–201.[ISI][Medline]
  32. Prelusky,D.B., Couts,R.T. and Pasutto,F.M. (1982) Stereospecific metabolic reduction of ketones. J. Pharmaceut. Sci., 71, 1390–1393.[ISI][Medline]
  33. Prelog,V. (1964) Specification of the stereospecificity of some oxidoreductuses by diamant lattice sections. Pure Appl. Chem., 9, 119–130.
  34. Trushin,N. and Hecht,S.S. (1999) Stereoselective metabolism of nicotine and tobacco-specific N-nitrosamines to 4-hydroxy-4-(3-pyridyl)butanoic acid in rats. Chem. Res. Toxicol., 12, 164–171.[ISI][Medline]
  35. Hecht,S.S., Hatsukami,D.K., Bonilla,L.E. and Hochalter,J.B. (1999) Quantitation of 4-oxo-4-(3-pyridyl)butanoic acid and enantiomers of 4-hydroxy-4-(3-pyridyl)butanoic acid in human urine: a substantial pathway of nicotine metabolism. Chem. Res. Toxicol., 12, 172–179.[ISI][Medline]
  36. Maser,E., Richter,E. and Friebertshauser,J. (1996) The identification of 11-ß-hydroxysteriod dehydrogenase as carbonyl reductase of the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone. Eur. J. Biochem., 238, 484–489.[Abstract]
  37. Maser,E. (1998) 11-ß-Hydroxysteroid dehydrogenase responsible for carbonyl reduction of the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in mouse lung microsomes. Cancer Res., 58, 2996–3003.[Abstract]
Received December 8, 1999; revised February 10, 2000; accepted February 29, 2000.