Stereoselective Metabolism of Nicotine and Tobacco-Specific N

While several metabolites of NNK can be quantified in human urine, ... determine human urinary levels of NNK and NNN metabolites resulting from the cr...
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Chem. Res. Toxicol. 1999, 12, 164-171

Stereoselective Metabolism of Nicotine and Tobacco-Specific N-Nitrosamines to 4-Hydroxy-4-(3-pyridyl)butanoic Acid in Rats Neil Trushin† and Stephen S. Hecht*,‡ American Health Foundation, Valhalla, New York 10595, and University of Minnesota Cancer Center, Minneapolis, Minnesota 55455 Received September 10, 1998

The carcinogenic tobacco-specific nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N′-nitrosonornicotine (NNN) are believed to play a role in cancers associated with the use of tobacco products. Urinary metabolites of NNK and NNN could be used as biomarkers for an individual’s ability to metabolically activate or detoxify these nitrosamines. While several metabolites of NNK can be quantified in human urine, no assay is available to determine human urinary levels of NNK and NNN metabolites resulting from the critical R-hydroxylation metabolic activation pathways. The major urinary metabolites resulting from R-hydroxylation of NNK and NNN in rodents are 4-oxo-4-(3-pyridyl)butanoic acid (keto acid) and 4-hydroxy-4-(3-pyridyl)butanoic acid (hydroxy acid). The major obstacle to the use of these metabolites as biomarkers of metabolic activation is the fact that they are also metabolites of nicotine, which is present at levels 1400-13000 times greater than those of the nitrosamines in cigarette smoke. However, the chirality of hydroxy acid could be useful in overcoming this problem. If different enantiomers of hydroxy acid were formed from nicotine versus the nitrosamines, and if the overall yield of hydroxy acid from nicotine were substantially smaller than that from the nitrosamines, then hydroxy acid might be useful as a urinary biomarker of NNK and NNN R-hydroxylation. To these ends, F-344 rats were administered either [5-3H]NNK, [5-3H]NNN, [5-3H]keto acid, or [2′-14C]nicotine. The levels of urinary hydroxy acid were determined by HPLC analysis. Its stereochemistry was determined by conversion to its methyl ester, reaction with (S)-(-)-R-methylbenzyl isocyanate, and separation and quantitation of the resulting diastereomers by HPLC. Urinary hydroxy acid accounted for 12% of the NNK dose and 31% of the NNN dose, but only 1 and 0.1% of the dose of keto acid and nicotine, respectively. Furthermore, metabolism of NNK produced mainly (S)-hydroxy acid in the urine, while metabolism of keto acid and nicotine gave predominantly (R)-hydroxy acid. Both enantiomers were present in the urine of NNN-treated rats. Therefore, in the rat, it is possible to distinguish the hydroxy acid derived from nicotine from that derived from the nitrosamines. If similar pathways occur in humans, (S)-hydroxy acid could potentially be developed as a urinary biomarker of NNK and NNN R-hydroxylation in smokers.

Introduction Several nitrosamines derived from tobacco alkaloids are carcinogenic to laboratory animals and are present in tobacco products (1-4). Among these, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)1 is a potent lung carcinogen in rodents and a likely causative factor in human lung carcinogenesis (1-4). N′-Nitrosonornicotine (NNN) is an esophageal carcinogen believed to play a role in cancer of the esophagus caused by tobacco products (1-4). NNK and NNN are widely accepted as causes of oral cancer in people who use smokeless tobacco products (3-7). The carcinogenic activities of NNK and NNN result from metabolism by R-hydroxylation, producing inter* To whom correspondence should be addressed: University of Minnesota Cancer Center, Box 806 Mayo, 420 Delaware St. SE, Minneapolis, MN 55455. Telephone: (612) 624-7604. Fax: (612) 6265135. E-mail: [email protected]. † American Health Foundation. ‡ University of Minnesota Cancer Center.

mediates that alkylate DNA (4). Some of the relevant metabolic pathways of NNK metabolism are illustrated in Scheme 1. Hydroxylation of the NNK methyl carbon yields R-hydroxymethylNNK (1) which spontaneously loses formaldehyde, producing the pyridyloxobutyl diazohydroxide 5. This intermediate or its derived diazonium ion or cyclic oxonium ion alkylates DNA or reacts with H2O, producing 4-hydroxy-1-(3-pyridyl)-1-butanone (keto alcohol), which is further oxidized in vivo to 4-oxo-4-(31 Abbreviations: diol, 4-hydroxy-4-(3-pyridyl)-1-butanol; hydroxy acid, 4-hydroxy-4-(3-pyridyl)butanoic acid; keto acid, 4-oxo-4-(3-pyridyl)butanoic acid; keto alcohol, 4-hydroxy-1-(3-pyridyl)-1-butanone; keto aldehyde, 4-oxo-4-(3-pyridyl)butanal; keto amide, 4-oxo-4-(3pyridyl)-N-methylbutyramide; lactol, 5-(3-pyridyl)-2-hydroxytetrahydrofuran; MBIC, R-methylbenzyl isocyanate; (R,S)-MMPB, methyl4(R)-[(S)-R-methylbenzylcarbamoyl]-4-(3-pyridyl)butanoate; NNAL, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol; NNAL-Gluc, [4-(methylnitrosamino)-1-(3-pyridyl)but-1-yl]-β-O-D-glucosiduronic acid; (R)NNAL-(S)-MBIC, 4-(methylnitrosamino)-1(R)-[(S)-R-methylbenzylcarbamoyl]-1-(3-pyridyl)butane; NNK, 4-(methylnitrosamino)-1-(3-pyridyl)1-butanone; NNN, N′-nitrosonornicotine; PCI-MS, positive ion chemical ionization mass spectrometry; pyridyl-THF, 2-(3-pyridyl)tetrahydrofuran.

10.1021/tx980213q CCC: $18.00 © 1999 American Chemical Society Published on Web 01/13/1999

Metabolism of Nicotine and Derived Nitrosamines

Chem. Res. Toxicol., Vol. 12, No. 2, 1999 165

Scheme 1. Metabolism of NNK and NNAL by r-Hydroxylationa

a

For more details, see ref 4.

pyridyl)butanoic acid (keto acid). Hydroxylation of the NNK methylene carbon yields intermediate 2 which spontaneously produces 4-oxo-4-(3-pyridyl)-1-butanal (keto aldehyde) and methane diazohydroxide (6). The latter ultimately methylates DNA and hemoglobin. Keto aldehyde is most likely converted to keto acid in vivo. In humans, rodents, and primates, NNK is extensively converted to its carbonyl reduction product 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), which is also a potent lung carcinogen and undergoes metabolic activation by R-hydroxylation in a fashion similar to that illustrated for NNK (4). Both R-hydroxylation pathways of NNAL ultimately yield hydroxy acid, a major urinary metabolite of NNK in rodents and primates (Scheme 1). As shown in Scheme 1, NNK-derived 4-hydroxy-4-(3pyridyl)butanoic acid (hydroxy acid) could be formed via NNAL R-hydroxylation or by reduction of keto acid. On the basis of previous work, the former route is more likely (8-11). NNN is also metabolically activated by R-hydroxylation (Scheme 2) (4). Hydroxylation of the 2′-carbon produces diazohydroxide 5 and results in alkylation of DNA and hemoglobin, and production of keto alcohol and keto acid. Hydroxylation of the 5′-carbon gives intermediate 9 which ring opens to diazohydroxide 10. This reacts with H2O, producing 5-(3-pyridyl)-2-hydroxytetrahydrofuran (lactol), which is oxidized in vivo to hydroxy acid. Thus, keto acid and hydroxy acid are major urinary metabolites of both NNK and NNN, resulting from the R-hydroxylation metabolic activation process. The levels of these urinary metabolites could reflect the extents of DNA adduct formation occurring by the same pathways. One goal of our research is to quantify urinary metabolites of NNK and NNN in humans. Urinary metabolites could be biomarkers of individual capacity to activate or detoxify these carcinogens, and thus, their levels could be related to cancer risk in people who use tobacco products. In work to date, we have developed assays for several urinary metabolites of NNK, including NNAL, its glucuronide, and the pyridine N-oxidation metabolites

of NNK and NNAL (12-14). Although hemoglobin and DNA adducts of NNK and NNN have been quantified in humans (4), there is currently no method for quantifying urinary metabolites as an indicator of the critical activation pathways of NNK and NNN. Urinary metabolites have many practical advantages over hemoglobin or DNA adducts as biomarkers of carcinogen metabolism in humans. The development of such an assay is the intent of this work. As noted above, keto acid and hydroxy acid are the major urinary metabolites of NNK and NNN resulting from the R-hydroxylation activation pathway. However, keto acid and hydroxy acid are also formed in nicotine metabolism (Scheme 2). They are produced by further metabolism of cotinine, via norcotinine and 5′-hydroxycotinine (15-24). Nicotine levels in cigarette smoke are 1400-13000 times greater than the combined level of NNK and NNN (25, 26). Therefore, even if keto acid were a minor nicotine metabolite, it would not be possible to distinguish nicotine-derived keto acid from that produced by metabolism of NNK or NNN in a smoker. But, in the case of hydroxy acid, its chirality presents a potential opportunity for distinguishing its origin. Hydroxy acid formation from nicotine is believed to occur exclusively by keto acid reduction, whereas hydroxy acid formation from NNK and NNN follows the several paths described above (4, 15-24). The following combination of results would allow us to potentially distinguish between hydroxy acid formed from nicotine and that formed from NNK and NNN: (1) conversion of nicotine to hydroxy acid via keto acid is a minor pathway; (2) reduction of keto acid to hydroxy acid is stereoselective; (3) reduction of keto acid to hydroxy acid is a minor pathway of NNK and NNN metabolism; and (4) production of hydroxy acid by R-hydroxylation of NNK and NNN proceeds with stereoselectivity opposite of that of keto acid reduction. If these conditions were fulfilled, it would be possible to identify a hydroxy acid enantiomer distinctive of nitrosamine metabolism. With these considerations in mind, we determined both the levels and enantiomeric composi-

166 Chem. Res. Toxicol., Vol. 12, No. 2, 1999

Trushin and Hecht

Scheme 2. Formation of Hydroxy Acid in the Metabolism of NNN and Nicotinea

a

For further details, see refs 4, 19, and 23.

tion of hydroxy acid in the urine of F-344 rats treated with radiolabeled NNK, NNN, keto acid, or nicotine.

Experimental Section Caution: NNK and NNN are carcinogens and mutagens, and nicotine is toxic. These compounds should be handled with extreme care, using appropriate protective clothing and ventilation at all times. Apparatus. HPLC analyses were carried out with a system consisting of two model 510 pumps (Millipore, Waters Division, Milford, MA), a Rheodyne model 7125 injector (Rheodyne Inc., Cotati, CA), and either a Gilson model 112 (Gilson Medical Electronics Inc., Middleton, WI) or a Shimadzu SPD 10AV (Shimadzu Scientific Instruments, Inc., Columbia, MD) UV detector. Radiochemical analyses were accomplished either with a Flo-one/Beta (Packard Instrument Co., Downer’s Grove, IL) radiochemical detector or by collection of fractions using an Isco Retriever III, model 328 fraction collector (Instrument Speciality Co., Lincoln, NE) and subsequent analysis on a Beckman LS 9800 liquid scintillation counter (Beckman Instrument Co., Fullerton, CA). Reverse phase HPLC was carried out with a 0.46 cm × 25 cm Microsorb-MV 5µ C-18 column (Rainin Instrument Co., Woburn, MA) with the following elution systems: (A) isocratic 60% triethylammonium phosphate (pH 7.25)/40% CH3OH at a flow rate of 1 mL/min, (B) isocratic 96% 20 mM sodium phosphate buffer (pH 7.0)/4% CH3OH at a flow rate of 1 mL/ min, (C) a linear gradient of 0 to 40% CH3OH in 60 mM sodium acetate buffer (pH 4.5) over the course of 40 min at a flow rate of 1 mL/min, (D) a linear gradient of 0 to 35% CH3OH in 20 mM sodium phosphate buffer (pH 7.0) over the course of 70 min at a flow rate of 1 mL/min, (E) a linear gradient from 0 to 65% CH3OH in 10 mM ammonium phosphate buffer (pH 5.4) over the course of 65 min at a flow rate of 1 mL/min, and (F) a linear gradient from 0 to 45% CH3CN in 20 mM sodium phosphate

buffer (pH 7.0) over the course of 90 min at a flow rate of 1 mL/min. Normal phase HPLC was carried out with a 0.46 cm × 25 cm Alltech Econosil silica 5µ column (Alltech Associates, Inc., Deerfield, IL) eluted isocratically with 78.4% hexane/19.6% CHCl3/2% CH3OH at a flow rate of 1 mL/min. In all cases, metabolite identities were determined by coelution of radioactivity with standards. MS spectra were obtained on a Hewlett-Packard model 5987 instrument (Hewlett Packard Co., San Fernando, CA). LC-MS/ MS was carried out with a Finnigan TSQ 700 system (Finnigan MAT/Thermoquest, San Jose, CA). 1H NMR spectra were obtained with a Bruker model AM 360 WB spectrometer (Bruker Instruments, Manning Park, MA). Chemicals. (R)-(+)-R-Methylbenzyl isocyanate [(R)-(+)MBIC] [96% enantiomeric excess (ee)], (S)-(-)-MBIC (96% ee), and (S)-(-)-nicotine were obtained from Aldrich Chemical Co. (Milwaukee, WI). Hydroxy acid was prepared as described previously (17). Keto acid, NNK, and NNN were synthesized (17, 27, 28) and provided by the American Health Foundation Organic Synthesis Facility (Valhalla, NY). Reagents used in the microsomal incubation were purchased from Sigma Chemical Co. (St. Louis, MO). [5-3H]NNK, [5-3H]NNN, and (S)-(-)[2′-14C]nicotine-di-(+)-tartrate were purchased from Chemsyn Science Laboratories (Lenexa, KS). [5-3H]NNK and [5-3H]NNN were purified using HPLC system D. They were >99% pure by HPLC analysis (system D). [2′-14C]Nicotine was purified using HPLC system A. The collected material was concentrated in vacuo to remove CH3OH and basified with Na2CO3, and the [2′-14C]nicotine was extracted into CHCl3. HPLC analysis (system A) indicated that the radiochemical purity of this material was >98%. [5-3H]Keto acid was isolated from the urine of rats treated with [5-3H]NNK in a previous study. The urine was treated with a 3-fold excess of CH3OH to precipitate protein. After filtration, the filtrate was concentrated to dryness and reconstituted in

Metabolism of Nicotine and Derived Nitrosamines

Figure 1. Structures of hydroxy acid enantiomers, (S,S)- and (R,S)-MMPB, and NNAL-MBIC diastereomers. 20 mM sodium phosphate buffer (pH 7). [5-3H]Keto acid was purified using HPLC systems B and C. It was collected from system B at 14.5-18 min and further purified using system C, in which its elution time was 34.5-38 min. [5-3H]Keto acid was then desalted by HPLC using a 0.46 cm × 25 cm Phenyl 5µ column (Rainin) with elution by H2O at a flow rate of 1 mL/ min. The radiochemical purity of [5-3H]keto acid was >98% as determined with HPLC system C. Methyl-4-[(S)-r-methylbenzylcarbamoyl]-4-(3-pyridyl)butanoate (MMPB). Hydroxy acid (300 mg, 1.7 mmol) was dissolved in 2 mL of CH3OH, and 0.12 mL of H2SO4 was added. The solution was heated at 70 °C overnight. After the solution was cooled to room temperature, 0.42 mL of 28% NH4OH was added, the mixture was centrifuged, and the supernatant was evaporated to near dryness. [Concentration of this solution under acidic conditions produces the lactone, 5-(3-pyridyl)tetrahydrofuran-2-one.] Two milliliters of 20 mM sodium phosphate buffer (pH 7.0) was added, and the methyl ester was extracted with CHCl3. The extracts were dried (Na2SO4) and concentrated, yielding 300 mg (1.5 mmol) of methyl ester. Conversion to the ester was nearly quantitative as judged by HPLC analysis using system D, in which the retention times of hydroxy acid and its methyl ester were 13 and 60 min, respectively. The methyl ester had the following spectral properties: 1H NMR (CDC13) δ 8.59 (1H, d, pyr-2H), 8.53 (1H, dd, pyr-6H), 7.75 (1H, m, pyr-4H), 7.29 (1H, dd, pyr-5H), 4.84 (1H, t, CHOH), 3.69 (3H, s, OCH3), 2.50 (2H, td, CH2CO), 2.09 (2H, dd, CHCH2); PCI-MS (NH3) m/z (relative intensity) 213 (M + 18, 100), 196 (M + 1, 46). It was used without further purification. It was dissolved in 8 mL of benzene, to which were added 1.39 mL (10 mmol) of triethylamine and 1 mL (7.6 mmol) of (S)-(-)-MBIC. The solution was heated under reflux overnight. Upon cooling, the reaction mixture was filtered, concentrated, and redissolved in CHCl3. The crude carbamate mixture was partially purified on a silica gel column with elution by 0.25-0.5% CH3OH in CHCl3. The MMPB diastereomers (Figure 1) were then collected separately by normal phase HPLC. (S,S)MMPB eluted at 26 min and (R,S)-MMPB at 28 min. Spectral data for (S,S)-MMPB were as follows: 1H NMR (CDCl3) δ 8.58 (1H, br s, pyr-2H), 8.53 (1H, d, pyr-6H), 7.6 (1H, d, pyr-4H), 7.3 (6H, m, pyr-5H and PhH), 5.71 (1H, m, CHO), 5.03 (1H, br s, NH), 4.79 (1H, m, CHCH3), 3.66 (3H, s, OCH3), 2.39 (2H, m, CH2CO), 2.19 (2H, m, CH2CH2CO), 1.50 (3H, d, CH3); PCI-MS

Chem. Res. Toxicol., Vol. 12, No. 2, 1999 167 (NH3) m/z (relative intensity) 360 (M + 18, 29), 343 (M + 1, 100), 139 (81), 91 (13). Spectral data for (R,S)-MMPB were as follows: 1H NMR (CDCl3) δ 8.61 (1H, br s, pyr-2H), 8.55 (1H, br s, pyr-6H), 7.64 (1H, d, pyr-4H), 7.3 (6H, m, pyr-5H and PhH), 5.67 (1H, dd, CHO), 5.04 (1H, s, NH), 4.77 (1H, m, CHCH3), 3.61 (3H, s, OCH3), 2.33 (2H, m, CH2CO), 2.17 (2H, m, CH2CH2CO), 1.45 (3H, d, CH3); PCI-MS (NH3) m/z (relative intensity) 360 (M + 18, 44), 343 (M + 1, 63), 139 (81), 91 (100). Rat Study. Male F-344 rats (Charles River Laboratories, Kingston, NY) were maintained on a diet of Purina 5018-NIH meal and were housed individually in stainless steel metabolism cages. The rats (average weight of 289 g) were divided into four groups of three rats each. Each rat received a single iv injection of one of the compounds (8.5 µmol/kg of body weight) in 0.5 mL of saline. The amount of radioactivity and specific activity of each compound were as follows: [5-3H]NNK, 0.92 µCi and 0.367 µCi/µmol; [5-3H]NNN, 12.5µCi and 5.34 µCi/µmol; [5-3H]keto acid, 0.74 µCi and 0.287 µCi/µmol; and [2′-14C]nicotine, 9.3 µCi and 4.01 µCi/µmol. Urine was collected for 24 h in tubes kept on dry ice. Analysis of Urine. (1) Total Hydroxy Acid. CH3OH (510 volumes) was added to a portion of the urine from each sample. The mixtures were centrifuged, and the supernatant was evaporated to dryness. The samples were reconstituted in 2 mL of 20 mM sodium phosphate buffer (pH 7.0). For the animals treated with NNK or NNN, aliquots of these samples were analyzed with HPLC system D. For the keto acid- and nicotine-treated animals, both UV and radioactive interference in the hydroxy acid region were too great to allow for direct analysis of this metabolite. Therefore, hydroxy acid levels were determined by HPLC analysis following derivatization to its methyl ester. The samples were injected onto the HPLC system, and the hydroxy acid region was collected using system D. The sample was evaporated to dryness and treated with acidic methanol as described above. The solution was made basic with NH4OH, concentrated to dryness, and redissolved in 2 mL of 20 mM sodium phosphate buffer (pH 7.0). It was analyzed using HPLC system D. (2) Hydroxy Acid Stereoisomers. For the NNK and NNN samples, a portion of the reconstituted urine was injected onto HPLC system D and the hydroxy acid region collected. The hydroxy acid was converted to its methyl ester as described above. Following ester formation, all samples were extracted into CHCl3, dried (Na2SO4), concentrated to dryness, and redissolved in 2 mL of benzene. MMPB diastereomers were formed by addition of triethylamine (70 µL) and (S)-(-)-MBIC (40 µL), and heating overnight at 70 °C. After cooling, the mixture was filtered, the benzene was evaporated, and the samples were redissolved in CHCl3. Analysis of (S,S)- and (R,S)MMPB was accomplished using normal phase HPLC. The effluent from each sample injection was collected, and the solvent was allowed to evaporate. The carbamate region was collected in 0.5 mL fractions, while the other portions of the run were collected in 1 mL fractions. The amount of radioactivity in each fraction was determined by liquid scintillation counting. Absolute Configuration of (S)-Hydroxy Acid. (S)-NNAL was obtained as described previously (29). Rat liver microsomes were prepared as described previously (30). (S)-NNAL (0.96 mM) was incubated for 2 h at 37 °C with rat liver microsomes (1.5 mg), glucose 6-phosphate (5 mM), glucose-6-phosphate dehydrogenase (0.8 unit), NADP+ (1 mM), and MgCl2 (3 mM) in 100 mM potassium phosphate buffer (pH 7.4), in a total volume of 1 mL. The reaction was stopped by addition of 200 µL each of 0.3 N ZnSO4 and 0.3 N Ba(OH)2. Following cooling on ice, the mixture was filtered through a Gelman LC PVDF syringe filter (Gelman Sciences, Ann Arbor, MI). Hydroxy acid was isolated and converted to MMPB by the methods described above, except that (R)-(+)-MBIC was used. MMPB was analyzed by LC-MS/ MS, using HPLC system E and selected reaction monitoring for m/z 343-178. (S)-NNAL was isolated and reacted with (R)-(+)MBIC; the product was analyzed by normal phase HPLC with isocratic elution with 60/40/2 hexane/CHC13/CH3OH.

168 Chem. Res. Toxicol., Vol. 12, No. 2, 1999

Trushin and Hecht Table 1. HPLC Retention Times of NNAL-MBIC and MMPB Diastereomers retention time (min) (R)-NNAL-(R)-MBIC and (S)-NNAL-(S)-MBIC (R)-NNAL-(S)-MBIC and (S)-NNAL-(R)-MBIC (R,R)-MMPB and (S,S)-MMPB (R,S)-MMPB and (S,R)-MMPB

normal phasea

reverse phaseb

38 [(Z)-isomer], 40 [(E)-isomer] 40 [(Z)-isomer], 43 [(E)-isomer] 27

76.9 [(E)-isomer], 77.7 [(Z)-isomer] 77.7 [(E)-isomer], 78.6 [(Z)-isomer] 64.8

29

65.6

a Conditions for MMPB are described in the Experimental Section. For analysis of NNAL-MBIC, the same column was eluted with 80/20/2 hexane/CHCl3/CH3OH, as described in ref 29. b NNAL-MBIC analyzed using HPLC system F and MMPB using system E.

Figure 2. Reverse phase LC-MS/MS analysis of diastereomeric MMPB formed from hydroxy acid produced in the rat liver microsomal metabolism of (S)-NNAL. Derivatization was carried out with (R)-(+)-MBIC. Selected reaction monitoring of m/z 343178 for (A) standard (R,R)- and (S,R)-MMPB, (B) MMPB produced from hydroxy acid formed in the metabolism of (S)NNAL, and (C) co-injection of the compounds described for panels A and B. The right y-axis shows MS intensity units; E+04 2.735 means 2.735 × 104.

Results In a recent study, we determined the absolute configuration of NNAL enantiomers using Mosher’s acid (29). The absolute configuration of hydroxy acid enantiomers (Figure 1) had not been previously established. This was accomplished here by metabolism of (S)-NNAL to hydroxy acid, using rat liver microsomes. The chiral center was not affected under these conditions, as determined by recovery and analysis of unreacted (S)-NNAL. Hydroxy acid formed in this reaction was converted to its methyl ester and derivatized with (R)-(+)-MBIC. The resulting MMPB diastereomers (Figure 1) were analyzed by reverse phase LC-MS/MS. As shown in Figure 2B, a single MMPB diastereomer was detected; this must be (S,R)-MMPB. This peak coeluted with the second of the two MMPB diastereomers produced in the derivatization of racemic hydroxy acid with (R)-(+)-MBIC (Figure 2A,C). Therefore, the second eluting peak in Figure 2A is (S,R)MMPB and the first eluting peak (R,R)-MMPB. The same order of elution is observed under the normal phase conditions used in the studies described below, in which derivatization was carried out with (S)-(-)-MBIC (Table 1). Therefore, in those studies, the first eluting peak is

Figure 3. Representative radiochromatograms obtained upon reverse phase HPLC analysis of (A) urine of rats treated with NNN, analyzed without derivatization, and (B) urine of rats treated with nicotine in which hydroxy acid was analyzed as its methyl ester.

(S,S)-MMPB and the second is (R,S)-MMPB. These results are consistent with those obtained upon derivatization of NNAL with MBIC, yielding NNAL-MBIC (Figure 1 and Table 1). The (S,S)- and (R,R)-diastereomers elute before the (S,R)- and (R,S)-diastereomers under both normal and reverse phase HPLC conditions. This is reasonable considering the similarities in structure of MMPB and 4-(methylnitrosamino)-1-(R-methylbenzylcarbamoyl)-1-(3-pyridyl)butane (NNAL-MBIC) (Figure 1), further supporting our assignment. Incubation of (R)-NNAL with rat liver microsomes under the conditions used above did not produce detectable amounts of hydroxy acid (data not shown). Rats treated with [5-3H]NNK, [5-3H]NNN, [5-3H]keto acid, or [2′-14C]nicotine excreted hydroxy acid in their urine. The urine of the rats treated with [5-3H]NNK or [5-3H]NNN was analyzed for hydroxy acid directly by radioflow HPLC (Figure 3A). Hydroxy acid in the urine of rats treated with [5-3H]keto acid or [2′-14C]nicotine was analyzed after conversion to its methyl ester, as illustrated in Figure 3B for the [2′-14C]nicotine-treated rats. The results of these studies are summarized in Table 2. It is apparent that nitrosamine metabolism to hydroxy acid is considerably more extensive than that of either keto acid or nicotine. The percentages of the total dose of NNK or NNN metabolized to hydroxy acid were 12.4 and 31.8, respectively, while the corresponding figures for rats treated with keto acid and nicotine were only 1.10 and 0.105. Thus, NNK-derived hydroxy acid levels were 12 and 125 times greater than those produced

Metabolism of Nicotine and Derived Nitrosamines Table 2. Excretion of Hydroxy Acid in the Urine of Rats Treated with NNK, NNN, Keto Acid, or Nicotinea % of dose

compound administered

excreted in urine

hydroxy acid in urine

NNK NNN keto acid nicotine

81.5 ( 14.6 66.6 ( 8.51 94.8 ( 16.6 71.0 ( 7.80

12.4 ( 3.2 31.8 ( 4.9 1.10 ( 0.11b 0.105 ( 0.026b

a

Chem. Res. Toxicol., Vol. 12, No. 2, 1999 169 Table 3. Stereoselectivity of Hydroxy Acid Formation from NNK, NNN, Keto Acid, and Nicotine in Ratsa compound rat (S,S)-MMPB (R,S)-MMPB administered no. (dpm)b (dpm)b NNK

1 2 3

940 487 1430

88 NDc 491

NNN

1 2 3

903 893 840

1630 1570 1540

keto acid

1 2 3

308 ND ND

2940 1790 2170

nicotine

1 2 3

114 379 236

2400 3050 1960

[5-3H]NNK,

Groups of three F-344 rats were treated with [5-3H]NNN, [5-3H]keto acid, or [2′-14C]nicotine by iv injection as described in the Experimental Section. Values are mean ( SD. b Determined as hydroxy acid methyl ester.

(S)-hydroxy acid (% of total) 91 100 74 mean ( SD, 88 ( 13 36 36 36 mean ( SD, 36 ( 0 9 0 0 mean ( SD, 3 ( 5 4.5 11 11 mean ( SD, 8.8 ( 3.8

a F-344 rats were treated with [5-3H]NNK, [5-3H]NNN, [5-3H]keto acid, or [2′-14C]nicotine by iv injection as described in the Experimental Section. Hydroxy acid was isolated from urine and reacted with (S)-(-)-MBIC. Diastereomeric MMPB was quantified by HPLC with liquid scintillation counting (see Figure 4A-D). b Values corrected on the basis of (S)-(-)-MBIC (96% ee). c ND means not detected (e100 dpm).

the (S)-isomer was below the limit of detection, while for nicotine, the (S)-isomer comprised a mean of 8.8% of the total hydroxy acid.

Discussion

Figure 4. Normal phase HPLC analysis of diastereomeric MMPB derivatives of hydroxy acid from urine of rats treated with (A) [5-3H]NNK, (B) [5-3H]NNN, (C) [5-3H]keto acid, and (D) [2′-14C]nicotine. Derivatization was carried out with (S)(-)-MBIC. The histogram represents radioactivity, and the UV trace represents standard (S,S)- and (R,S)-MMPB. There is a 30 s delay between detection by UV and radioactivity.

from keto acid and nicotine, respectively. The extent of NNN metabolism to urinary hydroxy acid was 30 and 300 times greater than that of keto acid or nicotine. The stereochemistry of the hydroxy acid enantiomers present in the urine was determined by conversion to their methyl esters followed by derivatization with (S)(-)-MBIC, yielding diastereomeric MMPB. The first and second eluting HPLC peaks were assigned as (S,S)MMPB and (R,S)-MMPB, respectively, on the basis of the considerations discussed above. The results of these analyses are summarized in Figure 4A-D and Table 3. The urine of the rats treated with NNK contained predominantly (S)-hydroxy acid; the level of the (R)isomer was below the limit of detection in one sample, and (S)-hydroxy acid comprised 74 and 91% of the total hydroxy acid in the other two urine samples. The urine of the rats treated with NNN contained substantial amounts of both (S)- and (R)-hydroxy acid. These results contrast sharply with those obtained in the analyses of urine from rats treated with keto acid or nicotine. In both cases, (R)-hydroxy acid was the major enantiomer detected. In two of the three keto acid samples, the level of

The results of this study demonstrate that, in rats, the conditions necessary for potentially distinguishing NNNor NNK-derived hydroxy acid from nicotine-derived hydroxy acid are fulfilled. Thus, we established the following: (1) hydroxy acid is a minor urinary metabolite of nicotine, accounting for only 0.1% of the dose; (2) in rats treated with keto acid or nicotine, urinary (R)-hydroxy acid is produced with high stereoselectivity; (3) reduction of keto acid to hydroxy acid is a minor pathway of NNK and NNN metabolism, which follows from the observed stereoselectivity; and (4) in rats treated with NNK, (S)hydroxy acid is the major enantiomer in urine, while both (R)- and (S)-hydroxy acid are urinary metabolites of NNN. Thus, if similar pathways exist in humans, urinary (S)-hydroxy acid could potentially be used as a biomarker of NNK and NNN R-hydroxylation. Quantitative aspects of this hypothesis are discussed further below. Our results are generally consistent with previous studies of the metabolism of nicotine, NNK, and NNN and provide some new insights about metabolic pathways in rats. McKennis and co-workers were the first to identify hydroxy acid as a metabolite of keto acid, nicotine, and norcotinine (15-17). Rats, dogs, and rabbits treated with keto acid excreted unchanged keto acid as well as hydroxy acid in the urine (17). Levels of hydroxy acid were not quantified directly, but on the basis of conversion to the corresponding lactone, 5-(3-pyridyl)tetrahydrofuran-2-one, it might have accounted for 1-10% of the keto acid dose. In rats and dogs, the (-)-lactone of unknown absolute configuration was the major product, whereas racemic lactone was isolated from the urine of rabbits (17). On the basis of our results, one could speculate that the absolute configuration of the (-)lactone is (R). In a previous study in which unlabeled keto acid was administered to rats, we did not detect hydroxy acid in the urine, presumably because of the

170 Chem. Res. Toxicol., Vol. 12, No. 2, 1999

relative insensitivity of our method (8). Schepers et al. (24) studied the metabolism of nicotine in non-pretreated and Aroclor-pretreated Sprague-Dawley rats. Amounts of keto acid (10% of the dose) and hydroxy acid (2%)were estimated in urine of non-pretreated rats; the corresponding amounts in Aroclor-pretreated animals were 14 and 2%, respectively (24). Since hydroxy acid was not well resolved from a larger peak in their chromatographic system, the estimated levels of this metabolite could be high. Kyerematen et al. (21, 22) treated male SpragueDawley rats with nicotine and identified keto acid as a urinary metabolite, accounting for 2% of the dose; hydroxy acid was not identified. Collectively, the available results indicate that hydroxy acid is only a minor urinary metabolite of nicotine in rats and that keto acid is its immediate precursor. Previous studies demonstrated that urinary keto acid comprised 17-40% of the NNK dose in rats (9-11, 31). Since conversion of keto acid to urinary hydroxy acid occurs in only 1% yield, it is unlikely that this reaction accounts for much of the hydroxy acid found in the urine of NNK-treated rats. The results of the present investigation are consistent with this supposition. Thus, 88% of the total hydroxy acid present in the urine of NNKtreated rats is (S)-hydroxy acid, whereas hydroxy acid formed from keto acid is predominantly (R)-hydroxy acid. Therefore, of the possible modes of hydroxy acid formation from NNK illustrated in Figure 1, the routes through NNAL clearly predominate. Thus, (S)-hydroxy acid found in the urine of NNK-treated rats is most likely formed from (S)-NNAL via R-hydroxylation either at the R-methylene carbon, resulting in lactol, or at the R-methyl carbon, yielding diol. Both are converted in vivo to urinary hydroxy acid (8). The pharmacokinetics of formation of NNAL enantiomers from NNK, and their further metabolism, is poorly understood at present. However, it is known that (S)-NNAL is the major enantiomer found in the urine of NNK-treated rats, and that (S)-NNALGluc is the predominant NNAL-Gluc diastereomer in rat urine (29). These results appear to be consistent with our finding that (S)-hydroxy acid predominates in the urine of NNK-treated rats. As shown in Scheme 2, there are two possible routes to hydroxy acid from NNN metabolism: via keto acid or via 5′-hydroxylation. Previous studies showed that urinary keto acid comprises 13-31% of the NNN dose in rats (8). Therefore, if the 1% yield of hydroxy acid from keto acid is considered, the major source of NNN-derived urinary hydroxy acid must be 5′-hydroxylation of NNN. The NNN used in this study was racemic. 5′-Hydroxylation could therefore produce two pairs of diastereomers of 5′-hydroxyNNN (9). However, asymmetry at the 5′position would be lost in the spontaneous ring opening of 9 to diazohydroxide 10. Diazohydroxide 10 would thus be comprised of equal amounts of (R)- and (S)-enantiomers, as would lactol. Oxidation of (R)- or (S)-lactol to hydroxy acid could proceed at different rates. Thus, our finding of 64% (R)-hydroxy acid and 36% (S)-hydroxy acid in the urine of the NNN-treated rats presumably results from more rapid oxidation of (R)-lactol than (S)-lactol. This requires further investigation. On the basis of the results obtained here, one could estimate the levels of hydroxy acid which might be present in a smoker’s urine, although we recognize that differences in smoke yields of nicotine versus nitrosamines based on individual smoking characteristics and

Trushin and Hecht

differences in metabolism between rats and humans could substantially alter these estimates (32, 33). A typical cigarette delivers about 1000 µg of nicotine, 150 ng of NNK, and 200 ng of NNN, although these values vary widely (25, 26, 34-36). Therefore, a smoker who smokes 20 cigarettes per day might be exposed to 20 mg of nicotine. On the basis of the rat studies, 0.1%, or 20 µg, would appear in the 24 h urine as hydroxy acid, and 8.8% of this, or 1.8 µg, would be (S)-hydroxy acid. Similarly, NNN and NNK together would produce about 0.9 µg of (S)-hydroxy acid in the 24 h urine. These calculations suggest that the amount of (S)-hydroxy acid produced from NNN and NNK could be large enough compared to that derived from nicotine to allow its use as a biomarker of NNN and NNK R-hydroxylation. Therefore, this concept was further pursued in a study of hydroxy acid levels in the urine of smokers, as described in the following paper.

Acknowledgment. This study was supported by Grant CA-44377 from the National Cancer Institute.

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