metabolism in the f344 rat of 4-( w-methyl- a/-nitrosamino)-1 ......metabolism in the f344 rat of...

[CANCER RESEARCH 40, 4144-4150, November1980]0008-5472/80/0040-0000 $02.00

Metabolism in the F344 Rat of 4-( W-Methyl- A/-nitrosamino)-1-(3-pyridyl)-1butanone, a Tobacco-specific Carcinogen1

Stephen S. Hecht,2 Ruth Young, and Chi-hong B. Chen3

Division of Environmental Carcinogenesis, Naylor Dana Institute for Disease Prevention, American Health Foundation, Valhalla, New York 70595

ABSTRACT

The metabolism of the tobacco-specific carcinogen, 4-(/V-methyl-A/-nitrosamino)-1-(3-pyridyl)-1 -butanone (NNK), was

studied in the F344 rat, in which it induces tumors of the nasalcavity, liver, and lung. When NNK was incubated with rat livermicrosomes and a reduced nicotinamide adenine dinucleotidephosphate-generating system, metabolites resulting from a-hydroxylation, carbonyl reduction, and /V-oxidation were isolated. a-Hydroxylation at the mÃ©thylÃ¨necarbon gave 4-oxo-4-(3-pyridyl)butanal, whereas a-hydroxylation at the methyl carbon gave myosmine and 4-hydroxyl-1-(3-pyridyl)butan-1-one.The formation of these products involved the intermediacy ofelectrophilic diazohydroxides or carbonium ions which may beproximate or ultimate carcinogens of NNK. Carbonyl reductiongave 4-(/V-methyl-/V-nitrosamino)-1-(3-pyridyl)butan-1-ol and/V-oxidation yielded 4-(/V-methyl-/V-nitrosamino)-1-(3-pyridyl-A/-oxide)-1 -butanone. When rats were gavaged with NNK, themicrosomal products of a-hydroxylation were not detected inthe 48-hr urine. Compounds which presumably resulted fromfurther oxidation or reduction of these products, 4-oxo-4-(3-pyridyl)butyric acid, 4-hydroxy-4-(3-pyridyl)butyric acid, and4-hydroxy-1-(3-pyridyl)butan-1-ol, were isolated. 4-(A/-Methyl-A/-nitrosamino)-1-(3-pyridyl)butan-1-ol and 4-(/V-methyl-A/-ni-trosaminoM -(3-pyridyl-/V-oxide)-1 -butanone were also urinary

metabolites.

INTRODUCTION

Tobacco and tobacco smoke contain relatively high concentrations of NNK4 (Chart 1,7) and NNN, which are derived from

the principle tobacco alkaloid, nicotine (10, 14). NNN, NNK,and a related tobacco-specific nitrosamine, A/'-nitrosoanata-bine, may be among the causative factors in the tobacco-

related cancers (21). Whereas the levels of NNK in tobaccoand mainstream and sidestream tobacco smoke are somewhatlower than those of NNN, it is apparently more carcinogenicthan NNN in mice and rats (12,13), as well as in Syrian goldenhamsters.5 NNK induces lung adenomas in strain A mice and

' This study was supported by Grant CA-12376 from the National CancerInstitute. This is Paper 31 of the series, "A Study of Chemical Carcinogenesis."

2 Recipient of National Cancer Institute Research Career Development Award

5K04CA00124. To whom requests for reprints should be addressed.3 Recipient of National Institute of Environmental Health Sciences Award ESO-

2236.' The abbreviations used are: NNK, 4-(W-methyl-N-nitrosamino)-1-<3-pyridyl)-

1-butanone; NNN, N'-nitrosonornicotine; NMR, nuclear magnetic resonance; MS,mass spectrum; GLC-MS, combined gas-liquid chromatography mass spectrom-etry; GLC, gas-liquid chromatography; TLC, thin-layer chromatography; HPLC,high-pressure liquid chromatography; m, multiplet; t. triplet; br s, broad singlet;s. singlet; NNK-1-N-oxide, 4-(W-methyl-W-nitrosamino)-1-(3-pyridyl-W-oxide>-1-

butanone.5 D. Hoffmann, A. Castonguay, A. Rivenson, and S. S. Hecht, manuscript in

preparation.Received April 15, 1980; accepted August 1, 1980.

nasal cavity, liver, and lung tumors in F344 rats (12, 13). Inboth of these experiments, tumors were induced in a highpercentage of the animals, indicating that NNK is a fairly potentcarcinogen. In previous studies on the tobacco-specific nitros-

amines, we have described the metabolism of NNN and haveobtained evidence supporting its activation by a-hydroxylation(2,11). Presently, we report the identification of metabolites ofNNK in the F344 rat. a-Hydroxylation, carbonyl reduction, and/V-oxidation of NNK have been observed.

MATERIALS AND METHODS

Apparatus

Melting points were determined on a Thomas Hoover capillary melting point apparatus and are uncorrected. IR spectrawere run on a Perkin-Elmer Model 267 grating IR spectropho-tometer. NMR spectra were determined with a Hitachi Perkin-Elmer Model R-24 spectrometer in CDCU solution and arereported as ppm downfield from tetramethylsilane as internalreference. MS and GLC-MS were run with a Hewlett-PackardModel 5982A dual-source instrument with a membrane separator. High-resolution MS's were obtained by Shrader Analyti

cal and Consulting Laboratories, Inc., Detroit, Mich., on an AEI-MS-30 instrument. GLC was done on a Hewlett-Packard Model

5830A instrument equipped with a flame ionization detectorand the following columns: Column A, 6-ft x 0.25-inch 10%Carbowax 20M-TPA on Gas-Chrom Q; and Column B, 6-ft x0.125-inch 10% UCW98 on Gas-Chrom Q with helium as

carrier gas. TLC was done with silica gel 60F glass plates (EMLaboratories, Elmsford, N. Y.) HPLC was carried out with aWaters Associates Model ALC/GPC-204 high-speed liquid

Chromatograph equipped with a Model 6000A solvent deliverysystem, a Model 660 solvent programmer, a Model U6K sep-

tumless injector, and a Model 440 UV/visible detector (WatersAssociates, Milford, Mass.). Elemental analyses were performed by Galbraith Laboratories, Inc., Knoxville, Tenn.

Cell disruption was performed with a Polytron homogenizer(Willems type; Kinematic GmbH, Lucerne, Switzerland). Cen-trifugation was done with a Sorvall RC2-B centrifuge and a

Spinco Model L ultracentrifuge.

HPLC Analyses

Solvent systems, gradients, and columns were used as follows. System A: two 3.9-mm x 30-cm Cie-ÃÃBondapakcolumns

(Waters Associates) were used in series. The gradient wasSolvent A for 10 min and linear to 60% Solvent B in 60 min at1 ml/min. Solvent A was 30 ml of 1 M CH3CO2H, 12 ml of 1 MNaOH, and 18 ml of 1 M NaCI in a total volume of 0.5 liter ofH2O, pH 4.5. Solvent B was CH3OH/H2O, 1/1. System B: thesame as System A, except the pH of Solvent A was adjusted to6.0 with 1 M NaOH. System C: a 3.9- x 30-cm ds-fiBondapak

4144 CANCER RESEARCH VOL. 40

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Metabolism of NNK

column was used. The gradient was linear from 40% SolventB to 100% Solvent B in 30 min at 2 ml/min. Solvent A was0.02% phosphate buffer, pH 5.5, and Solvent B was CH3OH/H2O, 70/30.

Chemicals

NADPH, glucose 6-phosphate, and glucose-6-phosphate de-

hydrogenase were obtained from Sigma Chemical Co., St.Louis, Mo. Aroclor 1254 was obtained from Analabs, Inc.,Hamden, Conn. Regisil RC-2 was procured from Regis Chemical Co., Morton Grove, III. Sodium borohydride and trifluoroac-

etic acid were purchased from Fisher Scientific Co., Springfield, N. J. Sodium cyanoborohydride, m-chloroperbenzoicacid, and N,A/'-dicyclohexylcarbodiimide were obtained from

Aldrich Chemical Co., Milwaukee, Wis.NNK and the following metabolites were synthesized as

described previously: NNK (Chart 1,7), 4-hydroxy-1-(3-pyri-dyl)butan-1-one (Chart 1, 14; Ref. 8), myosmine (Chart 1,73;Ref. 15), 4-(3-pyridyl)-4-oxobutyric acid (Chart 1,75; Ref. 17),and 4-hydroxy-4-(3-pyridyl)butyric acid (Chart 1,76; Ref. 17).

4-Hydroxy-1-(3-pyridyl)butan-1-ol (Chart 1, 17). NaBH4(0.38 g, 7.9 mmol) was added slowly with stirring to a solutionof 14 (0.21 g, 1.3 mmol) in 5 ml CH3OH containing 0.2 mlglacial acetic acid. The mixture was stirred for 2 hr at 25Â°and1 hr at 50Â°. The CH3OH was removed, and 10 ml H2O was

added. The resulting solution was neutralized with 10 N NaOHand extracted 4 times with 25 ml of ethyl acetate. The ethylacetate layers were combined, dried (Na2SO4), and concentrated, giving 0.19 g (90%) 77 as an oil which was >95% pureby TLC (silica gel; CHCI3/CH3OH, 10/1; RF 0.11). HPLC(System A) retention volume was 36 ml and HPLC (System B)retention volume was 46 ml. The spectral properties were thefollowing. IR (film): 3300 (broad), 1600, 1580 cm'1. NMR: S

8.48 to 8.13 (m, 2H), 7.76 to 7.42 (m, 1H), 7.30 to 6.95 (m,1H, pyridylâ€”H); 4.80 to 3.90 (t + br s, 3H, CHOH, CH2OH),3.50 (t, 2H, CH2OH), 1.95 to 1.30 (m, 4H,â€”CH2CH2). MSm/e: 167 (M +, 5), 122 (15), 108 (100), 80 (33).

C9H,3NO2Calculated: C 64.65, H 7.84, N 8.38Found: C 64.41, H 7.95, N 8.84

NNK-1-N-oxJde (Chart 1, 3). m-Chloroperbenzoic acid(0.107 g, 0.6 mmol) was added in one portion at 20Â° to a

solution of NNK (0.10 g, 0.5 mmol) in 5 ml CHCI3, and themixture was stirred at 20Â°for 24 hr. The reaction mixture was

washed twice with 25 ml saturated aqueous NaHCO3, dried(Na2SO4), and concentrated. The residue was purified by silicagel chromatography with elution by CHCI3 and 1% CH3OH inCHCI3 yielding 3 (0.09 g; 83%) which was pure by TLC (silicagel; CHCI3/CH3OH, 15/1 ; RF0.13). HPLC (System A) retentionvolumes were 45 ml (E isomer) and 47 ml (Z isomer) and HPLC(System B) retention volumes were 52 ml (E isomer) and 54 ml(Z isomer). The spectral properties were the following. IR(Nujol): 1690, 1590 cm-1. NMR: S8.68, (s, 1H), 8.30 (doublet,

1H), 7.8 to 7.2 (m, 2H, pyridyl-H); 4.20 (t, 1.5H,CH2â€”Nâ€”N=O, E isomer), 3.80 to 3.50 (s + t, 1.3H,CH2â€”Nâ€”N=O + CH3â€”Nâ€”N=O, Z isomer), 3.2 to 1.7 (m,4.3H, CH3â€”Nâ€”N=O, E isomer + CH2â€”C=O), 2.2 (quintet,2 H, CH2â€”CH2â€”CH2);MS m/e: 223 (M +, 8), 193 (100), 122

(82), 106 (45), 78 (44), 70 (53).

Calculated: C 53.81, H 5.87, N 18.82Found: C 53.66, H 6.01, N 18.65

4-Oxo-4-(3-pyridyl)butanal (Chart 1, 8). To a solution ofketo alcohol 14 (0.99 g, 6 mmol) in 20 ml of dry benzene and20 ml of dry dimethyl sulfoxide were added sequentially anhydrous pyridine (0.48 ml), redistilled trifluoroacetic acid (0.24ml), and /V.N'-dicyclohexylcarbodiimide (3.7 g, 18 mmol). The

reaction mixture was tightly stoppered and allowed to stand atroom temperature for 65 hr. After this time, 60 ml of benzeneand 50 ml of H2O were added, and the mixture was stirred andfiltered. The H2O layer was separated and extracted 3 timeswith 50 ml of CHCI3. The benzene layer was concentrated,redissolved in 200 ml of CHCI3, and washed twice with 50 mlof H2O. The CHCI3 layers were combined, dried (Na?SO4), andconcentrated, giving a residue which was purified by preparative TLC on 1-mm silica gel plates with elution by CHCI3/CH3OH, 15/1 (Rf 0.26). The aldehyde 8 was obtained as an oilin 20% yield. The spectral properties were the following. IR(film): 1720, 1690, 1590 cnr'. NMR: S 9.85 (s, 1H, CHO),

9.14 (br s, 1H); 8.71 (br s, 1H), 8.22 (m, 1H), 7.6 to 7.3 (m,1H, pyridylâ€”H); 3.5 to 2.7 (m, 4H, CH2â€”CH2).MS m/e: 135

(30), 121 (36), 106 (100), 78 (72).The 2,4-dinitrophenylhydrazone 70 was prepared from 8 by

reaction with 2,4-dinitrophenylhydrazine reagent (m.p. 212-214Â°). Purity was established by HPLC using System C. Re

tention volume was 88 ml. The spectral properties were thefollowing. MS m/e: 523 (MM ), 325 (50), 291 (100), 79 (61 ),78(67.1).

C2,H)7N9O8-H2OCalculated: C 46.58, H 3.53, N 23.28Found: C 46.38, H 3.21, N 22.87

4-(N-Methyl-N-nitrosamino)-1 -(3-pyridyl)butan-1 -ol (Chart1, 2). A mixture of NNK (0.22 g, 1.1 mmol) and NaBH4 (0.037g, 1 mmol) in 8 ml CH3OH was adjusted to pH 4 by addition of1 N HCI and stirred for 3 hr at room temperature. The CH3OHwas removed, the residue was dissolved in H2O, and the pHwas adjusted to 7. The resulting mixture was extracted withCHCI3 (5 x 25 ml), and the CHCI3 extracts were combined,dried (Na2SO4), and concentrated. The residue was purified bysilica gel chromatography with elution by CHCI3/CH3OH (15/1) yielding 0.22 g (95%) 2, pure by TLC (silica gel; CHCI3/CH3OH, 15/1; RF 0.22), HPLC (System A) retention volumeswere 49 ml (E isomer) and 51 ml (Z isomer) and HPLC (SystemB) retention volumes were 67 ml (E isomer) and 69 ml (Zisomer). The spectral properties were the following. IR (film):3600 to 3100 (broad), 1580cm-'. NMR: S 8.7 to 8.3 (m, 2H),

7.9 to 7.6 (m, 1H), 7.4 to 7.1 (m, 1H, pyridylâ€”H); 5.0 to 4.6(m, 1H, CHOH), 4.4 to 3.4 (t + s + m, 3.5H, CH2â€”Nâ€”Nâ€”O,E + Z isomer, CH3â€”Nâ€”N=O, Z isomer, OH), 2.99 (s, 2.5H,CH3â€”Nâ€”N=O, E isomer), 2.0 to 1.5 (m, 4H, CH2CH2). High

resolution MS m/e: 192.1168, C,0H,4N3O (4); 179.1191,C,oH15N2O(27); 161.1081, C10H13N2(20); 148.0753, C9H,0NO(89); 108.0447, C6H6NO(100); 106.0291, C6H4NO (60).

Metabolism of NNK

In Vitro

Liver microsomes were obtained from male F344 rats that

NOVEMBER 1980 4145



S. S. Hecht et al.

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Chart 1. Metabolism of NNK in the F344 rat. Compounds 2, 3, 8, Ã•3, and 14 were detected in vitro; 8 may also be converted to 14 under the incubationconditions. Compounds 2, 3, 15, 16, and 77 were detected in the urine. Brackets represent hypothetical intermediates. DNPH, 2,4-dinitrophenylhydrazine reagent.

had been given i.p. injections of Aroclor (500 mg/kg) 4 daysprior to sacrifice. Microsomes were prepared as describedpreviously (9). Incubations were carried out at 37Â°for 1 hr in

five 25-ml Erlenmeyer flasks. Each flask contained 2 ml ofmicrosomal suspension (12.0 mg/ml protein), 20 mg (0.1mmol) of NNK, 2.5 mg (0.003 mmol) of NADPH, 50 units ofglucose-6-phosphate dehydrogenase, 0.05 mmol of glucose6-phosphate, and 0.05 mmol of MgCI2. The total volume wasbrought to 10 ml with 0.1 M Tris-HCI buffer, pH 7. After 1 hr,the reaction was stopped by addition of ethanol (10 ml). Themixture was centrifuged at 10Â°to remove protein. In controls,

boiled enzyme was used. Three methods were used for analysis.

Method 1. A 75% aliquot of the supernatant was concentrated to remove ethanol, and the aqueous residue was extracted 4 times with CHCI3. The CHCI3 extracts were dried(Na2SOâ€ž),concentrated, and analyzed by TLC (silica gel;CHCI3/CH3OH, 15/1) and MS or by GLC and GLC-MS usingColumn A, programmed for 140Â°for 8 min and then 4Â°/min to200Â° at a flow rate of 40 ml/min. Identity of retention times

between reference compounds and metabolites was established by coinjection.

Method 2. A 5% aliquot of the supernatant was analyzed for2 and 3 by HPLC using System A. Identity of retention volumesbetween reference compounds and metabolites was established by coinjection.

Method 3. A 20% aliquot of the supernatant was treated with2,4-dinitrophenylhydrazine reagent, 4 ml of 0.15 M reagent per

flask, and allowed to stand overnight. The resulting mixturewas neutralized and analyzed for 10 by HPLC using System C.The identity of retention volumes of reference and metabolic70 was established by coinjection.

In Vivo

Two male F344 rats were given 450 mg NNK per kg intrioctanoin by gavage. The 48-hr urine was collected at â€”76Â°

and extracted 5 times with ethyl acetate. The extracts weredried (Na2SOâ€ž),concentrated, and analyzed by TLC (silica gel;CHCI3/CH3OH, 15/1) and HPLC (System A). MS were obtained on bands corresponding to standards. The extractedaqueous portion of the urine was lyophilized and sonicallydispersed in CH3OH. An aliquot of the CH3OH solution wasconcentrated, silylated with Regisil RC-2 for 20 min at 80Â°,andexamined by GLC and GLC-MS using Column B (150Â° for 8min and then 4Â°/min to 240Â°with a flow rate of 50 ml/min). A

portion of the ethyl acetate extract was also silylated andanalyzed by GLC-MS. Identity of retention times of referencecompounds and metabolites was established by coinjection.

Two male F344 rats were each given [1-14C] NNK6 (2.0 x107 dpm, 4.2 mCi/mmol) in trioctanoin by gavage. The 48-hrurine was collected at -76Â° and lyophilized. The residue was

sonically dispersed in CH3OH, and the resulting CH3OH solution was analyzed by HPLC using System B. Unlabeled standards were added as UV markers. Retention volumes of standards: 76, 18 ml; 75, 32 ml; 77, 46 ml; 3(E isomer), 52 ml; 3(2.isomer), 54 ml; 2(E isomer), 67 ml; 2(Z isomer), 69 ml; NNK(Eisomer) 79 ml; NNK(Z isomer) 81 ml.

RESULTS

Chart 1 summarizes the metabolism of NNK in the F344 rat.For the in vitro studies, liver microsomes from Aroclor-pre-

Ã¤A. Castonguay, S. S. Hecht, and D. Hoffmann, manuscript in preparation




Metabolism of NNK

S

15 20

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25 30

Chart 2. GLC trace of metabolites of NNK extracted with CHCI3 from micro-

somal incubations. The peaks eluting at 21.4 and 25.5 min were also observedin controls. See "Materials and Methods" for details.

treated rats were used in order to facilitate identification ofmetabolites. Analysis by TLC of CHCI3 extracts of incubationmixtures led to identification of 4-(N-methyl-A/-nitrosamino)-1 -(3-pyridyl)butan-1-ol (Chart 1, 2), NNK-1-N-oxide (Chart 1, 3),4-oxo-4-(3-pyridyl)butanal (Chart 1, 8), myosmine (Chart 1,73), and 4-hydroxy-1 -(3-pyridyl)butan-1 -one (Chart 1, 74). The

isolated metabolites, which were not observed in control incubations, had MS's identical to those of synthetic standards.

Analysis of the CHCI3 extracts by combined GLC-MS providedfurther confirmation for the presence of 8, 73, and 74, asshown in Charts 2 and 3. Formation of metabolites was quantified by GLC (73 and 14) and by HPLC (Chart 4, 2 and 3).GLC was not a reliable method for quantitation of 8, in contrastto 73 and 74. The formation of keto aldehyde 8 was thereforequantified by HPLC of the corresponding 2,4-dinitrophenylhy-

drazone 70. The results are summarized in Table 1. The effectsof Aroclor induction on the extents of formation of each metabolite are not known.

To determine urinary metabolites, F344 rats were gavaged

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NOVEMBER 1980 4147



S. S. Hecht et al.

with NNK or [1 -14C]NNK, and thÃ©48-hr urine was analyzed. An

ethyl acetate extract of the urine contained 2 which was identified by its NMR spectrum and MS after isolation by preparativeTLC. The ethyl acetate extract also contained NNK-1-A/-oxide(3), which was identified by its MS, and diol 77, which wascharacterized by GLC-MS of the extract after silylation. The

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Chart 4. HPLC trace of NNK-1 -N-oxide (Chart 1. 3) and 4-(N-methyl-N-nitro-samino)-1-(3-pyridyl)butan-1-ol (Chart 1, 2) formed in vitro from NNK. See

Materials and Methods' for details.

Table 1Formation of metabolites of NNK in vitro

NNK (96.6 /imol) was incubated for 60 min at 37Â°with liver microsomes fromAroclor-pretreated rats (24 mg protein) and an NADPH-generating system. Metabolites were quantified by GLC (Ã•3 and 14) or HPLC (2, 3, and 8).

Metabolite /Â«noiformed

4-(N-Methyl-W-nitrosamino)-1 -(3-pyridyl)butan-1 -ol (Chart 1, 2) 3.74NNK-1 -N-oxide (Chart 1, 3) 0.274-Oxo-4-(3-pyridyl)butanal (Chart 1. 8)a 1.84

Myosmine (Chart 1, J3) 0.614-Hydroxy-1 -(3-pyridyl)butan-1 -one (Chart 1, 14) 2.79

a As the 2.4-dinitrophenylhydrazone 10.

MS's of these metabolites are shown in Chart 5. Myosmine

(73), keto alcohol 14, and keto aldehyde 8 were not detectedin the ethyl acetate extracts of the urine. Silylation of theextracted aqueous portion of the urine resulted in identificationof keto acid 75 and hydroxy acid 76, as the correspondingtrimethylsilyl derivatives (Charts 6 and 7). Urine was analyzedby HPLC after administration of [1-14C]NNK, and the approxi

mate percentage of excretion of the dose of each metabolitewas determined as follows: 2 (10%); 3 (3%); 15 (38%); 76(14%); and 77 (4%). Less than 1% unmetabolized NNK waspresent in the urine.

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Chart 6. GLC trace of the silylated water-soluble components of urine fromrats treated with NNK. See "Materials and Methods" for details.

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Metabolism of NNK

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Chart 7. MS of reference compounds )5 and 76 (trimethylsilyl derivatives) and of 15 and 16 (trimethylsilyl derivatives) isolated from the urine of rats treated withNNK.

DISCUSSION

The results of this study provide evidence for 3 types ofmetabolic pathways for NNK in the F344 rat: a-hydroxylationto 8, 73, and 14; carbonyl reduction to 2; and /V-oxidation to3. Assignment of 8, 73, and 74 as products of metabolic a-hydroxylation is based on the chemistry of the unstable a-

hydroxynitrosamines and related model compounds (1, 7, 20,22). a-Hydroxylation of NNK gives the a-hydroxy-4-(N-methyl-A/-nitrosamino)-1-(3-pyridyl)-1-butanone intermediates 4 and

5, which are expected to decompose to diazohydroxides (7from 4 and 9 from 5) and aldehydes (formaldehyde from 4 andketo aldehyde 8 from 5). The chemistry of diazohydroxide 7,as generated from the nitrosourethane 6, was previously studied. The major product of decomposition of 7 was keto alcohol

14; myosmine (Chart 1, 73) was also detected (7). Thus,identification of 8, 73, and 14 as metabolites of NNK in vitroprovides strong evidence for a-hydroxylation of NNK. We didnot attempt to identify the other expected products of a-hydroxylation, formaldehyde and methanol. Compounds 8, 73,and 14 were not detected as urinary metabolites of NNK.However, keto acid 75, hydroxy acid 76, and diol 77 wereidentified in the urine of NNK-treated rats. These metabolitesprobably are formed, at least partially, by further oxidationand/or reduction of 8 and 14.

a-Hydroxylation of NNK is a likely activation process. Hy-

droxylation of the mÃ©thylÃ¨negroup, giving intermediate 5,presumably results in formation of diazohydroxide 9, which isa methylating agent. Diazohydroxide 9 and/or a methyl car-

bonium ion are formed in vivo from dimethylnitrosamine, A/

NOVEMBER 1980 4149



S. S. Hecht et al.

methyl-/V-nitrosourea, and related compounds (3, 16, 19).

Since these carcinogens methylate DNA, NNK can also beexpected to be a methylating agent. Hydroxylation of the methylgroup of NNK leads to diazohydroxide 7 and possibly tocarbonium Â¡on12. Previous studies have shown that nitrosou-

rethane 6, which isa precursor to 7 and 72, is highly mutagenictoward Salmonella typhimuhum TA 100 in the absence of liverhomogenate (2). Thus, the electrophilic intermediates 7 and12, as well as 9, may be ultimate carcinogenic forms of NNK.

Inspection of Chart 1 reveals a common pathway in themetabolism of NNK and the related tobacco specific nitrosa-mine, NNN. a-Hydroxylation of NNN at the 2' position gives 2'-hydroxy-A/'-nitrosonornicotine (Chart 1, 11) resulting in for

mation of 13 and 14 as metabolites of NNN (2). Both NNN andNNK induce nasal cavity tumors in F344 rats, when administered by s.c. injection, but NNK also gives liver and lung tumors(13). Since NNN and NNK both yield oxobutyldiazohydroxidesor carbonium ions such as 7 and 12, but NNK is also amethylating agent, it is tempting to speculate that the liver andlung tumors induced by NNK may relate to its methylatingability. This hypothesis is presently under investigation.

Reduction of the NNK carbonyl is apparently a major metabolic process in the F344 rat, since 2 was a prominent metabolite isolated in vitro and in vivo. Carbonyl reduction has beenreported previously in metabolic studies on nitrosamines, including nitrosobis(2-oxopropyl)amine and related compounds,and in investigations of 3-acylpyridines (4, 5, 18). /V-oxidationhas been commonly observed in the metabolism of pyridineand nicotine derivatives, including NNN (6, 11). The possibleroles of carbonyl reduction and /V-oxidation in the activation ordetoxification of NNK are currently being evaluated in bioas-says of 2 and 3.

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1980;40:4144-4150. Cancer Res Stephen S. Hecht, Ruth Young and Chi-hong B. Chen Carcinogen-nitrosamino)-1-(3-pyridyl)-1-butanone, a Tobacco-specific

N-Methyl-NMetabolism in the F344 Rat of 4-(

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