Jump to:

Product Design

"Tobacco-Specific Nitrosamines: Formation from Nicotine In Vitro and During Tobacco Curing and Carcinogenicity in Strain A Mice"

Date: Apr 1978
Length: 6 pages
1000145870-5874A
Jump To Images
snapshot_pm 1000145870-1000145874A

Abstract

Examines formation of tobacco specific nitrosamines from nicotine. States that tobacco specific nitrosamines could be related to incidence of tobacco related cancers. Considers nicotine to be the major precursor for the carcinogen NNN. Studies tumorigenic activity of NNN,NNK, and NNA in Strain A mice. Finds that NNK resulted in more lung adenomas per mouse than did NNN.

Fields

Author
CHEN, CB
HECHT, SS
HIROTA, N
Hoffmann, Dietrich, Ph.D. (Biochemist, American Health Foundation, Plaintiff's Expert)
Plaintiff
ORNAF, RM
Tso, T.C., Ph.D. (PM Tobacco Working Group)
Defense
Keyword
Animal testing
Carcinogenic (Cancer-causing)
Tumorigenic
Smoke Constituent
Nicotine
Nitrosamines (N-nitrosamines)
Named Organization
BELTSVILLE RESEARCH FARM
Eastman Kodak Co. (Kodak) (Cigarette filter mfg from 1950s to 1994.)
Manufacturers of quality control equipment for cigarette packaging
HEWLETT-PACKARD
JACKSON LAB
NEW ENGLAND NUCLEAR
NUCLEAR-CHICAGO
PACKARD INSTRUMENT
PURINA LAB
WATERS ASSOCIATES
ALDRICH CHEMICAL
Subject
nicotine technology
Test/Animal Subject (Testing)
Cancer (Health Effects)

Document Images

Text Control

Highlight Text:

OCR Text Alignment:

Image Control

Image Rotation:

Image Size:

Page 1: ajf08e00
S Tobacco-Specific Nitrosamines: Formation From Nicotine In Vitro and During Tobacco ~ Curing and Carcinogenicity in Strain A Mice'` z• 3' Stephen S. Hecht, 4.5 Chi-hong B. Chen, 4 Norio H'trota, 4 Raphael M. O'rnaf,'' T. C. Tso, 6 and Dietrich . C Hoffmann, 4 ABSTRACT-The formation of tobacco-specific nitrosamines from the major tobacco alkaloid nicotine was examined. De- tached leaf tobacco was fed either [2'-*'C]nicotine or [2'- '•C]nornicotine and' air cured. The cured leaf was then analyzed for [2'-"C]N'-nitrosonornicotine ([2'-"C]NNN). The yield of [2'- "C]NNN was 0.007% from nornicotine and 0.009% from nicotine. Because the ratio of nicotine to nornicotine in conventional nicotine-type tobacco is 20-100:1, nicotine is considered to be the major precursor for the carcinogen NNN in tobacco. The formation of other nitrosamines from nicotine In vitro was then studied. Reaction of nicotine with, NaNO2 gave rise to NNN, as well as to two other nitrosamines, 4-(N-methyl'~N-nitrosamino)-1- (3-pyridyl)-1-butanone (NNK) and 4-(N-methyhW-nitrosamino)-4- (3-pyridyl)butanal: (NNA). Analysis of market products revealed the presence of NNK (0.6-2.4 µg/g) in chewing tobacco and snuff. The tumorigenic activity of NNN, NNK, and NNA in strain A mice was studied. NNK induced more lung adenomas per mouse than did NNN, whereas NNA was less active than NNN. 1n addition, two cases of undifferentiated carcinoma of the salivary giands occurred in the NNN' experimentat groups.-J N'atI Can- cer 1!nst 60: 819-824, 1978. Tobacco-specific nitrosamines, which are derived from the tobacco alkaloids, could be related to the incidence of tobacco-related cancers. The prototype of tobacco-specific nitrosamines is NNN, which could be formed fromleither nicotine (text-fig. 1) or nornicotine. NNN!, which induces nasal cavity and esophageal tu- mors in rats (1„2), respiratory tract tumors in hamsters (3), and lung adenomas in mice (4, 5), has been detected both in unburned tobacco (0.3-88,6 ppm)land in tobacco smoke (40-250 ng/cigarette) (5-12). NNN was not pres- ent in fresh tobacco, but was formed during curing (5). Various lines of evidence suggested that the major precursor for NNN in, tobacco was nicotine (5, 6, 13). This question has now been examined in more detail. In this report, we present evidence that nicotine is the major precursor for the tobacco-specific nitrosamine NNN'_ As previously suggested (9), at least two other nitrosamines, NN!K and NNA (text-fig. 1), can also be formed from nicotine; one of these, NNK, has been detected in tobacco. The tumorigenic activity of NN!N, NNK, and NNA in strain A mice was examined. MATERIALS AND METHODS7 Apparatus,-GLC was done on a Hewlett-Packard model #5711 idstrument (Hewlett-Packard Co., Palo Alto,, Calif.) equipped with a flame ionization detector and' two columns: column I, 6-footx 1/8-inch 10% Car- bowax 20M-TPA on Gas-Chrom Q; and column II, 6- footx 1/8-inch 10% Carbowax 20M-2% KOH on Chro- mosorb W with helium (60 ml/min) as carrier gas. GLC- MS (EIMS and CIMS) was done with a Hewlett-Packard model 5982A dual-source instrument with use of a membrane separator (EIMS) and' helium (EIMS) or methane (CIMS) as carrier gas. For direct effluent counting, a Hewlett-Packard model 5711 GLC was cou- plled via an effluent splitter to a Packard model 894 Gas Proportional Counter (Packard Instrument Co., Inc.,. Downers Grove, 111.). HPLC was performed with a Waters Associates model ALC/GPC-202 high-per£orm- ance liquid chromatograph equipped with a model 6000A solvent delivery system, a model 660 solvent programmer, a modeli U6K septumless injector, and a model 440 UV/visible detector (Waters Associates, Inc., Milford, Mass.). HPLC columns were as follows: col- umn A, 6 mmx30 cm Microporasil (Waters Associates); column B, 6 mmx30 cm Microbondapak/C18 (Waters Associates); column C, 6 ftx ]/8 inch activity II-III Weelm basic alumina (38-75 µ) (M. Woelrri, Eschwege, Federal Republic of Germany). Liquid scintillation counting was done with a Nuclear-Chicago Isocap 300 scintillation system (Nuclear-Chicago Corp., Des Plaines, Ill.). Evaporations were done under reduced pressure with water temperatures below 60° C. Reagents.-A11 organic solvents were spectroquality. ABBREVIATIONS USED: NNN = N'-nitrosonornicotine; NNK = 4-(1V- methyl-N-nitrosamino)-1-(3-pyridyl)+1-butanone;l NNA = 4-(N=methyl- N-nitrosamino)-4-(3-pyridyl)butanal; GLC = gas-liquid phase chro- matography; GLC-MS = combined gas-liquid phase chromatography and mass spectrometry; EIMS = electrom impact mass spectrometry;,.e. CIMS = chemical ionization mass spectrometry; HPLC = high-per- O formance liquid chromatography; dpm = disintegrations per minute. O 0 '' Received July 1, 1977; accepted October 31, 1977. 10A = Supported by Public Health Service contract N0l-CP55666 from j4~b the Division,of Cancer Cause and Prevention; National Cancer Insti- Vi tute, and by contract 12-14-100-11031(34) from the Agricultural Re- ~^y~ search Service. V[J~ t ' Publication #XVi in "A Study of Tobacco Carcinogenesis." ~i" ' Division of EnvironmentallCarcinogenesis. Naylor Dana Institute 0 for Disease Prevention, American Health Foundation, Valhalla, N.Y. 10595. s Recipient of Public Health Service Career Development Award 5- K04-CA00124 from the National Cancer Institute. ° Tobacco Laboratory, Beltsville Agricultural' Research Center, Agricultural Research Service, U.S. Department of Agriculture. Belts- ville, Md. 20705. ' Mention of a trademark or proprietary product does not consti- tute a guarantee or warranty of the product by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products that may also be suitable. VOL. 60, NO. 4, APRIL 1978 819 J NATL CANCER INST
Page 2: ajf08e00
820 HECHT„ CHEN, HIROTA, ORNAF, TSO, AND HOFFMANN I ® Curing of tobacco leaves: Experimental leaf tobacco was hung in, the hood! under conditions similar to air curing. Environments were adjusted to prevent quick drying and thus provided conditions essential for bio- chemical changes that normally occur during air curing. This curing process was completed in 3 weeks. Analysis for [2'-14C]NNN: All analyses for NN N were done by a previously published method (12) with the use of [3H]NNN as internal standard (relative standard deviation, 5%). In a typical analysis (20-30 g tobacco), 1.1 x 106 dpm (0.08 µg) [3H]NNN was added. An aliquot, of the final HPLC eluent containing NNN was counted by dual channels to determine recovery of 3H. Another aliquot was injected into the combined GLC-effluent counter system (column I, 210° C); which was calibrated with known amounts of [2'-14C]NNN. [3H]NNN gave no response in this system. This allowed determination of [2'-14C]NNN content and, by splitting to the flame ionization detector, total NNN content. An aliquot of each 14C-treated tobacco sample was submitted for combustion analysis (New England Nu- clear) to determine total '"C in the cured tobacco sam- ples. Alkaloid analyses were done by published proce- dures (17). Reaction of nicotineandNaN(a2.-Nilcotine (1.0 g, 6.17 mmole) was dissolved in 1' ml H20, and the pH was adjusted to the desired~ value by addition of 1 N HCI. This was then added to the appropriate buffer to make a total volume of 20 ml. Buffer solutions were: pH 2, HCl-KCIk pH 3.4, citrate-phosphate; pH 4.5, citrate- phosphate; pH 7,phosphate-NaOH. In one experi- ment, ascorbic acid (10.0 g, 56:8mmole) was also added, and the total volume was 50 ml. NaNO2 (0.6 g, 8.7 mmole) was added in one portion and the resulting solutiom was stirred for 17 hours at 20° C. The reaction mixture was brought to pH 10 by addition of aq NaOH, saturated with NaCI, and extracted 6 times with equal volumes of CHC13. The combined CHC13 extracts were dried (Na2SO4) and concentrated to give a mixture that was dissolved in 1.0 ml MeOH for quantitation of nitrosamines by GLC. Aliquots were injected on column I at a column temperature of 220° C. The relative retention times of nicotine, NNN, NNA, and NNK under these conditions were 0.18, 1.00, 1.69, and 2.38, respectively (absolute retention time of NNN=13 min). The mass spectrum of each peak was determined by GLC-MS. The area under each peak was d'etermined~ by integration. Yields were calculated by comparisom to response curves constructed from standards. Tobacco analysis for NNK.-Tobacco products were obtained! on the open market (1975-76) in the New York metropolitan area. Snuff and fine-cut chewing tobacco were analyzed directly, without grinding. Other prod- ucts were ground in a blender before extraction. An aliquot was taken for water determination (18). Ground tobacco (80-100 g) was stirred at 20° C overnight with 700 ml aqueous ascorbic acid (5 mM) at pH 4.5 (citrate- phosphate buffer). The weakly basic (pyridine) fraction was obtained as previously described (7) except that ethyl' acetate was substituted for chloroform. This ma- terial was passed through a plug of activity II-III basic NiO t CH2O TEXT-FIGURE l.-Tobacco-specific nitrosamines that can be formed from nicotine. [2'-1 " C]nicotine, [2'-"C]klornicotine, and [2'-'4C]NNN were obtained from New England! Nuclear Corp., Bos- ton, Massachusetts. [3H]NNN was obtained by nitrosa- tion of [3H]nornicotine, as described in (12). The purity of the [2'-14C]nornicotine, ['2'-14C] nicotine, and [3H]NNN was reconfirmed by thin-layer chromatogra- phy on silica gel (14) and by GLC (column I, 220° Q. [2'-1"C]NNN was repurified before use by HPLC (col~ umn A, elution by a cyclohexane:chloroform:methanol ratio of 30:68.6:1.4). Nicotine was obtained from Ald- rich Chemical Company, Milwaukee, Wisconsin, and was redistilled before use; it contained less than 0.01% nornicotine according to GLC analysis (column 11, 165° Q. Nornicotine, N!NN, NNK, and N'NA were synthe- sized by methods reported in the literature (14-16). NNA was stored in CH2C12 at 0° C to prevent decompo- sition. Trioctanoin (Eastman Kodak Company, Roches- ter, N.Y.) was redistilled before use. Column chroma- tography was done with activity IT-III basic alumina (Woelm). (2'- "CJN'NN in tobacco fed [2'= "C]nicotine or [2'- Y'CJnornicotine.-Growing and selection of experimental tobacco: Leaves of Nicotiana tabacum cv. Burley 21 (1-2% nitrate) were used. The tobacco plants were grown in the fieU at Beltsville Research Farm, Beltsville, Mary- land, and regular Burley production practice was em- ployed. Wben~ they reached harvesting stage, two well- developed middle leaves from each plant were selected. These experimental leaves were primed (detached) im- mediately before feeding. Feeding of nicotine or nornicotine to tobacco leaves: Five experimental groups were used: 1) negative control (8 leaves); 2) cold nicotine control (8 leaves); 3) cold nornicotine control (8 leaves); 4) [2'-14C]nicotine (4.67 mCi/mmole; 2 groups of 4 leaves each); 5) [2'- 14C]nornicotine (7.75 mCi/mmole; 2 groups of 4 leaves each). In each case, the alkaloids were fed in solution buffered to pH 6 with tartaric acid; group 1 received only pH 6 buffer. All feeding was done in laboratory hoods. The appro- priate solution (2 ml) was placed in a small vial, and the base of a detached leaf was inserted into the solution. When the solution-had been completely absorbed~, three 2-ml portions of distilled water were added to the vial. The total feeding time for each leaf was approximately 24 hours. Ic -6 C_ J NATL CANCER INST VOL. 60, NO. 4, APRIL 1978
Page 3: ajf08e00
Y C TOBACCO-SPECIFIC NITROSAMINES 821 lated amounts of NN!K were determined by comparisonI TEXT-FIGURE 2.-Gas chromatogram of the NNN fraction isolated of peak heights to those obtained upon injection of from tobacco leaves fe& with [2'-"C]nicotine. See "Materials and standards. Methods" for conditions. alumina (2.5 g)Iwith elutnion by 10 mllof 30% isopropanol in cyclohexane to remove highly polar or insoluble materials. The eluent was concentrated and chromato- graphed by HPLC (column C) with the use of solvent program; 8 in 15 minutes: initial conditions, solvent B (25%); final conditions, solvent B(100~'0); solvent A, cyclohexane; solvent B, 30% isopropanol in cyclohex- ane; flow rate, 1.5 ml/minute. The part of the mixture having the same retention volume (20-31 ml) as stan- dard NNK wascollected. Several injections were neces- sary for a sample of this size. After concentration, the combined fractions were rechromatographed by HPLC (column A) with the use of solvent program 6 in 10 minutes: initial conditions, solvent B (30%); final condi- tions, solvent B (100%); solvent A, 3% cyclohexane in CHC13; solvent B, 3% isopropanol in chloroform; flow rate, 1.5 ml/minute. The peaks corresponding to E, and Z-N'N K were collected individually and concentrated for determination of mass spectra by direct inlet (EIMS and CIMS). The ratio E:Z in reference NNK was determined by nuclear magnetic resonance (115). Iso- Bioassas.-Strain A/J female mice, 6-8 weeks old (The Jackson Laboratory, Bar Harbor, Maine) were housed in polycarbonate cages in groups of 5 and kept under standard conditions (20±2° C; 50±10% relative humid- ity;, light-dark cycle, 12 hr and 12 hr)I. The animals were given Purina Laboratory Chow pellets and tap water ad libitum. The experimental groups were as follows: 1) un- treated control, 2) vehicle control (sallne), 3) vehicle control (trioctanoin), 4) positive control (urethan)S 5) N'NN (saline), 6) NNN! (trioctanoin), 7) NNA (saline), 8) NNK (trioctanoin). Each group consisted of 25 animals. Three times weekly, animals were given ip injections of the compounds as 01.5% solutions in saline (0.2 ml injected) or 1% suspensions in trioctanoin (0.11 ml'in- jected). A total of 22 inject2ons was given to~ each mouse. The total dosage was therefore 22 mg (0.12 mmole)/ mouse for NN!N, 22 mg (0.1i1 mmole)/mouse for NINK and N!NA, and 22 mg (0.25 mmole)/mouse for urethan.. After the injections were complete, the mice were kept without further treatment for an additional 30 weeks and then killed by cervical dislocation. At autopsy, lungs and other organs with macroscopic lesions were fixed in 10% Formalin. Lung tumors were counted, and the tissues were processed by standard procedures for his- tologic evalhation. The frequency of lung tumors in treated groups was compared to that in the appropriate control by Students"t-test. RESULTS Freshly detached tobacco leaves were fed either buffered [2'-14t?]nicotine or buffered [2'-"C]nornicotine. Control groups were fed unlabeled nicotine, unlabeled nornicotine, or buffer. The leaves from each group were cured and analyzed for NNN and [2'-14C]NNN. A gas chromatogram of the final NNN concentrate from [2'-"C]klicotine-fed tobacco~ is shown in text-figure 2. The results of the [2'-14C]nicotine and [2'-"C]nornicotine feeding stud}~~ are summarized iin table'1. N!NN levels ranged from 0.3 to 1.2 µg/g tobacco. The yields of [2'- '"C]NNN fromI [2'-14C]hlicotine and [2'-"C]nornicotine were 0.009 and 0.007%, respectively. To investigate the formation of other nitrosamines fromI nicotine, especially the nitrosaminoketone NNK and the nitrosaminoaldehyde NNA (text-fig. 1), model studies on the reaction of nicotine and NaNO2 were done. Nicotine and NaNO2 were allowed to react at room temperature for 17 hours, at various pH levels. The results are summarized in table 2. Yields were determined by GLC; the peaks corresponding in reten- tion time to NNN, NNK, and NNA had mass spectra identical to those of reference standards (14-16). The formation of the three nitrosamines was favored in the pH range 3.4-4.5. Ascorbic acid inhibited nitrosamine formationI from nicotine (19). Since NNA and NNK could be formed from nicotine under mild conditions, the possible presence of these substances inI tobacco was studied. Suitable analytic methods for NN!A could not be developed, inasmuch as it is very reactive and could not be determined in underivatized form; various derivatives of NNA (2,4- dinitrophenylhydrazone, dimethylhydrazone, and di- methone) were also not suitable for analysis by us. However, an analytic method was developed for NNK. The method was similar to techniques used previously for NNN. An HPLC' chromatogram of NNK in snuff tobacco is shown in text-figure 3. Mass spectra (EIMS and CIMS) obtained by collection of the peaks indicated were identical to those of reference standards. Values for NNK.in tobacco are summarized im table 3, along with NNN levels in the same tobacco~ products. NNK
Page 4: ajf08e00
822 HECHT, CHEN, HCROTA, ORNAF, TSO, AND HOFFMANN TABLE 1. Formation of NNN from nicotine and nornicotine in tobacco Treatment Amount fed, m / Analytical results° g g NNN,° pg/g, NNN, dpm Yield NNN, % None (negative control)° Leaves fed cold nicotine 0 0.4'. 0.4 - 1.2 - Leaves fed cold nornicotine Leaves fed [2'-1'C]nicotine 0.01 0.4 (5.5x10Qdpm)a 0.6 - 0.3 5.1x10+ - 0.009 Il.eavesfed'[2'-"C]nornicotine 0.01 (3.6x107dpm)° 0.6 2.4x10a 0.007 ° All values are based on weight' of cured tobacco;,analyses on groups 4 and 5 were run in duplicate. e[pH]NNN was used! as internal standard. ~ Neither nicotine nor nornicotine was fed. ' Determined by combustion ~ analysis of tobacco from groups 4 and 5. .~~ TABLE 2. Formation of NNN, NNK, and NNA from nicotine and N NO ° TABLE 3. NNK and NNN in commercial U.S. tobacco products ti a Y NNK content ° NNN'content " , , Tobacco product , Percentage yields ' µg/g µg/g pH Cigarett.e, non-filter ND° 1.3 NNN NNK NNA Cigar ND 0 3 2.0 0.1 ND° 0.2 Snuff 1.3° . 29.1 3.4 0.5 0.1 2.8 Chewing tobacco, scrap 0.6 3.5 4.5° 0.5 0.5 2.3 leaf 7.0 0.2 0.1 0.1 Chewing tobacco, fine 2.4 88.6 `Reactions were done at 20° C for 17 hr with NaNO,:nicotine=1.4:1. cut °Based on starting nicotine. ° Isolated amounts. ° Not' detected. ° Calculated by the isotope dilution method reported previously • Addition of ascorbic a cid preve nted'i formation of all th ree nitros- (6). amines. °ND=not detected (<0.1 µg(g): °'Average of three runs; sD=0.3 µg/g, 0 N-CHs lNJ \H-Q F IZ t 0 15 30 -f Minutes TEXT-FtGURE 3.-High performance liquidd chromatogram of the NNK fraction isolated from snuff tobacco. Both the E- and' Z- isomers of NNK were observed. See "Materials and Methods" for conditions. was found only in chewing tobacco and snuff, at levels lower than NNN. For comparative bioassay, NNN, NNA, and NNK were used for induction of lung adenomas in straim A mice. For reasons of solubility, NNK was used as a suspensiom in trioctanoin, whereas NNA was used in saline solution. NNN was used both in saline and trioctanoin (groups 5 and 6). The results are summa- rized in table 4. As judged by the multiplicity of lung tumors, both NNN and NNK showed significant (P<0.05) activity when compared to controls, and NNK was significantly (P<0.05) more active than NNN; NNA did not show significant tumorigenic activity. The posi- tive and negative controls gave the expected tumor yields (20). Histologic evaluation of the lung tumors revealed that they were all multiple adenomas; except the 6 adenocar- cinomas in experimental group 4 and the 1 ad'enocarci- noma in group 6 (table 4). Two undifferentiated carci- nomas of the salivary glands developed' in groups 5 and 6; one carcinoma had metastasized to the lungs and pleura. These tumors were poorly differentiated and were composed of spheroidal and spindle cell types; many mitotic figures were visible. DISCUSSION The yields of NNN from nicotine and from nornico- tine in the tobacco feeding experiment were approxi- mately equal. The amount of NNN actually detected (0.3 µg/g) in the [14C]nicotine-fed tobacco was lower than the theoretical amount (1.8 µg/g) expected based C i ~
Page 5: ajf08e00
824 HECHT, CHEN, HIROTA, ORNAF, TSO, AND HOFFMANN high speed liquid chromatography. Anal Chem 47:2046-2048, 1975 (8) BHARADWAJ VP, TAKAYAMA S, YAMADA T, et al: N'-nitrosonor- nicotine in Japanese tobacco products. Gann 66:585-586, 1975 33:3069-3085, 1973 ` (9) KLUS H, KUHN H: Untersuchungen iiber die nichtfl'uchtigen N- (21) SMITH PA, LOEPPKY RN: Nitrosative cleavage of tertiary amines. Nitrosamine der Tabakalkaloide. Fachliche Mitt Oesterr Ta- J Am Chem Soc 89:1147-1153, 1967 bakregie 16:307-317, 1975 (22) KEEFER LK, ROLLER PP: N-nitrosation by nitrite ion in neutral (10) : Die Bestimmung des Nornikotinnitrosamines im Rauch- and basic medium. Science 181:1245-1247, 1973 kondensat nornikotinreicher Zigaretten. Fachl Mitt Oesterr Tabakregie 14:251-257, 1973 (23) HECHT SS, CHEN CB, ORNAF RM, et al: Reaction of nicotine and sodium nitrite: Formation of nitrosamines and fragmentation (11) HOFFMANN D, RATHKAMP G, LIU YY: Chemical studies on of the pyrrolidine ring. J Org Chem. In press tobacco smoke. XXVI. On the isolation and identification of volatile and nonvolatile N-nitrosamines and hydrazines in (24) MURPHY PJ: Enzymatic oxidation.of nicotine to nicotine A''ts'' iminium ion. J Biol Chem 248:2796-2800, 1973 ,. cigarette smoke. IARC Sci Pub19:159-165, 1974 (25) NGUYEN TL GRUENKE LD CASTAGNOLI N: Metabolic N-de- (12) HOFFMANN D, DONG M, HECHT SS: Origin in tobacco smoke of , , methylation of nicotine Trapping of a reaction iminium N'-nitrosonornicotine, a tobacco-specific carcinogen. J Natl Cancer Inst 58:1841-1844, 1977 . species with cyanide ion. J Med Chem 19:1168-1169, 1976 6. (26) LI INSKY W K L C E l Ni (13) MIRVtsH SS, SAMS J, HECHT SS: Kinetics of nornicotine and J , EEFER , ONRAD , et a : trosation of tertiary amines and some bi l i i li i N l anabasine nitrosation in relation to N'-nitrosonornicotine oc- o og c mp cat ons. J at Cancer Inst 49:1239-1249 1972 currence in tobacco and to tobacco-induced cancer. J Natl Cancer Inst 59:1211-1213 1977 , (27) LIJINSKY W: Reaction of drugs with nitrous acid as a source of (14) Hu MW, BONDINELL WE, HOFFMANN D: Synthesis of carbon-14- carcinogenic nitrosamines. Cancer Res 34:255-258, 1974 labelled myosmine, nornicotine and N'-nitrosonornicotine. J Labelled Compd 10:79-88, 1974 (28) FINE DH, HUFFMAN F, ROUNBEHLER DP, et al: Analysis of N- nitroso compounds by combined high-performance liquid (15) HECHT SS, CHEN CB, DONG M, et al: Studies on non-volatile chromatography and thermal energy analysis. IARC Sci Publ nitrosamines in tobacco. Beitr Tabakforsch 9:1-6, 1977 14:43-50, 1976 (16) HECHT SS, CHEN CB, HOFFMANN D: Synthesis of N-nitrosami- (29) SHIMKIN MB, WEISBURGER JH, WEISBURGER EK, et al: Bioassay noaldehydes. Tetrahedron Lett 593-596, 1976 of 29 alkylating chemicals by the pulmonary-tumor response in (17) RATHKAMP G, Tso TC, HOFFMANN D: Chemical studies on strain A mice. J Natl Cancer Inst 36:915-935, 1966 tobacco smoke. XX. Smoke analysis of cigarettes made from Bright tobaccos differing in variety and stalk positions. Beitr Tabakforsch 7:179-189, 1973 (18) VON BETHMANN M, LIPP G, VAN NOOY H: Feuchtigkeitsbestim- mung im Tabak. Beitr Tabakforsch 1:19-20, 1961 (19) MIRVISH SS, WALLCAVE L, EAGEN M, et al: Ascorbate nitrite reaction. Possible means of blocking the formation of carcino- genic N-nitroso compounds. Science 177:65-67, 1972 (20) STONER GD, SHIMKIN MB, KNIAZEFF AJ, et al: Test for carcino- genicity of food additives and chemotherapeutic agents by the pulmonary tumor response in strain A mice. Cancer Res (30) LIJINSKY W, TAYLOR HW: The effect of substituents on the carcinogenicity of N-nitrosopyrrolidine in Sprague-Dawley ' rats.Cancer Res 36:1988-1990, 1976 (31) : Carcinogenicity of methylated nitrosopiperidines. Int J Cancer 16:318-322, 1975 (32) DRUCKREY, H, PREUSSMANN R, IvwNKOVIC S, et al: Organotrope carcinogene Wirkungen bei 65 verschiedenen N-Nitroso-Ver- bindungen an BD-Ratten. Z Krebsforsch 69:103-201, 1967 C C J NATL CANCER INST VOL. 60, NO. 4, APRIL 1978
Page 6: ajf08e00
t TOBACCO-SPECIFIC NITROSAMINES TABLE 4. Nitrosamines derived from nicotine: Bioassay in strain A mice 823 xperimental group Effective No, of ani- malfi No. of animals with lung adeno- mas" Percentage of animals with lung. tumors Lung tumors per animal° Other tumors Untreated control 25 1 4' 0.04±0!20 Vehicle control, saline 25 3 12 0.24±0:72 Vehicle control, trioctanoin 24 5 21 0.20±0!4b Urethan in saline 25 25 (6) 100 14.80±4.33 NNN in saline 21 16 76 1.74±1.37 Undifferentiated carcinoma of salivary NNN in trioctanoin 23 12 (1) 57 0.87'±1.011 gland§, 1 (metastasis' to lungs, pleuray, Undifferentiated carcinoma of salivary NNA in saline 25 9 36 0.44'±.65 glandfi;,1; malignant lymphoma, 1 NNK in trioctanoin 23 20. 87 2.61±1.85 ° No. of adenocarcinoma-bearing animals given in parentheses. ° Average±sD. on the yield of 0.009% from nicotine (19.7 mglg). Similar considerations applq to the control group (0.4 µg(g NNN; 22.3' mg/g nicotine). This indicates that the 14C-labeled compounds fed to the tobacco leaves may not have mixed completely with the existing alkaloid pool and that the yields of [14C]NNN may be higher than those under natural conditions. Nevertheless, leaves from groups 4 and 5 were treated under identical experimental conditions, and a comparison of percent- age yields of NNN in the 2 groups is therefore valid. Inasmuch as nicotine and nornicotine were equally effective as precursors for NNN formed during curing of tobacco in this experimental setting, NNN! in tobacco must arise principally from nicotine. This is true be- cause in normal tobacco leaf, nicotine concentration is generally 20-100 times as great, as that of nornicotine (6). Previous results also suggested~ that nicotine was the major precursor for NNN! in tobacco (5, 6, 13), The formation of NNK, NNA, and NNN from nico- tine in vitro probably proceeds at least partially through the intermediacy of cyclic iminiumi salts. This is the expected~ pathway for nitrosation of tertiary amines (21). Such salts can be hydrolyzed to second'ary amines that are nitrosated under acidic conditions or, as shown in previous studies, they can react directly with nitrite under neutral conditions to give nitrosamines (16, 22). Further evidence for this mechanistic pathway will be described separately (23). It is interesting, and perhaps significant„that these iminium species are demonstrated intermediates in t~he mammalian (and possibly the mi- crobial) metabolism of nicotine (24; 25). The yields of NNA, NNK, and N!NN from nicotine under the conditions described (table 2): are typical for the conversion of tertiary amines to nitrosamines (26, 27). The formation of all three compounds is suggestive of their presence in tobacco. ln fact, both NNN and NNK were detected in tobacco (table 3). The presence of NNA may also be demonstrated when suitable ana- lytic methods, such as combined HPLC and thermal energy analysis (28), are employed. The compalfative bioassayy data summarized in table 4 indicate that NNK was significantly more tumorigenic than NNN, when judgedl by number of tumors per animal in groups 6 and 8. NNA was significantly less active than NNN', under these conditions. The tumori- genic activity of NN!N in strain A mice is consistent with previous results, as is the higher activity when saline, rather than, trioctanoin, is the vehicle (4, 5, 29). Since salivary tumors are not ordinarily observed in strain A mice, these tumors were probably caused byy systemic administration, of NNN. The greater activity of NNK compared to NNN could be due to less substitution in the positions a to the nitrosamine functionality. Substi- tution in the a-position often results in decreased carci- nogenic activity for nitrosamines (30-32). The low activ- ity of N!NA may be due to the presence of a reactive aldehyde group; thiscompound may not reach cellular targets. The results of this study clearly indicate that nicotine is a significant precursor for carcinogenic tobacco-spe- cific nitrosamines, which are formed during the curing and processing of tobacco. These nitrosamines may be related to the incidence of tobacco-related cancers in man. Since such compounds are formed during curing, methods can and should be devised~ to significantly reduce their concentrations in both tobacco and tobacco smoke. REFERENCES (I)i HOFFMANN D, RAINERIi R,. HECHT SS, et al: A study of tobacco carcinogenesis. XIV. Effects of N'-nitrosonornicotine and N'- nitrosoanabasine in rats. J NatliCancer Inst 55c977-951, 1975 (2) SINGER GM, TAYLOR HW: Carcinogenicity of N'-nitrosonornic- otine in Sprague-Dawley rats: J Nad Cancer Inst 57:1275-1276, 1976 (3): HIILFRICH J, H.ECHT SS, HOFFMANN D: Effects of A"-nitrosonor- nicotine and N'-nitrosoanabasine in Syrian golden hamsters. Cancer Lett 2:169-176, 1977 (4)', BOYLAND E, ROE FJ, GORROD JW: Induction of pulmonary tumors in mice by nitrosonornicotine, a possible constituent of tobacco smoke. Nature [iLond]1202i1126; 1964 (:S)', HOFFMANN D, HECHT SS„ORNAF RM, et aL'.Chemical.studies on tobacco smoke. XLII. N'-nitrosonornicotine: Presence in to- bacco, formationI, and carcinogenicity. IARC Sci Publ! 14:307- 320, 1976 (6), HECHTSS,. ORNAF RM,. HOFFMANND:', Chemical studies on tobacco smoke. XXXIII. N'-nitrosonornicotine in tobacco: Analysis of possible contributing factors and biologic implica- tions: J Nad Cancer Inst 54:1237-1244, 1974 (7), : Determination of N'-nitrosonornicotine in tobacco by

Text Control

Highlight Text:

OCR Text Alignment:

Image Control

Image Rotation:

Image Size: