Carcinogenicity of N'-Nitrosonornicotine in Sprague-Dawley Rats 1 , 2 George M. Singer 3 ,4 and H. Wayne Taylor 3 ,5 ABSTRACT-N'·Nitrosonornicotine was added to the drinking water of outbred Sprague-Dawley rats. Adenocarcinomas of the olfactory epithelium occurred in all 15 rats, squamous papillomas of the esophagus in 1, squamous papillomas of the nonglandular stomach in 1, and a hepatocellular tumor in 1. The possible ingestion and subsequent in vivo nitrosation of nicotine and/or nornicotine could create a serious carcinogenic hazard to cigarette smokers.-J Natl Cancer Inst 57: 1275-1276, 1976.
The incidence of lung cancer among cigarette smokers is much higher than among nonsmokers (1). Despite the apparent carcinogenicity of cigarette smoke or its condensate ("tar"), few identified carcinogens have been found in appreciable quantities; e.g., two of the most prevalent polycyclic aromatic hydrocarbons, benzo[a]pyrene and chrysene, are present at 39 ng/cigarette (2) and 36.5 ng/cigarette (3), respectively. Recently, several N-nitrosamines have been identified in tar at significant levels: NNN, 140 ng/cigarette (4, 5); dimethylnitrosamine, 84 ng/cigarette (4); and ethyl methylnitrosamine, 30 ng/cigarette (4). Moreover, large quantities of dimethylamine and pyrrolidine have recently been discovered in tar (6). Nicotine is the most abundant amine, with nornicotine next, but at much lower levels in tobacco and tar (7). The nitro so derivatives of dimethylamine and pyrrolidine are established carcinogens (8), and nitrosation of either nicotine or nornicotine gives NNN (4, 9, 10), which has been reported to increase the incidence of lung adenomas in mice when administered ip (11). Although NNN has been identified in tar (4, 5) in substantial amounts, nitrosation in vivo of nicotine and/ or nornicotine could produce even larger amounts, especially in view of the recent findings that an appreciable portion of inhaled smoke is swallowed by an experimental animal model (Kendrick J, Rubin IB, Creasia DA, et al: Submitted for publication); even more relevant is that nor nicotine and nicotine are found in the saliva of smokers (12). We synthesized NNN [1-nitroso-2-(3-pyridyl)pyrrolidine] and administered it in drinking water to rats, since this route of delivery most closely approximates that by which smokers experience the greatest exposure to NNN if it is formed in vivo. During the course of this work, results of a similar study were reported (5).
minutes of reflux, the solution was poured into water (200 ml) and then was extracted twice with 100 ml chloroform. The chloroform solution was dried (anhydrous K2C03 ) and evaporated in a N2 stream. The residual oil weighed 6.45 g (overall yield, 42%) and gave only one spot on thin layer chromatography. Nitrosation of the nornicotine was performed by dissolving 7 g oil in 100 ml acetic acid (30%) and adding a solution of 37 g sodium nitrite/lOO ml water. After 1 hour at room temperature the solution was poured into 100 ml NaOH (10%) and extracted twice with 200 ml methylene chloride. The extract was dried and the solvent evaporated under N 2, which yielded 6.5 g NNN (78%) as a yellow oil: UV Amax (diluted HCI), 346 nm (E, 64); in (10), Amax (MeOH), 237 nm (7,580). Mass spectrum (70 eV): m/e, 177 (60%, M+), 160 (8%, M+-OH), 147 (47%, M+-NO), 118 (66%),105 (100%),104 (33%),78 (44%), 77 (14%). Animal treatment. -The treatment of rats followed our established protocol for evaluation of nitrosamine carcinogenicity (14, 15). Female Sprague-Dawley outbred rats, born and maintained in a closed colony in this laboratory, were housed 3 to a cage and fed Purina Lab Chow ad libitum. They were 8 weeks of age at the start of the experiment. NNN was fed as a drinking solution at a concentration of 354 mg/liter [equimolar with our concurrent pyrrolidine study (16)]. This solution was given at a rate of 20 ml/rat/day, 5 days each week for 44 weeks; each rat received a total of 1.55 g (8.8 mmoles). Tap water was provided on the remaining 2 days each week. The rats consumed all of the allotted drinking solution each day, and the total dose received was quantitated. The animals were allowed to die naturally or were killed when they became moribund; all were necropsied. All lesions and major organs were examined microscopically. RESULTS
The first rat died 31 weeks after treatment was started; all were dead at 46 weeks. Adenocarcinomas of the olfactory epithelium were present in all 15 rats; in addition, 1 had squamous papillomas in the esophagus, 1 had squamous papillomas in the nonglandular stomach, and 1 had a hepatocellular tumor. The hepatocellu-
MATERIALS AND METHODS
ABBREVIATIONS USED: NNN = N' -nitrosonornicotine; m/e = mass divided by elementary charge; M+ = molecular ion.
NNN.-Synthesis of NNN followed that described by Leete et al. (13), beginning with 3-pyridinecarboxaldehyde. In the final stage, 3-cyano-l-(3-pyridyl)propan-lone was ring-closed by reduction with Raney nickel, which gave a mixture of my os mine and nornicotine. For an increased yield of nornicotine, the mixture (9 g) was dissolved in ethanol (50 ml) and boiled under reflux under N 2. A solution of NaBH4 (4 g) in 1 N NaOH (40 ml) was added dropwise during 45 minutes. After 30
Received January 19, 1976; accepted May 11, 1976. Supported jointly by Public Health Service contract CP50200 from the Division of Cancer Cause and Prevention, National Cancer Institute, and the U.S. Energy Research and Development Administration with Union Carbide Corporation. 3 Biology Division, Oak Ridge National Laboratory, P. O. Box Y, Oak Ridge, Tenn. 37830. 4 Present address: Frederick Cancer Research Center, P. O. Box B, Frederick, Md. 21701. 5 Present address: Department of Pathology and Parasitology, School of Veterinary Medicine, Auburn University, Auburn, Ala. 36830.
VOL. 57, NO.6, DECEMBER 1976
1275
1
2
J NATL CANCER INST
1276
SINGER AND TAYLOR
lar tumor was present in the longest-living rat; the esophageal and gastric papillomas were found in animals that died in week 43 of the experiment. Adenocarcinomas in the nasal cavities arose from the olfactory epithelium of the posterior turbinates, invaded the cribriform plate, and displaced and destroyed the anterior portions of the brain. These tumors were essentially the same as those previously described (14). In addition to the 1 rat with papillomas of the nonglandular stomach, 4 had plaques of hyperkeratosis and pseudoepitheliomatous hyperplasia. These changes occurred in conjunction with small foci of superficial erosion in the nonglandular stomach. The trachea of another rat contained areas of marked basal cell hyperplasia and replacement of superficial columnar epithelium with rounded polyhedral cells. The laryngeal epithelium of this animal had undergone marked hyperkeratosis also. DISCUSSION
Our results and those of Hoffmann et al. (5) indicate that NNN is a potent carcinogenic nitrosamine, certainly more potent than its parent compound nitrosopyrrolidine (16). The difference between their results [3 esophageal carcinomas, 3 olfactory carcinomas, and 11 benign esophageal papillomas in a group of 20 male F344 rats (5)] and ours (15 olfactory carcinomas in 15 female Sprague-Dawley rats from the Oak Ridge colony) may be due to differing strain susceptibility and/or dose response. Our total dose, for comparison with our other chemical carcinogenesis studies, was 2.5 times that of Hoffmann et al. For reasons that are not clear, NNN is also one of the very few examples of carcinogenic nitrosamines with a polar (acidic or basic) center (the pyridine nitrogen). 4-Methyl-l-nitrosopiperazine, also a base (8, 15),N-nitrosoproline (8, 17),N-nitrosopipecolic acid, and N-nitrosoisonipecotic acid (Taylor HW, Lijinsky W: Unpublished data) are all noncarcinogenic; Nnitrososarcosine is a weak carcinogen (8), andN-nitroso4-picolyleth ylamine and N - nitrosotrimeth ylh ydrazine are carcinogenic (8). Various components of cigarette smoke may condense in the mouth and be swallowed with saliva in smokers [Kendrick J, Rubin IB, Creasia DA, et al: Submitted for publication; (12)]. By this mechanism, nicotine and nornicotine m"ay be nitrosated in the stomachs of smokers to form NNN. Although extrapolation of effects of nitrosamines from one species to another is not always reliable, the demonstrated respiratory carci-
J NATL CANCER INST
nogemClty of NNN in our rats certainly suggests that this compound should be considered a possible carcinogen in humans. REFERENCES (1) STEINFELD JL: The Health Consequences of Smoking. A Report of the Surgeon General, 1971. Washington, D.C., U.S. Govt Print Off, 1971 (2) HOFFMANN D, RATHKAMP G, RUBIN J: Chemical studies on tobacco smoke. II. Comparison of the yields of several selected components in the smoke from five major Turkish tobacco varieties. Food Cosmet Toxicol 5:37-38, 1967 (3) HECHT SS, BONDINELL WE, HOFFMANN D: Chrysene and methylchrysenes: Presence in tobacco smoke and carcinogenicity. J Nat! Cancer Inst 53: 1121-1133, 1974 (4) HOFFMANN D, RATHKAMP G, Lw YY: Chemical studies on tobacco smoke. XXVI. On the isolation and identification of volatile and nonvolatile N-nitrosamines and hydrazines in cigarette smoke. In N-Nitroso Compounds in the Environment (Bogovski P, Walker EA, eds.). International Agency for Research on Cancer, Scientific Publ No.9. Lyon, 1975, pp 159-165 (5) HOFFMANN D, RAINERI R, HECHT SS, et al: A study of tobacco carcinogenesis. XIV. Effects of N' -nitrosonornicotine in rats. J Nat! Cancer Inst 55:977-979, 1975 (6) SINGER GM, LIJINSKY W: Naturally occurring nitrosatable amines. II. Secondary amines in tobacco and cigarette smoke condensate. J Agric Food Chern 24:553-555, 1976 (7) NEURATH G: Stickstoffverbindungen des Tabakrauchles. Beitr Tabakforschung 5:115-133, 1969 (8) DRUCKREY H, PREUSSMANN R, IVANKOVIC S, et al: Organotrope Carcinogene Wirkungen bie 65 verschiedenen N-Nitrosoverbindungen on BD-Ratten. Z Krebsforsch 69: 103-201, 1967 (9) NEURATH G, DUNGER M:Darstellung und Eigenschaften von NNittrosoverbindungen der Tabakalkaloids. Beitr Tabakforschung 3:339-345, 1966 (10) Hu MW, BONDINELL WE, HOFFMANN D: Chemical studies on tobacco smoke. XXIII. Synthesis of carbon-14 labeled myosmine, nornicotine, and N' -nitrosonornicotine. J Labelled Compounds 10:79-88, 1974 (11) BOYLAND E, ROE FJ, GORROD JW: Induction of pulmonary tumours in mice by nitrosonornicotine, a possible constituent of tobacco smoke. Nature 202:1126, 1964 (12) SCHWEINSBURG F, SANDER J, SCHWEINSBERG E, et al: Untersuchungen iiber die Nitrosierbarkeit von Nicotin und Nornicotin und iiber die Frage der Bildung von N-Nitrosonornicotin in Magen von Rauchern. Z Krebsforsch 84:81-87, 1975 (13) LEETE E, CHEDEKEL MR, BODEM GB: Synthesis of myosmine and nornicotine, using an acyl carbanion equivalent as an intermediate. J Org Chern 37:4465-4466, 1972 (14) LIJINSKY W, TAYLOR HW: Tumorigenesis by oxygenated nitrosopiperidines in rats. J Nat! Cancer Inst 55:705-708, 1975 (15) - - : Carcinogenicity of methylated dinitrosopiperazines in rats. Cancer Res 35:1270-1273, 1975 (16) - - : Carcinogenicity of pyrrolidine and some derivatives in rats. Cancer Res 36: 1988-1990, 1976 (17) GREENBLATT M, LIJINSKY W: Failure to induce tumors in Swiss mice after concurrent administration of amino acids and sodium nitrite. J Nat! Cancer Inst 48:1389-1392,1972
VOL. 57, NO.6, DECEMBER 1976
The incidence of lung cancer among cigarette smokers is much higher than among nonsmokers (1). Despite the apparent carcinogenicity of cigarette smoke or its condensate ("tar"), few identified carcinogens have been found in appreciable quantities; e.g., two of the most prevalent polycyclic aromatic hydrocarbons, benzo[a]pyrene and chrysene, are present at 39 ng/cigarette (2) and 36.5 ng/cigarette (3), respectively. Recently, several N-nitrosamines have been identified in tar at significant levels: NNN, 140 ng/cigarette (4, 5); dimethylnitrosamine, 84 ng/cigarette (4); and ethyl methylnitrosamine, 30 ng/cigarette (4). Moreover, large quantities of dimethylamine and pyrrolidine have recently been discovered in tar (6). Nicotine is the most abundant amine, with nornicotine next, but at much lower levels in tobacco and tar (7). The nitro so derivatives of dimethylamine and pyrrolidine are established carcinogens (8), and nitrosation of either nicotine or nornicotine gives NNN (4, 9, 10), which has been reported to increase the incidence of lung adenomas in mice when administered ip (11). Although NNN has been identified in tar (4, 5) in substantial amounts, nitrosation in vivo of nicotine and/ or nornicotine could produce even larger amounts, especially in view of the recent findings that an appreciable portion of inhaled smoke is swallowed by an experimental animal model (Kendrick J, Rubin IB, Creasia DA, et al: Submitted for publication); even more relevant is that nor nicotine and nicotine are found in the saliva of smokers (12). We synthesized NNN [1-nitroso-2-(3-pyridyl)pyrrolidine] and administered it in drinking water to rats, since this route of delivery most closely approximates that by which smokers experience the greatest exposure to NNN if it is formed in vivo. During the course of this work, results of a similar study were reported (5).
minutes of reflux, the solution was poured into water (200 ml) and then was extracted twice with 100 ml chloroform. The chloroform solution was dried (anhydrous K2C03 ) and evaporated in a N2 stream. The residual oil weighed 6.45 g (overall yield, 42%) and gave only one spot on thin layer chromatography. Nitrosation of the nornicotine was performed by dissolving 7 g oil in 100 ml acetic acid (30%) and adding a solution of 37 g sodium nitrite/lOO ml water. After 1 hour at room temperature the solution was poured into 100 ml NaOH (10%) and extracted twice with 200 ml methylene chloride. The extract was dried and the solvent evaporated under N 2, which yielded 6.5 g NNN (78%) as a yellow oil: UV Amax (diluted HCI), 346 nm (E, 64); in (10), Amax (MeOH), 237 nm (7,580). Mass spectrum (70 eV): m/e, 177 (60%, M+), 160 (8%, M+-OH), 147 (47%, M+-NO), 118 (66%),105 (100%),104 (33%),78 (44%), 77 (14%). Animal treatment. -The treatment of rats followed our established protocol for evaluation of nitrosamine carcinogenicity (14, 15). Female Sprague-Dawley outbred rats, born and maintained in a closed colony in this laboratory, were housed 3 to a cage and fed Purina Lab Chow ad libitum. They were 8 weeks of age at the start of the experiment. NNN was fed as a drinking solution at a concentration of 354 mg/liter [equimolar with our concurrent pyrrolidine study (16)]. This solution was given at a rate of 20 ml/rat/day, 5 days each week for 44 weeks; each rat received a total of 1.55 g (8.8 mmoles). Tap water was provided on the remaining 2 days each week. The rats consumed all of the allotted drinking solution each day, and the total dose received was quantitated. The animals were allowed to die naturally or were killed when they became moribund; all were necropsied. All lesions and major organs were examined microscopically. RESULTS
The first rat died 31 weeks after treatment was started; all were dead at 46 weeks. Adenocarcinomas of the olfactory epithelium were present in all 15 rats; in addition, 1 had squamous papillomas in the esophagus, 1 had squamous papillomas in the nonglandular stomach, and 1 had a hepatocellular tumor. The hepatocellu-
MATERIALS AND METHODS
ABBREVIATIONS USED: NNN = N' -nitrosonornicotine; m/e = mass divided by elementary charge; M+ = molecular ion.
NNN.-Synthesis of NNN followed that described by Leete et al. (13), beginning with 3-pyridinecarboxaldehyde. In the final stage, 3-cyano-l-(3-pyridyl)propan-lone was ring-closed by reduction with Raney nickel, which gave a mixture of my os mine and nornicotine. For an increased yield of nornicotine, the mixture (9 g) was dissolved in ethanol (50 ml) and boiled under reflux under N 2. A solution of NaBH4 (4 g) in 1 N NaOH (40 ml) was added dropwise during 45 minutes. After 30
Received January 19, 1976; accepted May 11, 1976. Supported jointly by Public Health Service contract CP50200 from the Division of Cancer Cause and Prevention, National Cancer Institute, and the U.S. Energy Research and Development Administration with Union Carbide Corporation. 3 Biology Division, Oak Ridge National Laboratory, P. O. Box Y, Oak Ridge, Tenn. 37830. 4 Present address: Frederick Cancer Research Center, P. O. Box B, Frederick, Md. 21701. 5 Present address: Department of Pathology and Parasitology, School of Veterinary Medicine, Auburn University, Auburn, Ala. 36830.
VOL. 57, NO.6, DECEMBER 1976
1275
1
2
J NATL CANCER INST
1276
SINGER AND TAYLOR
lar tumor was present in the longest-living rat; the esophageal and gastric papillomas were found in animals that died in week 43 of the experiment. Adenocarcinomas in the nasal cavities arose from the olfactory epithelium of the posterior turbinates, invaded the cribriform plate, and displaced and destroyed the anterior portions of the brain. These tumors were essentially the same as those previously described (14). In addition to the 1 rat with papillomas of the nonglandular stomach, 4 had plaques of hyperkeratosis and pseudoepitheliomatous hyperplasia. These changes occurred in conjunction with small foci of superficial erosion in the nonglandular stomach. The trachea of another rat contained areas of marked basal cell hyperplasia and replacement of superficial columnar epithelium with rounded polyhedral cells. The laryngeal epithelium of this animal had undergone marked hyperkeratosis also. DISCUSSION
Our results and those of Hoffmann et al. (5) indicate that NNN is a potent carcinogenic nitrosamine, certainly more potent than its parent compound nitrosopyrrolidine (16). The difference between their results [3 esophageal carcinomas, 3 olfactory carcinomas, and 11 benign esophageal papillomas in a group of 20 male F344 rats (5)] and ours (15 olfactory carcinomas in 15 female Sprague-Dawley rats from the Oak Ridge colony) may be due to differing strain susceptibility and/or dose response. Our total dose, for comparison with our other chemical carcinogenesis studies, was 2.5 times that of Hoffmann et al. For reasons that are not clear, NNN is also one of the very few examples of carcinogenic nitrosamines with a polar (acidic or basic) center (the pyridine nitrogen). 4-Methyl-l-nitrosopiperazine, also a base (8, 15),N-nitrosoproline (8, 17),N-nitrosopipecolic acid, and N-nitrosoisonipecotic acid (Taylor HW, Lijinsky W: Unpublished data) are all noncarcinogenic; Nnitrososarcosine is a weak carcinogen (8), andN-nitroso4-picolyleth ylamine and N - nitrosotrimeth ylh ydrazine are carcinogenic (8). Various components of cigarette smoke may condense in the mouth and be swallowed with saliva in smokers [Kendrick J, Rubin IB, Creasia DA, et al: Submitted for publication; (12)]. By this mechanism, nicotine and nornicotine m"ay be nitrosated in the stomachs of smokers to form NNN. Although extrapolation of effects of nitrosamines from one species to another is not always reliable, the demonstrated respiratory carci-
J NATL CANCER INST
nogemClty of NNN in our rats certainly suggests that this compound should be considered a possible carcinogen in humans. REFERENCES (1) STEINFELD JL: The Health Consequences of Smoking. A Report of the Surgeon General, 1971. Washington, D.C., U.S. Govt Print Off, 1971 (2) HOFFMANN D, RATHKAMP G, RUBIN J: Chemical studies on tobacco smoke. II. Comparison of the yields of several selected components in the smoke from five major Turkish tobacco varieties. Food Cosmet Toxicol 5:37-38, 1967 (3) HECHT SS, BONDINELL WE, HOFFMANN D: Chrysene and methylchrysenes: Presence in tobacco smoke and carcinogenicity. J Nat! Cancer Inst 53: 1121-1133, 1974 (4) HOFFMANN D, RATHKAMP G, Lw YY: Chemical studies on tobacco smoke. XXVI. On the isolation and identification of volatile and nonvolatile N-nitrosamines and hydrazines in cigarette smoke. In N-Nitroso Compounds in the Environment (Bogovski P, Walker EA, eds.). International Agency for Research on Cancer, Scientific Publ No.9. Lyon, 1975, pp 159-165 (5) HOFFMANN D, RAINERI R, HECHT SS, et al: A study of tobacco carcinogenesis. XIV. Effects of N' -nitrosonornicotine in rats. J Nat! Cancer Inst 55:977-979, 1975 (6) SINGER GM, LIJINSKY W: Naturally occurring nitrosatable amines. II. Secondary amines in tobacco and cigarette smoke condensate. J Agric Food Chern 24:553-555, 1976 (7) NEURATH G: Stickstoffverbindungen des Tabakrauchles. Beitr Tabakforschung 5:115-133, 1969 (8) DRUCKREY H, PREUSSMANN R, IVANKOVIC S, et al: Organotrope Carcinogene Wirkungen bie 65 verschiedenen N-Nitrosoverbindungen on BD-Ratten. Z Krebsforsch 69: 103-201, 1967 (9) NEURATH G, DUNGER M:Darstellung und Eigenschaften von NNittrosoverbindungen der Tabakalkaloids. Beitr Tabakforschung 3:339-345, 1966 (10) Hu MW, BONDINELL WE, HOFFMANN D: Chemical studies on tobacco smoke. XXIII. Synthesis of carbon-14 labeled myosmine, nornicotine, and N' -nitrosonornicotine. J Labelled Compounds 10:79-88, 1974 (11) BOYLAND E, ROE FJ, GORROD JW: Induction of pulmonary tumours in mice by nitrosonornicotine, a possible constituent of tobacco smoke. Nature 202:1126, 1964 (12) SCHWEINSBURG F, SANDER J, SCHWEINSBERG E, et al: Untersuchungen iiber die Nitrosierbarkeit von Nicotin und Nornicotin und iiber die Frage der Bildung von N-Nitrosonornicotin in Magen von Rauchern. Z Krebsforsch 84:81-87, 1975 (13) LEETE E, CHEDEKEL MR, BODEM GB: Synthesis of myosmine and nornicotine, using an acyl carbanion equivalent as an intermediate. J Org Chern 37:4465-4466, 1972 (14) LIJINSKY W, TAYLOR HW: Tumorigenesis by oxygenated nitrosopiperidines in rats. J Nat! Cancer Inst 55:705-708, 1975 (15) - - : Carcinogenicity of methylated dinitrosopiperazines in rats. Cancer Res 35:1270-1273, 1975 (16) - - : Carcinogenicity of pyrrolidine and some derivatives in rats. Cancer Res 36: 1988-1990, 1976 (17) GREENBLATT M, LIJINSKY W: Failure to induce tumors in Swiss mice after concurrent administration of amino acids and sodium nitrite. J Nat! Cancer Inst 48:1389-1392,1972
VOL. 57, NO.6, DECEMBER 1976
