Europ. d. CancerVol. 11, pp. 97-99. Pergamon Press 1975. Printed in Great Britain
Carcinomas of the Esophagus in Rats Ingesting Diethylnitrosamine MELVIN D. REUBER Laboratory of Biology, National Cancer Institute, Bethesda, Maryland 20014, U.S.A. Abstract--Buffalo strain male and female rats 12 weeks of age ingested 0.0114% diethylnitrosamine (DEN) in the dietfor 26 weeks and were returned to the basal diet without D E N for the remainder of the experiment. Rats survived an average of 28 weeks. Carcinomas of the esophagus were observed in 9 of 14 male and 5 of 14female rats. The incidence of carcinomas of the esophagus was higher in male rats; however, the incidence of liver carcinomas was higher in female rats. The organ site of induction of carcinomas with the symmetrical and asymmetrical nitrosamines is discussed.
INTRODUCTION
diet for 26 weeks ad libitum. After that time rats were given the basal diet. Animals were weighed weekly. Surviving animals were killed by exsanguination 36 weeks after the start of the experiment. All organs, except the nasal cavity, were examined grossly and histologically. The esophagus was removed with the thyro-cricoid cartilage and the stomach. The esophagus was opened and lesions described, and the size and location recorded. Tissues were fixed in 4 % formaldehyde and stained routinely with hematoxylin and eosin. Special stains, when indicated, included periodic acidSchiff (PAS).
DRUCKREY et al. felt that the N-N-diethylnitrosamines had an affinity for the liver because they were metabolized by and induced carcinomas of the liver [1] and that the asymmetrical nitrosamines were specific for the esophagus and carcinomas in that organ [2, 3]. This report describes carcinomas of the esophagus, as well as liver, in Buffalo strain male and female rats ingesting diethylnitrosamine (DEN) in the diet. MATERIAL AND METHODS Inbred Buffalo male and female rats (National Institutes of Health Animal Production), 12 weeks of age, were used. They were given 0.0114% D E N in a semisynthetic diet. Morris diet # 272 contained 300 g commercial casein, 2275 g skim milk powder, 6152 g ground hard spring wheat, 200 g brewer's yeast, 200 g desiccated liver, N.P. (Wilson Laboratories, Chicago, Ill.) 140 g sodium chloride, 10 g ascorbic acid, 13 g ferric citrate, 100 g cod-liver oil, and 610 g corn oil. Animals ingested the carcinogen-containing
RESULTS Animals gained weight until about 2 weeks before death or termination of the experiment, at which time they lost 16-20 g. Both the males and females survived for an average of 28 weeks [range 25-34]. Nine of 14 male rats and 5 of 14 female rats developed carcinomas of the esophagus. The lesions measured 1.5-1-7 cm dia and were located 1.5-2.0cm (average 1.7) from the gastro-esophageal junction in male rats and half of the female rats; in the remaining half of the females the carcinomas were located at the
Accepted 27 September 1974. *Present address: 11014 Swansfield Road, Columbia, Maryland 21044, U.S.A. 97
98
Melvin D. Reuber
pharyngeal tracheal level of the esophagus. Histologically the carcinomas were a mixture of undifferentiated, poorly differentiated cells (Figs. 1 and 2) and well-differentiated squamous cells with keratin pearls. There was focal invasion of the submucosa. Bronchopneumonia, observed in 5 female and 3 male rats with carcinomas of the esophagus, probably resulted from obstruction of the esophagus and regurgitation into the lungs. Carcinomas of the esophagus occurred most often in rats with either small carcinomas of the liver or in rats without malignant tumors of the liver.
Lesions in organs other than esophagus Ten female rats had carcinomas and 3 sarcomas of the liver; as compared with 5 carcinomas and 2 sarcomas of the liver in male rats ingesting DEN. This higher incidence of malignant tumors of the liver in females was significant at the P = < 0.05 level. Female rats also had random malignant tumors in other organs. These were located in the uterus, brain, and ovary. Carcinomas were not observed in the oral cavity, tongue or squamous portion of the stomach. DISCUSSION Carcinomas of the esophagus were observed regularly by unsymmetrical nitrosamines in young BD strain rats and the authors concluded that the chemicals were specific inducers of carcinomas of that organ [2]. Ethyl-vinylnitrosamine, methyl-benzyl-nitrosamine, dimetyl-dinitroso-ethylenediamine, n-nitroso-sarkosin, nitroso-sarkosin-ethyl, n-nitrosopiperidine and n-methyl-n-nitrosoaniline all induced carcinomas of the esophagus; whereas rats given ethyl-isopropyl-nitrosamine and ethyl-nbutyl-nitrosamine had carcinomas of the liver in addition to esophageal carcinomas [1-4]. Carcinomas were observed only in the liver in rats given ethyl-ethanol-nitrosamine [2]. Ethylalkyl-nitrosamide and diazo-aceto-ester rarely produced carcinoma of the esophagus, but almost always induced squamous cell carcinoma of the stomach [5].
Buffalo strain rats ingesting D E N in the diet developed carcinomas of both the esophagus and liver. The carcinomas of the esophagus occurred in rats without hepatic carcinomas or in rats with small hepatic carcinomas. Carcinomas of the esophagus were either localized or diffuse in BD strain rats given unsymmetrical nitrosamines. In Buffalo strain rats given D E N carcinomas of the esophagus were focal and located either near the gastroesophageal junction or at the level of the pharyngeal tracheal bifurcation. The incidence of carcinomas of the esophagus was higher in male Buffalo strain rats and the incidence of hepatic carcinomas and sarcomas was higher in Buffalo strain female rats ingesting DEN. This finding would indicate that the chemical was first transformed into an active carcinogenic agent in the esophageal mucosa and later reached the liver for additional metabolism, and that the esophagus was more susceptible in males than in female rats. With diffuse localization in the esophagus in BD strain rats given unsymmetrical nitrosamines, it could be predicted that little of the chemicals or their metabolites would reach the liver. R F mice given D E N in the drinking water developed tumors of the lungs, liver, and stomach and a small number of microscopic tumors of the esophagus [6]. The authors stressed the importance of not relying on gross examination and of doing histological studies of the esophagus. Hamsters developed some liver tumors, but differed from rats and mice by developing tumors of the nasal cavity, trachea, bronchus and lungs regardless of the route by which D E N was given [7-10]. There are differences in the location of tumors in rats given nitrosamines. These differences are also observed in mice, rats and hamsters and may also be related to strain, dose, and route of administration, although to our knowledge there are no studies concerning route of administration in mice and rats. Until further studies are done the difference in location of the tumors in various organs in rats given symmetrical or asymmetrical nitrosamines can be considered to be quantitative rather than qualitative.
REFERENCES 1. 2.
H. ])RUCKREY, R. PREUSSMAN,D. SCHM.~HL and M. IV~iJLLER, Chemische Konstitution und carcinogene Wirkung bei Nitrosaminen. Naturwissenschaften 48, 134 (1961). H. DRUCKREY, R. PREUSSMAN, G. BLUM, S. IVANKOVIC and J. AFKHAM, Erzengung yon Korzinomen der Speiser6hre durch unsymmetrische Nitrosarnlne. Naturwissenschaften 50, 100 (1963).
2 ~
........
o,~.~
....
.............................
.~. ~......
Fig. 1. A small carcinoma of the esophagus that would have been di~cult to observe on gross examination of the esophagus. The carcinoma is well demarcated and is located beneath the surface that is covered by keratin. Hematoxylin and eosin, x 1 6 0 . Fig. 2. Histologically the carcinoma is undifferentiated. Cells have large nuclei with prominent nucleoli, a small amount of basophilic, cytoplasm, and grow in sheets. Hematoxylin and eosin, x 440.
(to face
p.
98)
Carcinomas of the Esophagus in Rats Ingesting Diethylnitrosamine 3. 4. 5. 6. 7. 8. 9. 10. 11.
N . P . NAPOLKOV and K. M. POZHARISSKI,Morphogenesis of experimental tumors of the esophagus, d. nat. Cancer Inst. 39, 903 (1967). H. DRUCKREY, R. PREUSSMAN,D. SCHMAHLand G. BLUM, Carcinogene Wirkung yon N-Methyl-N-Nitroso-Anilin. Naturwissenschaften 98, 722 (1961). H. DRUCKREY,R. PREUSSMAN,D. SCHM2~HLand M. M/.)LLER,Erzengung von Magenkrebs durch Nitrosamide an Ratten. Naturwissenschaften 48, 165 (1961). N . K . CLAPP and A. W. CRAIG, Carcinogenic effects of diethylnitrosamine in RF mice. J. nat. Cancer Inst. 39, 903 (1967). K. McD. HERROLD, Effect of route of administration on the carcinogenic action of diethylnitrosamine (N-nitrosodiethylamine). Brit. J. Cancer 18, 763 (1964). K. McD. HERROLD, Epithelial papillomas of the nasal cavity. Arch. Path. 78, 189 (1964). K. McD. HERROLD, Induction of olfactory neuroepithelial tumors in Syrian hamsters by diethylnitrosamine. Cancer (Philad.) 17, 114 (1964). K. McD. HERROLD and L. J. DUNHAM, Induction of tumors in the Syrian hamster with diethylnitrosamine (N-nitrosodiethylamine). Cancer Res. 23, 773 (1963). K. MaD. HERROLD,Epidermoid carcinomas of the esophagus and forestomach induced in Syrian hamsters by N-Nitroso-N-methylurethan. d. nat. Cancer Inst. 37, 389 (1966).
99
Carcinomas of the Esophagus in Rats Ingesting Diethylnitrosamine MELVIN D. REUBER Laboratory of Biology, National Cancer Institute, Bethesda, Maryland 20014, U.S.A. Abstract--Buffalo strain male and female rats 12 weeks of age ingested 0.0114% diethylnitrosamine (DEN) in the dietfor 26 weeks and were returned to the basal diet without D E N for the remainder of the experiment. Rats survived an average of 28 weeks. Carcinomas of the esophagus were observed in 9 of 14 male and 5 of 14female rats. The incidence of carcinomas of the esophagus was higher in male rats; however, the incidence of liver carcinomas was higher in female rats. The organ site of induction of carcinomas with the symmetrical and asymmetrical nitrosamines is discussed.
INTRODUCTION
diet for 26 weeks ad libitum. After that time rats were given the basal diet. Animals were weighed weekly. Surviving animals were killed by exsanguination 36 weeks after the start of the experiment. All organs, except the nasal cavity, were examined grossly and histologically. The esophagus was removed with the thyro-cricoid cartilage and the stomach. The esophagus was opened and lesions described, and the size and location recorded. Tissues were fixed in 4 % formaldehyde and stained routinely with hematoxylin and eosin. Special stains, when indicated, included periodic acidSchiff (PAS).
DRUCKREY et al. felt that the N-N-diethylnitrosamines had an affinity for the liver because they were metabolized by and induced carcinomas of the liver [1] and that the asymmetrical nitrosamines were specific for the esophagus and carcinomas in that organ [2, 3]. This report describes carcinomas of the esophagus, as well as liver, in Buffalo strain male and female rats ingesting diethylnitrosamine (DEN) in the diet. MATERIAL AND METHODS Inbred Buffalo male and female rats (National Institutes of Health Animal Production), 12 weeks of age, were used. They were given 0.0114% D E N in a semisynthetic diet. Morris diet # 272 contained 300 g commercial casein, 2275 g skim milk powder, 6152 g ground hard spring wheat, 200 g brewer's yeast, 200 g desiccated liver, N.P. (Wilson Laboratories, Chicago, Ill.) 140 g sodium chloride, 10 g ascorbic acid, 13 g ferric citrate, 100 g cod-liver oil, and 610 g corn oil. Animals ingested the carcinogen-containing
RESULTS Animals gained weight until about 2 weeks before death or termination of the experiment, at which time they lost 16-20 g. Both the males and females survived for an average of 28 weeks [range 25-34]. Nine of 14 male rats and 5 of 14 female rats developed carcinomas of the esophagus. The lesions measured 1.5-1-7 cm dia and were located 1.5-2.0cm (average 1.7) from the gastro-esophageal junction in male rats and half of the female rats; in the remaining half of the females the carcinomas were located at the
Accepted 27 September 1974. *Present address: 11014 Swansfield Road, Columbia, Maryland 21044, U.S.A. 97
98
Melvin D. Reuber
pharyngeal tracheal level of the esophagus. Histologically the carcinomas were a mixture of undifferentiated, poorly differentiated cells (Figs. 1 and 2) and well-differentiated squamous cells with keratin pearls. There was focal invasion of the submucosa. Bronchopneumonia, observed in 5 female and 3 male rats with carcinomas of the esophagus, probably resulted from obstruction of the esophagus and regurgitation into the lungs. Carcinomas of the esophagus occurred most often in rats with either small carcinomas of the liver or in rats without malignant tumors of the liver.
Lesions in organs other than esophagus Ten female rats had carcinomas and 3 sarcomas of the liver; as compared with 5 carcinomas and 2 sarcomas of the liver in male rats ingesting DEN. This higher incidence of malignant tumors of the liver in females was significant at the P = < 0.05 level. Female rats also had random malignant tumors in other organs. These were located in the uterus, brain, and ovary. Carcinomas were not observed in the oral cavity, tongue or squamous portion of the stomach. DISCUSSION Carcinomas of the esophagus were observed regularly by unsymmetrical nitrosamines in young BD strain rats and the authors concluded that the chemicals were specific inducers of carcinomas of that organ [2]. Ethyl-vinylnitrosamine, methyl-benzyl-nitrosamine, dimetyl-dinitroso-ethylenediamine, n-nitroso-sarkosin, nitroso-sarkosin-ethyl, n-nitrosopiperidine and n-methyl-n-nitrosoaniline all induced carcinomas of the esophagus; whereas rats given ethyl-isopropyl-nitrosamine and ethyl-nbutyl-nitrosamine had carcinomas of the liver in addition to esophageal carcinomas [1-4]. Carcinomas were observed only in the liver in rats given ethyl-ethanol-nitrosamine [2]. Ethylalkyl-nitrosamide and diazo-aceto-ester rarely produced carcinoma of the esophagus, but almost always induced squamous cell carcinoma of the stomach [5].
Buffalo strain rats ingesting D E N in the diet developed carcinomas of both the esophagus and liver. The carcinomas of the esophagus occurred in rats without hepatic carcinomas or in rats with small hepatic carcinomas. Carcinomas of the esophagus were either localized or diffuse in BD strain rats given unsymmetrical nitrosamines. In Buffalo strain rats given D E N carcinomas of the esophagus were focal and located either near the gastroesophageal junction or at the level of the pharyngeal tracheal bifurcation. The incidence of carcinomas of the esophagus was higher in male Buffalo strain rats and the incidence of hepatic carcinomas and sarcomas was higher in Buffalo strain female rats ingesting DEN. This finding would indicate that the chemical was first transformed into an active carcinogenic agent in the esophageal mucosa and later reached the liver for additional metabolism, and that the esophagus was more susceptible in males than in female rats. With diffuse localization in the esophagus in BD strain rats given unsymmetrical nitrosamines, it could be predicted that little of the chemicals or their metabolites would reach the liver. R F mice given D E N in the drinking water developed tumors of the lungs, liver, and stomach and a small number of microscopic tumors of the esophagus [6]. The authors stressed the importance of not relying on gross examination and of doing histological studies of the esophagus. Hamsters developed some liver tumors, but differed from rats and mice by developing tumors of the nasal cavity, trachea, bronchus and lungs regardless of the route by which D E N was given [7-10]. There are differences in the location of tumors in rats given nitrosamines. These differences are also observed in mice, rats and hamsters and may also be related to strain, dose, and route of administration, although to our knowledge there are no studies concerning route of administration in mice and rats. Until further studies are done the difference in location of the tumors in various organs in rats given symmetrical or asymmetrical nitrosamines can be considered to be quantitative rather than qualitative.
REFERENCES 1. 2.
H. ])RUCKREY, R. PREUSSMAN,D. SCHM.~HL and M. IV~iJLLER, Chemische Konstitution und carcinogene Wirkung bei Nitrosaminen. Naturwissenschaften 48, 134 (1961). H. DRUCKREY, R. PREUSSMAN, G. BLUM, S. IVANKOVIC and J. AFKHAM, Erzengung yon Korzinomen der Speiser6hre durch unsymmetrische Nitrosarnlne. Naturwissenschaften 50, 100 (1963).
2 ~
........
o,~.~
....
.............................
.~. ~......
Fig. 1. A small carcinoma of the esophagus that would have been di~cult to observe on gross examination of the esophagus. The carcinoma is well demarcated and is located beneath the surface that is covered by keratin. Hematoxylin and eosin, x 1 6 0 . Fig. 2. Histologically the carcinoma is undifferentiated. Cells have large nuclei with prominent nucleoli, a small amount of basophilic, cytoplasm, and grow in sheets. Hematoxylin and eosin, x 440.
(to face
p.
98)
Carcinomas of the Esophagus in Rats Ingesting Diethylnitrosamine 3. 4. 5. 6. 7. 8. 9. 10. 11.
N . P . NAPOLKOV and K. M. POZHARISSKI,Morphogenesis of experimental tumors of the esophagus, d. nat. Cancer Inst. 39, 903 (1967). H. DRUCKREY, R. PREUSSMAN,D. SCHMAHLand G. BLUM, Carcinogene Wirkung yon N-Methyl-N-Nitroso-Anilin. Naturwissenschaften 98, 722 (1961). H. DRUCKREY,R. PREUSSMAN,D. SCHM2~HLand M. M/.)LLER,Erzengung von Magenkrebs durch Nitrosamide an Ratten. Naturwissenschaften 48, 165 (1961). N . K . CLAPP and A. W. CRAIG, Carcinogenic effects of diethylnitrosamine in RF mice. J. nat. Cancer Inst. 39, 903 (1967). K. McD. HERROLD, Effect of route of administration on the carcinogenic action of diethylnitrosamine (N-nitrosodiethylamine). Brit. J. Cancer 18, 763 (1964). K. McD. HERROLD, Epithelial papillomas of the nasal cavity. Arch. Path. 78, 189 (1964). K. McD. HERROLD, Induction of olfactory neuroepithelial tumors in Syrian hamsters by diethylnitrosamine. Cancer (Philad.) 17, 114 (1964). K. McD. HERROLD and L. J. DUNHAM, Induction of tumors in the Syrian hamster with diethylnitrosamine (N-nitrosodiethylamine). Cancer Res. 23, 773 (1963). K. MaD. HERROLD,Epidermoid carcinomas of the esophagus and forestomach induced in Syrian hamsters by N-Nitroso-N-methylurethan. d. nat. Cancer Inst. 37, 389 (1966).
99
