Synthesis of N-Butyl-N-nitrosourethan (BNUR) BNUR was synthesized as follows:

Transcription

1 [GANN, 65, ; June, 1974] INDUCTION OF TUMORS OF THE FORESTOMACH, ESOPHAGUS, PHARYNX, AND ORAL CAVITY OF THE DONRYU RAT GIVEN N-BUTYL-N-NITROSOURE- THAN IN THE DRINKING WATER*1 Masaya TAKEUCHI,*2 Shozo KAMIYA,*3 and Shigeyoshi ODASHIMA*2 Department of Chemical Pathology*2 and Department of Synthetic Chemistry,*3 National Institute of Hygienic Sciences, Tokyo Three groups of Donryu rats of both sexes were given continuously 400, 200, or 100ppm solution of N-butyl-N-nitrosourethan (BNUR) as their drinking water. A high incidence of squamous cell carcinomas, 126 out of 150 rats (84%), and papillomas, 149 out of 150 rats (99%), arising from the upper digestive tract, was found in all three groups. Mean survival time of tumor-bearing rats was as short as 200 days in the group that received 400ppm BNUR solution, and the earliest development of squamous cell carcinoma was found in the forestomach of a rat killed on the 112th experimental day. The study on the morphogenesis of squamous cell tumors arising from the esophagus and forestomach was made in rats that were killed on the 4th, 8th, 12th, 16th, and 20th experimental week of the BNUR treatment. The result showed that the majority of these tumors developed either directly or from papillary down-growths of the squamous cell epithelium. It is well known that various kinds of unsymmetric dialkyl N-nitrosoamines induce only esophageal tumors in experimental animals.2) The carcinogenic activity of orally administered N-nitrosourethan derivatives has been reported by several investigators, and tumors of the fore- and glandular stomach of rats and mice were induced by N-methyl-N-nitrosourethan,1, 8,9) N-ethyl-N-nitrosourethan,9) and N-(2-chloroethyl)-N-nitrosourethan.11) However, carcinogenicity of N-butyl-N-nitrosourethan (BNUR) has not been previously reported. This report describes the results of two animal experiments; a study on the carcinogenicity and target organ of BNUR in rats given BNUR continuously for their life span, and the morphogenesis of squamous cell carcinomas arising from the esophagus and forestomach in rats given BNUR for 4 to 20 weeks. MATERIALS AND METHODS Rats Five-week-old Donryu rats, obtained from the Nippon Rat Co., Tokyo, were maintained on the basal diet CE-2 (CLEA Japan Inc., Tokyo) and tap water until they were 11 weeks old when the oral administration of BNUR was started. Four rats were housed in a hanging metal cage and kept in an airconditioned room. Synthesis of N-Butyl-N-nitrosourethan (BNUR) BNUR was synthesized as follows: CH3CH2CH2CH2-NH2 CH3CH2CH2CH2-NH-COOC2H5 NaOH NO CH3CH2CH2CH2-N-COOC2H5 H2SO4 Butylurethan: A solution of 22.0g (0.55mol) of NaOH in 200ml of water and 59.5g (0.55mol) of Cl-COOC2H5 was added dropwise into a mixture of 36.5g (0.50mol) of butylamine and 100ml of water in a three-necked flask, with stirring under ice-cooling. The ice bath was then removed, and the mixture was stirred for 2hr. The reaction mixture was extracted twice with ether, the ether layer was washed with 2% NaOH solution and then with ice water. After treatment with anhyd. Na2SO4, the ether was evaporated, and the residue was distilled under a reduced pressure. The product, butylurethan, was obtained N, Found: C, 57.42; H, 10.08; N, N-Butyl-N-nitrosourethan: A solution of 72.5g (three times the theoretical amount) of NaNO2 in 180 ml of water was added dropwise into an ice-cooled mixture of 50.8g (0.35mol) of butylurethan and 150 *1 This work was supported partly by a Grant-in-Aid for Scientific Research from the Ministry of Education. 65(3)

2 M. TAKEUCHI, ET AL. twice with ether, and the ether layer was washed with ice water and dried over anhyd. CaCl2. The product Torr. Yield, 57g (94%), Anal. Calcd. for C7H14O3N2: C, 48.26; H, 8.10; N, Found: C, 47.81; H, Experiment I Three experimental groups were given continuously 15-25ml/day/rat of 400 (Group A), 200 (Group B), and 100 (Group C) ppm BNUR solution, as their drinking water until they were autopsied. Eighty rats, 40 males and 40 females, were in each group. Dead or sacrificed rats were autopsied and examined grossly and histologically for the development of tumors in various organs. Experiment II Twenty-five male Donryu rats were given 20ml/day/rat of a 400ppm BNUR solution as their drinking water. Five rats each were killed on the 4th, 8th, 12th 16th, and 20th experimental week, for morphological studies on the development of squamous cell carcinoma of the esophagus and forestomach. RESULTS Experiment I Mean daily dose of BNUR given to the rats was 27, 16, and 8mg, and the average total dose was 5.3, 3.7, and 2.3g/kg body weight in each rat in Groups A, B, and C, respectively, calculated from the mean body weight of the rat and the mean volume of the BNUR solution consumed by the rat. Oral LD50 of BNUR in Donryu rat was approximately 900mg/kg. Incidence of Tumors and Mean Survival Time (Tables I and II) Since the earliest death of a rat with tumor was found on the 160th experimental day, the rats surviving for more than 160 days were arbitrarily selected as effective animals. Ninety (37.5%) out of 240 rats in all groups died of toxic effect of BNUR and of pneumonia before the 160th day. In some of these rats, neoplastic lesions of the forestomach and esophagus, and degenerative changes in the liver and spleen were found. As shown in Table I, tumors were limited to the upper digestive tract, i.e., oral cavity, pharynx, esophagus, and forestomach, except in the mammary glands where tumors were detected in 7 (5%) out of 150 rats. Total incidence of papillomas was 99% (149/150) and that of carcinoma was 84% (126/150). There was no sex difference or dose response in the incidence of these tumors. However, the mean survival time was prolonged in the groups given lower concentrations of BNUR. Incidence of tumors in the oral cavity, pharynx, esophagus, and forestomach is summarized in Table II. Total incidence of papillomas was 94% (141/150) in forestomach, 91% (137/150) in esophagus, 70% (98/139) in pharynx, and 36% (48/135) in oral cavity. Carcinomas were detected in 73% (110/150) in forestomach, 34% (51/150) in esophagus, 16% (22/139) in pharynx, and 10% (14/135) in oral cavity. There was no sex difference or dose response on the development of tumors in each organ. Table I. Tumor Incidence and Mean Survival Time Pap=papilloma, Ca=carcinoma 228 GANN

3 CARCINOGENICITY OF BUTYLNITROSOURETHAN IN RATS Table II. No. of Rats with Tumors in Upper Digestive Tract Pap=papilloma, Ca=carcinoma Percentage in parentheses Adhesion of the stomach to surrounding organs, due to the invasive growth of tumors, was occasionally associated with abscess and observed in 39 rats (26%). Dissemination of the tumors in the peritoneal cavity was found in 13 rats (9%) (Photo 2). Remote metastasis of the forestomach tumor was detected in 3 rats (2%), only in the lung in 2 rats, anti in the lung, adrenals, liver, and kidneys in the remaining 1 rat. Gross Findings of the Tumors Most of the forestomach tumors were papillary and granular, either rounded and elevated or broad and sessile. The serosal surface was collapsed and protruded at the tumorous lesions (Photo 1). In many cases, a large tumor nodule or multiple tumorous lesions occupied most of the mucosa with extension through the wall. Esophageal tumors were generally white and papillary with thin pedicles, but sometimes were broad and sessile. The location of tumors was often diffuse, but large tumor nodules were frequently found at the distal portion close to the gastroesophageal junction (Photo 1). Tumors of the pharynx, tongue, lip, and soft and hard palates were mainly white and papillary with a thin pedicle. Microscopic Findings of the Tumors All the malignant tumors of the upper digestive tract were squamous cell carcinoma (Photo 4). However, the pattern varied due to the different proportion of the tumor cell clusters to connective tissue, different shapes and sizes of the clusters, different degrees of keratinization, hemorrhage, necrosis, and inflammation. Tumor cells were arranged in sheets, masses, nests, and anastomaosing branching cords. In the central part of the tumor clusters, there was pearl formation consisting of concentrically arranged masses of keratin and parakeratotic material containing scattered eleidin granules and bordered by viable neoplastic cells. In the surrounding areas of tiny cancerous lesions, there were papillomatous and downgrowths of irregularly shaped squamous cell epithelium penetrating the muscularis mucosae (Photos 3 and 5). In the collapsed lesions of the forestomach, the serosal surfaces were adherent, the submucosal tissues thickened with blood vessels, inflammatory cells, and edema, and the epithelium revealed papillomatous changes associated with small ulcers and endophytic down-growths (Photo 3). Moreover, invasion of squamous cell carcinomas was frequently present (Photo 3). Most tumors of the esophagus, pharynx, and oral cavity were squamous cell papillomas with thin pedicles. In a few cases, however, strands of cancer cells invaded into the pedicles 65(3)

4 M. TAKEUCHI, ET AL. (Photo 6). Although the tiny cancerous lesions were usually found in the submucosa of the pedicle of the papilloma, large cancerous lesions were broad and sessile penetrating through the muscle layers. Cancer clusters were composed of keratinizing squamous epithelial cells in many cases, and in a few cases, they were composed of small spinous cells associated with keratinization (Photo 7). One of the seven cases of mammary tumor was tubulo-acinary adenocarcinoma and 6 were fibroadenomas. Besides the neoplastic lesions, there was minimal bile duct proliferation and centrolobular degeneration in the liver, inflammatory changes in the lung, and slight degeneration of tubuli in the testis. Experiment II Fourth Week A few foci of leucoplakia on the mucosa of the forestomach were found in 2 out of 5 rats. No proliferative changes were noted in other organs. Microscopically, the leucoplakia was composed of thickened spinous cell layer and edema in the lamina propria mucosae. Eighth Week Many small foci of leucoplakia diffusely distributed on the mucosa and a few small papillomas were found in the forestomach (Photo 8). Microscopically, diffuse thickening of spinous cell layers, irregular patterns of basal cell layer in some areas, and slight edema in propria were seen (Photo 11). The papillomatous formation consisted of papillary hyperplasia and vacuolisation of epithelial cells and hyperkeratosis (Photo 12). Twelfth Week The mucosa of the forestomach was covered by numerous foci of leucoplakia and partly raised up in folds. Microscopically, irregularity in shape of basal cell layers, hyperplasia and atypism of spinous cell layers, and hyperkeratosis were seen in most of the forestomach mucosa. The submucosal connective tissues in the folds was edematous, and the muscularis mucosae was pulled up toward the cavity of the stomach. The esophageal mucosa was slightly thickened and vacuolisation of basal cells and edema in the propria were occasionally observed. Sixteenth Week The number of foci of leucoplakia in the forestomach mucosa became innumerable, and small ulcers were found in 2 out of 5 rats (Photo 9). In one of these two, a large part of the forestomach protruded forming a lump in the cavity (Photo 10). Microscopically, hyperplasia and atypism of spinous cell layers, marked hyperkeratosis, and focal dyskeratosis were noted in all parts of the forestomach (Photo 13), and endophytic downgrowths of epithelial cells were found at the protruded lesions associated with surficial ulcer and small abscesses. A well-developed squamous cell carcinoma was found in one rat. In addition to the proliferative changes in the forestomach, multiple tiny nodules resembling hyperplasia of epithelium were found in the esophagus and pharynx of one rat each (Photo 10). Twentieth Week Grossly, the protruded lesions of the forestomach were detected in 3 rats. Microscopically, the mucosal surface of all these protruded lesions was covered by either papillary hyperplasia or papillomas, and endophytic down-growths of epithelial cells were sporadically detected in the submucosal connective tissue. Papillomatosis was found in the remaining part of the forestomach. In one of the other 2 rats, the entire part of the forestomach was thickened solidly and was irregularly shaped. This was a lesion of squamous cell carcinoma invading through all the coats of the wall (Photo 14). On the esophageal and/or pharyngeal mucosa a few papillomas (diameter, 3-8mm) with thin pedicles, and many granular foci of leucoplakia were observed. Microscopically, hyperplasia of spinous cell layers and slight irregularity of basal cell layers were found on these areas. 230 GANN

5 CARCINOGENICITY OF BUTYLNITROSOURETHAN IN RATS DISCUSSION The present studies showed that BNUR has a strong carcinogenic activity for the squamous cell epithelium of the upper digestive tract, i.e., the forestomach, esophagus, pharynx, and oral cavity, of the rat when it is administered continuously in their drinking water. Its remarkable features among various carcinogens having similar activity are the high incidence and short period for the induction of these tumors. The earliest detection of a carcinoma of the forestomach was made on the 112th experimental day or in the 16th week, and the average survival period was as short as 187 days in male animals given 400ppm BNUR solution. On the morphogenesis of squamous cell carcinoma of the esophagus, Napalkov and Pozharisski6) stated that the foci of leucoplakia develop to papilloma and finally to carcinoma in the rat given orally N-methyl-N-nitrosoaniline. On the other hand, Pozharisski7) recently proposed three different processes for the development of esophageal carcinomas in the rat given orally N-methyl-N-nitrosoaniline or with ethyl ester of N-nitrososarcosine. The first process is similar to that described by Napalkov and Pozharisski.6) In the second process, it develops through leucoplakia, dysplasia, basal cell type of carcinoma in situ, and then invasive squamous cell carcinoma. The third process is "straightway" carcinoma. Ito et al.4) suggested, however, that some of the hyperplasias and papillomas of the esophagus induced by N- nitrosopiperidine may be reversible. In our present studies, many of the tiny lesions of carcinoma in the esophagus were found either in the pedicle of a small papilloma or in the submucosa under leucoplastic lesions. Although a few small carcinomas consisted mainly of small spinous cells, foci of keratinization were always present. Therefore, it was difficult to find basal cell carcinoma, in situ or invasive, in our studies. In the forestomach, a solitary papilloma was seldom seen, and development of papillomatosis in the whole mucosa was common. A tiny carcinomatous lesion in the submucosa was found either continuing with the basal cell layer of papillomatous mucosa or branching from the down-growth which was composed of cells with irregularity in shape, size, and arrangement of keratinizing squamous cell epithelium (Photos 3 and 5). There was no association of these small carcinomas with either basal cell carcinoma or papilloma. Druckrey reported that some of nitrosoamine derivatives induced esophageal tumors in the rat not only by oral administration but also with subcutaneous or intravenous injection of the chemical.2) In the case of N-methyl-N-nitrosourethan (MNU), however, intraperitoneal or subcutaneous injection did not produce tumors in the forestomach of rats10) or mice,5) although oral administration did in rats.1,8,9) Shoenthal9) proposed the local action of MNU in producing tumors in the esophagus and forestomach, but Herrold3) stated a reverse view based on her experiment using hamsters. The present studies clearly showed that BNUR induced squamous cell carcinoma in a short period of time, and that the tumors developed earlier in the mucosa where BNUR solution stayed for a longer period, i.e., in the forestomach. Therefore, it is postulated that BNUR has a local action in tumorigenesis and also has a highly specific affinity for squamous cell epithelium. Thus, it may be expected that BNUR will be a useful carcinogen which induces a model disease of squamous cell carcinoma in experimental animals. REFERENCES (Received January 30, 1974) 1) Druckrey, H., Preussmann, R., Schmahl, D., Muller, M., Naturwissenschaften, 48, 165 (1961). 2) Druckrey, H., Preussmann, R., Ivankovic, S., Schmahl, D., Z. Krebsforscḥ, 69, 103 (1967). 65(3)

6 M. TAKEUCHI, ET AL. 3) Herrold, K. McD., J. Natl. Cancer Inst., 37, 389 (1966). 4) Ito, N., Kamamoto, Y., Hiasa, Y., Makiura, S., Marugami, M., Yokota, Y., Sugihara, S., Hirao, K., Gann, 62, 445 (1971). 5) Matsuyama, M., Suzuki, H., Nakamura, T., Brit. J. Cancer, 23, 167 (1969). 6) Napalkov, N., Pozharisski, K. M., J. Natl. Cancer Inst., 42, 927 (1969). 7) Pozharisski, K. M., "Pathology of Tumors in Laboratory Animals," p. 87 (1973). International Agency for Research on Cancer, Lyon. 8) Schoenthal, R., Nature, 188, 420 (1960). 9) Schoenthal, R., Nature, 199, 190 (1963). 10) Schoenthal, R., Bensted, J. P. M., Brit. J. Cancer, 22, 316 (1968). 11) Schoenthal, R., Bensted, J. P. M., Cancer Res., 31, 573 (1971). EXPLANATION OF PLATES Photo 1. A large tumor of the forestomach, multiple large and small tumor nodules in the esophagus, and several small papillary tumor nodules in the pharynx are seen. The entire wall of the esophagus is dilatated due to the multiple large tumor nodules at the distal portion. From a rat given 200ppm BNUR solution for 36 weeks and killed. Photo 2. Disseminated tumor nodules in the peritoneal cavity. The stomach is adherent to the surrounding organs and peritoneum. From a rat given 100ppm BNUR solution for 49 weeks and killed. Photo 3. A section of a protruded nodule of the forestomach. The surface of the nodule is covered by hyperkeratosis, papillomatosis, papillomas, and a small ulcer. In the submucosa, a few down-growths and carcinomatous lesions are found in highly thickened connective tissues with a number of blood vessels, edema, and inflammatory cells. From a rat given 400ppm BNUR solution for 24 weeks and killed. Photo 4. Squamous cell carcinoma of the forestomach. Irregularly shaped keratinizing cancer cell clusters. anastomosing with each other invade the serosa. From a rat given 100ppm BNUR solution for 47 weeks and killed. Photo 5. A number of irregularly shaped strands and clusters, branching and anastomosing with each other, are seen under papillomatosis and surrounding the endophytic down-growth. From a rat given 400ppm BNUR solution for 47 weeks and killed. Photo 6. A papilloma and some clusters of cancer cells invading into the submucosa in the pedicle of the papilloma in the esophagus. From a rat given 200ppm BNUR solution for 34 weeks and killed. Photo 7. Squamous cell carcinoma of the esophagus, composed of small spinous cells like a basal cell carcinoma with keratinizing masses. From a rat given 100ppm BNUR solution for 48 weeks. Photo 8. Early changes on the mucosa of the forestomach. Many small foci of leucoplakia and a few tiny papillomas are seen. From a rat given 400ppm BNUR solution for 8 weeks and killed. Photo 9. Innumerable foci of leucoplakia and a few ulcers on the mucosa of the forestomach and a few foci of leucoplakia in the esophagus are seen. From a rat given 400ppm BNUR solution for 16 weeks and killed. Photo 10. A tumor nodule with an ulcer in the forestomach and multiple fine nodules in the esophagus are seen. From a rat given 400ppm BNUR solution for 16 weeks and killed. Photo 11. Early changes in the forestomach include thickening of all layers of the epithelium, slight irregularity in basal cell layer, and edema of the lamina propria. From a rat given 400ppm BNUR solution for 8 weeks and killed. Photo 12. Papillary hyperplasia of the forestomach mucosa. From a rat given 400ppm BNUR solution for 8 weeks and killed. Photo 13. Papillomatosis and focal dyskeratosis of the forestomach. From a rat given 400ppm BNUR solution for 16 weeks and killed. Photo 14. Keratinizing squamous cell carcinoma of the forestomach invading through all the coats of the wall. From a rat given 400ppm BNUR solution for 20 weeks and killed. 232 GANN

7 CARCINOGENICITY OF BUTYLNITROSOURETHAN IN RATS 65(3)

8 M. TAKEUCHI, ET AL. 234 GANN

9 CARCINOGENICITY OF BUTYLNITROSOURETHAN IN RATS 65(3)

10 M. TAKEUCHI, ET AL. 236 GANN