Glandular Tumors of the Nasal Cavity Induced by Diethylnitrosamine in Syrian Golden Hamsters 1,2

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1 Glandular Tumors of the Nasal Cavity Induced by Diethylnitrosamine in Syrian Golden Hamsters 1,2 F. Stenback/ The Eppley Institute for Research in Cancer, University of Nebraska Medical Center, Omaha, Nebraska SUMMARY-The morphologic pattern and biologic characteristics of diethylnitrosamine (DEN}-induced glandular tumors of the nasal cavity in hamsters were studied. DEN induced papillary tumors of the surface epithelium, and acinar tumors of the submucous glands. Adenomas, and ultimately papillary adenocarcinomas with squamous metaplasia, were preceded by papillary hyperplasia of the respiratory epithelium. Most of the acinar tumors in the anterior nasal cavity were weakly mucin-producing, in contrast to those in the posterior part with mucosa covered by olfactory epithelium, which were strongly mucin-producing. The tubular type, a subtype, was composed of cylindrical cells with little mucin production. These findings show that the DEN-induced tumors originating from the surface epithelium and the submucous gland in Syrian golden hamsters are in many respects similar to such tumors in man.-j Natl Cancer Inst 50: , THE CARCINOGENICITY of diethylnitrosamine (DEN) for the respiratory tract, including the nasal cavity, has been demonstrated in several species (1-3). Herrold (4) showed that neuroblastomas of the olfactory epithelium are common in hamsters treated with DEN. Nasal papillomas induced in hamsters by DEN are histologically and biologically similar to those in humans (5). The induced adenomatoid nasal cavity tumors (2, 3, 6, 7) are of diverse histologic patterns. However, reports of their histogenesis and behavior have not been published. The hamster nasal cavity is an appropriate model for study of neoplastic alterations similar to those in humans and DEN is a possible environmental carcinogen (8, 9); therefore, it is of interest to describe in detail the glandular tumors induced by DEN in these animals and compare their morphologic and biologic characteristics with those in humans. Chemicals.-Subcutaneous injections of DEN (Eastman Organic Chemicals, Rochester, N.Y.) dissolved in 0.9% sterile N aci (Baxter Laboratories Inc., :Morton Grove, Ill.) were given I mg/week for 12 weeks. (Other aspects on the experiments are discussed separately.) The DEN solutions were prepared before each treatment. The experimental animals were examined and weighed weekly. Some animals were killed during the experiment, but most were left to die or were killed when moribund. All animals were autopsied except for a few lost through cannibalism. The entire skull was fixed in 10% formalin and decalcified. :Multiple frontal sections of the nasal cavity were stained with hematoxylin and eosin and special stains when necessary. RESULTS The nasal cavity of the Syrian golden hamster consists of 2 ectoturbinal and 4 endoturbinal spaces divided by a turbinal septum (fig. 1). The hamster has only 1 sinus maxillaris. The anterior one-third of the turbinals is covered by a columnar epithelium and the posterior two-thirds is covered MATERIALS AND METHODS Animals.-A total of 184 female Syrian golden hamsters from our colony, randomly bred since 1959, were divided into groups of 6, housed in plastic cages with sterilized granular cellulose bedding, and fed Rockland rat diet pellets and tap water ad libitum. The animals were 6-7 weeks old and averaged 95 g when the experiment started. 1 Received June 20, 1972; accepted October 10, Supported by grants WOO B from the American Cancer Society, and WOO A from Research Council Incorporated. 3 Part of the work reported here was done during the tenure of a Research Training Fellowship awarded to The International Agency for Research on Cancer, Lyon, France. 895

2 896 STENBACK by olfactory epithelium. In the anterior portion, the ciliated columnar epithelial cells are interspersed with mucus-secreting goblet cells; also observed in this area are alveolar submucosal glands containing neutral and acid mucopolysaccharides. The lamina propria mucosae of the epithelium in the posterior portion of the turbinal system contains the olfactory glands of Bowman, which are of rounded tubular-alveolar type with abundant acid and neutral mucopolysaccharides. These glands open to the surface through a narrow duct. The secretory portions are lined with low pyramidal serous cells. In the lower part of the anterior septum is Jacobson's vomeronasal organ. After a few weeks of DEN treatment, the initial change was necrosis of the epithelium of the nasal cavity. Hyperplasia and squamous metaplasia were observed later in 46 animals. The first tumors, polypoid lesions with a fibrous stalk supporting a columnar epithelium (fig. 2), were seen at the 39th week. The number of tumors gradually increased and a total of76 of 146 animals developed nasal cavity tumors: 6 squamous tumors, 22 neuroepithelial tumors, and 48 glandular tumors. No consistent relationship between nasal cavity tumors and tumors of other organs was found. All animals were dead by the 90th week; respiratory disorders were the most common causes of death. Animals without tumors occasionally showed accumulations of inflammatory cells in the submucosa, epithelial detachment and metaplasia, and necrotic debris in the nasal cavity. Goblet-cell increase and a mucin positive layer on the surface were observed with alcian blue-pas staining (fig. 2). The epitheliallayer gradually increased in thickness, with the histologic appearance of a regular hyperplasia, and later became polypoid in character. Adenomatous hyperplasia of the columnar epithelium, with increase in cell size and loss of cilia, was observed after lo weeks of treatment. Later the tumors formed polypoid masses composed of glandular structures containing mucin (fig. 3). Some of the large tumors almost filled the nasal cavity. Squamous metaplasia was a common feature of these polypoid masses. In the fully developed stage the tumors had multiple cystic spaces, which were lined with epithelial cells and contained mucous material (fig. 4). On a histologic and cytologic basis these papillary tumors were classified as carcinomas though they did not infiltrate the surrounding tissue, and no metastases were present. The morphologic alterations of the submucous glands followed 3 main lines. The glands of the anterior part of the nasal cavity showed in a few cases a dysplastic alteration of the epithelium (fig. 5). There were some tumors that consisted of acinic structures formed by mucin-producing cells with eccentric nuclei and a faintly eosinophilic cytoplasm (fig. 6). Osseous and fibrous metaplasia were also seen in the tumors. These "acinar" carcinomas became large in some animals, eroded the skull, and infiltrated the brain. Other neoplastic transformations started as an adenomatous hyperplasia made up of tubular structures that contained columnar cells without secretory activity (fig. 7). The fully developed tumors consisted of single or divided tubules. Mucous substances in significant amounts were visible only in the stroma (fig. 8). The tumor cells were large and cylindrical, with cytoplasm that was faintly mucin-positive. Even when these carcinomas were large they showed little infiltration and no metastases. Other patterns of neoplastic progression originated from the tubuloalveolar mucous glands which are located in the lamina propria mucosae of the posterior part of the nasal cavity and are covered by olfactory epithelium. The tumors began as small nodules that were sharply delimited and consisted of fairly normal glands arranged in alveolar structures; the latter were composed of cells with basal hyperchromatic nuclei and vacuolated cytoplasm. These cells contained abundant mucous material that stained positively for acid mucopolysaccharides, mainly hyaluronic acid; they also had diastase-reactive, PAS-positive material, possibly glycogen (fig. 9). A few of these carcinomas exhibited patterns of multiple secretory ducts accompanied by tubular structures covered by a regular epithelium; such sti uctures were devoid of mucin and thus constituted a tubular subtype (fig. 10). DISCUSSION The nasal cavity of the Syrian golden hamster differs from that of man (10) in that the cavity is long and narrow with a prominent Jacobson's organ. In the hamster and the rat (11, 12), the JOURNAL OF THE NATIONAL CANCER INSTITUTE

3 TUMORS OF THE NASAL CAVITY IN HAMSTERS 897 ethmoid region forms the posterior-superior part of the nasal cavity and contains turbinates instead of cells which occur in the human. Structurally the nasal cavities of the hamster and the rat are similar (11, 12); the main difference is the less prominent subseptal duct in the former. In man, adenomas and adenocarcinomas of the nasal or paranasal cavity are rare (13). The main types are: l) glandular groups of clear cells that resemble adenocarcinomas of the kidney or parathyroid gland; 2) neoplastic groups of goblet cells that produce varying amounts of mucus and are similar to adenocarcinoma of the digestive tract; and 3) an "oncocytic type" with a lymphoid component (13). Ringertz (14) separated the nasal cavity adenocarcinomas into 2 types: alveolar adenocarcinomas with large amounts of mucin and signet cells reminiscent of colloid cancer of the intestine and a pseudopapillary type with numerous goblet and cylindrical cells covering graceful papillae. He postulated that the pseudopapillary adenocarcinoma originated in the epithelium and crypts of the mucous membrane, and the alveolar adenocarcinoma originated in the glandular parenchyma. The tumors in this study bear some similarity to those mentioned in humans. The papillary types growing from the surface epithelium are polypoid and composed of glandular structures with less mucin content than their human counterpart. Adenomatous hyperplasia, frequently found in this study and related to later tumor development, emphasizes the significance of these preneoplastic lesions in the development of adenocarcinomas. Squamous metaplasia is common in this type of tumor, and at times it was difficult to distinguish adenocarcinomas with squamous metaplasia from squamous carcinomas with adenomatoid features. In serially sectioned tumors, some parts of the neoplastic process resembled a squamous tumor and other parts a glandular tumor; separate tumors of distinctly different morphologic character were infrequent. The tumors originating from the submucous glands resemble both the bronchial mucous gland tumors of humans (15) and the nasal alveolar adenocarcinoma (14). The various forms of alveolar tumors were differentiated mainly on the basis of the staining reaction, i.e., some showed a less intense reaction for mucopolysaccharides. The tubular and other forms were classified according to the degree of participation of the various elements in the tubuloalveolar submucous glands in the formation of tumors. No tumors similar to the "colonic" type in man (14) were found. Findings in this study support the opinion presented earlier on the significance of environmental carcinogens in the genesis of cancer of the nasal cavity (8, 9). This study suggests that DENproduced tumors in hamsters, derived both from the surface epithelium and the submucous glands, are in many aspects similar to tumors in man. REFERENCES (1) HOFFMAN E, GRAFFI A: Carcinome der Nasenhohle bei Mausen nach Tropfung der Riickenhaut mit Diathylnitrosamin. Acta Bioi Med Gdansk 12: , 1964 (2) THOMAS C: Zur Morphologie der Nasenhohlentumoren bei der Ratte. Z Krebsforsch 67:1-10, 1965 (3) MONTESANO R, SAFFIOTTI U: Carcinogenic response of the respiratory tract of Syrian golden hamsters to different doses of diethylnitrosamine. Cancer Res 28: , 1968 (4) HERROLD KM: Induction of olfactory neuroepithelial tumors in Syrian hamsters by diethylnitrosamine. Cancer 17 : , 1964 (5) HERROLD KM: Epithelial papillomas of the nasal cavity: Experimental induction in Syrian hamsters. Arch Pathol 78: , 1964 (6) DRUCKREY H, PREUSSMANN R, SCHMAHL D, et al: Chemische Konstitution und carcinogene Wirkung bei Nitrosaminen. Naturwissenschaften 48: , 1961 (7) HERROLD KM, DUNHAM Lj: Induction of tumors in Syrian hamster with diethylnitrosamine (N-nitrosodiethylamine). Cancer Res 23 : , 1963 (8) SANDERSj, BURKLE G: Induktion maligner Tumoren bei Ratten durch gleichzeitige Verfiitterung von Nitrit und sekundiiren Aminen. Z Krebsforsch 73 :54-66, 1969 (9) ZIPPIN C, TEKAWA IS, BRAGG KU, et al: Studies on heredity and environment in cancer of the nasopharynx. j Nat! Cancer Inst 29: , 1962 (10) HAM A W: Histology, 6th ed. Philadelphia, Lippincott, 1969 (11) KELEMEN G, SARGENT F: Nonexperimental pathologic nasal findings in laboratory rats. Arch Otolaryngol 44:24-42, 1946 VOL. 50, NO.4, APRIL 1973

4 898 STENBACK (12) KELEMEN G: The junction of the nasal cavity and the pharyngeal tube in the rat. Arch Otolaryngol 45: , 1947 (13) ASHJE, RAUM M: An Atlas ofotolaryngic Pathology, Washington, D.C., Armed Forces Institute of Pathology, 1949, pp 190 (14) RINGERTZ, N: Pathology of malignant tumors arising in the nasal and paranasal cavities and maxilla. Acta Otolaryngol (suppl) 27:1-405, 1938 (15) KREYBERG L: Histological lung cancer types: A morphological and biological correlation. Acta Pathol Microbiol Scand (suppl) 157:1-92, 1962

5 FIGURE I.-Transverse section of anterior hamster nasal cavity. J = Jacobson's organ, MS = maxillary sinus, NT = nasoturbinal space, MT=maxilloturbinal space, P=palatal process of maxilla, S=septum. Hematoxylin and eosion. X 15 FIGURE 2.-Adenoma in nasal cavity of hamster. Mucus-secreting cells on tumor surface and goblet cells in epithelium contain abundant mucosubstances. Alcian blue-pas. X 225 STENBACK 899

6 FIGURE 3.-Nasal cavity of hamster with tumor consisting of proliferating epithelium on fibrous stalk, and glandular structures apparently deriving from surface epithelium. Hematoxylin and eosin (H & E). X 80. FIGURE 4.-Papillary adenocarcinoma with cylindrical tumor cells. Note connections to the surface epithelium. Alcian blue. X 450. FIGURE 5.-Dysplastic alterations in epithelium of submucous gland of the anterior nasal cavity in DEN-treated hamster. H & E. X 200. FIGURE 5.-Adenocarcinoma of acinar type in nasal cavity. Note irregularly arranged cells infiltrating submucous tissues. H & E. X STENBACK

7 FIGURE 7.-Adenomatous hyperplasia of nasal cavity epithelium with ductular structures covered by a cylindrical epithelium. Hematoxylin and eosin (H & E). X FIGURE 8.-Adenocarcinoma of the acinar non-mucus-producing type in the nasal cavity consisting of tubular structures. Note abundant mucinous material only in stroma. Alcian blue-pas. X 450. FIGURE 9.-Adenocarcinoma of the acinar, mucus-producing type formed of cells strongly positive for acid mucopolysaccharides. Alcian blue-pas. X 180. FIGURE 1O.-Structures in nasal cavity adenocarcinoma of tubular subtype. H & E. X 115 STENBACK