ORIGIN OF PULMONARY TUMORS IN RATS INDUCED BY 4-NITROQUINOLINE 1-OXIDE. (Plates I-V)

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1 [GANN, 57, 1-7; February, 1966] UDC [616.24]: : ORIGIN OF PULMONARY TUMORS IN RATS INDUCED BY 4-NITROQUINOLINE 1-OXIDE (Plates I-V) Kazuo MORI, Jo HIRATSUKA, Shuhei SUZUKI, Ei-ichi KOIBUCHI, and Atsuo HASHIMOTO (Department of Medical Biology, School of Medicine, Showa University*) Synopsis Origin of pulmonary tumors in Buffalo rats induced by the subcutaneous injection of 4-nitroquinoline 1-oxide was investigated. Pulmonary tumors induced in 15 out of 70 rats were examined through serial sections to determine their relation to alveolar ducts, bronchus, and bronchiolus. For example, 19.3% of the tumors showed no relation to the bronchus, while 80.7% indicated direct relation to the bronchial epithelium. Several cases of focal metaplasia originating either from the alveolar area or from the bronchial epithelia were found. Recently, pulmonary tumors were induced in mice and rats by the subcutaneous injection of 4-nitroquinoline 1-oxide.6-8) Histogenetical observations on the site of the origin of tumors in mice were previously reported and it was demonstrated that some adenomas arise from alveolar lining cells and others from the epithelial cells in the area where the terminal bronchial cells pass into the alveolar ducts, while most of adenocarcinomas arise from the bronchial terminals.9,10) In the present work, the studies have been extended to examine the early changes in pulmonary tumors of rats induced by the administration of 4-nitroquinoline 1- oxide. EXPERIMENTAL The method of inducing pulmonary tumors of rats was similar to that reported in detail previously.7,8) Seventy Baffalo rats, male and female, received subcutaneous injections of 4-nitroquinoline 1-oxide in total of 10mg in a mixture of olive oil and cholesterol. The animals were sacrificed when found in poor condition and autopsies were performed. The lungs were fixed in neutral formaldehyde solution. Complete were cut and stained routinely with Hematoxylin and Eosin. Special staining was used for selected materials, if necessary. The sections were used in the search for areas of transitional changes between hyperplasia, adenomatous, squamous cell metaplasia, and cancerous lesions. * Hatanodai 1-chome, Shinagawa-ku, Tokyo

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3 In the experiment, 70 rats received the injection of 4-nitroquinoline 1-oxide, and 43 (61.2%) survived more than 210 to 400 days. In all the animals, multiple nodules of adenomatous proliferation occurred. Fifteen out of these 43 had various types of lung cancer. The types of the tumors were columnar, tubular, or papillary adenocarcinomas and epidermoid carcinomas, differentiated or undifferentiated (Photos 1-3). Metastases were found in the mediastinal and cervical lymph nodes, pericardium, and kidneys, as reported previously. The basis for the classification of the histogenesis of lung tumors lies in the histological relationship among the alveolar duct, bronchial epithelium, and tumors. The distribution and incidence of pulmonary tumors in the pulmonary lobes were examined first. Three lobes (I, II, and III) with multiple pulmonary tumors were selected. Numbers of sections for each of three series were 1804, 2618, and 1616, andthe numbers of various types of tumor nodules found in these lobes were 28, 33, and 22, respectively. The distribution of pulmonary tumors in the first lobe (I) containing 28 tumor nodules is shown in Fig. 1. From this graph, distribution of the pulmonary tumors induced is easily seen as occurring multicentrically throughout the lobe. Among 83 pulmonary tumors examined through the serial sections of the three lobes of the lung, 16 or 19.3% showed no relation to the bronchus or showed a secondary connection, while the remaining 67 or 80.7% indicated relation to the bronchial epithelial cells. Alveolar Origin Type A-a: Six out of 83 cases (7.2%) showed adenomas in the areas of the subpleura, protruding extensively on the surface of the lung. The growth began as focal hyperplasia and later transformed into adenomatous proliferation and, further, in some instance, into relatively large nodules which could be designated as adenomas. These adenomas sometimes developed into adenocarcinomas. It is suggested that the adenomas arose from the alveolar lining cells (Photo 4). Type A-m: In six out of 83 cases (7.2%), nests of squamous cell metaplasia were found in the alveolar structure and showed no relation to the bronchus or bronchiolus, or if they did, it was always considered to be secondary. Some showed well-differentiated extensive keratinization, while others, less. Very frequently, they were transformed into epidermoid carcinomas (Photo 5). Type A-a-c: In 4 out of 83 cases (4.8%), adenocarcinomas were found adjacent to or within the lesion of adenomas which originated from the alveolar lining cells, as described in Type A-a. Bronchial Origin There was another type of adenomatous growths which suggested the bronchial origin, although the epithelium itself did not show extensive hyperplastic changes. 3

4 Type B-a: Fourteen adenomas out of 83 cases (16.9%) suggested their origin from the terminal ends of bronchioli. The resemblance between tumor cells and bronchiolar epithelium is of course accentuated by the flattened state of the bronchial lining. It was found that the bronchial epithelia in the transitional area between neoplasms often lost mucin-secreting granules (Photo 6). Type B-m: In 5 out of 83 cases (6.0%), nests of squamous cell metaplasia were observed among the epithelial layer of bronchus. Since they frequently transform into epidermoid carcinomas, these lesions were not discarded from the experiment (Photo 7). Type B-a-c: Adenocarcinomas very often arose directly from the bronchial or bronchiolar membrane. As shown in Photos 8 and 9, the origin of cancer is immediately adjacent to the open-end of the terminal bronchioli. It is noteworthy that the lining of the bronchiolus showed no marked changes. Silver staining does not reveal any boundary between the neoplastic growths and their original membranes. As many as 45 out of 83 cases (60.0%) were found to belong to this type. These may be considered as the well-progressed form of Type B-a, because adenocarcinomatous growths are often found between adenoma and adenocarcinoma (Photo 10). Type B-ec: In 3 out of 83 cases (3.6%), epidermoid carcinomas were found directly adjacent to the bronchial epithelial cells (Photos 11 and 12). The result of the experiment is summarized in Table I, which contains data on three lobes examined serially. As shown in the lobe I (1804 sections), 3 developed adenomas and 2 adenocarcinomas, and they were of alveolar origin. In the lobe II, as many as Table I. Classification of the Origin of Pulmonary Tumors in Rats induced by Subcutaneous Injection of 4-Nitroquinoline 1-Oxide A: Alveolar origin a: Adenoma B: Bronchial origin m: Squamous cell metaplasia c: Adenocarcinoma ec: Epidermoid carcinoma 4

5 2618 sections were made. Two adenomas were seen and 4 nests of focal metaplasia of the alveolar origin. In the lobe III, cut into 1616 sections, there were I adenoma, 2 focal metaplasias, and 2 adenocarcinomas, and they were proved to be of alveolar origin. In total, 14 adenomas, 5 focal metaplasias, 45 adenocarcinomas, and 3 epidermoid carcinomas were found to be of bronchial origin, in lobes I, II, and III. Of two kinds of adenomas which arose from alveolar ducts, one was located in the subpleura and the other in deeper tissues of the lung. Earlier changes apparently began as the general increase in the cellularity and disorientation among cells. There were relatively larger and more definite areas of cellular aggregation. These hypercellular areas are hyperplastic foci. Distinction between such foci and adenoma was possible on the basis of the well-developed adenomatous pattern of tumor, and large and irregular cells comprising the neoplasma. In a few cases, both adenocarcinoma and epidermoid carcinoma were found transforming independently from each end of the same bronchiolus and thereafter these growths unite again according to the way of their growths, as shown in Photos 13 and 14. In some instances, adenocarcinomas which originated from associated metaplastic bronchoectatic epithelium were found with chronic purulent bronchitis. DISCUSSION By introduction of carcinogenic hydrocarbons or radioactive substances through the respiratory passage, it has been possible to induce cancer of the lung, mainly epidermoid carcinomas,2,3) many of which have been considered to be bronchogenic. Recently, cancers of the lung with metastases were induced in mice or rats in this laboratory by subcutaneous injection of 4-nitroquinoline 1-oxide. In rats, the induced cancers were adenocarcinomas and epidermoid carcinomas. One of the induced epidermoid carcinomas has been successfully transplanted to the same strain of rats, subcutaneously as well as intraperitoneally. Difference of opinion exists as to the histogenesis of pulmonary tumors, dispute being focussed on the histogenetical relationship among the alveolar duct, bronchial epithelium, and tumor. In the present experiment, three lobes from different lungs with multiple tumors were selected and serial sections were made. Among 83 tumors examined, 16 or 19.3% demonstrated no relation to the bronchus or showed only a secondary connection, while 67 or 80.7% indicated a direct connection to the bronchial epithelial cells. It is noteworthy that 45 out of 83 (54.0%) were adenocarcinomas of the bronchial origin. These results were similar to those in mice with lung tumors induced by 4-nitroquinoline 1-oxide.10) Apparently, there was no marked change in the epithelium of bronchus from which the cancer developed. Very often, mucin-secreting cells were not found in the transitional area between bronchus and neoplastic cells, as shown in Photo 6. Moreover, activity of alkaline phosphatase suddenly increased when tissue became cancerized, 5

6 although the activity was lower in the bronchial epithelium (Photo 16). Successful induction of lung cancer occurred when specific condition, not yet fully understood, for the deposit of carcinogen in the lung tissues was especially created, and importance of the deposition of a carcinogenic agent in the lung tissue was confirmed by Stanton and Blackwell.2) It is suggested that cholesterol in the solvent affected the carcinogenic potency of 4-nitroquinoline 1-oxide and its affinity to the lung tissue, in the present experiment. In spite of the fact that the majority of lung cancers in man are bronchial cancers which are often monocentric, it is suggested that the histogenesis in lung cancers in man may be closely related to those in animals.1,4,11,12,15) This work was supported by Grant DRG-739B from the Damon Runyon Memorial Fund for Cancer Research. (Received June 23, 1965) REFERENCES 1) Balo, J., "Lungenkarzinom und Lungenadenom," (1959). Verlag der Ungarischen Akademie der Wissenschaften, Budapest. 2) Blackwell, J.W.S., Brit. J. Cancer, 11, 181 (1957). 3) Howell, J.S., ibid., 15, 263 (1961). 4) Kreyberg, L., "Histological Lung Cancer Types", (1962). Norwegian Monographs on Medical Sciences, Oslo. 5) Kuschner, M., Laskin, S., Nelson, N., Altschuler, B., Am. J. Pathol., 34, 554 (1958). 6) Mori, K., THIS JOURNAL, 52, 265 (1961). 7) Idem, ibid., 53, 303 (1962). 8) Idem, ibid., 54, 415 (1963). 9) Mori, K., Hirafuku, I., ibid., 55, 205 (1964). 10) Mori, K., ibid., 55, 315 (1964). 11) Oota, K., Kyobu Geka, 8, 299 (1955). 12) Petersen, A.B., Hunter, W.C., Sneeden, V.D., Cancer, 2, 991 (1949). 13) Shabad, L.M., J. Natl. Cancer Inst., 28, 1305 (1962). 14) Stanton, M.F., Blackwell, R., ibid., 27, 335 (1961). 15) Tauchi, H., Goto, S., Nagoya Med. J., 1, 265 (1953). 6

7 EXPLANATION OF PLATES I-V Photo 4. Irregular hyperplasia and adenomatous proliferation with no relation to bronchus. Photo 13. Adenocarcinoma and epidermoid carcinoma found originating from each end of the Photo 14. PAS staining of the area where both adenocarcinoma and epidermoid carcinoma Photo 15. Alkaline phosphatase staining showing activity is less in the bronchus than in can- 7

8 GANN, Vol. 57 PLATE I

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12 GANN, Vol. 57 PLATE V