[CANCER RESEARCH 32, 1209-1217, June 1972]

Experimental Cancer of the Lung in Rabbits Induced byChemical Carcinogens1

Fumio Hirao, Tomoo Fujisawa, Eiro Tsubura, and Yuichi Yamamura

Third Department of Internal Medicine, Osaka University Medical School, Dojima-Hamadori 3-chome, Fukushimaku, Osaka, Japan

SUMMARY

Lung cancer was induced in rabbits by the instillation ofchemical carcinogens into the lower bronchus, with the use ofa bronchoscope. The rabbits were divided into 2 groups.Group 1 received a mixture of 3-methylcholanthrene (3-MCA)and 4-nitroquinoline 1-oxide in rabbit plasma, and Group 2received 3-MCA alone in distilled water. Doses of 40 mg of3-MCA and 0.4 mg of 4-nitroquinoline 1-oxide were givenevery 7 to 10 days.

The total doses of carcinogens required for 50% incidenceof lung cancer were 1000 ±100 mg of 3-MCA and 10 ±1 mgof 4-nitroquinoline 1-oxide in Group 1 and 1330 ±100 mg of3-MCA in Group 2.

Altogether, lung cancer was induced in 80 of 173 rabbitsthat received more than 4 doses of carcinogens and survivedfor more than 30 days. In 55 cases, métastasesand/or invasionof the chest wall and diaphragm were observed. Histologically,the cancers were of various types (e.g., squamous cellcarcinomas, adenocarcinomas, undifferentiated cell carcinoma,pleomorphic carcinomas, and mixed types).

The main bronchus, in rabbits of another group, wasswabbed every 1 to 2 days with cotton wool soaked in a 10%suspension of 3-MCA in Tween 60. Of the 65 rabbits thatsurvived for more than 60 days, only 2 developed lung cancerwith métastasesor invasion of tissues adjacent to the lung.

INTRODUCTION

We reported previously (12) on atypical squamousmetaplasia and adenomatous proliferation that we induced inthe bronchial submucosa of rabbits by swabbing the bronchusat intervals with a suspension of 3-MCA2 in Tween 60. The

changes observed were limited to the submucosa and did notextend to, or cause destruction of, the cartilage. However, afew rabbits showed atypical squamous metaplasia andadenomatous proliferation in the peripheral bronchi and wallsof the alveoli. These observations suggested that it might bepossible to induce cancer in the parenchyma of the lung.

Accordingly, we devised the new technique of instillingcarcinogens suspended in distilled water or rabbit plasma

1Supported by a Grant-in-Aid for Scientific Research from the

Ministry of Education of Japan. This study was presented in part at the10th International Cancer Congress in Houston, Texas, in May 1970.

'The abbreviations used are: 3-MCA, 3-methylcholanthrene;4-NQO,4-nitroquinoline 1-oxide.

Received May 14, 1971;accepted February 28, 1972.

directly into the lower bronchus of rabbits. This resulted in ahigh incidence of typical lung cancer.

This report describes the technique used for induction oflung cancer and histological findings on the cancers that wereinduced.

MATERIALS AND METHODS

Young rabbits approximately 50 days old, weighing 1 kg,and adult rabbits approximately 100 days old, weighing 3 kg,both male and female, were used. The animals were given food(ORC 5, rabbit-guinea pig diet, from Oriental Yeast Industries,Ltd., Osaka, Japan) and water ad libitum throughout theexperiment. The carcinogens used, 3-MCA and 4-NQO, weregiven in either normal rabbit plasma, distilled water, or Tween60. 3-MCA, 4-NQO, and Tween 60 were obtained from WakoPure Chemical Industries, Ltd., Tokyo, Japan. Animals wereanesthetized with ether (Sanraku Ocean Co., Ltd., Tokyo,Japan). A special bronchoscope (inner diameter, 4 mm; length,20 cm) was used for instillation of the carcinogens.

Two experiments were performed. In Experiment 1,carcinogens were instilled into the bronchus, and inExperiment 2 carcinogen was swabbed on the inside of thebronchus. In Experiment 1, animals were divided into 2groups. Group 1 contained 52 young rabbits and 63 adultrabbits, and Group 2 contained 18 young rabbits and 40 adultrabbits. Only animals that survived for more than 30 days wereexamined. Carcinogens were introduced into the right lung ofall rabbits in Group 1 and into the right lung of 45 rabbits andthe left lung of 13 rabbits in Group 2.

Rabbits in Group 1 received intrabronchial instillations of amixture of 40 mg of 3-MCA and 0.4 mg of 4-NQO suspendedin 0.4 ml rabbit plasma together with s.c. injections of amixture of 0.1 mg of 4-NQO and 4 mg of cholesterol(Daiichi-Kagaku Co., Ltd., Tokyo, Japan) in olive oil (WakoPure Chemical Industries, Ltd.). Group 2 received doses of 40mg of 3-MCA alone, suspended in 0.4 ml of distilled water. The1st 5 doses of carcinogens given to young rabbits were one halfthe amount given to adult rabbits. To instill the carcinogens,we anesthetized the animals and inserted a polyvinyl tube(diameter, 1.5 mm; length, 23 cm) into the lower bronchusthrough a bronchoscope. Instillations were repeated every 7 to10 days.

In Experiment 2, the superficial mucosa of the right mainbronchus of adult rabbits was swabbed with pieces of cottonsoaked in a suspension of 10% 3-MCA in Tween 60, which was

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fixed onto the tip of a stick with the use of a bronchoscope.This treatment was repeated every day or every other day.

To protect rabbits from infection, oxytetracyclinehydrochloride (Pfizer Taito Co., Ltd., Tokyo, Japan), 50mg/kg body weight, was given i.m. at the same time as werethe carcinogens. Administration of carcinogens was continuedunless the rabbits became severely ill or died.

Complete necropsies and histological examinations weredone on rabbits that died or became severely ill during theexperiment. All organs were examined macroscopically andwere then fixed in 10% formalin. Sections of these tissues werestained with hematoxylin and eosin, as a routine method, andwith periodic acid-Schiff, van Gieson stains, and silverimpregnation, when necessary.

RESULTS

Experiment 1

Instillation of Carcinogens into the Lower Bronchus.Rabbits that received more than 4 instillations of chemicalcarcinogens and that survived for more than 30 days wereexamined histologically. Table 2 shows that administration ofcarcinogens reduced the survival rate. As seen from Table 1, 80(approximately 46%) of 173 rabbits (90 females and 83 males)developed lung cancer. These included 42 cases of squamouscell carcinoma (approximately 50%), 22 cases ofadenocarcinoma (approximately 28%), 6 cases of squamouscell carcinoma mixed with adenocarcinoma, and 8 cases ofpleomorphic carcinoma. There was also 1 case each ofundifferentiated cell carcinoma and sarcoma.

Various benign pathological changes were also observed(e.g., proliferation of atypical squamous epithelium ofperipheral bronchioli, adenomatous hyperplasia, or adenoma),but only major pathological changes are recorded in Table 1.In this paper, adenomatous hyperplasia is defined as extensivehyperplasia extending from the terminal bronchi to alveoli,with an intact alveolar wall. Adenoma is defined as histologicalchanges with formation of a glandular lumen lined bycylindrical or cubic cells, but without infiltrative changes ordestructive growth.

The lung cancers showed various histological pictures. Insome, squamous cells carcinomas cornification, necrosis, orsomewhat poor differentiation was observed (Figs. 7 and 8).Most adenocarcinomas were composed of long cylindrical cellswith eosinophilic cytoplasm and a basally located nucleus, buta few were composed of low columnar cells (Figs. 9 and 10).Scirrhous changes were often observed in both squamous cellcarcinomas and adenocarcinomas. Many bizarre-shaped orgiant cells were found in pleomorphic carcinomas (Fig. 11).

Most of the lung cancers were induced in the lower part ofthe lung on the side where the carcinogen was instilled, andthey varied from 15 to 40 mm in diameter. In some rabbits,the lung was almost entirely replaced by tumor masses (Fig.4). The center of the tumor masses was intensively necrotic(Figs. 2 and 3). In most rabbits the tumor adhered tightly toeither the underlying diaphragm and/or the chest wall.Sometimes the diaphragm just below the tumor was so fibroticthat it was almost impossible to distinguish or separate it fromthe tumor mass.

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Experimental Lung Cancer in Rabbits

Table 2Survival of rabbits after intrabronchial instillation

of chemical carcinogens (Experiment I)

No. of survivors at the age ofApproximate

initial age No. of 30 40 50 60 70 80 90 100 110 120 130Group (days) rabbits0 wk wk wk wk wk wk wk wk wk wk wk

250100so100526318403258173426

25554914

1328201842918113151491729411151503102010

" Rabbits that died within 30 days after the start of the experiment were excluded.

In 1 rabbit, a sagittal section of the right lung revealeddisseminated tumors of from 1 to 25 mm in diameter (Fig. 1),and in the left lung many miliary métastaseswere alsoobserved.

Métastasesand Invasion of Tissues Adjacent to the Lung.Table 1 shows that, in 55 cases (approximately 70% of thetotal), métastasesto other organs and/or invasion of tissuesadjacent to the lung were observed.

Métastaseswere observed in the following areas: theparatracheal lymph nodes in 22 rabbits (Fig. 13); the kidneysin 7 rabbits (Figs. 6, 14); the mediastinum and chest wall in 4rabbits (Fig. 5); and the collateral lung and diaphragm in 3rabbits. There was 1 case each of metastasis to the liver (Fig.16), adrenals, retroperitoneum, and omentum.

The diaphragm was the most frequently affected of thetissues adjacent to the lung (40 cases, i.e., 50% of the total).Cancer cells infiltrating the fibrotic muscle layer of thediaphragm were scirrhoid. Invasion of the muscle layer of thechest wall (Fig. 12) was noted in 14 rabbits.

Effects of the Dose of Chemical Carcinogen andExperimental Period on the Incidence of Lung Tumors. Theminimum carcinogenic doses required for the induction oflung cancer were 380 mg 3-MCA and 3.8 mg. of 4-NQO inGroup 1 and 400 mg of 3-MCA in Group 2. On the basis of therelationship between the dose and the incidence of cancer, thedoses required for 50% incidence of lung cancer productionwere estimated as 1000 ±100 mg of 3-MCA and 10 ±1 mg of4-NQO in Group 1 and 1330 ±100 mg of 3-MCA in Group 2.

In the early period of the experiment (within 60 days), themajor pathological change was adenomatous hyperplasia,followed by the appearance of adenoma. No lung cancerdeveloped by Day 60 after the 1st instillation of carcinogens.Métastasesor invasion into tissues adjacent to the lung were1st observed after Day 120 and were frequently found afterDay 300.

Experiment 2

Swabbing the Bronchial Mucosa with Chemical Carcinogen.The bronchial mucosa was swabbed at intervals with asuspension of 10% 3-MCA in Tween 60. Only 2 of the 65rabbits that survived for over 60 days developed lung cancer.These 2 rabbits were killed on Day 995 after the start of theexperiment. In 1 rabbit, the lung to which the carcinogen hadbeen applied was replaced by a tumor mass with intensivenecrosis, and métastaseswere found in the paratracheal lymph

nodes, retroperitoneum, and adrenals (Fig. 15), with invasionof the chest wall and diaphragm. Histologically, the cancer wasdefined as pleomorphic. In the other rabbit, the tumor masswas in the middle to lower part of the lung and invaded boththe diaphragm and pleura. It was identified as a poorlydifferentiated squamous cell carcinoma.

DISCUSSION

It is difficult to induce lung cancer experimentally inanimals, but recently there have been reports of the inductionof lung tumors in mice, rats, and hamsters (1-11, 14-29,31-38).

In humans, main bronchial cancer is the most common typeof lung cancer. In an attempt to induce cancer in this site inrabbits, we repeatedly swabbed the main bronchus with asuspension of 3-MCA in Tween 60 (12). Repeated applicationof the carcinogen was presumed to be crucial for its prolongedaction on the main bronchus. However, after more than 100applications, many rabbits still showed no carcinomatouschanges and the bronchial mucosa tended to showdesquamation. However, some rabbits showed growth ofatypical squamous epithelium and adenomatous hyperplasia inthe terminal bronchi or alveolar walls. This suggested that theterminal bronchi or alveolar walls might be more susceptiblesites.

Accordingly, we tested the effect of instilling carcinogensonto the lung parenchyma, using a polyvinyl tube inserted intothe lower bronchus of rabbits. In a previous paper (13), whichwe believe was the first report of induction of lung cancer inrabbits, we stated that 5 of 32 rabbits developed lung cancerwith métastases or invasion of neighboring tissue whencarcinogens were instilled into the lower bronchus.

To obtain a high incidence of induction of lung cancer, atechnique was required by which the carcinogens wouldremain in the lung for a long time. For this purpose,Andervont (1), Kuschner et al. (15) and Laskin et al. (16)tested the effect of sticking thread impregnated withcarcinogen on the lung or of inserting pellets of chemicalcarcinogens or radioactive substances into the bronchi of miceand rats. The importance of the local persistence of acarcinogen was confirmed by Stanton and Blackwell (31), whoinduced multiple areas of pulmonary infarction in rats whilegiving them 3-MCA. Shabad et al. (30) administeredcarcinogen in a suspension with casein and India ink, while

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Hirao, Fujisawa, Tsubura, and Yamamura

Saffìotti et al. (28) used crystals of ferric oxide to retaincarcinogen in the lung for a long time.

The duration of the local persistence of a carcinogen seemsto depend on the vehicle in which it is administered.Previously (12), we detected the autofluorescence of 3-MCA inthe alveolar walls and terminal bronchi for as long as 10 daysafter its instillation, when given in distilled water or plasma.However, no autofluorescence could be detected after 10 dayswhen the 3-MCA was given in Tween 60. The reason for thismay be that, when 3-MCA was given in distilled water orplasma, the latter fluids were rapidly absorbed so thatinsoluble crystals of 3-MCA were deposited on the walls of thealveoli and peripheral bronchi, while when 3-MCA was given inTween 60, both were absorbed together into the circulation.

Previously, it was thought to be very difficult to induceexperimental lung cancer in rabbits. However, induction isnow possible by instilling 3-MCA, in either distilled water orplasma, into a given site in the lower bronchus through abronchoscope.

ACKNOWLEDGMENTS

We gratefully acknowledge the helpful advice of Professor TomMiyaji and Dr. Kunio Uematsu of the Department of Pathology, OsakaUniversity Medical School, Osaka, Japan, and of Dr. YasuyukiAkamatsu of the Department of Pathology, Medical College of Georgia,Augusta, Georgia.

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8. Grant, G. A., Carter, R. L., Roe, F. J. C., and Pike, M. C. Effects ofthe Neonatal Injection of a Carcinogen on the Induction of Tumorsby the Subsequent Application to the Skin of the SameCarcinogen. Brit. J. Cancer, 22: 346-358, 1968.

9. Gross, P., Pfitzer, E. A., Watson, J., de Treville, R. T. P., Kaschak,M., Tolker, E. B., and Babyak, M. A. Experimental Carcinogenesis.Bronchial Intramural Adenocarcinomas in Rats From X-rayIrradiation of the Chest. Cancer, 23: 1046-1060, 1969.

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C5, BL Mice Exposed to a Cigarette Smoke and Air Mixture. Brit.Med. J.,4: 637-641, 1967.

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Y. Experimental Cancerous Changes in the Lung Induced byChemical Carcinogens in Rabbits. Gann, 58: 427-434, 1967.

13. Hirao, F., Fujisawa, T., Tsubura, E., Akamatsu, Y., and Yamamura,Y. Experimental Lung Cancer in Rabbits Induced by ChemicalCarcinogens. Gann, 59: 497-505, 1968.

14. Hueper, W. C., and Payne, W. W. Experimental Cancers in RatsProduced by Chromium Compounds and Their Significance toIndustry and Public Health. Am. Ind. Hyg. Assoc. J., 20: 274-280,1959.

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16. Laskin, S., Kuschner, M., Nelson, N., Altschuler, B., Harley, J. H.,and Daniels, M. Carcinoma of the Lung in Rats Exposed to the(3-Radiation of Intrabronchial Ruthenium106 Pellets: 1. Dose-Response Relationships. J. Nati. Cancer Inst., 31: 219-231, 1963.

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Experimental Lung Cancer in Rabbits

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///rao, Fujisawa, Tsubura, and Yamamura

Fig. 1. Gross appearance of the right lung in a sagittal section, with disseminated tumors, 1 to 25 mm in diameter. This was classifiedhistologically as a pleomorphic carcinoma. Rabbit P-133, female, Experiment 1, Group 1; experimental period, 529 days.

Fig. 2. Gross appearance of a primary squamous cell carcinoma, with intensive necrosis of the lower part of the right lung in sagittal section.This tumor adhered tightly to the underlying diaphragm and liver. Rabbit Y-104, male, Experiment 1, Group 2; experimental period, 524 days.

Fig. 3. Gross appearance of a primary squamous cell carcinoma of the right lung in sagittal section. The central and lower parts of the lung areoccupied by tumor, and it is impossible to differentiate the diaphragm from the lung. Necrotic lesions are observed in the center of the tumor.Rabbit N-3, female, Experiment 1, Group 2; experimental period, 720 days.

Fig. 4. Gross appearance of a primary squamous cell carcinoma of the right lung in sagittal section. The tumor occupies almost all of the lung.There is extensive invasion of the tumor into the diaphragm (arrows). Rabbit P-47, male, Experiment 1, Group 1; experimental period, 399 days.

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Experimental Lung Cancer in Rabbits

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Fig. 5. Disseminated metastatic tumors, 2 to 8 mm in diameter, in the chest wall on both sides. Rabbit P-I33.Fig. 6. Gross appearance of the kidneys in saggital section, with metastatic tumors, 1 to 10 mm in diameter. Rabbit P-133.Fig. 7. Squamous cell carcinoma with intensive necrosis of the lung. Rabbit Y-104. H & E, X 100.Fig. 8. Squamous cell carcinoma of the lung. Lower portion of photograph, squamous cancer cells infiltrating the pleura. Rabbit ¿-25,female,

Experiment 1, Group 2; experimental period, 438 days. H & E, X 100.

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Mimo, Fujisawa, Tsubura, and Yamamura

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Fig. 9. Tubular adenocarcinoma of the lung. Rabbit Y-l 7, male, Experiment 1, Group 1; experimental period, 368 days. H & E X 100.Fig. 10. Tubular adenocarcinoma of the lung. In some areas of this specimen, pleomorphic structures are observed. Rabbit Y-37, male,

Experiment 1, Group 1; experimental period, 724 days. H & E, X 100.Fig. 11. Pleomorphic carcinoma of the lung showing bizarre-shaped and giant tumor cells. The tumor cells aie extremely variable in size and

shape. Rabbit P-133. H & E, X 40.Fig. 12. Infiltration of squamous cancer cells from the lung into the muscle layer of the chest wall. Rabbit Y-104. H & E, X 100.

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Experimental Lung Cancer in Rabbits

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Fig. 13. Metastatic squamous cell carcinoma in the peripheral sinuses of the paratracheal lymph nodes. Rabbit Y-75, male. Experiment 1, Group1; experimental period, 532 days. H & E, X 100.

Fig. 14. Metastatic squamous cell carcinoma in the kidneys. Rabbit N-15, female, Experiment 1, Group 2; experimental period, 644 days.

H & E, X 100.Fig. 15. Metastatic pleomorphic carcinoma in the adrenals. Rabbit NM-13, male, Experiment 2; experimental period, 995 days. H & E, X 100.Fig. 16. Metastatic squamous cell carcinoma in the liver. Rabbit Y-4, male, Experiment 1, Group 1; experimental period, 480 days. H & E, X 40.

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1972;32:1209-1217. Cancer Res   Fumio Hirao, Tomoo Fujisawa, Eiro Tsubura, et al.   Chemical CarcinogensExperimental Cancer of the Lung in Rabbits Induced by

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