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The Production of Malignant Tumors of the Lung and Pleura in Dogs from Intratracheal Asbestos Instillation and Cigarette Smoking EDWARD W. HUMPHREY. MD, STEPHEN L. EWING, MD, JOHN V. WRIGLEY, MD, WILLIAM F. NORTHRUP, III, MD, THOMAS E. KERSTEN, MD, JOHN E. MAYER. MD, AND RICHARD L. VARCO, MD Nine dogs were given yearly inlratracheal instillations of crocidolite asbeslos for periods up to three years. The maximum dose latalled 66 mg/kg. In addi¢ion, seven of Ihese dogs smoked nine cigaretles per day, live days per week for six years. A malignan! pleural and/or peritoneal mes~}lhdioma de- veloped in six of the.~ dogs, and adenocar¢inoma o1' the lung developed in tbur, one af which had areas of squamous differentiation. The first animal died of a malignant tumor six years after the onset of e×p~sure, and the last animal died eight years after the onset. Caocer 47:1994-199~, 1981. TWO TYPES OF NEOPLASIA in humans have been definitely associated with inhalation of asbestos fibers: carcinoma of the lung and pleural mesothelioma. Two o~her types, gastrointestinal carcinoma and peritoneal mesothelioma, may have such an associa- tion. The. relationship between carcin~)ma of the lung and asbestos inhalation was first noted in the United States by Lynch and Smith in 1935,:' but the magnitude of this risk was compiled by Se]ikoff~ in a study of 632 insulation workers with more than 20 years elapsed since the onset of asbestos exposure. These men had a death rate from cancer of the lung or pleura 6.8 times that of the United States white male population of similar age, In a later report,~= he found the risk of death from carcinoma of the lung in asbestos workers who smoked cigarettes to be 92 times that of men who neither smoked nor had been ex- posed to asbestos dust, but he found no cases of cancer of the lung among the 87 asbestos workers who had never smoked cigarettes regularly. In the majority of instfinces, lung cancer in persons exposed to asbestos is peripheral in location and is an adeno- carcinoma, although squamous ceil carcinoma of the larger bronchi can also occur. The increased risk of developing carcinoma of the lung in humans who are cigarette smokers is now From the Deparlments of Surgery and Palhology. Universily of Minnesota and Ihe Minneapolis Velerans Administration Medical Cenler. Address for reprints: Departmenl of Surgery ill2), S4th St. & 481h Ave.. So.. Minneapolis. MN 55417. ~ccepted for publication April 28. 1980. well known, but considerable difficulty has been en- countered in producing carcinoma of the hmg from cigarette smoke in large animals. Auerbach et al.~ and Hammond etal.~ claim to have found invasive kmeu after bronchioloalveolar tumors in 8 of 12 dogs "" " 2.4 years of smoking. These dogs gradually increased theii- rate of smoking unfiltered cigarettes to nine per day and continued at this rate for !.8 years. None of these tumors had metastasized al the time the dogs were killed. Several forms of asbestos have been administered to a variety of small animals, but at the time this study was initiated, there was no report in the medical literature on the combined effect of asbestos installa- tion and cigarette smoke in large animals. It seemed to the authors that such a combination mighl be used to develop a model for the production of cancer of the lung in dogs. Method Preparathm of Asbestos A supply of high grade industrial crocidolite (blue asbestos) was obtained from the North American Asbestos Company. The asbestos was washed three times with distilled water. Each time it was passed through a 16 mesh screen and collected on Whatman #52 paper. Aiiquots of 2.5 g of the washed asbestos with 500 ml of distilled water were homogenized in a blender for 30 minules and then examined by using phase-contrast microscopy and an ocular mi- crometer. The fiber length varied from 2 to 90/am in length and 0.2 to 2.0 #m in diameter. The asbestos 0008-543X/gI/IMIS/1994 5;0.85 P Ameri~m Cancer Society Ti04231119

INTRATRACHEAL ASBESTOS AND CIGARETTE SMOKE TABLE I. Experimental F,'atures Humphrey et al. 1995 Years from Pleural mesothelioma, first exposure Asbeslos Smoking Pcribronchiolar greates! diame[er of to death exposure exposure fibrosis average lesion (cm) Adenoc-',rclnom-'.. greates! diameter Di~tan! fcmJ metastasis Dog I 5 -r + ~r 0 0 0 Dog 2 6 + ~ + 0.5 0 0 Dog 3 7 + + + i.0 0.4 0 Dog 4 7 + + + 0 0 0 Dog 5 '7 + + + 0. I 8.0 adenocarcinoma Dog 6 8 + + + 3.0 2.0 0 Dog 7 9 + + + 0 0 0 Dog 8 9 0 0 0 0 0 0 Dog 9 5 + 0 + 0.3 0 mcsolhelloma Dog I0 8 ÷ 0 + 1.5 4.0 0 was collected on Whatman #52 paper, dried, and stored until used. AMmal Piwcednres Eight nine-month-old purebred male beagles were used for this investigation. In addition, ten mongrel dogs were used for preliminary testing of acute tol- erance to three different doses of asbestos. All dogs underwent a tracheostomy with the place- ment of a number 5 or 6 nylon tracheostomy tube. A modified leather dog collar was used to hold the tracheostomy tube in place, and a plastic wound shield was fitted over the tracheostomy site until the wound was well healed. After healing, the tra- chegstomy tubes were changed three times a week. Thd dose of asbestos to be injected was sus- pended in 5 cc of saline. Using a 10 cc syringe with a 10 cm length of sterile vinyl tubing, we injected the asbestos suspension into the trachea: the tra- cheostomy opening was plugged for 30 seconds. It is estimated that less than 10% of the total volume was lost during the administration. The procedure for cigarette smoking was that described by Hammond et al.4 The dogs were placed in a stall that was adjustable to the animal's size. Each was fitted with a leather harness that could be fastened to the stall. The smoking machine was a motor-driven unit that drew smoke from the cigarette into a chamber during one phase and delivered this smoke to the dog during the second phase. The machine was cycled so that the dog had time to take several breaths of air between puffs of smoke. A thermister was placed near the tracheostomy end of the tubing to monitbr the temperature of the smoke delivered to the dog. Unfiltered, high-tar cigarettes were used. Experimental Protocol In a preliminary study, the ten mongrel dogs were given 4.75, 3.0, or 1.75 medkg body weight of asbestos once each week for three to five consecutive weeks. One month later, eight of the ten dogs were killed. The two remai.ning dog~ in this group again received asbestos 4.75 mg/kg each week. for three consecutive weeks each year for two years. Each received a total of 52.25 mg/kg. These Iwo dogsdid not smoke cigarettes. Beginning in 1970, three months after the tra- cheostomy was made, seven of Ihe beagles received 4.75 mg/kg body weight of asbestos into the trachea once each week for five consecutive weeks. At yearly intervals for the en~uing three years, the dogs were given asbestos 4.75 mg/kg/week for three consecutive weeks for a total dose of 66 mg/kg. One month after the first course of asbestos, the seven beagles were started on a regimen of cigarette smoking. Beginning with one" cigarette a day. their exposure to cigarette smoke was gradually increased so that after two months they were smoking nine cigarettes a day for five days a week except during the month of August when the additional courses of asbestos were administered. This smoking regimen was continued for six years. One beagle received neither asbestos nor any exposure Io cigarette smoke. The one dog that smoked and did not die and the control dog were killed after nine years. Twice each year we secured blood counts and chest roentgenograms. At death, autopsies were performed on all animals. We fixed the lungs by gravity in- flation with formalin, and the tissues were stained with hematoxylin and eosin for light microscopy. Sections of all tumors were also stained for matin with aician blue-PAS, and the pleural tumors were stained with alcian blue with and without hyaluronidase. Results In the preliminary group of eight mongrel dogs killed one month after the administration of varying amounts of asbestos, there was some evidence of acute bronchitis. Throughout all regions of the lung. T104231120

1996 CANC~:K April 15 1981 V~I. 4-/ Fl6. I. Areas of fibrosis adjacent to lerminai hronchioles (×40). there were focal granulomata with asbestos fibers present, both lying free and also within foreign body giant cells. No dog in this group died as a result of asbestos administration. The findings in the series of beagles and the two surviving mongrel dogs are presented in Table 1. Dogs I through 7 were the beagles exposed to both asbestos and cigarette smoke, dog 8 was'the beagle having a tracheostomy only. and dogs 9 and 10 were lhe two mongrel dogs from the preliminary study exposed Io asbestos only. The pertinent pathologic findings follow. Peribronchh~lar Fibrosis Gross find#tg~': All nine dogs exposed to asbestos had areas.of fibrosis up to ! cm in greatest dimension in the hilar area of each lung. Distinct peripheral areas of fibrosis were not easily recognized. Mh'rost'opk'findings: These nine dogs had extensive focal areas of fibrosis around many bronchi, bron- chioles, and alveolar ducts, but the area of fibrosis w~,s most prominent around the respiratory and ter- minal brouchioles (Fig. I). The fibrosis rarely ex- tended out into the alveolar sepia. There were nu- merous asbestos fibers Iocaled free or partially en- gulfed by multinucleated giant cells in areas of perihronchiolar fibrosis. The fibrosis was slightly more pronounced in the diaphragmatic lobes than the apical lobes. Me.~'otheHoma Gross .[[lldill.~s: Font of lhe seven dog~ exposed to asbestos and cigarelte smoke and both dogs ex- posed to only asbestos had extensive mesotheliomas of the pleural surface. These lesions were pre- dominanlly papillary. They were much more extensive on the parietal pleural surface (up to 3 cml than on the visceral pleural surface (up Io 0.5 cm). Three of the dogs also had diffuse papillary mesotheliomas of the pericardium (up to 1 cm). One dog in addition had diffuse papillary peritoneal mesotheliomas (up to 0.4 cm) as well as extensive metastatic disease. Figure 2 is a photograph of the lungs and purietal plenra of dog 10. The parietal pleura has wide.,,pread involvement wilh papillary mesothelioma, while the visceral pleura is relatively uninvolved. This dog also had an adenocarcinoma in the left lung. Microscopic findings: Five of the six dogs with mesotheliomas had predominantly epithelial mesothe- iiomas. Each of these had both a diffuse papillary surface proliferation (Fig. 3A} as well as solid invasive FIG. "2_. Posterior view of malignanl mesolheliom:l of parietal pleura and adenocarcinoma of left Itmg tdog Itn. Exlensiv¢ malignant papillary ep.;th¢lial meso~heliorna,~ arixing in the parietal pleura Ihcavy arrowL ~ 4 cm diameter :denocarcinoma i~. prc~nl in the lefl diaphragmatic lobe flight arrow). Ti04231121

INTRATRACIIEAL ASBESTOS AND CIGARETTE SMOKE "" Htlnzphre.~, el ol. 1997 FIG. 3. Epilhelial meso- Ihelioma. A. Predominanlly papillary surface prolifera- lion (x40). B. Solid in- vasive areas (x64). areas (Fig. 3B). The cuboidal epithelial cells were proliferating over cores of fibrous tissue. In the solid areas, a tubulopapillary pattern was present with the cuboidal cells forming glandular structures. The sixth dog had multiple invasive mixed epithelial- fibrous mesotheliomas. The pattern was that of a cellular spindled tumor, witl~ oval to round nuclei that focally had clusters of round cells with an epithelial appearance. The metastatic mesothelioma had extensive plugging of lymphatics and blood vessels in the lung. ~one of these lesions contained mucin with the alcian blue-PAS stain. Alcian blue positivity decreased by hyaluronidase was not found in the cytoplasm of the cells or glandular lumens. Carchtoma Grossfind~ngs: Three of the seven dogs exposed to asbestos and cigarette smoke and one of the dogs ex- posed only to asbestos had grossly recognizable neoplasms which ranged in size from 0.4-8.0 em (aver- age 3.8 cm). They were circumscribed, white, and firm: all abutted on the pleural surface. Two were in the left diaphragmatic lobe, one in the left cardiac lobe, and one in the left apical lobe. Figure 4A shows a tumor, 8 cm in diameter, in .the left diaphragmatic lobe of dog 5. This dog also had widespread distant metastatic disease. Microscopic findings: The four neoplasms were composed of predominantly columnar shaped cells that contained slightly enlarged nuclei with prominent nucleoli, mitosis was easily found, and focal areas of necrosis were present. The cells were arranged in a glandular pattern illustrated by Figure 4B. Each tumor also contained are~is that were papillary. The stroma varied from a thin alveolar septal wall to thicker fibrous septa. There was focal intracytoplasmlc mucin present in three of the tumors. The tumors in two of the dogs were composed entirely of the histologic pattern of a well to moderately differentiated adeno- carcinoma, but in a third tumor, focally solid areas with inlercellular bridges were present, indicating some squamous differentiation. These latter three tumors had microscopic localized aerogenous ex- tension around the tumor but had not metastasized to lymph nodes or to adjacent pulmonary parenchyma. The fourth tumor contained in addition to the well- differentiated adenocarcinoma, a highly undifferentiated component in the primary illustrated in Figure 4C, which was also the pattern present in the widespread metastatic deposits. Cytologically, the nuclei were more pleomorphic and contained numerous mitoses. The cells were arranged in a spindled pattern.'which in areas had a pseudosareomalous appearance repre- senting an anaplastic transformalion of the adeno- carcinoma. The localized areas of alveolar cell hyperplasia associated with the peribronchiolar fibrosis were not considered to be neoplastic. Only lesions that formed T!04231122

C^NCErt April 15 1981 Vol. 47 FJ6.4, A. Eight centimeter diameter adenoearcinoma in the left diaphragm.atie lobe of dog 5. B. Glandular areas Ix 160). C. Area with both adenocareinoma and an undifferentiated eomponeat (×I60L This undifferentiated component was present in the metastasis. . confluent tumor masses with papillary areas were considered to be malignant. Other Varyin~ degrees of microscopic rupture of alveolar septa were seen. but the dog not exposed to cigarette smoke and asbestos fibers also had a comparable amotmt of microscopic rupture of alveolar septa. Very little fibrosis or thickening of alveolar septa or thickening of walls of small arteries was seen. No pleural plaques or microscopic thickening of the pleura was seen away from the mesotheliomas. Discussion Asbestos is the generic name for a group of naturally occuring silicate fibers. Two general types are described: Serpentine, of which chrysotile is the most important example, and amphiboles, of which cro- cidolite (blue asbestos) and amosite are the most im- portant. Chrysotile is the most common commercially used asbestos, while crocidolite, lhe fiber employed in this experiment, is the second most commonly used. Crocidolite is a sodium ferrosoferric silicate mined principally in the North West Cape and Trans- vaal areas of South Africa. A high incklence of pleural mesotheliomas among persons who worked with crocidolite or who lived in the vicinity of crocidolite mines or mills has been previously re- ported.~,~ Previous efforts to produce malignant tumors with asbestos have been confined to small animals. Schmahl"' produced sarcomata in rats after the subcutaneous injection of asbestos of an undisclosed type. Wagner"" reported the development of mesotheliomas in rats after the intrapleural injection of chrysotile and crocidolite, and Smith et al.~s produced mesothcliomas in hamsters with intrapleural injections of amosite. Reeves et al.r exposed groups of rabbits, guinea pigs. and gerbils to atmospheres containing 48 mg/m"~ of crocidolite, amosite, and chrysotile on four days/ week for 18 months, but no tumors were seen. The same author exposed rats to inhalation of the three major types of asbestos at a concentration o1"50 m~m:~ for 6 to 24 months,r The rats exposed to crocidolite had a 14% incidence of lung carcinoma, while the rats exposed lo chrysotile and amosite had a 5~ total incidence of malignancies. Wagner"-I exposed rats to atmospheres ofamosite, anthophyilite, crocidolite, and two samples of chrysotiles at an average concentra- tion of 11.5 mg/m"~ for periods between one day and two years. Both adehocarcinoma and squamous carcinoma" of the lung were found as well as l l mesotheliomata. Two of the latter tumors occurred in rats with onl.x one day's exposure to asbestos: however, he found no evidence of a difference between chry, sotile and the amphiboles in carcinogenicity for rats. Shabad~a produced epidermoid carcinomas, retlculo- sarcomas, lung papillomas, or pleural mesotheliomas in 12 of 22 rats given intratracheal instillation of chrysotile plus benzo(a)pyrene but there were no lung tumors of'mesotheliomas in rats given chrysotile alone. Similar findings for the intratracheal instillation of crysotile and benzo(a)pyrene in the hamster were reported by Smith et al.t~ In 1970, when this experiment was designed, the standard for industrial air dust pollution below which asbestosis was not thought to develop was 5 mppcf tmillion particles per cubic foot} or 1.5 mg/m:L~ This T!04231123

No. 8 ~NTKATRALIIEAL ASBESTOS AND CIGARETTE SMOKE ][ttnlphrey el td. 1999 dust is not all asbeslos. From the data of Ayer," air containing dust at 1 mppcf averaged ten asbestos fibers/ml, o1" approximately 28% asbestos. To calculale a lifetime exposure to asbestos for humans working in an atmosphere of dust at 5 mppcf, we have assumed that a man's alveolar ventilation will be 2.4 m'~/8 hour work shift, five shifts/week for 45 weeks/year. To correspond with the findings of Selikoff, we used a career length of 20 years,t~ For a 70 kg human, this yields a career pulmonary exposure to asbestos of 65 mg/kg. The amount of asbestos instilled into the trachea of these dogs, 66 mg/kg, is very close to this figure. Weill et al.t~ reported that personnel of two asbestos plants with a cumulative exposure of more than 100 mppcf-years, a value similar to the above, had an in- crdase in the standard mortality ratio for respiratory malignancies, and that exposure to crocidolite fibers as opposed to chrysotile increased this risk. in this total group of nine dogs exposed to asbestos, a malignant pleural mesothelioma developed in six, and a lt,ng adenocarcinoma developed in four. The incidence of spontaneous primary lung tumors in dogs is low: Surert~ quotes it to be 5.6/100,000, the majority of which were malignant. According to the same author, 75~. were of bronchiolar origin. Brodeya reported adenocarcinoma to be the most common histologic type in dogs and found at autopsy that 50~ had metastasized. In 9882 canine necropsies, Nielsen and Horava" found 12 bronchial adenocar- cinomas, only one of which had metastasized. Although the first animal to die of a malignant tumor did so six years after the first exposure, the rest of the dogs died during the seventh and eighth years. Death occurred from one to two years after the tumors first became apparent on the chest x-ray. The adenocarcinoma in dog 5, for example, was first noticed in the left lower lobe 20 months before death o1" five years after the first exposure. Wagner~ found no mesothelioma in rats earlier than 355 days after asbestos inhalation, and most lung carcinomas were found after two years. In Auerbach's study, the dogs were sacrificed after 2.4 years, possibly too soon for mal_ignancies to have developed. I"-'~'~hree of the seven dogs exposed to both asbestos and cigarette smoke did not develop a malignancy. One died at tire years of multiple small bowel perfora- tions, one al seven years of pneumonia, and one was killed at nine years. Both dogs that were exposed to asbestos alone did develop a malignant tumor. Because of the effectiveness of l his dose of asbestos in producing tumors, it is difficult to evahlate the role ~of cigarette smoke. If, as has been postulated, the ergism between cigarette smoke and asbestos is due to the impairment of bronchial clearing by smok- ing and subsequent retention o1" asbestos in the lung. this dose of asbestos may of itself have overwhelmed Ihe clearing mechanism and so obscured such syner- gism. Possibly a lesser total dose of asbestos might permit the detection of an added effecl from cigarettes: however, the incidence of malignant pulmonary tumors and of fibrosis following the intratracheal instillation of crocidolite should make this a useful method in the nine A study of lung disease in larger animals. REFERENCES 1. Auerbach O, Hammond EC. Kirman D. G;trfinkel L. I1 Pulmonary neoplasms. Arch Em'irtm tleahh 1970: 21:754-768. 2. Ayer HE, Lynch JR, Fanney JH. A comp;trison of impinger and mefnbrane filter techniques for evaluating air samples in asbestos plants. Ann NY Acad Sci 1965: 132:274-287. 3. Brodcy RS. Respiratory tract neoplasms in domestic animals. Arch Am Call Vet Sarg 1973; 2:28-35. 4. Hammond EC, Auerbach O. Kirman D. Garfinkel I.. Effects of cigarette smoking on dogs. I. Design of experiment, mortality, and findings in lung parenchyma. Arch ~'nt'icott lh'ahh 1970; 21:740-753. 5. Lynch KM, Smith WA. Pulmonary a.',bestosis: carcinoma of the lung in asbeslo-silicosis. Am J Ctttlt'er 1935: 24:56-64. 6. Nielsen SW, Horava A. Primary pulmonary tumor.,, of the dog. A report of sixleen eases. Am J Vt,! R,~,s 1960: 21:813-830. 7. Reeves AL, Puro HE, Smilh RG. Inhalation carcinogenesis from various forms of asbestos. E~vlron Re.s 1974: 8:17~t-202. 8. Reeves AL. The carcinogenic effee! of inhaled asbestos fibers. Ann CIh~ lab St'i 1976: 6:459-466. 9. Roach SA. Measurement of airborne asbestos dust by rostra- meats measuring different parameters. Attn NY Acad St.i 1965; 132:306-315. 10. Schmahl D. Cancerogene Wirkung van Asbestos bei Implanta- tion an Ratten. Z grebsforseh 1958: 62:561. I1. Selikoff J J, Churg J, Hammond EC. Asbestos exposure and neoplasia. JAMA 1964: 188:22-26. 12. Selikoff IJ, Hammond EC. Chary J. Asbestos exposure, smoking, and neoplasia..lAMA 1968: 204:106-110. 13. Shabad LM, Pylev LN, Krivosheeva LV. Kulagina TF. Nemenko BA, Experimental studies on asbestos carcinogenicity. .l Nail Cancer last 1974: 52:I 175-1187. 14. Sluis-Cremer GK. Asbestosis in Sonth Africa--cerlain geo- graphical and environmental considerations. Am~ 1965: 132:215-234. 15. Smith WE. Miller L, Chary J. Selikoff IJ. Pleu~.tl reation and mesothelioma in hamsters injected with a~bestos. I%,.. Am A~oc Caot.er Res 1964: 5:59. 16. Smith WE. Miller L. Churg J. An experimental model for study of carcinogenesis in the respiratory ~ract. In: Nette.',heim P. Hanna MG. Deatherage JW. eds. Morph~logy of Experimenlal Respiratory Carcinogenesis. Oak Ridge: U. S. Alomic Energy Commission 1970. 17. Suler, PF: Pulmonary neoplasia. In: Ettingcr SJ. ed. Textbuok of veterinary internal medicine. Phihdelphia: WB S;mndcrs. 1975. 18. Weill Ha Hughes J. Waggenspack C. Influence or dose and fiber type on respiratory maligmmcy risk in a.,,bestos cement manufacturing. Am Ret" Rcspir DN 1979: 120:345-3.';4. 19. Wagner JC, Sleggs CA. Marchand R. Diffuse plcural mesothelioma and asbeslos exposure in Northwestern Cape Ih'o~ lace. Br J Indu.¢ Med 1960: 17:260. 20. Wz~gner JC. Experimental production of me.',uthclial tumors of the pleura by implantation of dusts in I:~horutoLv animals. Nttture 1962: 196:180. 21. Wagner JC. Berry G. S "kidmore .IW. "rimbrelt V. The effects of the inhalalion of asbestos in rals. Br3 Cum'er 1974: 29:252-269. T!04231124