The Transport and Localization of Benzo(Â¿/)Pyrenã©-Hematiteand Hematite-210Po in the Hamster Lung Following Intratracheal Instillation1

[CANCER RESEARCH 34, 1344^1352, June 1974]

The Transport and Localization of Benzo(Â¿/)pyrenÃ©-Hematiteand Hematite-210Po in the Hamster Lung following Intratracheal Instillation1

Ann R. Kennedy and John B. Little

Department of Physiology, Harvard School of Public Health, Boston, Massachusetts 02115

SUMMARY soot in the urban atmosphere (2). Intratracheal instillations of 210Poadsorbed onto hematite carrier particles to mimic When administered intratracheally adsorbed onto hema adsorption to soot produced combined epidermoid and tite carrier particles, 210Poand benzo(a)pyrene (BP) induce adenocarcinomas in the peripheral lung of hamsters, while lung tumors of different histologies and apparent sites of BP-hematite produced mainly epidermoid carcinomas in origin. Hematite-210Po produces combined epidermoid and the major bronchi and trachea (10, 16). This difference in adenocarcinomas in the peripheral lung, while BP-hematite the histology and apparent sites of origin of the induced produces mainly epidermoid carcinomas of the major tumors suggests either that the cells in the lung are bronchi and trachea. To determine whether cells in the lung differentially sensitive to these 2 types of carcinogen or that are differentially sensitive to the two types of carcinogen or the carcinogens are reaching and acting at different sites in whether the carcinogens are reaching and acting at different the lung despite the fact that they were administered in the sites, we have studied their transport and localization same manner. To investigate this question, we have studied following intratracheal administration on carrier particles: the transport and localization of these carcinogens in the 210Polocalization by freeze-dry autoradiography and BP by lung. ultraviolet light fluorescence microscopy of frozen sections. Conventional histolÃ³gica! techniques were inappropriate The results show that 210Po represents a firmly bound in these localization experiments since tissue processing insoluble carcinogen which delivers its primary dose to the involved liquid steps which might lead to translocation of peripheral lung from the particles that are retained in the the carcinogen; techniques not requiring liquid preparation alveolar duct region. BP, on the other hand, is a loosely steps were therefore sought. Freeze-dry autoradiography, a bound soluble carcinogen which leaves the carrier particles technique originally developed by Stumpf and Roth (23) and enters upper airway epithelial cells during clearance of and modified for use with the delicate, inflated lung (6) was the particles on the mucous ciliary escalator. We thus used for the study of 210Po localization. Since BP is conclude that BP induces central tumors because a signifi autofluorescent, it can be identified directly on histological cant amount of the carcinogen reaches the upper airway sections by UV fluorescence microscopy (16). Frozen epithelial cells, while 210Po induces peripheral tumors sections seemed to be the most appropriate histological because the major radiation dose is delivered to the alveolar medium for the study of BP localization by autofluores- region. cence since frozen sections would most closely preserve the i/i vivo localization of the carcinogen. The localization of 210Poand BP following intratracheal INTRODUCTION administration adsorbed onto hematite carrier particles is 210Po,a naturally occurring Â«-emittingradionuclide, and described in this report, and the significance of their BP,2 a chemical carcinogen, are potent lung carcinogens in deposition and localization in the lung in relation to the tumors induced by either agent is discussed. Syrian golden hamsters following intratracheal administra tion on hematite carrier particles (10, 16, 17). Thea-radiation emitted by 210Po is very densely ionizing; for each a MATERIALS AND METHODS disintegration, 5.3 MeV of energy is deposited in a track length of only 37 jtm in tissue. 210Po has been found in Male Syrian golden hamsters, 100 to 125 g, obtained cigarette smoke (15) and cigarette smokers' lungs (15). BP, from Dennen Animal Industries, Inc., Gloucester, Mass., were given intratracheal instillations of either 0.2 fid 210Po also found in cigarette smoke (1), is commonly adsorbed to or 3 mg BP, both bound to 3 mg hematite particles suspended in 0.2 ml 0.9% NaCl solution as described by 'This research was supported by Contract NOI CP 33273 from the Little et al. (10). Instillations were given to hamsters in an National Cancer Institute and Grant ES-00002 from the National Institutes of Environmental Health Sciences. upright position on a slanted board as described by Saffiotti 2The abbreviation used is: BP, benzo(a)pyrene. el al. (16). Animals were killed by Brevital sodium (Eli Lilly Received December 12, 1973: accepted March 4, 1974. and Co., Indianapolis, Ind.) overdose and exsanguination

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from a renal artery. The trachea was clamped before Single Instillation opening the thoracic cavity to prevent lung collapse. Lungs were removed en bloc and frozen rapidly by placing them Zero to 1 Hr. Hematite-210Po was present overlying the for 1 min in methyl butane cooled to the viscous stage by epithelium throughout the airways in the lung, although in a liquid nitrogen. patchy distribution. In some branches, airway epithelium To study the localization of hematite-210Po, freeze-dried from the major bronchi to the bronchioles, including the frozen lung sections (2 to 4 /urnthick) were prepared for dry respiratory bronchioles and alveolar duct regions, was mourit autoradiography; stains used in this study were coated with hematite-210Po (Fig. 1). Occasional single celestine blue-Van Gieson's and hematoxylin-eosin. This tracks were seen in peripheral alveolar areas. These may technique has been described in detail (6) and involves no represent ionic 2lÂ°Po(not particle associated) or 210Po liquid steps during processing and autoradiography. To associated with minute dispersed particles. These single study the localization of BP-hematite, frozen sections (2 to 4 tracks declined rapidly in number during the 1st week, then Â¿Â¿mthick)were cut, stained with thionin or hematoxylin- gradually disappeared. No cells (macrophages) containing eosin, and immediately examined by UV fluorescence hematite-210Po were seen in the bronchioles or bronchi. microscopy. The white-yellow fluorescence of the BP could One to 4 Hr. Same as 0 to 1 hr, except that clumps of be easily distinguished using this technique (Figs. 4 to 7). hematite-210Po appeared on or overlying the ciliated epithe Animals used for the hematite-210Po localization study were lium of the trachea. sacrificed immediately after a single instillation and hourly Four to 6 Hr. The epithelium from the trachea to the for the 1st 8 hr, daily for the 1st week, weekly for the 1st 2 bronchioles still contained overlying hematite-210Po, but it months, and at 4 and 8 months; after the 2nd, 4th, and 6th was more patchy in nature. A few cells (macrophages) instillations sacrifices were made at 3 and 7 days; and at 10, containing hematite-210Po have now appeared in the bron 14, and 21 days after the 7th instillation. Animals used for chioles. the BP-hematite localization study were sacrificed after a Six to 12 Hr. Some airways still contained patches of single instillation at the same times as for the hematite- hematite-210Po. More macrophages containing hematite- 210Postudy up to 1 month after instillation. 210Po were found within the airways than at 4 to 6 hr. Sixteen to 48 Hr. Very few patches of hematite-2lÂ°Po were left in the airway lumina. Many macrophages contain RESULTS ing hematite-210Po were found within the airways (Fig. 2). Many such cells containing hematite-210Po were present in Hematite-210Po the esophagus. Two to 5 Days. The peak numbers of cells containing Throughout the study, 210Po activity remained closely hematite-210Po were found in the airway lumina (Fig. 2). associated with the visible hematite particles in the lung. As Patches of extracellular hematite-210Po remained in the the a particle emitted by 210Po travels only 37 /Â¿min tis alveolar duct regions but not in the larger airways. Much of sue, the distribution of radiation dose was closely related the hematite-210Po in the alveolar region appeared to be to the distribution of hematite particles and aggregates. The hematite-2 IOPo was present in a distinctly nonhomo- engulfed by macrophages. This finding was confirmed in geneous distribution in the lung. In general, within a histo- separate serial sacrifice studies (A. R. Kennedy and J. B. Little, unpublished data) utilizing glycol methacrylate (plas- logical section of a given lobe, apical regions contained less hematite-210Po than basal regions, probably an effect of tic)-embedded lung tissue; the thin l-^m sections allowed high resolution in the identification of individual cells types. gravity on the flow of hematite during instillation in the Six to 7 Days. Rapid decline in the number of macro upright position. At any given time after instillation, there phages containing hematite-210Po in the airways; patches of was a great variation in the amount of hematite-2lÂ°Po hematite-210Po remained in alveolar duct regions. appearing in different lobes of the same lung; some lobes Two to 8 Weeks. Very little macrophage associated were completely free of hematite-210Po while others had hematite-210Po remained in airways; patches of hematite- large amounts of activity, although the hematite did become 210Po in alveolar duct regions were somewhat reduced in more evenly distributed among the lobes following multiple intensity. instillations. Even within a single histological section, the Four to 8 Months. Airways were free of hematite-210Po. initial deposition of hematite was much greater in some Although the alveolar distribution was variable, all remain branches of the respiratory tree than others. At all times ing hematite-210Po was in the alveolar duct regions. studied, the hematite-210Po was patchy in nature. At early Throughout the 8-month period most a tracks remained times, hematite lined the airways as far downward in the associated with visible hematite deposits. There was no bronchial tree as the respiratory bronchiole-alveolar ducts evidence of dissociation of 210Pofrom the carrier particles. (Fig. 1). At later times, the only retained hematite was in the repiratory bronchioles and alveolar duct regions of the lung parenchyma (Fig. 3). Hematite-210Po was rarely seen in the Multiple instillations blood or lymph. In general, multiple instillations resulted in an increase in A qualitative description of areas where hematite-2lÂ°Po both the amount of hematite-210Po in the alveolar duct localized with time is as follows. regions (Fig. 3) and the number of alveolar duct regions

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Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1974 American Association for Cancer Research. A. R. Kennedy and J. B. Little containing hematite-210Po. With each successive instillation, sacrificed at these times. BP-hematite remained in alveolar there was an increase in the number of single tracks duct regions. During the 1st week, there were many appearing in the alveolar areas. These single track* gradu macrophages containing BP-hematite in the airways. ally disappared after the instillation period. Beyond 2 days, the epithelium of the larger airways in most animals was totally free of overlying BP-hematite clumps Benzo(a)pyrene and no BP free of particles remained within epithelial cells. The deposition of BP-hematite was the same as for At later times, small amounts of BP free of particles were hematite-2IOPo, but unlike 210Po, BP left the hematite seen in epithelial cells of the large airways in only 2 animals (sacrificed 2 and 4 weeks after instillation). This free BP was particles and appeared to enter adjacent epithelial cells. associated with rare clumps of BP-hematite which remained Most of this elution of BP from the particles occurred soon overlying the epithelium. after instillation. After the 1st 2 days, very little BP free of particles was seen in either the peripheral lung or larger airways. BP elution from the hematite carrier particles resulted in a significant dose of BP to the larger airway DISCUSSION epithelial cells during clearance of hematite-carcinogen on the mucous ciliary escalator. The only specific cell types We have previously found that intratracheally adminis tered hematite-2lÂ°Po gives rise to peripheral bronchiole- which could be identified using the frozen section technique were mast cells which, due to metachromatic granules, alveolar lung tumors while intratracheally administered stained red with thionin. Mast cells were commonly seen as BP-hematite induces primarily central tumors of the major a circular array of cells in the medial aspect of the trachÃ©al bronchi and trachea (10). From the results of this local Ca and bronchial adventitia. Mast cells never contained BP- tion study, it appears that the 2 carcinogens in large p irt hematite or BP free of particles. reach these 2 different regions of the lung despite their A qualitative description of areas where BP-hematite and administration in an identical manner. After intratracheal BP free of particles localized with time is as follows. administration, most of the hematite-210Po is deposited on the airways and is quickly cleared out of the lung. Owing to Single Instillation the short track length (37 um), little radiation probably reaches critical cells in the larger airways during clearance Zero to 1 Hr. The initial deposition of BP-hematite was of particle bound 210Po. The only carcinogen which is identical to that of hematite-210Po. BP-hematite lined the retained and delivers a long-term dose of radiation is the bronchi, bronchioles, and respiratory bronchiole-alveolar smaller fraction originally deposited in the alveolar duct duct regions in patchy areas of the lung. A section of the region (Fig. 3). BP, however, appears to be more loosely lung obtained immediately following instillation of BP- bound to the hematite as, unlike 210Po, it is seen free of hematite is shown in Fig. 4. BP is almost entirely associated hematite particles in the upper airway epithelium soon after with visible hematite particles at this time. Note auto- instillation. We propose that there is a direct route of entry fluorescence of elastic tissue (9, 25) in lung (Fig. 4b). to hematite-adsorbed soluble polynuclear hydrocarbon car One to 15 Hr. BP free of particles began accumulating in cinogens, such as BP, into upper airway epithelium during epithelial cells in some parts of trachea (Fig. 5), bronchi clearance of the particle-bound carcinogen on the mucous (Fig. 6), bronchioles (Fig. 7), and respiratory bronchiole- ciliary escalator. alveolar duct regions. BP was found in the epithelium of The initial pattern of hematite-carcinogen deposition is airways only in regions where BP-hematite aggregates were the same for the 2 carcinogens. Based on the present results, clearly present overlying the epithelium, although BP-hema the following route of hematite-carcinogen through the lung tite was not necessarily directly over the segment of is proposed. Hematite, after intratracheal instillation, epithelium containing free BP. In general, larger concentra spreads out over the surface of the airway epithelium like a tions of BP were found at airway bifurcations. By 4 hr after film and travels down the airway in this form. (This can be instillation, large concentrations of BP were seen in trachÃ©al demonstrated by coating a clean microscope slide with epithelium, connective tissue, and cartilage. Interestingly, mucus and then injecting hematite-210Po or BP-hematite on no BP free of hematite was found in the epithelium of some it. They form a film on the slide.) Most of the film spreads airways which nevertheless contained significant quantities itself over the airways, but some reaches the respiratory of BP-hematite overlying the epithelium. By 5 to 6 hr, bronchiole-alveolar duct region where it settles into the macrophages containing BP-hematite began to appear in proximal alveoli. The large fraction of the hematite which is the larger airways. deposited on the mucous ciliary escalator is rapidly cleared Fifteen to 48 Hr. Large numbers of macrophages with (almost completely by 48 hr), whereas the hematite reaching BP-hematite were seen in bronchioles, bronchi, and trachea. the alveoli is trapped in these cup-shaped structures, where BP free of hematite particles was seen outside the macro it appears to be phagocytized by macrophages. An alveolar phages in airway lumina and within adjacent epithelial cells macrophage may remain in situ for a long period, or it may of airways. migrate to the terminal bronchiole from which it is carried Forty-eight Hr to 1 Week to 1 Month. The distribution of upward on the mucous ciliary escalator (24). In the present BP-hematite was the same as for the hematite-210Po animals study, the maximal number of hematite-containing macro-

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Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1974 American Association for Cancer Research. Localization of Intratracheally Administered Carcinogens phages in the airway lumina was seen at 2 to 5 days after not have seen the BP-hematite lining the airways after instillation. instillation due to their method of lung tissue processing; When longitudinal sections of airways were studied, it conventional techniques utilizing liquid steps of fixation, was clear that the hematite film did not penetrate very far dehydration and clearing wash out almost all the material in into the alveolar region; only the 1st 3 to 5 alveoli of the the airways (6). alveolar ducts contained hematite. When the anatomy of the On the basis of this localization study, it is proposed that hamster lung is considered, it is not surprising that the a loosely particle-bound carcinogen delivers a significant major amount of hematite retained in the lung was in the carcinogenic dose to the upper airways during clearance of alveolar duct region. The tracheobronchial tree of the the particles by the mucous ciliary escalator, whereas a hamster is tube-like in nature until the level of the respira firmly bound carcinogen delivers its primary dose peripher tory bronchioles and alveolar ducts. Any material deposited ally where the particles are deposited and retained. Thus the on the ciliated epithelium is rapidly removed from the lung; physical properties of a carcinogen may play an important the alveoli of the respiratory bronchioles and the alveolar role in the location and type of lung tumors that it will ducts are the first nonciliated structures and, therefore, the induce. How the carcinogen is administered also appears to first to trap the hematite. The alveolar duct region clearly play an important role. Administered in 0.9% NaCl solution contained most of the trapped hematite, probably because without carrier particles, the bulk of the carcinogen would the respiratory bronchioles are very short in the hamster, be expected to be deposited and retained in the alveolar containing only a few alveoli (7). region, consistent with the findings of peripheral lung It is possible that the 0.9% NaCl solution in which the tumors induced by 210Po-0.9% NaCl solution in hamsters hematite-210Po (or BP-hematite) is suspended continues (11) and BP-0.9% NaCl solution (gelatin) in hamsters (5). through to the peripheral alveoli in the lung, depositing ionic Feron (4), on the other hand, recently reported that 36 210Po(i.e., not bound to hematite) in these regions. Presum weekly instillations of 1 mg BP in 0.9% NaCl solution ably, cilia retain particulate matter but not liquids. Both resulted in primarily malignant trachÃ©altumors, but it is of 210Po (10) and BP (16) are firmly bound to the hematite in interest that his "particles" of BP were very large, up to 80 the original solution, so very little free 210Poor BP would be firn. In his experiment, the BP itself was probably acting as expected to reach the peripheral lung. a carrier particle (19) and was cleared as a dust on the Although the initial deposition of hematite-carcinogen is mucous ciliary escalator. similar, the physical properties of the 2 carcinogens appear Like BP, methylcholanthrene bound to carrier particles to determine largely their subsequent localization. 2lÂ°Po has produced tracheobronchial lesions in a number of remains firmly bound to the particles and does not enter the studies (13, 14, 20-22). Recently, Schreiber et al. (18) found upper respiratory epithelium as the particles pass during peripheral (rather than upper airway) tumors following clearance. BP, on the other hand, does appear to enter the instillations of methylcholanthrene suspended in 0.9% NaCl upper airway epithelium as it is eluted off the carrier solution and gelatin. They ascribed the difference in tumor particles during clearance. That elution of BP from hema location to the possible effects of genetic differences in tite carrier particles (of the size used in this study) does animals used, microbiological status of the species tested occur has been firmly established in vitro (2, 3, 8) and in (Schreiber et al. (18) used specific pathogen-free animals), vivo (in the lung) (16). However, it has been assumed that and the difficulty in determining the origin of tumors in elution of BP from soot does not occur in the tracheobron animals with end-stage disease. Our findings suggest that chial tree (2). properties of the carcinogen, such as solubility and vehicle From the results of a previous BP-hematite localization of administration (particles or solution), may be sufficient study utilizing UV fluorescence microscopy, Saffiotti et al. to explain such differences in tumor location as Schreiber et (16) proposed that following their initial peripheral deposi al. (18) have described. tion, the BP and hematite carrier particles penetrate the alveolar epithelium and enter the interstitial spaces of the lung where they are engulfed by interstitial macrophages. REFERENCES Later, the soluble carcinogen is eluted out of these macro phages by a mechanism described by Falk et al. (2, 3) and 1. Bentley, H. R., and Burgan, J. G. Polynuclear Hydrocarbons in Kotin (8) and diffuses upward through the interstitial fluids Tobacco and Tobacco Smoke. Part 1.3.4 Benzopyrene. Analyst, 83: of the lung, reaching the larger airway epithelium from the 442-447, 1958. basal membrane side. Although this route may also occur, if 2. Falk, H. L., Kotin, P., and Markul, I. The Disappearance of it were the major mechanism by which BP reached the Carcinogens from Soot in Human Lungs. Cancer, //:482 489, 1958. bronchial and trachÃ©alepithelium, we would have expected 3. Falk, H. L., Miller, A., and Kotin, P. Elution of 3,4 Benzpyrene and to see BP fluorescence throughout the lung parenchyma and Related Hydrocarbons from Soots by Plasma Proteins. Science, 127: not localized to discrete regions associated with overlying 474-475, 1958. BP-hematite in the airways. From our localization study, 4. Feron, V. J. Respiratory Tract Tumors in Hamsters after Intratra- cheal Instillations of Benzo(a)pyrene Alone and with Furfural. Cancer therefore, it appears that the primary route of entry of BP Res., 32: 28 36, 1972. into upper airway epithelial cells is through the elution of 5. Henry, M. C., Port, C. D., Bates, R. B., and Kaufman, D. G. the carcinogen from the hematite particles during clearance Respiratory Tact Tumors Induced by Benzo(a)pyrene. Cancer Res., on the mucous ciliary escalator. Saffiotti et al. (16) would 33: 1585-1592, 1973.

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6. Kennedy, A. R., and Little, J. B. Autoradiography Using Dry 1968. Mounted Freeze-Dried Sections for Localization of Carcinogens in the 17. Saffiotti. U., Montesano, R., Sellakumar, A. R., Cefis, F., and Lung. J. Histochem. Cytochem., 22: 361 367, 1974. Kaufman, D. G. Respiratory Tract Carcinogenesis in Hamsters 7. Kleinerman, J. Some Aspects of Pulmonary Pathology in the Syrian Induced by Different Numbers of Administrations of Benzo(a)pyrene Hamster. Progr. Exptl. Tumor Res., 16: 287 299. 1972. and Ferric Oxide. Cancer Res., 32: 1073-1081, 1972. 8. Kotin, P. Exogenous and Endogenous Factors in the Pathogenesis of 18. Schreiber, H., Nettesheim, P., and Martin, D. H. Rapid Development Lung Cancer. Acta UniÃ³.Intern. Contra Cancrum, 75:613-618, 1959. of Bronchiolo-Alveolar Squamous Cell Tumors in Rats after Intratra 9. LaBella. F. S.. and Lindsay, W. G. The Structure of Human Aortic cheal Injection of 3-Methylcholanthrene. J. Nati. Cancer Inst., 49: Elastin as Influenced by Age. J. Gerontol., 18: 111 118. 1963. 541-554, 1972. 10. Little J. B., Grossman, B. N., and O'Toole, W. F. Respiratory 19. Sellakumar, A. R., Montesano, R., Saffiotti, U., and Kaufman, D. G. Carcinogenesis in Hamsters Induced by Polonium-210 Alpha Radia Hamster Respiratory Carcinogenesis Induced by Benzo(a)pyrene and tion and Benzo(a)pyrene. In: P. Nettesheim, M. G. Hanna, Jr., and J. Different Levels of Ferric Oxide. J. Nati. Cancer Inst., 50: 507-510, W. Deatherage. Jr. (eds.). Morphology of Experimental Respiratory 1973. Carcinogenesis, pp. 383-392. Atomic Energy Commission Symposium 20. Shabad, L. M. Experimental Cancer of the Lung. J. Nati. Cancer Series 21 Oak Ridge, Tenn: Atomic Energy Commission, 1970. Inst., 28: 1305-1332, 1962. 11. Little, J. B., Grossman, B. N., and O'Toole, W. F. Factors Influencing 21. Shabad, L. M. Dose Studies in Experimentally Induced Lung Tumors. the Induction of Lung Cancer in Hamsters by Intratracheal Adminis Environ. Res., 4: 305-315, 1971. tration of 210Po. In: C. L. Sanders, R. H. Busch, J. E. Ballou, D. D. 22. Shabad, L. M., and Pylev, L. N. Morphologic Lesions in Rat Lungs Mahlum, (eds.), Radionuclide Carcinogenesis, pp. 119-137. Atomic Induced by Polycyclic Hydrocarbons. In: P. Nettesheim, M. G. Energy Commission Symposium Series 29 Richland, Wash.: Atomic Hanna, Jr., and J. W. Deatherage, Jr. (eds.), Morphology of Atomic Energy Commission, 1973. Experimental Respiratory Carcinogenesis, pp. 227-242. Atomic En 12. Little, J. B., Radford, E. P., Jr., McCombs, H. L., and Hunt, V. R. ergy Commission Symposium Series 21. Oak Ridge, Tenn: Atomic Distribution of Polonium in Pulmonary Tissues of Cigarette Smokers. Energy Commission, 1970. New Engl. J. Med., 273: 1343-1351, 1965. 23. Stumpf, W. E., and Roth, L. J. High Resolution Autoradiography 13. Niskanen, K. O. Observations on Metaplasia of the Bronchial with Dry Mounted Freeze-Dried Frozen Sections. Comparative Study Epithelium and Its Relation to Carcinoma of the Lung. Acta Pathol. of Six Methods Using Two Diffusible Compounds 3H-Estradiol and Microbio!. Scand. Suppl. 80: 1 80, 1949. 3H-Mesobilirubinogen. J. Histochem. Cytochem., 14: 274-287, 1966. 14. Pylev, L. N. Induction of Lung Cancer in Rats by Intratracheal 24. Task Group on Lung Dynamics. Deposition and Retention Models Insufflation of Carcinogenic Hydrocarbons. Acta UniÃ³Intern. Contra for Internal Dosimetry of the Human Respiratory Tract. Health Cancrum, 19. 688 691, 1963. Phys., 12: 173-207, 1966. 15. Radford, E. P., Jr., and Hunt, V. R. Polonium-210: Volatile Radioele 25. Thomas, J. D., Elsden, D. F., and Partridge, S. M. Degradation ment in Cigarettes. Science, ÃŒ43:247249, 1964. Products from Elastin. Nature, 200: 651-652, 1963. 16. Saffiotti, U., Cefis, F., and Kolb, L. H. A Method for the Experimen 26. Udenfriend, S. Fluorescence Assay in Biology and Medicine, p. 278. tal Induction of Bronchogenic Carcinoma. Cancer Res., 28: 104 124, New York: Academic Press Inc., 1969.

Fig. I. a, low-power view of lung immediately following instillation of hematite-210Po. Note film of hematite-2'Â°Polining entire longitudinal section of bronchial tree and particular concentration of hematite-2'Â°Poin adjacent alveolar ducts (arrows). H & E, x 50. b, higher-power view of bronchus in a. Note heavy concentration of hematite-2'"Po on ciliated epithelium, x 200. Fig. 2. Hematite-210Po in macrophages within a small bronchus 48 hr after instillation of hematite-2'Â°Po.H & E, x 400. Fig. 3. a, low-power view of lung showing patchy nature of hematite-210Po. Seven days after 6th instillation. A bronchus is seen at top left: terminal bronchiole is in center of field. Celestine blue-Van Gieson's x 50. b, higher-power view of a showing a respiratory bronchiole-alveolar duct region extending from a terminal bronchiole. z'Â°Poa tracks can be seen in epithelial cell at center left and surrounding hematite particles, x 400. Figs. 4 to 7. These figures are all from frozen sections stained with thionin for UV fluorescent photomicrograph and restained with hematoxylin-eosin for tungsten light photomicrograph. Fig. 4. Comparison of same field in lung immediately after instillation of BP-hematite using tungsten light (a) and UV fluorescent light (6). A bronchus is seen in the center field, a blood vessel (artery) at bottom right. Note BP-hematite on ciliated epithelium (arrows); very little BP free of hematite particles is seen in epithelial cells at this time. Note autofluorescence of elastic tissue (9, 24) (Fig. 56) in blood vessel at bottom right, x 425. Fig. 5. Section of trachea 2 hr after BP-hematite instillation as seen by tungsten light (a) and UV fluorescent light (b). a, BP-hematite appears in airway lumen: cartilage is seen at bottom left, x 350. b, only a small portion of the BP has eluted off the hematite carrier particles and entered the epi thelial cells at this time as indicated by faint fluorescence (arrows). Elastic (9, 24), and to some extent collagenous (25), fibers found in cartilage (bot tom left) are known to be autofluorescent. x 400. Figs. 6 and 7. Two different fields of lung 2 hr after instillation as seen by tungsten light (a) and UV fluorescent light (b). Fig. 6. a, section of small bronchus with aggregates of BP-hematite on left and right of airway (arrows), x 385. b, BP free of hematite particles is clearly seen in bronchial epithelial cells as indicated by intense fluorescence of epithelial lining, x 385. Fig. 7. a, section of a bronchiole with BP-hematite aggregate in center of airway (arrow), x 450. b, intense fluorescence indicates presence of free BP in bronchiolar epithelial cells, x 350.

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Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1974 American Association for Cancer Research. The Transport and Localization of Benzo(a)pyrene-Hematite and Hematite- 210Po in the Hamster Lung following Intratracheal Instillation

Ann R. Kennedy and John B. Little

Cancer Res 1974;34:1344-1352.

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