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Airway epithelial cell changes in rats exposed to 0.25 ppm ozone for 20 months
Exp Toxic Patho11994; 46: 1-6 Gustav Fischer Verlag Jena
Departments of Pathology') and Hygiene 2), Yokohama City University School of Medicine, Yokohama, Japan
Airway epithelial cell changes in rats exposed to 0.25 ppm ozone for 20 months TAKAAKI ITOI), YOSHIAKI lKEMI 2), KAORU OHMORI 2), HITOSHI KITAMURA I), and MASAYOSHI KANISAWA 1) With 6 figures and 4 tables Received: July 27, 1992; Revised: August 3, 1993; Accepted: September 25, 1993 Address for correspondence: TAKAAKI ITO MD, Department of Pathology, Yokohama City University School of Medicine, 3-9 Fuku-Ura, Kanazawa-ku, Yokohama 236, Japan. Key words: Ozone, bronchial epithelium; Bronchial epithelium, ozone; BrdU-labeling index; Airway epithelia, ozone; Bromodeoxyuridine (BrdU); Bronchiolar epithelium, ozone; Neuroepithelial bodies, bronchial; Endocrine cells, bronchial.
Summary
Introduction
The present study was designed to characterize and quantify morphologic changes occurring in rat intra-pulmonary airway epithelia after long-term exposure to a high ambient level of ozone. Fifteen rats were exposed to 0.25 ppm ozone for 20 months (5 hr/day, 7 days/week) and a control group of 15 rats were exposed to filtered room air. Many intra-cellular brown pigmented granules (presumed to be lipofuscin) were seen in both the control and exposed animals; however, more of these granules were observed in the bronchial and bronchiolar epithelia of the exposed animals. To detect DNA synthesis that occurred in airway epithelial cells during the repair process induced by ozone toxicity, bromodeoxyuridine (BrdU) was injected intraperitoneally one hour before animals were sacrificed; the BrdUlabeling index was evaluated immunohistochemically. There was no difference in the BrdU-labeling index between the groups, the airway epithelia of the ozone-exposed animals exhibiting tolerance to ozone toxicity. However, the epithelial populations in the airways were altered by ozone exposure, and regional differences were seen in the changes. In the exposed animals, mucous cells increased in the lobar bronchus. Calcitonin gene-related peptide-immunoreactive pulmonary endocrine cells and neuroepithelial bodies were more frequently observed in the terminal bronchioles of the exposed animals than in the controls, although there were no differences in the lobar bronchus. Moreover, one adenoma in the peripheral lung was found in the exposed animals, while no tumorous lesions were detected in the controls. We conclude that exposure to a high ambient concentration of ozone induces morphologic changes in rats; these changes are related to the cellular toxicity of ozone and to the pathological responses of the epithelia, which responses reflect functional modifications in the respiratory tract.
Ozone, a major constituent of photochemical smog, produces pulmonary changes at ambient levels in humans and animals (24, 26, 39). It has been postulated that ozone gives rise to free radicals that can react with proteins and unsaturated fatty acids in cell membranes, and with nucleic acids, to cause cell injury (26, 28, 34). Ozone, even at near-ambient concentrations (0.1-0.5 ppm), induces morphologic changes in all parts of the respiratory tract in animals; the intensity of the changes varies depending on the ozone concentration, the exposure period, the respiratory tract level, the cell type, and the animal species. In the upper respiratory tract, ciliated cells appear to be most vulnerable, and damage to these cells has been observed in the nasal tissue of monkeys exposed to 0.15 and 0.3 ppm ozone for 6 days (13), in the trachea of rats exposed to 0.5 ppm ozone for 7 days (32), and in monkeys (8, 25). After longer exposure, secretory cell hyperplasia has been found (13, 16). In the smaller airways, the terminal bronchiole is particularly sensitive to ozone (1, 2, 8, 32, 35), injuries to ciliated cells here occurring after 2-hour exposure to 0.5 ppm ozone (35). The bronchiolar damage is followed by reparative processes as shown by cell renewal (9, 10) and bronchiolar cell hyperplasia (8, 25, 36). However, these changes are subtle at lower ozone concentrations (30, 32). A morphometrical study has shown loss of surface area in cilia and in Clara cells after 6-week exposure to 0.25 ppm ozone (1), while no remarkable change was reported after 90-day exposure to 0.2 ppm ozone (2). In the longer-term studies, the results vary; according to Gross and White (12), the bronchiolar epithelium did not change in rats exposed to 0.5 ppm for 6 and 12 months, while, in other studies, Exp Toxic Pathol46 (1994) 1
nodular epithelial hyperplasia was shown in the bronchioles of rats exposed to 0.5 ppm ozone for 180 days (27) and for 12 months (15). To our knowledge, no morphologic study of the effects of much longer exposure to ambient levels of ozone has been reported. However, one precise morphometrical study on the changes of rat bronchioloalveolar junction exposed to 1.0 ppm ozone for 20 months has recently been reported, in which extension of bronchiolar Clara cells and ciliated cells into alveolar ducts is well documented with various morphologic techniques including Clara cell immunohistochemistry and confocal microscopy (29). The present study was undertaken to examine the morphologic changes occurring in the intra-pulmonary airway epithelia of rats exposed to a relatively high ambient ozone concentration (0.25 ppm) for a much longer period than has hitherto been studied (20 months). We focused on the vestiges of airway epithelial cell injury induced by ozone exposure by counting lipofuscin granules (7) and monitoring epithelial regenerative activity in terms of bromodeoxyuridine incorporation (20). We also examined changes in airway epithelial cell populations, i.e., in airway mucous cells and endocrine cells, since mucous cell hyperplasia in the nose has been shown to be induced by ozone inhalation (13, 16) and since pulmonary endocrine cells have been shown to react as receptor cells when presented with gaseous stimuli (21, 33).
Material and methods Animals and Ozone Exposure Male Sprague-Dawley rats,S weeks of age, were obtained from a specific pathogen-free colony (Nihon Clea, Tokyo, Japan). They were divided into a control group (n = 15) and an exposure group (n =15). The animals were housed three per cage and were provided with water and a commercial diet ad libitum. The animals in the exposure group were kept in a stainless chamber with a volume of 4.2 m3, and were exposed to ozone at an average concentration of 0.25 ppm for 5 hours (from 11 :00 a.m. to 4:00 p.m.) per day, 7 days a week, for 20 months. The animal in the control group were exposed to an atmosphere of filtered air. The ozone used in this study was produced by the silent electric discharge method, using an ozone-generator (Type 0-3-2, Nippon Ozone Co., Tokyo, Japan), and it was mixed with filtered air. The mixed gas was admitted into the chamber, and the concentration of ozone was continuously monitored with a chemiluminescence type ozone meter (Type 807; Kimoto Electric Co., Tokyo, Japan) as reported previously (18).
Tissue Preparation On the day the animals were sacrificed, they received an intraperitoneal injection of 5-bromo-2' -deoxyuridine (BrdU; Sigma, St. Louis, MO; 20 mg/kg body weight) at 9:00-10:00 a.m., one hour before sacrifice. The animals were anesthe2
Exp Toxic Pathol46 (1994) 1
tized with pentobarbital before sacrifice. After thoracotomy, the lungs were infused, via the trachea, with 0.1 M phosphate-buffered 4 % paraformaldehyde (pH 7.3). Fixation of the lung was done at first under 30 cm fixation pressure for ten minutes in situ, and then the lungs were immersed in the same fixative at room temperature overnight. The fixed left lungs were cut to make a slice containing the lung hilus and lober bronchus. The tissue slices of the left lung were washed in the phosphate buffer solution (pH 7.3), dehydrated, and embedded in paraffin. Paraffin sections (4-llm-thick) were stained with hematoxylin alone, hematoxylin and eosin (HE), or Alcian bluePAS stains. For immunostaining for the detection of pulmonary endocrine cells, de-paraffinized sections were incubated with rabbit anti-calcitonin gene-related peptide (CGRP) antibody (Amersham, Buckinghamshire, England), followed by the use of the avidin-biotin-peroxidase complex (ABC) method, employing an ABC kit (Vector Laboratories, San Mateo, CA). To detect S-phase epithelial cells, the sections were incubated with mouse anti-BrdU antibody (BectonDickinson, Mountain View, CA), followed by the application of the ABC method. After being washed, the sections were reacted with a mixture of hydrogen peroxide and diaminobenzidine as a peroxidase substrate solution, and were counter-stained lightly with hematoxylin. Part of the right lung tissue was cut into small cubes and was fixed in a 0.1 M cacodylate-buffered 2 % glutaraldehyde solution. After postosmication with 1 % osmium tetroxide, the tissues were embedded in Epon and Araldite Resin mixture. Thin sections were stained with uranyl acetate and lead citrate and examined under an electron microscope (Hitachi H-600).
Morphometrical study In the present study, quantitative study was focused on the proximal half of the lobar bronchus and terminal bronchiole which was directly continuous with the alveolar duct. Before the following morphometrical studies, the lengths of the lobar bronchus and terminal bronchiole were measured with an image analyzer (Model G/A, Muto Kogyo Co., Tokyo, Japan), and the numbers of the airway epithelial cells per unit length were calculated to avoid bias due to modulation of cell size by ozone exposure. In the sections stained with hematoxylin alone, the number of brown pigmented granules in the lobar bronchus and the terminal bronchioli were counted; average numbers were estimated after 1,000 bronchial and bronchiolar cells were observed in each animal. To evaluate BrdU-positive cells (S-phase cells), 2,000 nuclei were observed in the lobar bronchus and terminal bronchioli of each animal, and the labeling index was calculated. When BrdU-positive cells were counted, epithelial areas adjacent to bronchus-associated lymphoid tissues with massive lymphocytic infiltration were omitted. In the Alcian blue-PAS-stained sections, the mucous cell number was calculated after examination of 1,000 bronchial and bronchiolar epithelial cells. The number of CGRP-positive grouped endocrine cells (neuroepithelial bodies; NEBs), and the number of CGRP-positive endocrine cells constituting the NEB were counted, and the density of the bronchial and bronchiolar NEBs and endocrine cells per unit airway length was evaluated (19). Comparisons were made using the two sample t-test to determine statistical significance.
Results
Fig. 1. Adenomatous lesion of the peripheral lung in an ozone-exposed rat. Columnar cells with foamy cytoplasm line the alveolus. HE stain, x500.
No remarkable pathological changes were found in the HE sections of the lungs of the exposed animals, except for one small adenomatous lesion in the peripheral lung (fig. 1). The cell sizes in the lobar bronchus and terminal bronchiole were similar between two animal groups; the average cell numbers in the lobar bronchus were 224 ± 20 in the control animals and 226 ± 32 in the exposed ones, and those in the terminal bronchiole were 186 ± 17 in the control and 191 ± 20 in the exposed. There were no emphysematous changes, nor nodular bronchiolar hyperplasia. There were scattered mononuclear cell infiltrations, largely composed of small lymphocytes, along the lobar bronchi in the groups. In the sections stained with hematoxylin alone, coarse irregularly-shaped brown pigmented granules were seen in the airway epithelial cells (fig. 2). These were mainly
Fig. 2. Many granules are seen in the supra-nuclear region of the bronchial epithelial cells in an ozone-exposed rat. Hematoxylin stain, x 900. Fig. 3. Electron micrograph of the bronchial epithelium of an ozone-exposed rat. Lysosomes with heterogeneous structures are prominent in a ciliated cell, x32,000. Exp Toxic Patho146 (1994) 1
3
Table 1. Numbers of pigmented granules (average ± SD/cell).
Table 2. BrdU-Iabeling index (average ± SD %).
Animal Group
Bronchus
Terminal Bronchiolus
Animal Group
Bronchus
Control
1.02 ± 0.32*
0.62 ± 0.22**
Control
6.0 ± 3.5 x 10-2 10.0 ± 8.0 x 10-2
Ozone-exposed
1.36 ± 0.33*
0.86 ± 0.27**
Ozone-exposed
6.5 ± 5.0 x 10-2
* significantly different, p < 0.01. ** significantly different, p < 0.05.
Terminal Bronchiolus
9.5 ± 5.5 x 10-2
Table 3. Numbers of mucous cells (average ± SD/cell). Animal Group
Bronchus
Terminal Bronchiolus
Control
2.73 ± 1.42*
Ozone-exposed
5.68 ± 2.29*
o o
* significantly different, at p < 0.01.
located in the supra-nuclear areas, and often showed PAS-positive reaction. The average number of these pigmented granules in the control group was 1.02 per bronchial cell and 0.62 per bronchiolar cell, while in the exposed group, the average numbers were 1.36 and 0.86, respectively, i.e., they were significantly increased in the latter group (table 1). Electron microscopically, lysosomal granules with heterogeneous contents were observed in the supra-nuclear areas (fig. 3); more of these were seen in ciliated cells than in non-ciliated cells. In the lobar bronchus and terminal bronchiole, the BrdUpositive cells were all columnar (fig. 4); the labeling index did not differ for the control and the exposed groups (table 2). Alcian blue and/or PAS-positive airway epithelial cells increased significantly in the lobar bronchus of the ozone-exposed group (fig. 5, table 3). Cells positive for PAS alone were seen much more frequently than Alcian blue-positive cells, and the proportion of these mucous cells appeared to be similar in both groups. There was no mucous cell metaplasia in the terminal bronchiole in either group. CGRP-positive airway epithelial cells were present as NEB (fig. 6), and no solitary CGRP-positive airway endocrine cells were observed. The numbers of CGRPpositive NEBs and CGRP-positive endocrine cells were significantly larger in the terminal bronchiole in the exposed group than in the control group, but this was not the case in the lobar bronchus (table 4).
@ Fig. 4. Nuclear staining is seen in a columar cell (arrow) of the bronchus in a control rat lung. Note that cytoplasmic granules are rarely seen. BrdU immunostain, x600. Fig. 5. Many mucous cells are seen in the lobar bronchus of an ozone-exposed rat. Alcian blue-PAS stain, x600. Fig. 6. CGRP-immunoreactive neuroepithelial body in the terminal bronchiole of an ozone-exposed rat. CGRP immunostain, x600. 4
Exp Toxic Pathol46 (1994) 1
Discussion In the present study, we have shown the morphologic changes that occurred in the rat airway epithelium after long-term exposure to a high ambient ozone concentration; we focused on changes related to cellular injury,
Table 4. Numbers of CGRP-positive neuroepithelial bodies (NEBs) and airway endocrine cells (average ± SD/cm of airway). Terminal Bronchiolus
Bronchus Animal Group
NEB
Endocrine Cell
NEB
Endocrine Cell
Control Ozone-exposed
1.42 ± 2.00 1.11 ± 1.32
10.24 ± 14.51 5.94± 7.02
0.48 ± 0.62* 1.29 ± 0.90*
3.35 ± 4.83** 7.35 ± 5.05**
*, ** significantly different, at p < 0.05, respectively.
epithelial pathological responses and tumorigenesis related to ozone exposure. Numbers of lipofuscin granules in the rat lung increase with age and their ultrastructure becomes more complex (17). Apart from ageing, increases in lipofuscin granules can be engendered by various noxious influences, such as starvation, wasting diseases, vitamin E deficiency and so on (11). In the present study, the number of brown pigmented granules, which were thought to be lipofuscin, increased in the ozone-exposed animals. This was assumed to be related to some injurious effect of long-term ozone exposure, although ozone exposure does not always affect the concentration of lipofuscin granules (7). To assess the injurious effects of prolonged exposure to ozone on the airway epithelium, we examined the BrdUlabeling indices for the lungs, since epithelial damage is followed by cell renewal (9, 10). No significant difference was seen between the control and exposed animals, and ozone tolerance was confirmed in the older animals. No difference in cell size between the two animal groups could be explained by the similar phenomenon. The airway epithelial cell population was changed by the long-term ozone exposure, and the numbers of mucous and endocrine cells were altered. As expected from previous studies (13, 16), we also found mucous cell hyperplasia in our study. Mucous cell hyperplasia is a wellknown feature in the airways of smokers (38), and has been induced experimentally by proteolytic enzmye treatment (3-5). The mechanisms by which proteases derived from neutrophils (3) and macrophages (5) cause mucous cell hyperplasia remain unknown, but this hyperplasia would seem to be reasonable, since mucous products are thought to protect the epithelium from oxidant damage (6). In ozone-induced mucous cell hyperplasia, inflammatory reactions evoked by ozone exposure could contribute to the condition through the release of proteases. We found that pulmonary endocrine cells, particularly CGRP-positive cells, were increased in the small airways in the ozone-exposed animals, but that there were no increases of endocrine cells in the lobar bronchus. Such regional differences in the reaction of endocrine cells to a toxic gas have been reported in the lungs of rats exposed to nitrogen dioxide, but in that case the increases of these
cells were seen in the larger airways (21). The airway endocrine cell is thought to function as a recepto-effector cell (23), but the effects of noxious stimuli transmitted through the airways on this cell type, and its reactions, have not been well documented. Further investigations are needed. Despite the vast literature that documents the effects of both acute and chronic ozone exposure in both humans and animals, few studies have addressed on the possible relationship between respiratory tumorigenesis and ozone exposure. In vivo studies have revealed that higher concentrations of ozone increase the number of pulmonary adenomas in mice (14, 22), and modify the incidence of chemically-induced lung tumors (14, 22). In an in vitro study using rat tracheal epithelium, ozone was shown to enhance the incidence of preneoplastic transformation of the epithelium (37). Although the experimental scale of the present study was too small to lead to statistical analysis, our result of one adenoma in 15 ozone-exposed rats, since the incidence of spontaneous lung adenomas in rats is about 1 % (31), might support the idea that ozone is potentially tumorigenic. Acknowledgements: The authors thank Mr. M. IKEDA, Mr. H. MITSUI and Mr. T. SUZUKI for their excellent technical assistance.
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4. CARDOZO C, PADILLA ML, CHOI H-SH, et al.: Goblet cell hyperplasia in large intrapulmonary airways after intratracheal injection of cathepsin B into hamsters. Am Rev Respir Dis 1992; 145: 675-679. 5. CHRISTENSEN TG, KORTHY AL, SNIDER GL, et al.: Irreversible bronchial goblet cell metaplasia in hamsters with elastase-induced panacinar emphysema. J Clin Invest 1977; 59: 397-404. 6. CROSS CE, HALLIWELL B, ALLEN A: Antioxidant protection: a function of tracheobronchial and gastrointestinal mucus. Lancet 1984; 1: 1328-1330. 7. CSALLANY AS, MANWARING JD, MENKEN BZ: Ozonerelated fluorescent compounds in mouse liver and lung. Environ Res 1985; 37: 320-326. 8. DUNGWORTH DL, CASTLEMAN WL, CHOW CK, et al.: Effect of ambient levels of ozone on monkeys. Fed Proc 1975; 34: 1670--1674. 9. ELSAYED NM, ELLINGSON AS, TIERNEY DF, et al.: Effects of ozone inhalation on polyamine metabolism and tritiated thymidine incorporation into DNA of rat lungs. Toxicol Appl Pharmacol1990; 102: 1-8. 10. EVANS MJ, JOHNSON LV, STEPHENS RJ, et al.: Cell renewal in the lungs of rats exposed to low levels of ozone. Exp Mol Patho11976; 24: 70--83. 11. GHADIALLY FN: Ultrastructural pathology of the cell and matrix. Butterworths, London 1982. 12. GROSS KB, WHITE HJ: Functional and pathologic consequences of a 52-week exposure to 0.5 ppm ozone followed by a clean air recovery period. Lung 1987; 165: 283-295. 13. HARKEMA JR, PLOPPER CG, HYDE DM, et al.: Effects of an ambient level of ozone on primate nasal epithelial mucosubstances. Quantitative histochemistry. Am J Patho11987; 127: 90--96. 14. HASSEIT C, MUSTAFA MG, COULSON WF, et al.: Murine lung carcinogenesis following exposure to ambient ozone concentrations. J Natl Cancer Inst 1985; 75: 771-777. 15. HIROSHIMA K, KOHNO T, OHWADA H, et al.: Morphological study of the effects of ozone on rat lung. II. Longterm exposure. Exp Mol Patho11989; 50: 270--280. 16. HOTCHKISS JA, HARKEMA JR, HENDERSON RF: Effect of cumulative ozone exposure on ozone-induced nasal epithelial hyperplasia and secretory metaplasia in rats. Exp Lung Res 1991; 15: 589-600. 17. IKEDA H, TAUCHI H, SATO T: Fine structural analysis of lipofuscin in various tissues of rats of different ages. Mech Ageing Dev 1985; 33: 77-93. 18. IKEMI Y, OHMORI K, ITO T, et al.: Effect of the exposure of rats to ozone on the osmotic resistance of their erythrocytes. Res Commun Chem Pathol PharmcoI1992; 78: 109-112. 19. ITO T, NAKATANI Y, NAGAHARA N, et al.: Quantitative study of pulmonary endocrine cells in anencephaly. Lung 1987; 165: 297-304. 20. JOHNSON NF, HOTCHKISS JA, HARKEMA JR, et al.: Proliferative responses of rat nasal epithelia to ozone. Toxicol Appl Pharmacol1990; 103: 143-155. 21. KLEINERMAN J, MARCHEVSKY AM, THORNTON J: Quantitative studies of APUD cells in airways of rats. The effects of diethylnitrosamine and NO z. Am Rev Respir Dis 1981; 124: 458-462. 22. LAST JA, WARREN DL, PECQUET-GOAD E, et al.: Modification by ozone of lung tumor development in mice. J Natl Cancer Inst 1987; 78: 149-154. 6
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23. LAUWERYNS JM, COKELAERE M, THEUNYNCK P: Neuroepithelial bodies in the respiratory mucosa of various mammals: a light optical, histochemical and ultrastructural in vestigation. Z Zellforsch Mikrosk Anat 1972; 135: 569-592. 24. LIpPMANN M: Health effects of ozone. A critical review. JAPCA 1989; 39: 672-695. 25 . MELLICK PW, DUNGWORTH DL, SCHWARTZ LW, et al.: Short term morphologic effects of high ambient levels of ozone on lungs of rhesus monkeys. Lab Invest 1977; 36: 82-90. 26. MENZEL DB: Ozone: an overview of its toxicity in man and animals. J Toxicol Environ Health 1984; 13: 183204. 27. MOORE PF, SCHWARTZ LW: Morphological effects of prolonged exposure to ozone and sulfuric acid aerosol on the rat lung. Exp Mol Patho11981; 35: 108123. 28. MUSTAFA MG, TIERNEY DF: Biochemical and metabolic changes in the lung with oxygen, ozone, and nitrogen dioxide toxicity. Am Rev Respir Dis 1978; 118: 1061-1090. 29. PINKERTON KE, DODGE DE, CEDERDAHL-DEMMLER J, et al.: Differentiated bronchiolar epithelium in alveolar ducts of rats exposed to ozone for 20 months. Am J Patho11993; 142: 947-956. 30. PLOPPER CG, DUNGWORTH DL, TYLER WS: Pulmonary alterations in rats exposed to 0.2 and 0.1 ppm ozone: a correlated morphological and biochemical study. Arch Environ Health 1979; 34: 390-395. 31. REZNIK G: Spontaneous primary and secondary lung tumors in the rat. In: Comparative respiratory tract carcinogenesis. Vol. 1 (ed REZNIK-SCHULLER HM). CRC Press, Boca Raton 1983, pp 95-116. 32. SCHWARTZ LW, DUNGWORTH DL, MUSTAFA MG, et al.: Pulmonary responses of rats to ambient levels of ozone. Effects of 7-day intermittent or continuous exposure. Lab Invest 1976; 34: 565-578. 33. SPRINGALL DR, COLLINA G, BARER G, et al.: Increased intracellular levels of calcitonin gene-related peptidelike immunoreactivity in pulmonary endocrine cells of hypoxic rats. J Pathol 1988; 155: 259-267. 34. STEINBERG JJ, GLEESON JL, GIL D: The pathobiology of ozone-induced damage. Arch Environ Health 1990; 45: 80--87. 35. STEPHENS RJ, SLOAN MF, EVANS MJ, et al.: Early response of lung to low levels of ozone. Am J Pathol 1973; 74: 31-58. 36. TEPPER JS, COSTA DL, LEHMANN JR, et al.: Unattenuated structural and biochemical alterations in the rat lung during functional adaptation to ozone. Am Rev Respir Dis 1989; 140: 493-501. 37. THOMASSEN DG, HARKEMS JR, STEPHENS ND, et al.: Preneoplastic transformation of rat tracheal epthelial cells by ozone. Toxicol Appl Pharmacol 1991; 109: 137-148. 38. THURLBECK WM, MALAKA D, MURPHY K: Goblet cells in the peripheral airways in chronic bronchitis. Am Rev Respir Dis 1975; 112: 65-69. 39. WRIGHT ES, DZIEDZIC D, WHEELER CS: Cellular, biochemical and functional effects of ozone: new research and perspectives on ozone health effects. Toxicol Lett 1990; 51: 125-145.
Departments of Pathology') and Hygiene 2), Yokohama City University School of Medicine, Yokohama, Japan
Airway epithelial cell changes in rats exposed to 0.25 ppm ozone for 20 months TAKAAKI ITOI), YOSHIAKI lKEMI 2), KAORU OHMORI 2), HITOSHI KITAMURA I), and MASAYOSHI KANISAWA 1) With 6 figures and 4 tables Received: July 27, 1992; Revised: August 3, 1993; Accepted: September 25, 1993 Address for correspondence: TAKAAKI ITO MD, Department of Pathology, Yokohama City University School of Medicine, 3-9 Fuku-Ura, Kanazawa-ku, Yokohama 236, Japan. Key words: Ozone, bronchial epithelium; Bronchial epithelium, ozone; BrdU-labeling index; Airway epithelia, ozone; Bromodeoxyuridine (BrdU); Bronchiolar epithelium, ozone; Neuroepithelial bodies, bronchial; Endocrine cells, bronchial.
Summary
Introduction
The present study was designed to characterize and quantify morphologic changes occurring in rat intra-pulmonary airway epithelia after long-term exposure to a high ambient level of ozone. Fifteen rats were exposed to 0.25 ppm ozone for 20 months (5 hr/day, 7 days/week) and a control group of 15 rats were exposed to filtered room air. Many intra-cellular brown pigmented granules (presumed to be lipofuscin) were seen in both the control and exposed animals; however, more of these granules were observed in the bronchial and bronchiolar epithelia of the exposed animals. To detect DNA synthesis that occurred in airway epithelial cells during the repair process induced by ozone toxicity, bromodeoxyuridine (BrdU) was injected intraperitoneally one hour before animals were sacrificed; the BrdUlabeling index was evaluated immunohistochemically. There was no difference in the BrdU-labeling index between the groups, the airway epithelia of the ozone-exposed animals exhibiting tolerance to ozone toxicity. However, the epithelial populations in the airways were altered by ozone exposure, and regional differences were seen in the changes. In the exposed animals, mucous cells increased in the lobar bronchus. Calcitonin gene-related peptide-immunoreactive pulmonary endocrine cells and neuroepithelial bodies were more frequently observed in the terminal bronchioles of the exposed animals than in the controls, although there were no differences in the lobar bronchus. Moreover, one adenoma in the peripheral lung was found in the exposed animals, while no tumorous lesions were detected in the controls. We conclude that exposure to a high ambient concentration of ozone induces morphologic changes in rats; these changes are related to the cellular toxicity of ozone and to the pathological responses of the epithelia, which responses reflect functional modifications in the respiratory tract.
Ozone, a major constituent of photochemical smog, produces pulmonary changes at ambient levels in humans and animals (24, 26, 39). It has been postulated that ozone gives rise to free radicals that can react with proteins and unsaturated fatty acids in cell membranes, and with nucleic acids, to cause cell injury (26, 28, 34). Ozone, even at near-ambient concentrations (0.1-0.5 ppm), induces morphologic changes in all parts of the respiratory tract in animals; the intensity of the changes varies depending on the ozone concentration, the exposure period, the respiratory tract level, the cell type, and the animal species. In the upper respiratory tract, ciliated cells appear to be most vulnerable, and damage to these cells has been observed in the nasal tissue of monkeys exposed to 0.15 and 0.3 ppm ozone for 6 days (13), in the trachea of rats exposed to 0.5 ppm ozone for 7 days (32), and in monkeys (8, 25). After longer exposure, secretory cell hyperplasia has been found (13, 16). In the smaller airways, the terminal bronchiole is particularly sensitive to ozone (1, 2, 8, 32, 35), injuries to ciliated cells here occurring after 2-hour exposure to 0.5 ppm ozone (35). The bronchiolar damage is followed by reparative processes as shown by cell renewal (9, 10) and bronchiolar cell hyperplasia (8, 25, 36). However, these changes are subtle at lower ozone concentrations (30, 32). A morphometrical study has shown loss of surface area in cilia and in Clara cells after 6-week exposure to 0.25 ppm ozone (1), while no remarkable change was reported after 90-day exposure to 0.2 ppm ozone (2). In the longer-term studies, the results vary; according to Gross and White (12), the bronchiolar epithelium did not change in rats exposed to 0.5 ppm for 6 and 12 months, while, in other studies, Exp Toxic Pathol46 (1994) 1
nodular epithelial hyperplasia was shown in the bronchioles of rats exposed to 0.5 ppm ozone for 180 days (27) and for 12 months (15). To our knowledge, no morphologic study of the effects of much longer exposure to ambient levels of ozone has been reported. However, one precise morphometrical study on the changes of rat bronchioloalveolar junction exposed to 1.0 ppm ozone for 20 months has recently been reported, in which extension of bronchiolar Clara cells and ciliated cells into alveolar ducts is well documented with various morphologic techniques including Clara cell immunohistochemistry and confocal microscopy (29). The present study was undertaken to examine the morphologic changes occurring in the intra-pulmonary airway epithelia of rats exposed to a relatively high ambient ozone concentration (0.25 ppm) for a much longer period than has hitherto been studied (20 months). We focused on the vestiges of airway epithelial cell injury induced by ozone exposure by counting lipofuscin granules (7) and monitoring epithelial regenerative activity in terms of bromodeoxyuridine incorporation (20). We also examined changes in airway epithelial cell populations, i.e., in airway mucous cells and endocrine cells, since mucous cell hyperplasia in the nose has been shown to be induced by ozone inhalation (13, 16) and since pulmonary endocrine cells have been shown to react as receptor cells when presented with gaseous stimuli (21, 33).
Material and methods Animals and Ozone Exposure Male Sprague-Dawley rats,S weeks of age, were obtained from a specific pathogen-free colony (Nihon Clea, Tokyo, Japan). They were divided into a control group (n = 15) and an exposure group (n =15). The animals were housed three per cage and were provided with water and a commercial diet ad libitum. The animals in the exposure group were kept in a stainless chamber with a volume of 4.2 m3, and were exposed to ozone at an average concentration of 0.25 ppm for 5 hours (from 11 :00 a.m. to 4:00 p.m.) per day, 7 days a week, for 20 months. The animal in the control group were exposed to an atmosphere of filtered air. The ozone used in this study was produced by the silent electric discharge method, using an ozone-generator (Type 0-3-2, Nippon Ozone Co., Tokyo, Japan), and it was mixed with filtered air. The mixed gas was admitted into the chamber, and the concentration of ozone was continuously monitored with a chemiluminescence type ozone meter (Type 807; Kimoto Electric Co., Tokyo, Japan) as reported previously (18).
Tissue Preparation On the day the animals were sacrificed, they received an intraperitoneal injection of 5-bromo-2' -deoxyuridine (BrdU; Sigma, St. Louis, MO; 20 mg/kg body weight) at 9:00-10:00 a.m., one hour before sacrifice. The animals were anesthe2
Exp Toxic Pathol46 (1994) 1
tized with pentobarbital before sacrifice. After thoracotomy, the lungs were infused, via the trachea, with 0.1 M phosphate-buffered 4 % paraformaldehyde (pH 7.3). Fixation of the lung was done at first under 30 cm fixation pressure for ten minutes in situ, and then the lungs were immersed in the same fixative at room temperature overnight. The fixed left lungs were cut to make a slice containing the lung hilus and lober bronchus. The tissue slices of the left lung were washed in the phosphate buffer solution (pH 7.3), dehydrated, and embedded in paraffin. Paraffin sections (4-llm-thick) were stained with hematoxylin alone, hematoxylin and eosin (HE), or Alcian bluePAS stains. For immunostaining for the detection of pulmonary endocrine cells, de-paraffinized sections were incubated with rabbit anti-calcitonin gene-related peptide (CGRP) antibody (Amersham, Buckinghamshire, England), followed by the use of the avidin-biotin-peroxidase complex (ABC) method, employing an ABC kit (Vector Laboratories, San Mateo, CA). To detect S-phase epithelial cells, the sections were incubated with mouse anti-BrdU antibody (BectonDickinson, Mountain View, CA), followed by the application of the ABC method. After being washed, the sections were reacted with a mixture of hydrogen peroxide and diaminobenzidine as a peroxidase substrate solution, and were counter-stained lightly with hematoxylin. Part of the right lung tissue was cut into small cubes and was fixed in a 0.1 M cacodylate-buffered 2 % glutaraldehyde solution. After postosmication with 1 % osmium tetroxide, the tissues were embedded in Epon and Araldite Resin mixture. Thin sections were stained with uranyl acetate and lead citrate and examined under an electron microscope (Hitachi H-600).
Morphometrical study In the present study, quantitative study was focused on the proximal half of the lobar bronchus and terminal bronchiole which was directly continuous with the alveolar duct. Before the following morphometrical studies, the lengths of the lobar bronchus and terminal bronchiole were measured with an image analyzer (Model G/A, Muto Kogyo Co., Tokyo, Japan), and the numbers of the airway epithelial cells per unit length were calculated to avoid bias due to modulation of cell size by ozone exposure. In the sections stained with hematoxylin alone, the number of brown pigmented granules in the lobar bronchus and the terminal bronchioli were counted; average numbers were estimated after 1,000 bronchial and bronchiolar cells were observed in each animal. To evaluate BrdU-positive cells (S-phase cells), 2,000 nuclei were observed in the lobar bronchus and terminal bronchioli of each animal, and the labeling index was calculated. When BrdU-positive cells were counted, epithelial areas adjacent to bronchus-associated lymphoid tissues with massive lymphocytic infiltration were omitted. In the Alcian blue-PAS-stained sections, the mucous cell number was calculated after examination of 1,000 bronchial and bronchiolar epithelial cells. The number of CGRP-positive grouped endocrine cells (neuroepithelial bodies; NEBs), and the number of CGRP-positive endocrine cells constituting the NEB were counted, and the density of the bronchial and bronchiolar NEBs and endocrine cells per unit airway length was evaluated (19). Comparisons were made using the two sample t-test to determine statistical significance.
Results
Fig. 1. Adenomatous lesion of the peripheral lung in an ozone-exposed rat. Columnar cells with foamy cytoplasm line the alveolus. HE stain, x500.
No remarkable pathological changes were found in the HE sections of the lungs of the exposed animals, except for one small adenomatous lesion in the peripheral lung (fig. 1). The cell sizes in the lobar bronchus and terminal bronchiole were similar between two animal groups; the average cell numbers in the lobar bronchus were 224 ± 20 in the control animals and 226 ± 32 in the exposed ones, and those in the terminal bronchiole were 186 ± 17 in the control and 191 ± 20 in the exposed. There were no emphysematous changes, nor nodular bronchiolar hyperplasia. There were scattered mononuclear cell infiltrations, largely composed of small lymphocytes, along the lobar bronchi in the groups. In the sections stained with hematoxylin alone, coarse irregularly-shaped brown pigmented granules were seen in the airway epithelial cells (fig. 2). These were mainly
Fig. 2. Many granules are seen in the supra-nuclear region of the bronchial epithelial cells in an ozone-exposed rat. Hematoxylin stain, x 900. Fig. 3. Electron micrograph of the bronchial epithelium of an ozone-exposed rat. Lysosomes with heterogeneous structures are prominent in a ciliated cell, x32,000. Exp Toxic Patho146 (1994) 1
3
Table 1. Numbers of pigmented granules (average ± SD/cell).
Table 2. BrdU-Iabeling index (average ± SD %).
Animal Group
Bronchus
Terminal Bronchiolus
Animal Group
Bronchus
Control
1.02 ± 0.32*
0.62 ± 0.22**
Control
6.0 ± 3.5 x 10-2 10.0 ± 8.0 x 10-2
Ozone-exposed
1.36 ± 0.33*
0.86 ± 0.27**
Ozone-exposed
6.5 ± 5.0 x 10-2
* significantly different, p < 0.01. ** significantly different, p < 0.05.
Terminal Bronchiolus
9.5 ± 5.5 x 10-2
Table 3. Numbers of mucous cells (average ± SD/cell). Animal Group
Bronchus
Terminal Bronchiolus
Control
2.73 ± 1.42*
Ozone-exposed
5.68 ± 2.29*
o o
* significantly different, at p < 0.01.
located in the supra-nuclear areas, and often showed PAS-positive reaction. The average number of these pigmented granules in the control group was 1.02 per bronchial cell and 0.62 per bronchiolar cell, while in the exposed group, the average numbers were 1.36 and 0.86, respectively, i.e., they were significantly increased in the latter group (table 1). Electron microscopically, lysosomal granules with heterogeneous contents were observed in the supra-nuclear areas (fig. 3); more of these were seen in ciliated cells than in non-ciliated cells. In the lobar bronchus and terminal bronchiole, the BrdUpositive cells were all columnar (fig. 4); the labeling index did not differ for the control and the exposed groups (table 2). Alcian blue and/or PAS-positive airway epithelial cells increased significantly in the lobar bronchus of the ozone-exposed group (fig. 5, table 3). Cells positive for PAS alone were seen much more frequently than Alcian blue-positive cells, and the proportion of these mucous cells appeared to be similar in both groups. There was no mucous cell metaplasia in the terminal bronchiole in either group. CGRP-positive airway epithelial cells were present as NEB (fig. 6), and no solitary CGRP-positive airway endocrine cells were observed. The numbers of CGRPpositive NEBs and CGRP-positive endocrine cells were significantly larger in the terminal bronchiole in the exposed group than in the control group, but this was not the case in the lobar bronchus (table 4).
@ Fig. 4. Nuclear staining is seen in a columar cell (arrow) of the bronchus in a control rat lung. Note that cytoplasmic granules are rarely seen. BrdU immunostain, x600. Fig. 5. Many mucous cells are seen in the lobar bronchus of an ozone-exposed rat. Alcian blue-PAS stain, x600. Fig. 6. CGRP-immunoreactive neuroepithelial body in the terminal bronchiole of an ozone-exposed rat. CGRP immunostain, x600. 4
Exp Toxic Pathol46 (1994) 1
Discussion In the present study, we have shown the morphologic changes that occurred in the rat airway epithelium after long-term exposure to a high ambient ozone concentration; we focused on changes related to cellular injury,
Table 4. Numbers of CGRP-positive neuroepithelial bodies (NEBs) and airway endocrine cells (average ± SD/cm of airway). Terminal Bronchiolus
Bronchus Animal Group
NEB
Endocrine Cell
NEB
Endocrine Cell
Control Ozone-exposed
1.42 ± 2.00 1.11 ± 1.32
10.24 ± 14.51 5.94± 7.02
0.48 ± 0.62* 1.29 ± 0.90*
3.35 ± 4.83** 7.35 ± 5.05**
*, ** significantly different, at p < 0.05, respectively.
epithelial pathological responses and tumorigenesis related to ozone exposure. Numbers of lipofuscin granules in the rat lung increase with age and their ultrastructure becomes more complex (17). Apart from ageing, increases in lipofuscin granules can be engendered by various noxious influences, such as starvation, wasting diseases, vitamin E deficiency and so on (11). In the present study, the number of brown pigmented granules, which were thought to be lipofuscin, increased in the ozone-exposed animals. This was assumed to be related to some injurious effect of long-term ozone exposure, although ozone exposure does not always affect the concentration of lipofuscin granules (7). To assess the injurious effects of prolonged exposure to ozone on the airway epithelium, we examined the BrdUlabeling indices for the lungs, since epithelial damage is followed by cell renewal (9, 10). No significant difference was seen between the control and exposed animals, and ozone tolerance was confirmed in the older animals. No difference in cell size between the two animal groups could be explained by the similar phenomenon. The airway epithelial cell population was changed by the long-term ozone exposure, and the numbers of mucous and endocrine cells were altered. As expected from previous studies (13, 16), we also found mucous cell hyperplasia in our study. Mucous cell hyperplasia is a wellknown feature in the airways of smokers (38), and has been induced experimentally by proteolytic enzmye treatment (3-5). The mechanisms by which proteases derived from neutrophils (3) and macrophages (5) cause mucous cell hyperplasia remain unknown, but this hyperplasia would seem to be reasonable, since mucous products are thought to protect the epithelium from oxidant damage (6). In ozone-induced mucous cell hyperplasia, inflammatory reactions evoked by ozone exposure could contribute to the condition through the release of proteases. We found that pulmonary endocrine cells, particularly CGRP-positive cells, were increased in the small airways in the ozone-exposed animals, but that there were no increases of endocrine cells in the lobar bronchus. Such regional differences in the reaction of endocrine cells to a toxic gas have been reported in the lungs of rats exposed to nitrogen dioxide, but in that case the increases of these
cells were seen in the larger airways (21). The airway endocrine cell is thought to function as a recepto-effector cell (23), but the effects of noxious stimuli transmitted through the airways on this cell type, and its reactions, have not been well documented. Further investigations are needed. Despite the vast literature that documents the effects of both acute and chronic ozone exposure in both humans and animals, few studies have addressed on the possible relationship between respiratory tumorigenesis and ozone exposure. In vivo studies have revealed that higher concentrations of ozone increase the number of pulmonary adenomas in mice (14, 22), and modify the incidence of chemically-induced lung tumors (14, 22). In an in vitro study using rat tracheal epithelium, ozone was shown to enhance the incidence of preneoplastic transformation of the epithelium (37). Although the experimental scale of the present study was too small to lead to statistical analysis, our result of one adenoma in 15 ozone-exposed rats, since the incidence of spontaneous lung adenomas in rats is about 1 % (31), might support the idea that ozone is potentially tumorigenic. Acknowledgements: The authors thank Mr. M. IKEDA, Mr. H. MITSUI and Mr. T. SUZUKI for their excellent technical assistance.
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