of asbestos exposure in man is the frequent presence in the lungs of numerous 'asbes- tos bodies'; these are elongated, irregularly shaped, golden-coloured.
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Laboratory Animals (1986) 20,132-137.
Asbestos bodies in rat lung following intratracheal instillation of chrysotile P. T. C. HARRISON & J. C. HEATH Strange ways Research Laboratory, Worts Causeway, Cambridge CBI 4RN, United Kingdom Summary Asbestos bodies in rat lung have rarely been reported, and just one previous record of their formation from chrysotile fibres in the rat is known. This paper illustrates the production of numerous true asbestos bodies in the lungs of Lister hooded rats after a single small intratracheal dose of lightly milled chrysotile. The demonstration of these bodies is particularly useful because uncoated chrysotile fibres in lung tissue cannot normally be visualized by light microscopy; the detection of asbestos bodies, and therefore of asbestos fibres, provides a means of directly relating asbestos exposure to observed tissue lesions. The asbestos bodies detected in the present study were nearly always associated with small pulmonary fibrotic lesions. The bodies ranged in length from 5 to 80 11m and were up to 5 11m in diameter. Small spheres, rods and bodies the shape of a comma were common; larger beaded structures were somewhat rarer. The bodies were visible in
Similar bodies have been seen in the lungs of guinea pigs and hamsters following inhalation or intratracheal instillation of any of the main commercial varieties of asbestos (Gross & De Treville, 1967; Suzuki & Churg, 1969a, b; Botham & Holt, 1971, 1972a, b). Only very rarely, however, have they been identified in rat lung, and in laboratory animals, as in humans, asbestos bodies are formed much less readily from chrysotite than they are from amphibole fibres (Churg & Warnock, 1980; Holmes, Morgan & Davison, 1983). Thus Holmes and Morgan (1980) and Botham and Holt (1972b), for example, have described 'rare and atypical' asbestos bodies in rat lung following inhalation of anthophyllite and crocidolite, but with regard to experiments using chrysotile there is only one known report of asbestos bodies being found in rat lung (Vorwald, Durkan & Pratt, 1951). Holt, Mills and Young (1964) found no bodies after long-term inhalation of chrysotile dust by rats, and similarly
tissue sections stained routinely with modified Azan
none was found by Wagner et af. (1974) in a series of
stain and with haematoxylin and eosin, but their detection and localization was enhanced by the use of Perls' Prussian blue stain in association with a pale eosin counterstain.
detailed experiments on the effects of inhalation of all four main asbestos types (chrysotile, crocidolite, amosite and anthophyllite). Gross and De Treville (1967), using the intratracheal method to administer chrysotile (Canadian) fibres, induced asbestos body formation in several species of laboratory animal but again not in rats. This paper, we believe, gives the first full lightmicroscope description of asbestos bodies formed from chrysotile fibres in rat lung, and as far as we know is the first record of asbestos body formation in rats following intratracheal instillation of chrysotile asbestos.
Keywords: Rats; Lung; Asbestos In experimental studies on the biological effects of asbestos fibres in laboratory animals, it is valuable to be able to relate observed pathological changes to lesions seen in human cases of asbestos exposure. In this respect the production of focal and interstitial fibrosis, pleural plaques and tumours of the lung and mesothelium is particularly significant. A further characteristic of asbestos exposure in man is the frequent presence in the lungs of numerous 'asbestos bodies'; these are elongated, irregularly shaped, golden-coloured bodies which stain positively for iron and have an asbestos fibre core. In humans they occur in various sizes, between 10 and 60 11m in length and 0·5-25 ~lm in diameter, and in a variety of shapes (Pooley, 1972). Characteristically they resemble a string of oval beads, often with a bulbous end. Received 30 September 1985. Accepted /1 November 1985.
Materials and methods Observations recorded in this paper represent preliminary findings for a series of experiments undertaken in this laboratory involving over 2000 animals in which potential carcinogenic effects of chrysotile asbestos fibres, alone and in combination with particulate metals, are under investigation. Lister hooded rats of the Strange ways strain were used in the experiments. This strain of rat was used because all the pilot studies on metal-induced tumours had been done in this laboratory using this
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strain. The animals each received a single 2 mg dose of IUCC standard reference chrysotile 'B' (Canadian)· suspended in 0-4 ml of sterile Tyrode's solution delivered by intratracheal instillation according to the method of Saffiotli, Cefis and Kolb (1968). Before administration the asbestos was milled for 3 min, at high speed, in a 'Spex' agate ball mill (Glen Creston Ltd, Stanmore, Middlesex, UK). The rats, which were approximately 10 weeks old at the time of treatment, were anaesthetized by intraperitoneal injection of Brietal Sodium (methohexitone sodium: Lilly, Basingstoke, Hants,
UK). Euthanasia was by ether inhalation. The age at death of the rats reported on ranged from 18 to 36 months. Full post mortem examinations were performed and pieces of lung tissue were excised and fixed in Carnoy's fluid. Paraffin sections of 6 ~tm thickness were cut and stained with haematoxylin and eosin (H&E), Heidenhain's Azan substitute stain (azocarmine replaced by carminic acid) or Perls' Prussian blue. Slides were examined and photographed using a Leitz Orthoplan photomicroscope.
Results Asbestos bodies described here were first seen during high power (c. 400x) microscopic examination of some interesting lung sections with unusual areas of pulmonary fibrosis. These fibrotic areas either were focal and specifically associated with bronchiolar walls (often with accumulated macrophages and occasionally involving a local proliferation of bronchiolar or alveolar duct smooth muscle cells) or were more diffusely structured and parenchymal, resembling small chronic inflammatory lesions. Similar lesions have been described in rats exposed to chrysotile in previous experimental studies of asbestosis (Holt et al., 1964; Gross & De Treville, 1967; Lemaire et al., 1985). They were similar also to the small-airway lesions in asbestosexposed human lungs described by Wright and Churg (1984). In the present investigation it was found that whenever asbestos bodies were seen (denoting the presence of otherwise invisible chrysotile fibres) they were nearly always associated with such areas of fibrotic change. . Figs 1-3 are low to medium power photomicrographs showing some of the typical fibrotic lesions described above. Fig. 2 is an example of bronchiolitis fibrosa obliterans, a lesion which has already been associated with experimental asbestos exposure (Cadieux, Masse & Sirois, 1983; Lemaire et al., 1985). Figs 4 and 5 show free asbestos bodies in situ 'Provided by Dr J. C. Wagner of the MRC Pneumoconiosis Unit, Penarth, UK.
Ng. I. Several pulmonary Ilbrotic loci, largely associated with bronchiolar walls. (H&E; 60X) in these areas (plump fibrocytes, abundant collagen and occasional macrophages can be seen), and Fig. 6 shows similar bodies stained with Perls' Prussian blue for haemosiderin. Prussian blue with a light eosin counterstain is recommended as an excellent method for detecting and localizing asbestos bodies in lung sections. The bodies in the photomicrographs range in length from 5 to 80 J.lm and are up to 5 J.lm in diameter (most are less than 20 J.lm long). They are generally similar in size and shape to bodies formed from chrysotile fibres in hamster lung (Suzuki & Churg, 1969a) and guineapig lung (Botham & Holt, 1971). They are also comparable in size with human asbestos bodies (Pooley, 1972) but are mostly at the smaller end of the size range. Single small round globules, simple rods, or bodies the shape of a comma are common; beaded structures and long bodies are somewhat rarer. The preponderance of simple forms should be stressed; at first sight these may be mistaken for plant spores or other adventitious inclusions or may be overlooked altogether. The asbestos bodies observed in this study were sometimes fully or partially engulfed by phagocytes
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Fig. 2. Bronchiolar fibrotic polypoid ingrowth (hronchiolitis fibrosa ohliterans) with dense collagen and a central area of macrophage accumulation with asbestos bodies. (Awn Slain; 120X)
but more usually appeared to be entirely extracellular or enmeshed in collagen.
Discussion The prevalence of asbestos bodies in human lungs as indicative of previous asbestos exposure is well documented; indeed, their abundance may be used as a rough quantitative measure of the degree of environmental or occupational asbestos exposure (Churg & Warnock, 1977). The paucity of asbestos bodies found to date in experimental rat studies, therefore. would seem to detract somewhat from the usefulness of the rat in investigations of the biological effects of asbestos since it suggests that in this respect the rat does not mimic the human response. It also, perhaps, implies a different pathological reaction to the fibres. Further, lack of asbestos bodies makes the quantification and localization of asbestos fibres in the lung more difficult; this is especially true of chryostile fibres which are virtually
Harrison
& Heath
Fig. 3. Focal fibrotic lesion which contains a profusion flf small asbestos bodies. (H&E; 152x)
impossible to see in lung tissue using light microscopy. The mechanisms involved in the formation of asbestos bodies are well known (e.g. Davis, 1964; Holt & Young, 1967; Suzuki & Churg, 1969a, b; Pooley, 1972). Various explanations for the rarity of such bodies in the rat (and other species), as revealed by Vorwald, Durken and Pratt (1951), have been proposed, notably by Botham and Holt (1972a) who suggested that asbestos fibres and the ferruginous material required for the production of asbestos bodies are cleared more readily from rat lung than, for example, from guineapig lung. II is possible that the type of chrysotile used and the method of administration of the asbestos are important here. Also, there may be real and marked differences in the mean sizes and the shapes of bodies detected in different species so that in comparative studies smaller more insignificant bodies such as the spherical ones observed in the present study are inadvertently overlooked or underestimated. Even if no fundamental importance is attached to
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bodies in rat lung
Fig. 4. A beaded asbestos body with clubbed ends, enmeshed in dense collagen. (Azan stain; 760x with use of a lOOx oil immersion objective and a large aperture (NA )·25) oil condenser.)
Fig. 6. Focal profusion of asbestos bodies of various shapes. (Perls' stain, no counterstain.) the presence or absence of asbestos bodies per se, the demonstration of these bodies is certainly useful in indicating the whereabouts in the lung of areas of
chrysotile fibre deposition. With this in mind the following suggestions success of the present bodies.
Fig. 5. A long beaded asbestos body with obvious fibre core. (H&E; 760x with use of a IOOx oil immersion objective and a large aperture (NA ),25) oil condenser.)
are made concerning the study in detecting asbestos
(1) The use of an intratracheal method for fibre administration rather than an inhalation technique may be important. (2) The use of Perls' Prussian blue stain with an eosin counterstain considerably aids the detection and localization of the asbestos bodies (which are frequently associated with unusual focal fibrotic lesions). (3) An overall microscope magnification factor of at least 250x is normally required to detect the bodies which are mostly of simple form and very small (less than 5 !lm x 20 ~lm). (4) It is conceivable that differences exist in the responses of different strains and varieties of rat; if this is so then the use of Lister hooded rats is recommended.
Harrison
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A further interesting observation is that asbestos bodies and associated fibrotic changes have been seen in rats at various times after treatment - up to 36 months in the present study after a single intratracheal dose. To conclude, this study has demonstrated, for the first time, asbestos body formation in rat lung after intratracheal instillation of chrysotile. It is of considerable advantage to be able, using light microscopy, to deduce the whereabouts in the lung of instilled chrysotile fibres, especially when studying subsequent pulmonary lesions; the formation and subsequent observation of asbestos bodies enables this to be done.
& Heath
Acknowledgements This work forms part of a programme of research commissioned by !JIe Health and Safety Executive (HSE). Special thanks are due to Dr D. Haines, retired DeputY'Director of Pathology and Research, HSE, for help, encouragement and advice in the early stages of the project. The invaluable assistance of Mr D. Rogers (technician), Mr W. Stebbings and colleagues (animal facilities), Mrs M. Farmer (histology) and Mr C. Green (photographic processing and printing) is gratefully recorded.
References Botham, S. K. & Holt, P. F. (1971). Development of asbestos bodies on amosite. ehrysotite and eroeidolite fibres in guinea-pig lungs. Journal of Pathology 105, 159-167: Botham, S. K. & Holt, P. F. (1972a). Asbestos-body formation in the lungs of rats and guinea-pigs after inhalation of anthophyllite. Journal of Pathology ]07, 245-252. Botham, S. K. & Holt, P. F. (1972b). The effects of inhaled crocidolites from Transvaal and North-West Cape mines on the lungs of rats and guinea pigs. British Journal of Experimental Pathology 53, 612-620. Cadieux, A., Masse, S. & Sirois, P. (1983). Effect of asbestos on the metabolism of vasoactive substances in isolated perfused guinea pig lungs. Environmental Health Perspectives 51, 287-291. Churg, A. & Warnock, M. L. (1977). Correlation of quantitative asbestos body counts and occupation in urban patients. Archives of Pathology and Laboratory Medicine 10], 629-634. Churg, A. & Warnock, M. L. (1980). Asbestos fibres in the general population. American Review of Respiratory Diseases 122, 669-678. Davis, J. M. G. (1964). The ultrastructure of asbestos bodies from guinea-pig lungs. British Journal of Experimental Pathology 45, 634-641. Gross, P. & De Treville, R. T. P. (1967). Experimental asbestosis. Studies on the progressiveness of the pulmonary fibrosis caused by chrysotitc dust. Archives of Environmental Health ]6, 638-649. Holmes, A. & Morgan, A. (1980). Clearance of anthophyllite fibres from the rat lung and the formation of asbestos bodies. Environmental Research 22, ]~21. Holmes, A., Morgan, A. & Davison, W. (1983). Forma-
tion of pseudo-asbestos bodies on sized glass fibres in the hamster lung. Annals of Occupational Hygiene 27.
301-313.
.
Holt, P. F .• Mills, J. & Young, D. K. (1964). The early effects of chrysotile asbestos dust on the rat lung. Journal of Pathology and Bacteriology 87, 15-23. Holt, P. F. & Young, D. K, (1967). The mechanism of production of asbestos bodies from anthophyllite fibres. Journal of Pathology and Bacteriology 93, 696-699. Lemaire, I., Nadeau, D., Dunnigan, J. & Masse, S. (1985). An assessment of the fibrogenic potential of very short 4T30 chrysolite by intratracheal instillation in rats. Environmental Research 36, 314-326. Pooley, F. D. (1972). Asbestos bodies, their formation, composition and character. Environmental Research 5, 363-379. Saffiotti, U., Cefis, F. & Kolb, L. (1968). A method for the experimental induction of bronchogenic carcinoma. Cancer Research 28, 104-124. Suzuki,Y. & Churg, J. (1969a). Formation of the asbestos body. A comparative study with three types of asbestos. Environmental Research 3, 107-118. Suzuki, Y. & Churg, J. (1969b). Structure and development of the asbestos body. American Journal of Pathology 55, 79-91. Vorwald, A. J., Durkan, T. M. & Pratt, P. C. (1951). Experimental studies of asbestosis. Archives of Industrial Hygiene and Occupational Medicine 3, 1-43. Wagner, J. C., Berry, G., Skidmore, J. W. & Timbrell, V. (1974). The effects of the inhalation of asbestos in rats. British Journal of Cancer 29, 252-269. Wright, J. L. & Churg, A. (1984). Morphology of small-airway lesions in patients with asbestos exposure. Human Pathology ]5, 68-74.
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in der Rattenlunge als Foige der intratrachealen
Instillation von Chrysotil
P. T. C. HARRISON &J. C. HEATH
Zusammenfassung Ober Asbestk6rperchen in der Rattenlunge ist selten berichtet worden, und es ist nur ein friiherer Berieht iiber ihre Bildung aus Chrysotilfasern in der Ratte bekannt. In diesem Papier wird die Produktion von zahlreiehen eehten Asbestk6rperchen in den Lungen von mit einer ListerHaube bedeck ten Ratten nach einer einzigcn klcincn intratrachealen Dosis von leicht zermahlenem Chrysotil erlautert. Der Nachweis dieser K6rperchen ist besonders niitzlieh. weil unbeschichtete Chrysotilfasern im Lungengewebe normalerweise nicht mit dem Lichtmikroskop sichtbar gemacht werden k6nnen; der Nachweis von Asbestk6rperchen und somit von Asbestfasern liefert eine M6glichkeit, die Asbestmenge, der die Ratte ausgesetzt worden ist, direkt mit den beobachteten Gcwebeliisionen
in Zusammcnhang zu bringen. Die mit der vorlicgcnden Untersuchung nachgewiesenen Asbestk6rperehen waren fast immer mit kleinen fibr6sen Pulmonalliisionen assoziiert. Die Lange der K6rperchen lag zwischen 5 und RO flm und ihr Durchmesser betrug bis zu 5 ftIll. Es waren hiiufig klcine Kugeln, stiibchen und kommaf6rmigc K6rpcrchen anzutreffen; gr6Bere perlenschnurf6rmige Strukturcn waren etwas seltener. Die K6rperchen waren in routinemiiBig mit einer modifizierten Azanftirbung und mit Hamotoxylin und Eosin gefarbten Gewebeschnitten zu erkcnnen, ihr Nachweis und ihrc Lokalisicrung wurdcn jedoch durch die Anwendung der PreuBischblaufiirbung nach Perl in Vcrbindung mit einer blassen Gegcnfiirbung mit Eosin verbessert.
