Intrapulmonary distribution of inhaled chrysotile and crocidolite asbestos

Br. 1. exp. Path. (I984) 65, 467-484

Intrapulmonary distribution of inhaled chrysotile and crocidolite asbestos: ultrastructural features Y. Oghiso, E. Kagan and A.R. Brody* Department of Pathology, Georgetown University, Washington DC and *Laboratory ofPulmonary Function and Toxicology, National Institute of Environmental Health Sciences, North Carolina, USA

Received for publication

I

3

October I983

Summary. Although all commercial types of asbestos can cause pulmonary fibrosis, little is known about ultrastructural differences in the evolution of pulmonary lesions induced by amphiboles and serpentines. The present study was designed to compare the histological and ultrastructural effects produced by chronic inhalation of either crocidolite (amphibole) or chrysotile (serpentine) asbestos in the rat. Animals, exposed by intermittent inhalation for 3 months, were killed after 2 to i6 months. When inhaled, both types of asbestos caused thickened alveolar duct bifurcations associated with macrophage aggregates. Crocidolite inhalation also produced subpleural collections of alveolar macrophages and lymphocytes. Electron microscopy revealed some similarities, but also distinct differences, in the pulmonary distribution of inhaled chrysotile and crocidolite. Whereas both asbestos varieties were identified within the pulmonary interstitium, only crocidolite was detected inside alveolar macrophages. Chrysotile fibres were seen infrequently within the vascular compartment. Microcalcifications were noted after chrysotile inhalation, but were never observed following crocidolite exposure. Both asbestos types induced slight pulmonary fibrosis. These findings indicate that crocidolite and chrysotile produce different pathogenetic features, although both are fibrogenic. Keywords: asbestos, asbestosis, lung ultrastructure, crocidolite, chrysotile

It has long been recognized that asbestos inhalation can induce pleural and diffuse interstitial pulmonary fibrosis (Becklake I976; Selikoff & Lee I978). There is considerable evidence, both from clinical sources and from experimental studies, that all commercial types of asbestos can evoke pulmonary fibrosis (Holt et al. I965; Suzuki & Churg I970; Botham & Holt I972; Wagner et al. I974; Craighead & Mossman I982). Never-

theless, it is not clear whether all asbestos types are equally fibrogenic in vivo (Becklake I982). One study has suggested that amphiboles may be more fibrogenic than chrysotile to humans (Regan et al. I97I). In another study, which involved experimental asbestos inhalation in the rat, greater fibrogenicity was shown for anthophyllite, when compared with other asbestos fibre types (Wagner et al. I974). These findings have

Correspondence: E. Kagan, Department of Pathology, Georgetown University Medical Center, 3900 Reservoir Road NW, Washington DC 20007, USA.

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Y. Oghiso et a]. 468 not, however, been repeated and the issue of ville Corporation, Jeffrey Mine, Quebec, a fibre gradient for asbestosis requires further Canada. elucidation. Serpentine and amphibole varieties of asbestos differ markedly in their physical and Exposure conditions. Inhalation exposures aerodynamic properties (Timbrell 1973). were performed under contractual agreePrevious studies of short-term chrysotile ment by ToxiGenics, Decatur, IL. Three inhalation in the rat have demonstrated the groups of 4-week-old weanling animals initial deposition of chrysotile fibres at alveo- (mean body weight; I 24 g) were housed and lar duct bifurcations (Brody et al. I 98 I), with exposed in 8.o m3 capacity stainless steel and subsequent translocation of fibres into the glass inhalation chambers. One group of rats interstitium (Brody & Hill I982). The chry- (crocidolite-exposed group) received a timesotile caused the formation of characteristic weighted average concentration of II.2 microcalcifications, in association with in- mg/m3 of crocidolite asbestos. A second terstitial fibres, within i month after a single group of rats (chrysotile-exposed group) i-h inhalational challenge (Brody & Hill received a time-weighted average concentraI982). It is not known whether chronic tion of I0.7 mg/m3 of chrysotile asbestos. asbestos exposure is associated with different The asbestos fibres had been previously fibre distribution patterns from those seen characterized (Rendall I980; Brody et al. after acute asbestos inhalation. Although I98I). A third group of rats (sham-exposed some studies have shown differences in the group) was employed as a control group. clearance and retention patterns of inhaled These animals were exposed to clean air only amphiboles and chrysotile asbestos in the rat and were used as a reference group for (Morgan et al. I 9 77; Middleton et al. I 9 79), histological comparisons by light microscomparisons of the intrapulmonary distribu- copy. The three groups of animals each tion of different types of inhaled asbestos received an intermittent inhalation exposure have not been made at the ultrastructural regimen of 6 h a day, 5 days a week, for 9I level. consecutive days. Details of aerosol geneThe present study was undertaken to ration and monitoring have been described address the question of fibre distribution in a previously (Kagan et al. I983a). rat model of chronic exposure to crocidolite and chrysotile asbestos. Both histological and ultrastructural features of the lungs Animal killings. Similar numbers of rats from were compared after 3 months of inhala- the three experimental groups were killed tional exposure. simultaneously at selected times. For light microscopy studies, rats were killed from 2 to I6 months after the cessation of inhalation Materials and methods exposure. For ultrastructural studies, animals were killed at 8 months and I6 months Animals. Male inbred albino Fischer-344 after terminating the exposure regimen. The strain rats were obtained from Charles River lungs of I2 animals from each group were Breeding Laboratories, Kingston, N.Y. studied by conventional light microscopy. Six animals from each asbestos-exposed Asbestos samples. A UICC standard reference group were killed for transmission electron preparation of crocidolite asbestos was microscopy studies. The rats were all exsanobtained from Mr R.E.G. Rendall, National guinated under anaesthesia by intramuscuCentre for Occupational Health, Johannes- lar injection of ketamine hydrochloride (Brisburg, South Africa. Chrysotile asbestos (Lot tol Laboratories, Syracuse, NY; 50 mg/kg AX 3 73 8-M) was obtained from Johns-Manbody weight).

Lung ultrastructure after asbestos exposure 469 Tissue preparation for light microscopy. The Assessment of asbestos fibre distribution. In lungs were inflation-fixed in situ, by the order to obtain a semi-quantitative assessintratracheal instillation of approximately ment of the respective distribution patterns I0 ml of io% neutral buffered formalin or after crocidolite and chrysotile inhalation, Bouin's fixative. The fixative was introduced 20 microgrids from the lungs of six crocidoliat a pressure of 20 cm through a tracheal te-exposed animals and 20 microgrids from cannula without opening the chest. Tissues the lungs of six chrysotile-exposed rats were were preserved in formalin for i week or in examined similarly. Randomization of the Bouin's fixative for 3 h. observations was achieved, by systematiFor histological examinations of the lungs, cally inspecting all of the tissue contained in sagittal sections of each lobe (right -anterior, each microgrid. A scoring system was right middle, right posterior, accessory and devised, to evaluate the numbers of asbestos left lobe) were dissected with sharp razor fibres in four pulmonary anatomical locablades. Tissue samples were all embedded in tions: alveolar macrophages, epithelium, inparaffin and routinely processed for histolo- terstitium and vasculature. Scoring at each gical analysis. Five-micron-thick sections of these locations was as follows: i =no asbestos fibres observed; 2 = I-I0 asbestos were prepared, and stained with haematoxylin and eosin (H & E), Prussian blue, Masson's fibres observed; 3=more than I0 asbestos fibres observed. If the same fibre was trichrome and reticulin stains. observed in more than one location, it was Tissue preparation for transmission electron assigned a separate score for each location. microscopy. The lungs were prepared for transmission electron microscopy by vascu- Results lar perfusion-fixation, as previously described (Brody et al. I98I). After perfusion, the lungs were removed en bloc and im- Macroscopical appearances of the lungs mersed in 2.5% fresh glutaraldehyde fixative No abnormalities were noted on gross inat 40 C for 24 h. Approximately two or three spection of the lungs of chrysotile- and tissue blocks of about 3 x 3 x I mm were sham-exposed animals. In contrast, multiple dissected from each lobe (right anterior, right discrete, darkly pigmented macules were middle, right posterior, accessory and left consistently observed on the pleural aspects lobe) with sharp razor blades, and washed of the lungs of crocidolite-exposed rats several times with phosphate buffer. After throughout the entire period of study. These post-fixation with I% OS04 in 0.2 M S-colli- pigmented foci had a bilateral distribution, dine buffer, tissue blocks were dehydrated affecting all the lobes of the lungs with through ethanol and propylene oxide, and exception of the accessory lobe. embedded in Epon 8I2. Thick (i ,um) sections were cut with glass knives, and mounted on glass slides for staining with Light microscopical appearances of the lungs 0. I% toluidine blue in borax. These sections Thickening of alveolar duct bifurcations has were prepared to select sites of terminal been described after chrysotile inhalation bronchiolar and alveolar duct bifurcations. (Brody et al. I98I), due to a significant These regions were trimmed and then thin- increase in the proportion of macrophages at sectioned by diamond-knife ultramicrotomy these sites (Warheit et al. I984). In the (Ultracut, American Optical Corporation, present study, similar thickening of alveolar Buffalo, NY). Ultra-thin (6o nm) sections duct bifurcations was a striking feature were then stained with uranyl acetate and noted in the lungs of both groups of asbestoslead citrate, before viewing under a JEOL exposed rats (Fig. i), but not in shamiooS transmission electron microscope. exposed rats. Occasionally, focal intra-alveo-

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Fig. i. Photomicrographs of the lung from a chrysotile-exposed rat. (a) An alveolar duct bifurcation shows slight thickening (arrow). H & E, x 125. (b) Higher magnification of Fig. ia. Accumulation of macrophages is seen at the thickened bifurcation. H & E, x 300.

Lung ultrastructure after asbestos exposure

47I

Fig. 2. Photomicrograph ofthe lung from a chrysotile-exposed rat, showing an intra-alveolar collection of macrophages and multinucleated giant cells. H & E, x 300.

lar macrophage clusters were evident at sites distal to alveolar duct bifurcations (Fig. 2). These macrophage accumulations were seen as early as 2 months after the cessation of asbestos inhalation and persisted for at least I6 months after exposure had been terminated. Multinucleated giant cells were also evident in these regions and were observed in both groups of asbestos-exposed animals. Multinucleated forms were not observed within the lungs of sham-exposed animals. Asbestos fibres were never visualized by light microscopy within the alveolar macrophages of rats exposed to chrysotile. On the other hand, crocidolite fibres were readily detectable in many of the alveolar macrophages from crocidolite-exposed rats (Fig. 3). Surprisingly, macrophages laden with phagocytosed crocidolite fibres were seen as long as i 6 months after removal from asbestos challenge. Although typical ferruginous bodies were not observed after either crocidolite or chrysotile exposure,

occasional crocidolite fibres were coated with haemosiderin. A feature noted in crocidolite-exposed animals, but not in chrysotile-exposed animals, was the accumulation of subpleural aggregates of alveolar macrophages (Fig. 3) with occasional multinucleated giant cell forms. Lymphocytes were also commonly observed in these lesions. These cellular aggregates were only noted in areas corresponding to the darkly pigmented areas seen on gross inspection of the lungs. Numerous crocidolite fibres were found within the alveolar macrophages and giant cells in these regions (Fig. 3). All sections were stained within reticulin and Masson's trichrome stains in order to evaluate the presence of fibrosis. When comparisons of asbestos-exposed animals were made with the lungs of sham-exposed rats, only a minimal increase in collagen (trichrome-positivity) was noted in the thickened regions at alveolar duct bifurcations. These

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Fig. 3. Photomicrograph of the lung from a crocidolite-exposed rat. A subpleural lesion is seen, composed of macrophages and lymphocytes. Many macrophages are seen to contain crocidolite fibres. H & E, x 300.

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Fig. 4. Electron micrographs of the lungs from chrysotile-exposed rats. (a) Rectangular structures (arrows) are seen in a vacuolated alveolar macrophage (AM). AS, Air space; II, type II epithelial cell. Bar= i um, x io 6oo. (b) Rectangular structures are detected within the interstitial cell (IC), which also contains haemosiderin granules in lysosomal vacuoles (arrows). Co, Collagen. Bar= I ym, X 21 200.

Fig. 5. Electron micrographs of the lungs from chrysotile-exposed rats. (a) Segmented chrysotile asbestos fibres (arrowheads) are seen within an interstitial cell (IC) and within collagen matrix (Co). Bar = i Pm, X 21 200. (b) An asbestos fibre (arrow) is seen traversing through interstitial collagen (Co) as well as a type II cell (II) and the basement membrane (Bm). C, Capillary; En, endothelial cell. Bar= i pm, X 2I 200.


475

Fig. 6. Electron micrograph of the lung from a chrysotile-exposed rat. An annular form of interstitial microcalcification is seen (upper right). Segmented chrysotile fibres (arrowheads) are also observed in close apposition to the microcalcification. Co, Collagen. Bar = i pm. X 2I 200.

areas of fibrosis first appeared 8 months after the cessation of asbestos exposure and progressed slightly over the ensuing 8 months of the study. The subpleural lesions of crocidolite-exposed rats showed some increase in reticulin content 8 months after removal from asbestos challenge. Little fibrosis was noted in these areas at the termination of the study. By light microscopy, both types of asbestos inhalation were associated with minimal generalized increase in interstitial fibrous tissue.

Electron microscopical appearances of the lungs Chrysotile-exposed animals. Chrysotile asbestos fibres were never observed within alveolar macrophages. Fibres were, however, occasionally seen traversing epithelial cells (Fig. 5b). A striking finding in chrysotile-exposed

animals, was the frequent presence of elongated, rectangular, membrane-bound cytoplasmic inclusions within alveolar macrophages (Fig. 4a). These inclusions measured approximately 0.4 to 6.6 pm in length and 0.03 to 0.2 um in diameter. Similar inclusions were noted within interstitial macrophages, but not within epithelial cells (Fig.

4b). On several occasions, chrysotile fibres were identified within the alveolar interstitium. In this location, these asbestos fibres could be seen within interstitial macrophages and collagen (Fig. 5a). Chrysotile fibres traversed several anatomical regions, passing from epithelial cells, through the basement membrane and into the alveolar interstitium (Fig. 5b). The interstitial distribution of chrysotile was noted throughout the entire observation period of the study and

-rO. Fig. 7. Electron micrographs of the lungs from chrysotile-exposed rats. (a) Long asbestos fibres (arrows) are seen traversing through collagen (Co) as well as basement membrane (Bm), a capillary endothelial cell (En) and a circulating blood monocyte (Mo). Bar = i pm, x io 6oo. (b) Fragmented chrysotile fibres (arrowheads) are seen in the basement membrane of a capillary (C). A blood monocyte (Mo) contains an ingested microcalcification (arrow). AS, Air space; II, type II cell. Bar= i gm, x 8ooo.

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Fig. 8. Electron micrographs of the lungs from crocidolite-exposed rats. (a) Alveolar macrophages (AM), containing phagocytosed crocidolite asbestos fibres (arrowheads), form an aggregate within the air space (AS). Bar= 5 um, X 2250. (b) Intra-alveolar aggregates of alveolar macrophages contain numerous phagocytosed crocidolite fibres. Bar = 2 pm, x 4250.

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Fig. 9. Electron micrograph of the lung from a crocidolite-exposed rat. Severely vacuolated alveolar macrophages are seen within the alveoli. One macrophage has an atrophic nucleus. C, Capillary. Bar= 5 pm, X2100.

was centered primarily in areas surrounding alveolar duct bifurcations. Characteristic interstitial microcalcifications were observed in tissue adjacent to alveolar duct bifurcations. These microcalcifications, which have been described previously (Brody & Hill I982), usually exhibited a concentric annular arrangement (Fig. 6). In some instances, the microcalcifications contained a central electron-lucent core which contained fragments of chrysotile fibres. Chrysotile fibres were frequently observed in close opposition to these microcalcifications within interstitial structures (Fig. 6). Microcalcifications were seen more frequently i 6 months after withdrawal from exposure than in animals killed after 8 months. Occasionally, chrysotile fibres penetrated endothelial basement membranes and cytoplasm (Fig. 7a). On one occasion, a chrysotile asbestos fibre was noted within

the cytoplasm of a circulating blood monocyte (Fig. 7a). Similarly, a microcalcification was seen within a blood monocyte (Fig. 7b).

Crocidolite-exposed animals. A striking feature seen after crocidolite inhalation was the frequent demonstration of numerous crocidolite fibres within alveolar macrophages (Fig. 8). Several alveolar macrophages from crocidolite-exposed animals also showed extreme degenerative cytoplasmic vacuolation (Fig. 9). The rectangular cytoplasmic inclusions observed in the macrophages from chrysotile-exposed rats were only infrequently noted in the lungs of crocidoliteexposed rats. Typical ferruginous bodies were not observed, although some alveolar macrophages contained crocidolite fibres embedded within electron-dense cytoplasmic inclusions (Fig. io). Energy dispersive

Lung ultrastructure after asbestos exposure . X ,t

479

Fig. io. Electron micrograph of the lung from a crocidolite-exposed rat. An alveolar macrophage (AM) has fragmented crocidolite asbestos fibres (arrows) embedded within electron-dense cytoplasmic inclusions. Bar= I pm, X 1 2 700.

X-ray spectrometric analysis of these inclusions showed clear peaks for iron (Fig. ii), suggesting the early development of intracellular ferruginous bodies. Crocidolite fibres were also observed within interstitial structures (Fig. I 2). When seen in this location, these fibres were located within the cytoplasm of interstitial cells or extracellularly. Microcalcifications were never observed following crocidolite exposure. Assessment of asbestos fibre distribution patterns in different pulmonary locations The respective distribution patterns of inhaled crocidolite and chrysotile within various intrapulmonary locations are summarized in Table i. The data shown were pooled

6-4 eV Fig. i i. Elemental analysis of an electron-dense cytoplasmic inclusion in the lung of a crocidoliteexposed rat. A characteristic Ka peak for iron is seen at 6.40 eV on the X-ray spectrum.

480

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Y. Oghiso et al.

i.

Fig. I2. Electron micrograph of the lung from a crocidolite-exposed rat. Crocidolite fibres (arrows) are occasionally seen within interstitial cells, fibroblasts (Fb) or collagen fibres (Co). Bar= i 4um, x 7500.

from animals killed 8 and i 6 months after asbestos exposure had ceased. Although there were some similarities in the anatomical distribution patterns of inhaled chrysotile and crocidolite, distinct differences were also noted. Thus, both asbestos varieties were sighted within the pulmonary interstitium and within pneumocytes, although crocidolite was observed somewhat more frequently within the epithelium. On the other hand, only crocidolite fibres were observed within alveolar macrophages. Approximately 6o% of these phagocytic cells had ingested crocidolite, and a considerable proportion (28%) contained more than IO crocidolite fibres per cell. Interestingly, asbestos fibres were seen infrequently within the pulmonary microvasculature. This finding was noted exclusively after chrysotile inhalation.

Discussion It is generally believed that the lesions which result from amphibole and serpentine asbestos inhalation are morphologically indistinguishable (Becklake I976; Selikoff & Lee I978; Craighead et al. I982). This assumption has largely been based on early studies of asbestos inhalation in experimental animals and humans (Wagner et al. I974; Holt et al. I966). These observations, however, were based on histological analyses using light microscopy. To our knowledge, no ultrastructural studies have compared the effects of different types of asbestos inhalation in the lungs. The present study, which has employed both light microscopy and transmission electron microscopy, has demonstrated certain


48I

Table i. Distribution patterns of inhaled chrysotile and crocidolite asbestos in various intrapulmonary locations'

Scoreb (quantity of observed fibres) Chrysotile-exposed group Anatomical location Alveolar macrophages Epitheliumc

Crocidolite-exposed group

I

2

813 (ioo) 6ii

I7

0

(97)

(3)

(o)

3

I

2

3

0

0

451

(o)

333

(o)

(4I)

(3I)

308 (28)

6io

51

(92)

(8)

(o) 8 (I)

0

Interstitiumd

569

63

6

585

67

(Io) 9

(I)

(89)

(Io)

Vasculaturee

(89) 6ii

o

648

0

0

(98)

(2)

(o)

(ioo)

(o)

(o)

a The data were derived from the inspection of 20 microgrids for each asbestos-exposed group. The values for alveolar macrophages represent the total number of macrophages exhibiting a given score category. The values for the other anatomical locations represent the total number of instances any component of those locations was identified within a given score category. The figures in parentheses represent the respective frequencies (%) ofeach category at a given anatomical location. b Score of i =no asbestos fibres observed; 2 = I-I0 asbestos fibres observed; 3 = more than io asbestos fibres observed. c Includes type I and type II epithelial cells. d Includes interstitial cells, basement membrane and collagen. e Includes endothelium, capillary spaces and blood monocytes.

differences in the distribution of inhaled crocidolite and chrysotile asbestos in the lungs of rats. Crocidolite fibres were located almost exclusively within alveolar macrophages, and were still present in abundance in this location I6 months after asbestos inhalation had ceased. Occasionally, crocidolite fibres were observed penetrating epithelial cells or were found within interstitial structures. On the other hand, chrysotile fibres were never observed inside alveolar macrophages, but were most prevalent within the interstitium. There is no obvious explanation for our inability to detect chrysotile within these mononuclear phagocytes. It is, however, noteworthy that chrysotile fibres were also not detected within alveolar macrophages obtained by bronchoalveolar lavage from the same cohort of chrysotile-

exposed rats (Kagan et al. I 98 3a). Chrysotile asbestos has a tendency to progressively 'leach' out of tissues (Becklake I982). Since phagocytic uptake of fibres by alveolar macrophages is one of the earliest tissue responses to inhaled chrysotile (Brody et al. I98I), this 'leaching' effect may possibly explain why chrysotile fibres were not detected within macrophages long after exposure had ceased. Interestingly, similar numbers of crocidolite and chrysotile fibres were detected in the interstitial compartment (Table I). It is conceivable that the differences in these fibre distribution patterns reflect intrinsic physical and chemical dissimilarities between amphibole and serpentine asbestos. These differences may facilitate the preferential translocation of chrysotile into the interstitium.

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Characteristic rectangular inclusions were frequently noted within alveolar macrophages and interstitial cells after chrysotile inhalation. Identical structures were previously identified within macrophages obtained by bronchoalveolar lavage from chrysotile-exposed rats (Kagan et al. I98 3a). The exact nature and origin of these structures remains obscure. Necrotic alveolar macrophages were seen after both types of asbestos inhalation, although the changes were more severe after crocidolite exposure. This finding indicates that crocidolite can be cytotoxic to macrophages in vivo, despite its apparent poor cytotoxic potential after shortterm exposure in vitro (Bey & Harrington I97I; Miller 1978). It is of interest that microcalcifications were frequently identified in the lungs of chrysotile-exposed rats. Similar structures were previously described within i month after short-term chrysotile inhalation in the rat (Brody & Hill I982). Our findings, and those of Barry et al. (I983), indicate that these microcalcifications are also seen after chronic exposure to chrysotile, and that their frequency increases with time after withdrawal from asbestos exposure. As has been noted previously (Brody & Hill I982), the microcalcifications often contained a central chrysotile core. Extracellular chrysotile fibres were usually located in close juxtaposition to these structures. Microcalcifications were never observed in the lungs of crocidoliteexposed rats, but intracellular crocidolite fibres were surrounded by iron-rich electrondense material. The iron content was proven by X-ray energy spectrometry. This finding, plus the dense nature of the material, suggests that early ferruginous body formation is ongoing. In the lungs of two chrysotile-exposed rats, chrysotile and a microcalcification were observed within blood monocytes. Intracapillary chrysotile has been identified previously (Brody et al. I98 i). However, to our knowledge, this is the first report of asbestos or a microcalcification in a circulating mononuclear phagocyte. Ferruginous bodies

have been demonstrated in anatomical sites remote from the lungs in asbestos workers (Goodwin & Jagatic I970; Auerbach et al. I980). Although the mode of translocation of asbestos from the lungs to other organs has not been elucidated, it is conceivable that circulating plasma and monocytes may provide a means of transportation of fibres which have penetrated the pulmonary microvasculature. Thickening of alveolar duct bifurcations was a persistent feature of the lung histology seen in both groups of asbestos-exposed rats. These prominent bifurcations appeared to consist, in part, of macrophages in the interstitium and on the surface of the lesions. Studies of short-term particulate inhalation have shown that the preferential accumulation of alveolar macrophages tended to parallel the initial deposition patterns of the particulates at these sites (Brody & Roe I983; Warheit et al. I984). The persistent aggregations of these phagocytic cells, long after chronic asbestos exposure has ceased, are not readily explained. We have recently demonstrated that asbestos inhalation is associated with enhanced release in vitro of an alveolar macrophage-derived chemoattractant (Kagan et al. I983b). It is therefore conceivable that these macrophage aggregates result from the continual release of such chemotactic factors within the milieu of the lower respiratory tract. Subpleural collections of alveolar macrophages, replete with asbestos fibres, were a prominent feature in the lungs of crocidoliteexposed animals. These subpleural macrophage aggregates were not observed after chrysotile inhalation. In a study of inhaled radiolabelled asbestos, Morgan et al. (I977) showed that amphiboles (crocidolite and amosite) tended to preferentially translocate towards subpleural regions many months after inhalation had ceased. The mechanisms controlling the subpleural distribution of crocidolite are, however, not known. Although interstitial fibrosis developed after both chrysotile and crocidolite inhala-

Lung ultrastructure after asbestos exposure tion, this was minimal and essentially confined to regions contiguous with alveolar duct bifurcations. Other investigators, using either undefined or dissimilar strains of animals (Davis I963; Holt et al. I964; Wagner et al. I974), have shown that extensive asbestos-related fibrosis can be induced in the rat. These differences may reflect genetic variations in susceptibility to asbestosmediated fibrosis within different strains. In this regard, it is of interest that genetic factors have recently been shown to determine susceptibility to bleomycin-induced fibrosis in mice (Schrier et al. I983). This study has shown that differences exist between the intrapulmonary distribution of inhaled chrysotile and crocidolite asbestos fibres. These observations could have relevance to the pathogenesis of lung injury produced in humans by different commercial types of inhaled asbestos.

Acknowledgements This work was supported by US Environmental Protection Agency Grant R 8082520I. The authors thank Mr D. Gibson for preparing histological sections, and Ms E. Rusnock and Ms R.M. Rubino for preparing electron microscopical specimens and photomicrographs. The authors are also grateful to Mr R.E.G. Rendall for providing the crocidolite asbestos used in the study. References AUERBACH 0., CONSTON A.S., GARFINKEL L., PARs V.R., KASLOW H.D. & HAMMOND E.C. (I980) Presence of asbestos bodies in organs other than the lung. Chest 77, I 33-I 3 7. BARRY B.I., WONG K.C., BRODY A.R. & CRAPO J.D. (I983) The reaction of rat lungs to inhaled chrysotile asbestos following acute and subchronic exposures. Exp. Lung Res. 5, 1-22. BEcKLAE M.R. (1976) Asbestos-related diseases of the lung and other organs. Their epidemiology and implications for clinical practice. Am. Rev. resp. Dis. 114, I87-227. BECKLAXE M.R. (I982) Asbestos-related diseases of the lungs and pleura. Current clinical issues. Am. Rev. resp. Dis. 126, I87-I94.

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