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f' 1 asbestc Characterization of the Bronchoalveolar Cellular Response jn Experimental Asbestosis V Different Reactions Depending on the Fibrogenic Potential1-3 PLAINTIFF'S EXHIBIT m'/te1 * * bestos siructu auiocla ilepho? with a I siillatic IRMA LEMAIRE4 Introduction Pulmonary fibrosis results from the in ability of the lung to repair its altered structure. The mechanisms underlying the onset of fibrosis are unknown, but valuable insights into the pathogenesis of pulmonary fibrosis may be gained by studying animal models of the disease. Asbestos has been shown to cause pul monary fibrosis in humans (1, 2) and in laboratory animals (3-11). Using inhala tion or intratracheal instillation as the method of exposure, various investiga tors have demonstrated the histologic and physiologic similarities of asbestosinduced fibrosis in animals and asbesto sis in humans (1-11). Inhalation exposure provides a physiologic model system that allows for the natural clearance of in haled minerals, but it often requires months of experimentation and necessi tates elaborate and expensive facilities. On the other hand, intratracheal instil lation has some advantages because it is rapid, easily performed, and satisfacto ry in reproducing the histopathologic fea tures of fibrosis in various animal models (8-11). Depending on their concentration, length, and aerodynamic diameter (12), asbestos fibers may reach the alveoli and eventually the pulmonary interstitium (13, 14). Cells of the bronchoalveolar compartment are among the first to re act to this insult, and it is assumed that an orderly sequence of changes in their state and function must take place in or der to protect the lung integrity. During these responses cells may interact with others or with humoral components, leading to nonspecific reactions as well as to more specific events involved in the fibrotic process. Recently, in establishing a rat model of the disease, we have shown that as bestos fibers with similar chemical com position but different fiber length caused lung lesions that differed considerably SA-452 SUMMARY Analysis of bronchoalveolar cell types and structure was performed during the devtl. opment of asbestos-induced lung Injury In the rat. Animals received single Intratracheal rnjectio of one of the following: saline (control), U1CC chrysotlle B asbestos (5 mg), or very short 4T30chrysctfl, fibers (5 mg). Bronchoalveolar lavage (BAL) was performed at various Intervals after Instillation Analysis of BAL fluid showed significant Increase In Inflammatory cells In response to asbestot which persisted longer In animals treated with chrysotlle B. Presence of numerous mitotic figures In BAL fluid of treated animals suggests that macrophage replication may contribute in part to thlt response. Differential cellular analysis Indicated that after Injection of long chrysotlle fibers, whldt causes fibrotic lesions within 7 days, polymorphonuclear leukocytes (PMN) appear as early l3 1 In significant concentration (40%) In the bronchoalveolar compartment and persist through Dm 7 after treatment. From Day 7 to Day 21, multlnucleated cells (MGC) were found in lavage fluid (| to 8%). Most of these cells were blnucleated, and none had more than 3 nuclei. By contrast n. posure to very short chrysotlle fibers caused only a very transient influx of PMN on Day 1. By g,_ 7, there was a significant increase In MGC, which persisted through Day 21, at which time no flbntsls was apparent. Although most of these cells were blnucleated, many cells had 3 or more nucitl The giant cells were predominantly of the foreign body type, with MGC of the Langhans type ^ present. Our data demonstrate that In this rat model, lung alveolitis and fibrosis are assodstid with different bronchoalveolar cellular reactions and raise the question of a possible role forPItx and MGC In modulating macrophage function Bnd lung response to injury. AM REV RESPIR DIS 1985; 131.-144.14 In, Rats we of keta kg). Th. the asbc cd in a: of6 rat.imraira, U1CC:: chijso:: odialh lion. Ft: iniena.' is deser mlar a Animals biul (3C levered.. lungs we ml PBS tape fh: lo: 10 rr.. In PBS a co'smoc island 5 Cells wer. her, and blue cxcl both in nature and localization (10). In the present study, we compared the progressive bronchoalveolar cellular re sponse to these 2 asbestos samples with different fibrogenic potential. Our data demonstrate that bronchoalveolar cell al terations correlate with differences in lung injury, and further document some aspects of alveolar cell response. Methods Animals Male Wistar rats weighing 250 to 300 g were purchased from Charles River Canada Inc. (St-Constant, Quebec). These animals were derived from a pathogen-free colony, shipped behind filter barriers, and housed in isolated temperature-controlled quarters. They were given Purina Chow and water ad libitum and were used 1 wk later. No control animals or animals injected with asbestos fibers showed evidence of infection when evaluated by histochemical staining. Asbestos Fibers The U1CC standard sample of chrysotile B was obtained from the National Research In stitute for Occupational Diseases, Johannes burg, South Africa. None of the fibers pt ent in this sample was smaller than 2 pm,tf 21% were longer than 10 pm (15). Veryito 4T30 chrysotile fibers were isolated fromQ* bee 4T30 chrysotile, as described by Jolico* and coworkers (16). More than 98% ofti* fibers were smaller than 3 pm, with smaller than 0.5 pm and 100% smaller tk 8 pm. Except for differences in mean tap and in specific surface area (short 4T3U (Received in original form April 23,19M nf< revised form September 4, 1984) 1 From the Laboratory of Cellular Phyltf* and Immunology, Pulmonary Research Faculty of Medicine, Universite de Sherttf* Sherbrooke, Quebec, Canada. ' 'Supported by Grant No. MT-7310in** Medical Research Council of Canada and l|* stitut de recherche en same et ensecuriti du Quebec: 1 Requests for reprints should be additud'J Dr. Irma Lemaire, Laboratory ofCellularft*" ogv and Immunology, Pulmonary Research* Faculty of Medicine, Universite de Shert* Sherbrooke, Quebec, Canada JlH SN4. , 4 Scholar of the Fonds de la Recherche O I*] du Quebec. Z>: Diffcrcm. (romeyte x 104 cei: Min. A; The occur (MGC) in UtrJ by :: proporlio: luelci. In tv CYalu: ealeu HKlei v,:;: fcxlei ir. a Ctt> ford Vtttllsarc IW ngni. tbrtios.|r Emitted u Asbc Hiviolngi; vrrvcd ,, t*nuIc. j: 144 yftrms AND BRONCHOALVEOLAR LAVAGE CELLS 145 >nse in irrng the dewt heal Injection 4T30chryotS ter instillation. si ;e to asbestos mitotic flgum e in part to this a fibers, whict as early as Off st through 0t( lavage fluid (5 y contrast, * Dayl.ByOsj i me noflbiosis -*nucW. , esbo are associilsd e role for 985: 131:1a*-1** ; 23. 19& i'v,, ) lular PhV^S Research S, . de Sherbl^ T-7310 Wj* u nadai aannddVWj icuritedu i- -4drt*s<tf P< v i.-J-jI'cfl^it 'de Sherb^.H5N4- ^ cherche n " Bi/g- uiCC, 26.8 mVg) these 2 chrysotile asfcjtos samples had similar chemical and structural properties (10). Each sample was autoclaved for 45 min and suspended in ster ile phosphate-buffered saline (PBS) (pH, 7.4) with a Dounce glass homogenizer prior to initillation into the animals. Induction of Pulmonary Fibrosis Kits were lightly anesthetized with a mixture of ketamine-xylazine (85%-15%, 100 mg/ kj). The trachea was exposed surgically, and the asbestos suspension or saline was inject ed in a final volume of 0.5 ml. Three groups of 6 rats each received, respectively, a single Intratracheal injection of saline (control), UICC chrysotile B (5 mg), and very short 4T30 chrysotile (5 mg). Animals were killed peri odically (1, 7, 14, and 21 days) after instilla tion. Fifteen animals were killed at each time interval and evaluated for histologic changes, U described previously, and by bronchoalveolar analyses. Bronchoalveolar Lavage Animals were injected with sodium pentobar bital (30 mg/rat). The abdominal aorta was levered, and the trachea was cannulated. The nmgs were lavaged with a total volume of 50 ml PBS in 5-ml aliquots. After recovery, laJage fluid was centrifuged at 500 g at 4 C for 10 min. The cell pellet was washed twice la PBS and finally resuspended in Dulbec- s modified Eagle medium (DMEM) (Grand hand Biological Co., Grand Island, NY). Wls were counted in a hemocytometer cham ber, and viability was determined by trypan blue exclusion. Differential Cellular Analysis Differential counts of lavage cells were made cy,ocentrifuge smears prepared with 2.5 lO4 cells and stained with Wright-Giemsa Multinucleated Giant Cells (MCo nTenCe rnult`nucleatecl Siunt cells UieH u *n bronchoalveolar cells was quanti- 8ron . 0unting 10* cells and calculating the C|rVonbu f ceHs that contained 2 or more ei- In addition, the proportion of MGC j evaluated by the macrophage-fusion in- hucle'3 ?tec* ky dividing the number of Sticlc' MGC by the total number of Crau,r *n,at *east cells, as described by rd and coworkers (17). j. Statistical Analysis The*1 expressed as mean values SEM. *5bestSlEn'^'Cance differences between terrain!i"treated and control groups was de ed using Student's t test (p < 0.05). Results pjj j^bfstos-induced Lung injury bse e'C ev'c*ence of lung injury was ^ within 7 days after asbestos ex- re- The histopathologic changes in the lung were essentially the same as we described previously (10). Treatment with UICC chrysotile B asbestos, which con tains a high proportion of long fibers (21% smaller than lOftm), caused typi cal fibrotic lesions consisting of a focal fibroblastic proliferation in peribron chiolar tissues, which distorted and ob structed small airways. The predominant lesions were located in and around ter minal bronchioles, with some lesions lo cated at bifurcations of the distal airways. By contrast, animals treated with the same dose of very short 4T30 chrysotile fibers (98% smaller than 3 pm) had lung lesions confined to the alveolar struc tures. It was characterized by multifocal septal thickening and alveolar distortions caused by interstitial mononuclear cell infiltration. The small airways were nor mal, and no evidence of fibrosis was found 60 days after exposure to this type of asbestos fibers. Bronchoalveolar Cell Recovery at Various Stages after Asbestos Exposure Lavage fluid recovery was 45 ml for con trol animals and, respectively, 44 and 40 ml for rats treated with very short 4T30 chrysotile andchrysotile B. .For compar ison, values of total cell counts in lavage fluid of asbestos-exposed groups were corrected for lavage fluid recovery, and all values were expressed as cell counts per 45 ml lavage fluid recovered. The BAL fluid of saline-injected animals con tained an average of 5.9 x 106 cells 1 day after instillation, and no significant change was seen in the number of BAL cells of these animals on subsequent days after instillation. However, there was a significant increase in the total number of cells recovered from the lavage fluid of animals exposed to very short 4T30 fibers (12.5 x 106) and chrysotile B (9 IO*L SAL CELLS Fig 1. Total cell counts of lavage fluid at various times after intratracheal iniection of saline, very short 4T30 chrysotile and UICC chrysotile 8 Values are mean s SEM in 5 animals. Significantly different Irom control ( = p < 0.05). Fig. 2. Differential analysis of BAL cell populations at various times after asbestos exposure. Values are mean SEM in 5 animals. Significantly different from control (' = p < 0.05). x I06 cells) on Day 1 after treatment (fig ure 1). Higher cell counts were observed on Days 7 and 14 in these 2 groups of animals and returned to control values by Days 21 and 60, respectively, after treatment. Thus, the accumulation of in flammatory cells was more persistent in rats treated with long chrysotile fibers. Differential Cellular Analysis and Kinetics of Cell Populations of Lavage Fluid Lavage fluid obtained from salineinjected animals consisted of 97% mac rophages with no eosinophils and very few polymorphonuclear leukocytes (PMN) (1%) and lymphocytes (1%). No change in the cell populations of these animals occurred throughout the study. Intratracheal injection of UICC chryso tile B asbestos caused a significant in crease in PMN, which began to appear in the lavage fluid on Day 1 after treat ment. The infiltration of PMN (44% of the total cells) was still present on Day 7 and returned to normal by Day 14 (fig ure 2). By contrast, a very transient in crease in PMN seen on Day 1 only oc curred in response to very short 4T30 chrysotile fibers (figure 2). The absolute number of macrophages (total cells times differential percentage of macrophages) increased significantly in response to both asbestos samples (from 5.9 x 10* in the control group to 15.5 x I06 and 11.2 x 106, respectively, in the chrysotile B and 4T30 chrysotile groups on Day 14). l * 146 u,M*US ASBESTOS Fig 5. DsMils (3 or jssesios-e Iji cells Values 3's rep-eseni * dex). S'S" 0 05). Fig. 3. Presence of mitotic figures in lavage fluid of asbestos-exposed rats. Cytocentrifuge smears were prepared and stained with Wnght-Giemsa. A. Metaphase-anaphase (arrow). 6 and C. Telophase (arrows): original magnification: x 1,310. As illustrated in figure 3, microscopic evi dence of cell replication was found in lung lavage of rats exposed to asbestos. The occurrence of mitotic figures, includ ing anaphase and telophase in lavage flu id of these animals, was 3 to 5 times high er than in control animals, and suggests that local macrophage replication may contribute in part to this response. From Day 7 to Day 21, changes in the morphol ogy of lung free cells were seen, and oth er cell populations consisting of MGC appeared in the bronchoalveolar milieu of rats treated with both asbestos sam ples (figure 2). Peak accumulation of MGC was seen 14 days after asbestos ex posure and made up 7 and ll^o of total cells, respectively, after treatment with UICC and 4T30 chrysotile compared with 1% in control animals. No signifi cant changes in lymphocyte and eosino phil concentrations were observed at any given interval after asbestos exposure. * X' 'jA..*# e * * ** . !r < 1 * i s* ' *0 * I % ** ** / *V ** *m *^76,.u.m1 * Analysis of Muliinucleated Cells Present in Lavage Fluid Microscopic analysis of cell preparations revealed the presence of numerous cell aggregates in lavage fluid of animals ex posed to very short chrysotile fibers (fig ure 4B) compared with that seen in con trol animals (figure 4A). Aggregates were also present in rats treated with chryso tile B asbestos, although to a lesser ex- Fig. 4. Microscopic evidence of cell aggregates cell contact in cytocentrifuge smears of lavage Huxj} rats exposed to asbestos. Cell preparations stained with Wright-Giemsa. A. Control. B ana { Presence of cell aggregates in BAL fluid of ratstr* ed with very short 4T30 fibers (B, arrows) ana uc chrysotile B fibers (C, arrow); original magnificat x 130 D and E. Evidence of cell contact behwrl and more bronchoalveolar cells in lavage fluid d bestos exposed rats; original magnification: x t.tj i i tent (figure 4C). With respect to cell sc: shape, and surface membranecharacir istics, considerable heterogeneity was a; parent in these preparations, and mar cells showed vacuolate cytoplasm aolong cytoplasmic extensions. In addimx evidence of cellular contact between 3? I more bronchoalveolar cells was cor j monly seen in BAL fluid of animals ire* ed with short 4T30 fibers (figures4Dm-' 4E), wh: lhai the arises fr; as many 7 nuclei The distr the num: control a days aft-: binuclea: er in be animals respeetiu chrysotil: group (!.` am cells most cxe. of rats efibers, u: total cells index, a rr. uy, was s. treated u: in animal; and in cc The MGC types c majority ; 'hole cyt. the foreig; fox had ni >'-fihecei: (,11 and 6C hans type (Mng a ra: cj;ly and sc Liar alteral:|e asbesic feme poter jjSESTOS AND BRONCHOALVEOLAR LAVAGE CELLS 147 l*..;> regales vage fluid ^ ations n*1 i. B aJidC of rats w< r) and .agmlicalise ,t peiwee*'* e fluid el * ocelli ;haract(' $aiioe n 24 vsf4T30 n 141 crvysoiiie Q II 79 NUMBER OF NUCLEI Rg S. Distribution of binucleated and multinucleated C*fls (3 or more nuclei) in lavage fluid ol control and Mbestos-exposed rats 14 days after instillation. The I01 cells were counted by 2 different observers. Vilues are expressed as percent of total cells and represent mean SEM of 5 animals (f.i. fusion in dex). Significantly different from control (' = p < 0.05). 4E), which lends support to the concept that the formation of MGC probably arises from cell fusion. Some cells had as many as 16 nuclei, but cells with 3 to 7 nuclei were more commonly observed. The distribution of MGC in relation to the number of nuclei was determined in control and asbestos-treated animats 14 days after instillation. The number of binucleated cells was significantly high er in both groups of asbestos-treated animals (9.6 and 7.4% of total cells, respectively, for short 4T30 and UICC chrysotile B fibers) than in the control group (1.7% of total cells). However, gi ant cells with 3 or more nuclei were al most exclusively present in lavage fluid f rats exposed to very short asbestos fibers, where they represented 2.5% of total cells (figure 5). Similarly, the fusion index, a measure of cellular multinuclear'ty. was significantly higher in animals treated with very short fibers (14.1) than ln animals treated with chrysotile B (7.9) jtod in control animals (2.4) (figure 5). he MGC seen in lung lavage displayed lypes of nuclear arrangements: the majority had large nuclei spread in the th r ^P'hsr11 (figure 6A) typical of fewh C'en body tyPe giant cells, but a had nuclei arranged at the periphery the cell in an orderly fashion (figures , and 6C) characteristic of the Langns type giant cell. y Discussion ^ lng a rat model we have compared the lu|rly and sequential bronchoalveolar cel,jiar derations after exposure to chryso- -i-stos fibers with different fibrolc Potential (10). A common finding in our study was the significant increase in total bronchoalveolar cells in both groups of asbestos-exposed rats. How ever, the nature and the duration of the inflammatory cell accumulation differed after exposure to these 2 chrysotile prepa rations. Even though an early and sig nificant influx of PMN in the bronchoal veolar compartment occurred in response to both asbestos samples, the BAL fluid infiltration of PMN persisted longer in rats with fibrotic lesions and was only very transient in animals without fibro sis. This phenomenon preceded and par alleled the appearance of the fibrotic le sions and disappeared at a later stage of the disease. Similar increases in BAL neu trophils have been reported in experimen tal asbestosis (18, 19) and silicosis (20), and these cells have been implicated in the pathogenesis of pulmonary fibrosis (21). However, the role of PMN in fibro- ?6un Fig. 6 Morphology of multinucleated cells present in lavage fluid of rats exposed to very short 4T30 chryso lite libers. Cytocentrifuge smears were stained with Wrignt-Giemsa. A. Nuclei spread in the whole cytoplasm. B and C. Nuclei forming a ring at the periphery of the cytoplasm: original magnification: x 1,310 genesis remains controversial. Whereas some investigators have demonstrated that wound healing occurs normally in the absence of PMN (22), others have presented data suggestive of a restrain ing role for PMN in the fibrotic process (23, 24). In this regard, it is worth men tioning that neutrophils have the poten tial to counteract collagen deposition by producing collagenase (25) and to inhibit fibroblast proliferation by releasing tox ic mediators for fibroblasts (26). it is pos sible that PMN accumulation in BAL flu id of asbestos-treated animals may be sec ondary to the initial lung injury. Their presence may represent an index of the extent of lung injury rather than a deter minant factor in the development of fibro sis. Alternatively, PMN accumulation in BAL fluid, although transient, may modulate adjacent cell functions and in terfere in the initial stage of the disease process. Our results also demonstrate that chrysotile B asbestos caused a more sus tained increase in bronchoalveolar ceils than did very short 4T30 chrysotile fibers. This increase was due largely to macro phages, and there may be a relationship between the persistence of macrophage accumulation and the severity of the le sions observed. Similar increases in mac rophages have.been reported after as bestos inhalation in rats (18) or in tratracheal injection in sheep (27) and guinea pigs (28). It correlates well with the histopathologic finding of a macrophagic accumulation in lung lesions of experimental asbestosis (10, 11, 29). Al though higher macrophage number in the bronchoalveolar milieu may be attribut ed to recruitment of blood monocytes, our finding of mitotic figures in BAL flu id of animals exposed to asbestos sup ports the assumption that macrophage replication may also contribute to this phenomenon. The proliferative capaci ty of the alveolar macrophage has already been demonstrated (30), and macrophage division was observed after exposure to N02 (31) and in some chronic inflamma tory lung disorders (32). Whether mito genic factor(s) for alveolar macrophages are present in BAL fluid of asbestosexposed rats, as has been reported in oth er inflammatory exudates (33), is not known and will require further investi gation. A striking observation in our study was the morphologic heterogeneity of mac rophages in the bronchoalveolar milieu of asbestos-exposed animals. The cells showed considerable range in size and shape, many with vacuolate cytoplasm **o `-EMaihj | *S8: and long cytoplasmic extensions. In ad dition, a significant proportion of the cells were multinucleated. These cells have been previously found in histologic sections of lungs from animals inhaling chrysotile asbestos (5). More recently, 2 separate reports described briefly their presence in lung lavage of rats after in halation of chrysotile asbestos (34, 35). Our study confirms these findings and further documents some aspects of gi ant cell formation after asbestos ex posure. Although it is generally believed that MGC formation occurs by fusion of macrophages (36, 37), the mechanisms responsible for this process are not elu cidated. There is evidence to suggest that increases in relative amounts of phos pholipids, especially in lysolecithin, may favor cell fusion (38). However, chryso tile preparations that contain 15 to 21% of fibers longer than 10 pm (15) and that reportedly caused significant increase in pulmonary surfactant after intratracheal injection in rats (34) did not promote for mation of cells with 3 or more nuclei in our study. Possibly, giant cells formation may be the result of macrophages simul taneously attached to the same ingestible material (39). In this case, variations in the degree of fusion caused by endocytosis of UICC chrysotile B and very short 4T30 chrysotile fibers may be related to differences in cytotoxicity (40, unpub lished observations) or specific surface area (10). Alternatively, MGC formation may result from the in vivo release of lymphokines, such as macrophage fusion factor (41) selectively by sublethal con centration of very short 4T30 chrysotile fibers. Our results also demonstrate that both foreign body and Langhans giant cells were present in BAL fluid of animals treated with very short chrysotile fibers. As suggested by Mariano and Spector (36), these 2 morphologic variants prob ably represent different phases in the life of the same cell rather than distinct cell types. Study of the kinetics of MGC for mation revealed that although these cells were absent 1 day after asbestos instilla tion, they were found in significant num bers on Day 7. The reason for the initial delay before the appearance of MGC in BAL fluid of animals treated with very short chrysotile fibers is not known. Pos sibly the presence of great numbers of PMN 1 day after instillation may inhibit macrophage fusion. Similarly, the ab sence of giant cell formation after instil lation of chrysotile B fibers may be relat ed to a more persistent infiltration of PMN in BAL fluid of these animals. An other possible explanation for the delay in MGC formation may be that relative ly "aged" macrophages are necessary for the formation of such cells (36). The exact role of MGC in chronic in flammatory reactions is unknown. Previ ous data have demonstrated that these cells have a relatively short life span and a higher content of lysosomal hydrolases (42). On the basis of those characteris tics, it was suggested that MGC may rep resent an efficient disposal system for un wanted macrophages that might possess injurious potential (36). Interestingly, the presence of MGC in BAL fluid is as sociated with a less persistent increase in inflammatory cells and the absence of fibrosis. Another possible role for MGC may be related to their lower contact in hibition of locomotion (42). This prop erty may encourage and facilitate surface exploration of adjacent cells and enable them to modulate macrophage activity and involvement in inflammatory reac tions. In conclusion, our results demonstrate that bronchoalveolar cell populations are different, depending on the nature of lung injury induced by asbestos. The in flammatory cell response associated with lung fibrosis in our rat model is charac terized by a more sustained increase in PMN and alveolar macrophages and the absence of giant cells with 3 or more nuclei seen in the alveolitis produced by very short fibers. The role of PMN and MGC in such lung lesions remains to be clarified. In light of the important role of cell cooperation in many physiologic processes, one must question the sig nificance of the presence of PMN and MGC on macrophage function. This model will hopefully be useful in study ing the interactions between these vari ous cell populations and may prove valu able in providing new insights into the mechanisms of pulmonary fibrosis. Acknowledgment The writer thanks Dr. N. Briere for the use of his microscope and G. Hebert for excel lent secretarial help. The competent techni cal assistance of H. Beaudoin, C. Grondin, and C. Dubois is acknowledged. . References 1. Hourekane DO. McCaughcy WTE. Patholog ical aspects of asbestosis. Postgrad Med J 1966; 42:613-22. 2. Tukiainen P. Taskincn E. Korhola O. Valle M. Tru cut needle biopsy in asbestosis and silicosis: correlation of histological changes with radiologi cal changes and pulmonary function in 41 patients. Br J Ind Med 1978; 35:292-304. 3. Wagner JC, Berry G, Skidmore JW, Ttmbrell V. The effects of the inhalation of asbestos in . Br J Cancer 1974; 29:252-69. ts 4. Davis JMG. The use of animal models r studies on asbestos biocffects. Ann NY Acad 1979; 330:795-8. * 5. Holt PF, Mills J, Young DK. The early efft~ of chrvsotile asbestos dust on the rat lung, j pa.rj Bacteriol 1964; 87:15-23. no1 6. Goldstein B, Webster I, Rendall REG, MJ. The effects of asbestos-cement dust inhala' tion on baboons. Environ Res 1978; 16:216-25 7. Hiett DM. Experimental asbestosis: an jnvt, ligation of functional and pathological distu bances. II. Results for chrysotile and amosite J posures. Br J Ind Med 1978; 35:135-45. 8. Burger BF, Engelbrecht FM. The biologica]ef fects of the international standard reference asbestn." samples (UICC) on the lungs of rats. J AfrMf! J 1970; 44:1271-4. `* 9. Wright GW, Kuschner M. The influence of y^ ing lengths of glass and asbestos fibres on tjssi]j response in guinea pigs. In: Walton WH, ed |c haled particles. Vol 4. Pergamon Press, 1977:455.72 10. Lemaire 1, Nadeau D, Dunnigan J, Massey An assessment of the fibrogenic potential of VWl short 4T30 chrysotile by intratracheal insti|lai,0! in rats. Environ Res 1985; 36: in press. ` 11. Begin R, Masse S, Bureau VIA. Vlorpholop- features and function of the airways in earl; u bestosis in the sheep model. Am Rev Respir D. 1982; 126:870-6. " 12. Timbrell V. The inhalation of fibrous dura Ann NY Acad Sci USA 1965; 132:255-73. 13. Brody AR, Hill LH, Adkuis B, O'Connor Ru Chrysotile asbestos inhalation in rats: deposino: pattern and reaction of alveolar epithelium and js. monarv macrophages. Am Rev Respir Dis 191 123:670-9. " 14. Barry BE, Wong KC, Brody AR, Crapo JD Reaction of rat lungs to inhaled chrysotile asbesten following acute and subchronic exposures. Et,* Lung Res 1983; 5:1-22. 15. Timbrell V. Characteristics of the lnternatiori Unton Against Cancer Standard Reference Sit pics of Asbestos. In: Pneumoconiosis. 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