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Asbestos Exposure and Retention as Determinants of Airway Disease and Asbestos Alveolitis1'*3 - PLAINTIFF'S EXHIBIT 13J'3/S RAYMOND BEGIN, SERGE MASSE, PATRICK SEBASTIEN, JUDITH BOSSE, MAREK ROLA-PLESZCZYNSKI, MOKTAR BOCTOR, YVAN COTE, DANIEL FABI, and DANIEL DALLE Introduction Recently, it has been well documented that inhalation of asbestos dust (or other mineral dust) can produce a small air ways disease in exposed workers that is distinct from airway disease associated with cigarette smoking (1-5). This as bestos airway disease has been thought to cause significant air-flow limitation whether the workers were cigarette smok ers (1, 6-13) or lifetime nonsmokers (14). Furthermore, in humans and in animal models with either asbestos alveolitis (early asbestosis) or well-established as bestosis, similar airway dysfunction has been reported (15-18). This suggests that air-flow limitation in asbestos workers could be a marker of early parenchymal pulmonary damage and should preclude further exposure. In a previous study (15) of a sheep model of early asbestos peribronchiolar alveolitis with restricted lung volumes, we found a concomitant small airways disease that significantly limited air flow. Morphologic features and function of the small airways were studied in detail only after the initial alveolitis, which did not permit a study of the relationship of as bestos airway disease and parenchymal pulmonary damage. To provide additional information on the relationship of asbestos airway dis ease and early asbestosis, we obtained repeated bronchopulmonary function and morphologic data in 15 sheep ex posed to asbestos dust for 3 yr. The data clearly document that air-flow limitation occurred in all exposed sheep, whereas only 9 of 15 developed asbestos alveoli tis. Further analyses of BAL fiber con tent suggested that whereas airway dis ease is related to exposure dose, the as bestos alveolitis is primarily related to alveolar retention of the fibers. Methods Animats Twenty-six sheep weighing 25 to 40 kg were 1176 SUMMARY To evaluate the relationships ot asbestos exposure, retention, airway response, ant] the asbestos alveolitis, we exposed 2 groups of sheep every 2 wk tor 3 yr to either 100 ml phosphate, buffered saline (PBS) or 100 mg UICC chrysotlle fibers in 100 ml PBS. The sheep were evaluated periodically by pulmonary function tests (PFT), chest radiograph (CR), bronchoalveolar lavage (BAl) and transbronchia! lung biopsy (TLB). At Month 24 of the study, all asbestos-exposed sheep had significant increases in lung resistance and upstream resistance. However, only 9 ot the 16 asbestos- exposed sheep had significant changes In TLB, CR, Cst, and VC, which clearly separated them from the other $ sheep In these parameters. The 2 groups, however, had similar air-flow limitation At lung biopsy, all asbestos-exposed sheep had significant peribronchiolar fibrosis, with significant alveolitis only In the group ot 9 sheep with radiographic and functional changes of early asbestosis. The 9 sheep also had significant changes In BAL cellularity and biochemical profile, which differen tiated them from the other 6 asbestos-exposed sheep. Analysis of BAL fiber content at that point revealed that despite identical exposure, the group with Interstitial lung disease had significantly more fiber retention (p < 0.01). The data demonstrate that whereas asbestos airway disease appears to be primarily an exposure-dose-related response, the lung response appears to be mere closely related to alveolar retention of the dust. am rev respir oi$ i986; nanm-m used in this study. They were prepared and accustomed to the pulmonary techniques as previously reported (19-21). Experimental Design The flock was divided into a group of 11 sheep exposed to phosphate-buffered saline (PBS) only and a group of 15 sheep exposed to 100 mg UICC Canadian chrysotile asbestos fibers in 100 ml PBS every 2 wk. These fibers were relatively uniform and well characterized (22), 92^0 being less than 0.25 um in diameter and 20 um in length. Exposures were carried out after nasotracheal intubation via repeated slow infusions of the suspension in the tra chea at 2-wk intervals. The animals were stud ied prior to exposure and at 3- to 6-month intervals after exposure by chest radiographs (CR), pulmonary function tests (PFT), bron choalveolar lavage (BAL) analyses, and transbronchial lung biopsies (TLB). Pulmonary Function Tests The methods used in PFT assessment of the sheep have been published (15,19-21). Briefly, transpulmonary pressure was monitored with a nasoesophageal 7-ml balloon catheter and an airway catheter connected to a HewlettPackard 270 differential pressure transducer (Hewlett-Packard, Waltham, MA), Gas flow at the airway opening was measured by con necting the cuffed endotracheal tube to a Fleisch no. 2 pneumotachygraph (Dynasciences. Blue Bell, PA) attached to a flow integrator recorder system and a Mink data processing system (Digital Equipment, Mon treal, Quebec), for on-line analysis and stor ing of the data. Each PFT measurement was obtained after 3 inspiratory capacity meas urements at a constant volume; TLC was de fined as the lung volume at a transpulmonary Chest Radiograph Each sheep was positioned on a mobile cart with a wooden board and a grid cassette un der the thorax. The X-ray source was placed at a 30-degree caudal angle 2 feet from the cassette. The intubated animal was held at TLC using a giant syringe, and radiographs were taken at exposure factors 80 kV, 20 mAs, and 0.02 s. Each radiograph was scored ac cording to the International Labor Organi zation classification of radiographic profu sion of parenchymal opacities (23). (Received in originalJorm .November 2S, 1985 erd in revised Jorm May 27, 1986) 1 From the Unite de Recherche Puimonairt Universite de Sherbrooke, Sherbrooke, and the Dus Disease Unit, McGill School of Occupational Health, Montreal, Quebec, Canada. J Supported by 1RSST Quebec, the Medical Re search Council of Canada, and Societe de Thoracologie du Quebec. 3 Requests for reprints should be addressed to R. Begin, M.D., CHUS. Sherbrooke. Qurt*Canada JiH 5N4. \- e$TOS EXPOSURE ano retention AS DETERMINANTS OF AIRWAY DISEASE & 117 of t 35 5 cm H,0. and RV was mercially (Cappcll Laboratories, Downing- variance for experiments luning repeaie. L/jned as the lung volume at a transpulnio- town, PA). Lactate dehydrogenase, alkaline measurements on the same subjects. W'hei i 'jry pressure of-35 5 cm H,0. The static j r' 0jratory lung compliance (Cst) was deterJ %ied ^ multiple-step syringe deflation be- phosphatase, and P-giucuronidase were mea sured by standard methods (25-27). All results were expressed per milliliter of BAL fluid. a significant effect was detected, a Kruskal Wallis test was used to determine which grou; means were significantly different (29). Differ \ `aten TLC and FRC. For the measurement I '^maximal expiratory flow rates, the animal Electron Microscopy ences with p < 0.05 were considered signifi cant. i anesthetized with pentobarbital (initial .-He, 50mg/kg) and curarized with succinyl- The analytical transmission electron micro scope was used to look for the presence of Results 'esponse, #nd fRl phosphate 'ere evaluated Menage (&Ai), ed sheep had * 16 asbestos, parated them iow limitation, ith significant ly asbestosij. /hich ditfererv t at that point 3 significantly tease appears more closely 6; 134:n76-tm n jests I Moline (initial dose, 1 mg/kg), placed in a Tjjt integrated-flow plethysmograph, and ven- chrysotile fibers in lung lavages. All samples were prepared in a "clean room," using filtered ':ia!ed with a Harvard pump (Harvard Ap Iparatus Co., South Natick, MA) between arocedures. Total maxima! expiratory flow ^lume (MEFV) curves were obtained by sud' f,n|y connecting the airway opening to a large chemicals and carefully cleaned glassware. Thirteen milliliters were isolated from the third effluent of lavage and allowed to react at room temperature with sodium hypochlorite, in or der to digest the biologic material. An ali : ^uum source pressurized at - 200 mm Hg quot of the suspension, corresponding to 5 .i,j; the animal lungs were inflated to a nans* ^imonary pressure of 35 cm H20 (TLC). The ml of BAL, was filtered on a polycarbonate membrane filter 25 mm in diameter with a ^w-volume curves were replotted in relation pore size of 0.2 pm pore size (Nucleopore aabsolute lung volume and transpulmonary Corp.. Pleasanton. CA). Particles collected -ressure, the effective driving pressure produc at the upper surface of the membrane were es Vmax at any lung volume, and the up- embedded in a carbon film and subsequently I ,.r"jam resistance (Rus) was plotted as the ra- transferred onto 200-mesh grids (28). Several .:o0f transpulmonary pressure over Vmax at I [-ne volumes between near TLC and FRC. grid openings were observed in the transmis sion mode at a screen magnification x 10,000. fh* above were expressed in relation to abso Chrysotile fibers were identified by their mor lute lung volume. Diffusion capacity (Dlco) phologic features and their elemental com Is obtained by a passive rebreathing method using a gas mixture of KKo helium, 0.30To I arbon monoxide, and 2l-o oxygen in nitro} ten, a Collins catherometer (Warren E. Col- position as determined by energy-dispersive X-ray spectrometry (PGT system IVR; Prince ton Gamma Tech, Princeton, NJ). The length of each individual fiber encountered was mea J lias, Braintree, MA), and a Beckman infrared sured directly on the screen to the nearest 0.2 arbon monoxide analyzer (Beckman Instru pm: diameter was measured using an eyepiece ments, Fullerton, CA). For the Dlco test, the graticule. lAimals were passively ventilated with a 2.5-L fringe at a rate of 30 breaths/min with a I-L Statistical Analysis Subsets of Asbestos-exposed Sheep At Month 24 and after, the group of 15 sheep exposed to asbestos had signifi cantly higher pathology scores, higher ra diographic scores, lower VC, lower static lung compliance, and lower arterial Po: (data not shown), and these changes were accentuated in the year after. Within the group, these changes were the effect of disease limited to some of the sheep. We then separated the group of asbestosexposed sheep into 2 subsets: a subset of 6 sheep who had minimal or no alveoli tis shown by TLB and CR scores of 0/0 or 0/1, which were within the 95% toler ance limit of PFT of saline-exposed sheep; this subset is reported as without asbestos alveolitis hereafter. The second subset of asbestos-exposed sheep was composed of 9 sheep with changes shown by TLB, CR, and PFT consistent with those of the initial asbestos alveolitis pre viously reported in this model (15, 20); this subset is reported as with asbestos alveolitis hereafter. cessment of the 19-21). Briefly, monitored wish >n catheter and i to a Hewlett- ure transducer VIA). Gas flow asured by con ceal tube to a graph (Dyna:hed to a flow d a Mink data -lipment, Mon- tidal volume Transbronchial Lung Biopsy Under topical local anesthesia and without fluoroscopic guidance, TLB were obtained following the technique previously published (13). Subsegmental bronchi were randomly selected, and the TLB sample was immedi ately placed in 10% buffered formaldehyde fixative and processed as routinely done for human lung tissues. All sections were stained with hematoxylin-eosin, and selected biop sies were also stained with Masson-trichrome. In the presentation of the results, values of the data for each group of sheep are followed by the standard error as an index of disper sion. The data were evaluated by analysis of Pathologic Findings In the saline-exposed sheep, the lung tis sue remained normal. In the asbestos- -.lysis and storasuremetu was :apacity meate; TLC was de- anspulmonary Bmchoalveolor Lavage and Fluid Analysis Most of the techniques in BAL procedures and analyses have been previously described 119-21). The BAL effluent was passed through layers of cheesecloth to remove mucus, and die cells were pelietized by centrifugation. Cells were counted in a hemocytomeier, and ber2S, i985ar-d cell viability was determined by the trypan ciue exclusion technique. Cytocentrifuge \ Pulmotiaift ke. and the Dust ` Occupational da. caJfe,ete * >t addressed to -ooke, Quebec smears served to identify the cellular popula tions recovered with the Wright--Giemsa and naphthyl acetate esterase stains. In the super natant, albumin and IgG were measured by toe immunochemical methods of KillingNorth and Savory (24), using the Behring lalet nephelometer instrument (Behring LN j odular system; Hoechst Behring, Frankfurt, J WG). For sheep albumin and IgG, specific J antiserum raised in rabbits was obtained com Fig. l. Representative histopathologic features of the airway lesions and surrounding iyng tissue Tne lett panel is from an asbestos-exposed sheep with severe airway disease characterized by pentvoncn.c;a- iejcocyte ac cumulation. fibroblastic proliferation, severe airway distortion, but minimal alveolitis. The ngn: sane: is from a sheep with less severe airway disease associated with diffuse leukocyte infiltration of tne su-'cunamg atveoh and Interstitium (hematoxylm-eosm stain; magnification; x 63). 1178 BEGIN. UASS. SEBact exposed sheep without alveolitis, there was a diffuse peribronchiolar fibrotic process with minimal alveolitis (figure 1, left panel). In the sheep with alveolitis, there was the same peribronchiolar pro cess and in addition extensive accumu lation of macrophages and leukocytes in the alveoli and interstitium adjacent to the airway lesions (figure 1, right panel). Chest Radiographs AH CR of control animals were without infiltrates or pleural lesions throughout the experiment. In the asbestos-exposed sheep without alveolitis, the same obser vations (scores of 0/0 or 0/1) were recorded until Month 24 (figure 2, left panel) where 1 of the 6 sheep had a I/O infiltrate in 2 lung fields, which appeared in 2 other sheep at Month 36. In the asbestos-exposed sheep with alveolitis, scores of parenchyma] opacities of 1/0 or greater were present in each of the 9 sheep (figure 2, right panel), and the av erage score went from 2.7 1.7 at Month 24 to 7.3 2.7 at Month 36 (p < 0.05) (figure 2). Lung Volumes Between the 3 groups of sheep, the only significant lung volume difference was for VC, which was significantly lower in the asbestos-exposed sheep with alveoli tis from Month 24. For control sheep at Month 24 VC was 3.05 0.05 L, for the group without alveolitis VC was 2.99 0.05 (NS), and for the group with alveo litis, VC was 2.80 0.06 (p < 0.05). Fig. 2. Representative chest radiographs of exposed sheep with airway disease {let: pane/) and a sheep airway and interstitial lung disease {right pane/) The left panel radiograph is essenitally normal, whereas* * right panel radiograph shows diffuse reticulonodular infiltrates of the lung parenchyma. ** firmed on the full Rus/lung voiume curves (lower panels). Alveolitis In contol sheep, there was no significant change in BAL cells and biochemical analyses over time in any of the param eters. In the 6 asbestos-exposed sheep without alveolitis, the only significant changes were slight increases in BAL al bumin from Month 30 and IgG from Month 24. In the 9 asbestos-exposec sheep with alveolitis, there were signifi cant changes in all BAL parameters which cleariy differentiated those shee; from the sheep exposed to the same cu muiative asbestos exposure but withotr alveolitis (p < 0.05). These changes seen from Month 12 for lactate dehydrog enase, from Month 24 for neutrophils, and from Month 30 for total BAL cells, macrophages, and lymphocytes (the timr (TATIC LUMC COMMLIAMCC e V PULHOHARY KECHANICS uaac eiutaws .< u ,, ru Diffusion Capacity For control sheep at Month 24 Dlco was 23 0.6 ml/min/mm Hg, for the group without alveolitis Dlco was 23 1.4 (NS), and for the group with alveolitis Dlco was 19 0.8 (p < 0.05). ii u ma MONTH UPSTREAM RESISTANCE , I MONTH 12 Pulmonary Mechanics In the upper panels of figure 3, the time course of static lung compliance (Cl), lung resistance (Rl), and upstream lung resistance (Rus) at low lung volume are presented. In the lower panels of the same figure, the full upstream resistancecurves at selected times are presented. It can be seen that both groups of asbestos-ex posed sheep had lower Cl than did con trol sheep, with values for the sheep with alveolitis showing a significantly steeper rate of fall of Cl (p < 0.05). The air-flow resistance increased significantly and similarly for both groups of asbestosexposed sheep in the large (Rl) and small airways (Rus, 40*70), which was con Os I r w * m. II 2.1 ClTtM U MONTH 1* l.l II LI U LfTSH CNTH M LI U LT*e CONTMO. 6MCCP sO I r < JlA l.l LI t.l l.l iim < o.* B M < 0.O6 }* Fig. 3. Results of pulmonary mechanics test presented ferine 3 groups of sheep in the upDer panels, with se*** upstream resistance/lung volume curves m tne tower panels. 'N \*&*. JjgESTOS EXPOSURE AND RETENTION AS DETERMINANTS OF AIRWAY OlSEASE 1179 ASBESTOS DUST RETENTION Fiber length distribution, V. eep with j fe&s the . o ;po$ed ignifi. neters, sheep me cuithout is were ydrogophils, -cells, le time { \ j ; i wac* <v> a Fig. 4. Results of analysis of fit>er content of lung lavage at Month 24. The left upper panel is a bar diagram d fiber counts in million per milliliter of BAL effluent. The tight upper panel is a fiber length-distribution diagram of the fiber analysis of BAL showing similar length distribution in the 2 groups of exposed sheep. The middle left panel shows a fiber tS u long, the middle right panel an asbestos body 32 u long by 0.75 u wide, and the left tower panel shows bundles of fibers i u long, alt on a background of a 0.2-u Nueleopore- membrane. The r^ntlower panel shows the characteristic X-ray energy spectrometric pattern of a chrysotiie fiber used to ascertain oe nature of the fibrous materials observed on BAL. j course of these have been reported | separately, in relation to our analysis of f ?aliium-6" lung uptake in the process t '*). /touirrt .!TI select* RAL Fiber Analyses to figure 4, the resuits of mineraJogic anafoes of BAL fluid obtained at Month ^ as an index of asbestos dust alveolar Mention are presented. In control sheep, fiber counts averaged 1.8 1 x lOVml, jad all were shorter than 1 p. in length, to the sheep with airway disease only, we found 4.56 035 x 10* fibers/ml, and in the sheep with airway disease and al veolitis, we found 19.2 4.4 x 10s fibers/ml (p < 0-01). Analysis of size dis tribution did not differentiate the ex posed groups; a few asbestos bodies were observed in the 2 asbestos-exposed groups. Discussion This study showed that all sheep exposed to a cumulative exposure dose of 5 g of asbestos dust developed diffuse airway lesions that caused significant air-flow limitation. Furthermore, this study dem onstrated an individual heterogeneity of lung tissue response to the same exposure dose, with 40% of the sheep having dis ease limited to the airways and 60% also having a concomitant alveolitis. Analy sis of BAL fiber content in the 2 groups of exposed sheep showed a fourfold difference between the 2 groups without significant difference in the type, size dis tribution, width, or aspect ratios of the fibers. Although the relationship of asbestos exposure and airway disease has been a controversial subject during the last de cade, recent reports have contributed sub stantially to clarifying the issue. In the animal model, it has been shown that as bestos fibers are primarily deposited and retained at the bifurcation sites of the pe ripheral airways where the initial airway lesion is rapidly established (31). In as bestos workers, these airway lesions have been observed in the absence of asbestosis (interstitial lung disease) and have been associated with higher asbestos con tent of the lungs (32). However, because similar pathologic changes of the small airways have been observed in associa tion with a variety of mineral dust ex posures (4), the specificity of the lesions appears to be related to mineral dust ex posures, including asbestos. Our data on 15 sheep exposed chronically to asbestos dust confirmed the existence in all of these animals of airway lesions that caused significant air-flow limitation in dependent of the development of inter stitial lung disease. In this animal model, it is clear that the peripheral airway le sions were more severe than those reported in asbestos workers in terms of intensity of the bronchiolar disease and its effects on lung function. The airway lesions in this multiple intratracheal as bestos instillation mode! differ from the human disease associated with asbestos inhalation, as the animals were subjected to more intense and less frequent ex posures. This exposure produces a more intensely inflammatory airway lesion in sheep and more severe airway dysfunc tion than that reported in lifetime non smoking asbestos workers (14). Indeed, in a recent study of 34 lifetime nonsmok ing, long-term miners and millers of the Quebec asbestos industry, we found a 30% reduction in air-flow conductance at low lung volumes, which was consis tent with the concept of an asbestos air way lesion. However, this peripheral air way dysfunction did not reduce the expi ratory flow rates on the flow volume in the lifetime nonsmoking, chrysotiie miners and millers (33). 1180 DCClf. MASSE. StU4S7/Ni T *1 Is this airway lesion a precursor of in terstitial lung disease? In humans, this airway lesion was observed at total lung burden substantially lower (50%) than that of patients with asbestosis and may be linked to pleural changes (32). Its rela tionship to the development of intersti tial lung disease has not been reported. In our sheep with alveolitis at Month-24, significant changes in several parameters of interstitial lung damage preceded the airway disease documented at Month 18: lung compliance was reduced from Month 9 (figure 3), *7Ga lung uptake and lung lavage fibronectin increased from Month 6 (30), and lung lavage LDH in creased from Month 12. In the sheep without alveolitis at Month 24, only 3 of 6 developed interstitial lung disease despite continuous exposure for the fol lowing year. Thus, on the basis of these data, the asbestos-induced airway disease cannot be seen as a precursor of intersti tial lung disease but should be consid ered as a relatively independent process. The asbestos-induced alveolitis, which in our view constitutes the initial lung tis sue reaction leading to asbestosis (15,20, 21), has been further characterized in this study. In a recent report on the same groups of sheep, we have reported serial measurements by BAL and scan of 6,Ga lung uptake during the induction of the disease process and have found that the uptake of the marker occurred before the disease process could be detected by other methods (30). The enhanced 67Ga lung uptake correlated best with the excessive accumulation of BAL fibronectin. These observations confirmed our previous reports on *'Ga lung uptake as a useful indicator of early lung damage in as bestos workers (34) and further suggested that macrophage activity producing ex cessive amounts of fibronectin was pri marily responsible for the enhanced up take of the marker. The present study further investigated the heterogeneity of lung tissue response in our sheep with similar dust exposures, 60% of them developing the alveolitis at Month 24. This observation in the ani mal model is similar to the clinical ob servation of individual susceptibility of humans to develop the disease (35). To investigate the individual susceptibility factor, immunologic background and clearance capacity have been considered. Reports on human immunologic histo compatibility have failed to document definite markers identifying the more susceptible workers (36), and our own work in progress with some 200 asbestos workers tested appears to agree with these earlier reports, in the sheep, histocom patibility testing is not currently feasible. Alveolar dust clearance capacity in hu mans has not been directly studied in asbestos-exposed workers as a deter minant factor for individual susceptibil ity to the disease. Lung tissue burden of fibers have been found to be increased in asbestos workers compared with that in the general population (37). In the ex posed workers, it has been documented that workers with isolated airway disease have twice the fiber content of the lungs of workers without airway disease but only 50% the fiber content of patients with asbestosis (31). In the lung tissue of patients with asbestosis, the lung fiber burden appears to correlate with severity of the disease (38-41). Analysis of BAL fiber content in workers with asbestosis have yielded results in the same range as that of our sheep with asbestos alveoli tis, but lower values were found in ex posed workers without asbestosis (28, 42). Because in human studies, the amount of exposure is difficult to deter mine with precision, it has not been pos sible to compare groups of workers with similar exposures but different disease ac tivity. Given that all our exposed sheep had similar exposures, BAL fiber content can be seen as an index of alveolar dust reten tion (43) and hence assesses the in dividual alveolar clearance capacity. In the sheep with alveolitis, we found a four fold increase in the number of fibers re tained in the alveolar space without difference in type, size distribution, width, or aspect ratio of the fibers. These data in the sheep model clearly link the individual susceptibility to develop the asbestos alveolitis to alveolar retention of the dust and thereby to the individual dust clearance capacity. To verify the rel evance of these findings to the human condition, we have initiated alveolar clearance studies in asbestos workers. In conclusion, this study on the sheep model of asbestos-induced lung injury has shown that chronic asbestos dust ex posure can induce airway disease with sig nificant air-flow limitation in the absence of interstitial lung disease. Within the exposed population, there is a heteroge neity of lung tissue response, and the susceptibility to develop interstitial lung disease appears to be related to the in dividual capacity of alveolar dust clear ance. References 1. 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