Document v1wj0L63edDEgpmM3399X0BBw

Deposition, Clearance, and Translocation of Chrysotiie Asbestos from Peripheral and Central Regions of the Rat Lung Patrick G. Coin,*** Victor L. Roggli.* and Arnold R. Brody* ^Laboratory of Pulmonary Pathobiologv. National Institute ofEnvironmental Health Sdcin.es. fsearch Triangle Park. North Carolina 27709; and TDeparimcnr of Pollutions. Duke Cntversits and Durham Veterans Administration Medical Centers. Durham. North Carolina 27710 Received September 3. 1991 We investigated the pulmonary deposition, clearance, and translocation of chrysotiie asbestos in the context of our previously developed model of asbestosis tn the rat. Adult male rats were exposed for 3 hr to an aerosol of chrysotiie asbestos. Subgroups were sacrificed up to 29 days postexposure and the lungs of the animals fixed. Peripheral and central regions of the left lung were resected, digested, and analyzed for fiber content by scanning electron microscopy. Pulmonary deposition did not differ between peripheral and central regions. There was no evidence of translocation of fibers from central to peripheral regions. The average diameter of retained fibers decreased over time, consistent with lon gitudinal splitting. The average length of retained fibers increased over time, consistent with slower clearance of longer fibers. We employed a novel counting scheme to ensure accurate fiber number measurements, allowing the calculation of clearance rates for fibers 0.5 to Ift iim in length. Fibers of length 2= 1ft pm were cleared slowly, if at all. These findings could have important implications for the pathogenesis of asbestos-related pleural disease. Mans fibers arc deposited in the peripheral region, and the longest (16 pm) will persist there for extended periods. C 1991 Academic Pre\>. Inc INTRODUCTION Asbestos exposure causes disease in three regions of the respiratory system: carcinoma in the bronchial tree, asbestosis^(i.e . fibrotic scarring) in the small airways and parenchyma, and both mesothelioma and fibrosis in the pleural re gion. Upon tnnaiation. hber$ are deposited in both the bronchial tree and the parenchyma..In order to cause disease in the pleural region.- fibers must -presum ably be translocated there after deposition. The mechanism and kinetics of this nghess are unknown (Hillerdal. 1980). ^Pvious work suggested that few fibers would be deposited in parenchymal regions adjacent to the visceral pleura. There is enhanced deposition of asbestos fibers at first alveolar duct bifurcations (Brody et al., 1981). but these anatomic sites do not occur immediately adjacent to the visceral pleura. Deposition of fibers decreases with increasing airway pathlength and bifurcation number in the tra cheobronchial and pulmonary regions of the rat lung (Pinkerton et at.. 1986). Warheit and Hartsky (1990) observed that deposition of carbonyl iron particles at alveolar duct bifurcations decreased distaily from the terminal bronchioles toward the pleura, and the same pattern appears to hold for asbestos fibers (Brody and Roe, 1983). Experimental work suggests that, in order to cause pleural disease, fibers must be present in the pleura or immediately adjacent regions of the pulmonary paren- 97 0013-9J51'92 S5.00 Cops right r 1992 bs Academic Prev*. Inc. AH right* of reproduction m any form reserved. 98 COIN. ROGGU. AND BRODY chyma. For example, direct injection of fibers into the pleural or peritc ities causes a high incidence ofjnesothelioma (StantonTr"a7T7-l9Hl~;Jaurt 1987). suggesting that the mesothelium itself is the target tissue in tha Malignancy may also arise in mesenchymal cells underlying the viscei (Hill et al., 1990). In addition, some fibrotic pleural disease may be aeon of inflammation in this subserosal layer (Dodson and Ford. 1985; Lap 1991). Even though postdepositional movement (translocation) of fibers is i in the genesis of pleural disease, there have been few investigations translocation to the pleural region. Some studies in humans suggest tha fibers are translocated to the subpleural pulmonary parenchyma as w visceral and parietal pleura (Churg and Wiggs, 1987: Sebastien et al.. 198( and Kohyama. 1991). Viallat et al. (1986/ recovered short (0.3-1.3 p length) chrysotile fibers from the pleural cavity of rats in two peaks 7 tc after an intratracheal instillation. Morgan et al. (1977) exposed rats for 3 aerosols of radiolabeled amphibole asbestos and found that the label was trated in "hot spots" adjacent to the visceral pleura at 104 and 139-da exposure. They suggested that these concentrations were due to translot fibers from the central regions of the lung toward the peripheral (sul regions. This process is often called "pleural drift." . The goals of the work presented here are to investigate whether plet occurs with chrysotile asbestos and to study the changes in fiber din during the translocation process. In addition, this study seeks to desc clearance of chrysotile fibers from the rat lung. There are two reasons First, macrophage-mediated clearance of particles from pulmonary to bronchial region competes against transepitheliai movement of particle interstitium (Ferin and Feldstein, 1978: Greenspan et al.. 1988). Thus ar standing of clearance is a prerequisite for the investigation of fiber transli which may occur via the interstitium. Second, recent experimental w shown that longer 05 p.m) asbestos fiber preparations are more potent i of fibrosis than are shorter fibers (Adamson and Bowden. 1987a. 1987b; I al.. 1986: Davis and Jones. 1988). Differential clearance of these fiber p tions may explain some of their differential pathogenicity. Our findings r here show that both long and short chrysotile fibers are deposited by inhal peripheral and central regions of the rat lung. Subsequently, short fib cleared preferentially, and longer fibers remain in both regions of the lun MATERIALS AND METHODS Experimental design. Inhalation exposure methods have been describee ously (Warheit et al.. 1984a.b). Briefly. 23 adult male rats (Sprague-C specific pathogen free) of age 8-10 weeks were exposed for 3 hr to an aer 10 mg lrespirable)/'m3 chrysotile asbestos. This is approximately 5000 fib (j.m in length/enr. Subgroups of four or five animals were sacrificed imme after exposure (0 days) and at 1.8. 15. and 29 days postexposure. Four une animals were sacrificed 26 days after the exposure to serve as controls. A were sacrificed with an intraperitoneal injection of sodium pentobarbit: DEPOSITION OF CHRYSOTILE IN RAT LUNG 99 chest was opened and the pulmonary vasculature perfused with phosphate- buffered saline via the right ventricle. Lungs were fixed in situ by intratracheal instillation of 1% paraformaldehyde/1 % glutaraldehyde in sodium cacodvlate buffer, pH 7.2. at a pressure of 15 cm of water. Lungs were stored in fixative until dissection. Lung dissection. The left lung was separated into peripheral and central regions under a dissecting microscope. The hilar portion of the left bronchus was dis carded. The apical (--1 cm caudal to apex) and diaphragmatic (~2 mm cranial to diaphragmatic pleura) regions were resected as peripheral tissue. The remaining part of the left lung was cut transversely into ~2-mm-thick slices. Each slice was pinned to a dissecting board and the visceral pleura and a I- to 2-mm-thick band of subpleural tissue was cut from the slice. The core of each slice comprised the central tissue. Peripheral and central tissue were separately minced into ~2-mm cubes with scissors. Excess fixative was removed by blotting, and tissue was placed into polypropylene centrifuge tubes. The wet weight of peripheral and central tissue was recorded for each animal. Control lungs were dissected in this manner as well. For studies of deposition and translocation, fiber number and mass were di vided by a normalization factor. This factor was calculated for a peripheral sample as PKP + O, where P and C are the wet weights of peripheral and central tissue, respectively. Peripheral tissue typically comprised 40% of the wet weight of the left lung: therefore the normalization factor was typically 0.4 for peripheral and "or central tissue. ' ssue digestion. Lung tissue was digested by a modification of the method of ./illiams et al. (1982). All reagents were filtered through 0.2-fi.m pore size mem brane filters prior to use. Twenty milliliters of 12% sodium hypochlorite (NaOCl. Worth Chemical, Charlotte. NO was added to each tube containing tissue. The tube was vortexed periodically and digestion allowed to progress for 30-45 min. The digest from each peripheral or central sample was divided equally and filtered onto two 25-mm-diameter. 0.2-p.m pore size capillary pore filters (Nuclepore Corp.. Pleasanton. CA). Lipids and other undigested debris were extracted by filtering a sequence of reagents: deionized water, isopropanol. 8% oxalic acid. 12% NaOCl, and a final rinse with deionized water. Filters were allowed to dry overnight in a desiccator. The effect of the digestion procedure on fiber size distributions is described in Table 1. Portions of a suspension (in isopropanol) of 100 pg chrysotile asbestos/ml TABLE I Effect of Digestion Procedure on Fiber Number. Mass, and Size Fiber number Fiber mass (pg) Average fiber length (pml Average fiber diameter (pm) Standard suspension 8.82 x I07 7.07" 2.47 0.118 Spiked digest 1.00 x 10" 5.76" 2.33 0.108 " Recovery of standard suspension from tissue digest is 81CJ based on mass (5.76 p.g/7.07 pgj. 100 COIN, ROGGLJ, AND BRODY were either filtered directly or added to 1 g of lung tissue from an un' animal. The tissue was digested as described above, and the fibers were e; as described below. Recovery of chrysotile from the tissue was 81% b fiber mass. The digestion process caused a slight ( -- 10%) increase in fiber and slight decreases in both average fiber length and average fiber diam< Microscopy. One wedge was cut from one of the two filters for each sarc mounted on a carbon disk with carbon paint. The sample was sputter coal gold for 2 min. then examined with a JEOL JSM-35 scanning electron micr. 20-kV accelerating voltage, 15-mm working distance, 0 tilt, 5 sec/frame sc and magnification 4000-32.OOQx on the cathode ray tube (CRT, see belo Fiber number and dimensions. The fiber number was determined by c the number of fibers present in fields (defined on the CRT by a rectangular randomly selected by traversing of the filter surface. Fibers were defined particle having aspect ratio 5=3:1 and having the morphology of a chrysotil In practice, almost all fibers encountered had aspect ratio 55:1. The N Institute of Occupational Safety and Health "B rules" were used for co whereby the number of fiber ends protruding into a defined field is count the result is divided by two to give the fiber number (Schneider, 1979). Succ fields were selected until at least 40 ends were counted in each lengtl stratum (see below). The number of fields needed to reach this criteric recorded and used as the basis for the calculation according to the formui (fiber ends) x (filter area) Fiber number = 2 x (No. fields) x (field area) x (aliquot) "Aliquot" is the fraction of the sample on a given filter, here having the val (based on two filters per sample). Fiber dimensions were measured by classification of each fiber encoui into one of 48 length-diameter cells (Table 2). Each fiber was classified by at a given magnification using a transparent reticle with calibrated lines. L was based on the measurement of the fiber along its curve. The lowest length boundary (0.5 |im) was chosen on the basis of our confidence of fiber iden tion at 32,000x (32Kx). Fibers shorter than 0.5 p.m are present. Diameters classified at a screen magnification of 32Kx using a reticle with calibrated c: Generally, diameter was classified near the middle of a fiber, since the er chrysotile fibers tend to be splayed. Reticles were calibrated against a grid oi lines^mm (Ernest Fuilam, Latham. NY). Stratified counting procedure. Starting at low magnification (IQOx), a st: point was chosen near one edge of the filter. The magnification was then che to 32Kx and fiber ends of length (L) 0.5 L < 1 p.m were counted and diameters classified. The number of ends in each diameter class was tabulate an eight-channel laboratory counter. A separate counter was used to tabular number of fields. A new field was then chosen by moving the stage an arbi distance along the horizontal axis. Fields more than 30% obscured by debris not counted (less than 10% of all fields). New fields were chosen until the c criterion (40 ends) was reached or the edge of the filter was encountered. It deposition of chrysotile in rat lung 101 TABLE 2 Length and Diameter Classification ^Magnification used for length classification: 32.000X Diameter (<1)" class (pm) d < 0.07 0.07 d < 0.10 0.10 ad < 0.14 0.14 d < 0.20 0.20 d < 0.28 0.28 d < 0.40 0.40 d < 0.57 d s* 0.57 0.5 s L <J 0.75/0.06'' 0.75/0.085 0.75/0.12 0.75/0.17 0.75/0.24 0.75/0.34 0.75/0.49 0.75/0.69 I6.000X I6.000X 8.000 x Length (L) class (pm) I * L <2 2 J. < 4 4 L < 8 1.5/0.06 1.5/0.085 1.5/0.12 1.5/0.17 1.5/0.24 1.5/0.34 1.5/0.49 1.5/0.69 3/0.06 3/0.085 3/0.12 3/0.17 3/0.24 3/0.34 3/0.49 3/0.69 6/0.06 6/0.085 6/0.12 6/0.17 6/0.24 6/0.34 6'0.49 6-0.69 8.000 x 4.000x 8 L. < 16 1 2'0.06 12/0.085 12/0.12 12/0.17 12/0.24 12'0.34 12/0.49 12/0.69 L s= 16 24/0.06 24/0.085 24/0.12 24/0.17 24/0.24 24/0.34 240.49 24/0.69 " Diameter classes are based on a geometric progression: each class boundary is \ 2 * the value of tht next lowest boundary 10.07. 0.1.0.14.. . .). h Length classes are based on a geometric progression: each class boundary is twice the value of the next lowest boundary (0.5. 1. 2.. . .1. r The values in each cell arc the length/diameter values used for calculations of average length, average diameter, and average mass. latter case, the stage was repositioned along the vertical axis and the horizontal traversing continued in the opposite direction. For other length classes fibers were identified and classified by length at the appropriate magnification (see Table 2) and then diameters were classified at x. A separate eight-channel tabulator was used for each length class. Count- vas continued for each length class until the criterion of 40 ends was reached, is manner the statistical uncertainty due to sampling error was uniform across all length classes. Calculations. A microcomputer-based spreadsheet program was used to per form all calculations. Fiber mass was based on the fiber number in each lengthdiameter cell assuming a cylindrical morphology: Mass = 2 TtNijLjdj'p/4. v (2) Here Ny is the fiber number (calculated from formula (1) above) in the cell of length class f. diameter classy. L, is the arithmetic midpoint of the /th length class, and dj is the arithmetic midpoint of the /th diameter class (Table 2). The bulk density of chrysotile (p) is 2.55 grn/cm3. The average fiber length and diameter were calculated according to the formulas below, where iVlotaI is the total number of fibers in the sample: average length = (3) 102 COIN. ROGGU. AND BRODY average diameter = The formulas are approximations, since all fibers in a given size < assumed to have the same dimensions. However these formulas do re actual joint length-diameter distribution of the fibers. We have tested the validity of the counting procedure by preparing a su (in isopropanol) of chrysotile fibers of known mass concentration and th ing a small aliquot for microscopic measurements. The mass calculated b scopic measurements, using formula (2). was 7.1 p.g. while the mass kno gravimetric measurements was 12.2 p.g. Statistical analysis. The Systat microcomputer package was used for s tical analysis (Wilkinson. 1986). The clearance data (fiber number and ma log-transformed before analysis to equalize the variance at different time RESULTS Fiber Deposition in Peripheral and Central Regions Both total (0 days postexposure) and pulmonary (1 day postexposure) tion were uniform between peripheral and central regions: i.e.. normaliz number and mass .were similar in each region (Table 3). Likewise, avera TABLE 3 Deposition in Peripheral and Central Regions __ , Total deposition (0 day postexposure) n=5 Pulmonary depos 11 day postexpos n=4 Peripheral IP) Central tC) Peripheral (P) C( Fiber number" (normalized) Fiber mass (pg>" (normalized! Average fiber* length (pmi Average fiber* diameter ipmi P'C ratio (mean'1 = semi fiber number P'C ratio (mean* = semi fiber mass SC-P (mean* r semi average length SC-P (mean* = semi average diameter Means across animals 1.31 s 10" 1.10 x 10s 9.10 x 10' 8. 8.60 8.22 8.56 . 1.47 1.57 1.65 _ 0.123 0.126 Summary statistics for each antmal 0.133 1.32 2 0.32 (.05 = 0.14 1.25 2 0.38 1.00 s 0.23 0.100 2 0.107 -0.053 2 0.116 0.003 = 0.008 0.003 2 0.006 " Geometric mean across animals. * Arithmetic mean across animals. DEPOSITION OF CHRYSOTILE IN RAT LUNG 103 length and diameter were similar in the two regions (Table 3). In order to compare the deposition between the regions in a-statistically valid manner, we formed summary statistics for each animal, i.e., ratios of peripheral/central for fiber num ber and mass, and central-peripheral differences for average fiber length and diameter (Table 3). None of these parameters was statistically different from 1.0 (number and mass ratios) or 0.0 (dimensional differences). Translocation Our working model for the translocation process is shown in Fig. 1. According to this model, if translocation occurs from central to peripheral regions, there will be an increase in the ratio of peripheral to central fibers over time. We therefore calculated the ratio of peripheral fiber mass to central fiber mass (both normalized to tissue wet weight as described under Materials and Methods) for each animal from l to 29 days postexposure (Fig. 2). There was no significant trend in the ratio over time. We also analyzed the trend of fiber mass and number ratio for each central compartment peripheral compartment C(time) ktrC(t) translocation P(time) kclrC(t) clearance to tracheobronchial region Flo. 1. Translocation model. Pill and C(r) represent fiber burden in the peripheral and central regions as a function of time postexposure (rl. The arrows represent (luxes. The flux out of a com partment is assumed to be proportional to the burden in that compartment. The flux from the central to the peripheral region is therefore k,,Gn. where klr is a constant. Clearance to the tracheobronchial region is assumed to follow the same first-order rate for each compartment, i.e.. fcc!rPt ft and kcWCu\. Mass balance gives the following solutions for Pin and Ctrl, where P,, and C,, are the initial burdens: Cin = . Pin = IA) + c0(l - ko". This model predicts that the ratio PMlClt) will increase with time postexposure. Other models can give similar trends of lung burden in the two regions. In particular, a model can be formulated with no translocation but with slower clearance (rate kp) from the peripheral region than from the central (rate *>: On = Pm = where kp < kc. This model also predicts that the ratio PlihOn will increase with time postexposure. Generally, biologic data will not allow differentiation between the two models. 104 COIN. ROGGLI. AND BRODY Fig. 2. Translocaiion: Plot of the ratio of normalized peripheral fiber mass to normalized fiber mass over time. The points are arithmetic means of the ratio, with standard errors indie the bars. The solid line is a linear regression of P C ratio versus time. The slope of the lin significantly different from zero ip = 0.J9I. The dashed curve is the trend expected for a trans rate 209? of the overall clearance rate. This corresponds roughly to the upper 95tr confidence the slope of the linear regression. length class, from 0:5-1 (am to 5*16 ptm. Again, there were no statistically icant trends (data not shown). We performed a sensitivity analysis by applying the linear regression plot Fig. 2 to the translocation model. The 95% confidence limit of the slope linear regression corresponds roughly to a translocation rate of 20% of the o clearance rate (half-life ~ 13 days) in terms of Fiber mass. Thus this exper put an upper bound on the translocation rate of -0.01 day ~ `. correspondin process with a half-life of 73 days (half-life = In 2/translocation rate). The 0-day time point was excluded from calculations a priori because the burden then includes Fibers in the tracheobronchial region. We were only ested in translocation and clearance in the slow-clearing or pulmonary re Inspection of the data, however, showed that none of our conclusions wou altered substantially by inclusion of the 0-day time point. Clearance For analysis of clearance, we summed peripheral and central Fiber numbi length-diameter cell for each animal. Therefore clearance rates are based o fiber burden for the entire left lobe. Again, we exclude the time point at 0 postexposure, but its inclusion would not alter any conclusions substantial! In previous work, we plotted length and diameter distributions of chrys fibers retained in the lung at different times postexposure and showed tha tained ftbers are progressively narrower and longer as time increases (Roggli Brody, 1984). Log-probability plots of length and diameter conFtrm this ir. present study (data not shown). The changes in size distributions are also af ent in micrographs of digests (Fig. 3). A plot of total Fiber mass versus DEPOSITION OF CHRVSOTILE IN RAT LUNG 105 (Pftstexposure can be fit with a two-compartment clearance model (Fig. 4). in agreement with previous work from this laboratory (Roggli and Brody. 1984). The length-stratified counting scheme permits a more informative analysis. Fig ure 5 shows plots of fiber number versus time postexposure for each length class separately. These plots show" that the number of fibers of all classes <8 pm in length decreased over time. In contrast, fiber number in the 8- to 16-p.m class did not change significantly up to 29 days postexposure, and the number of fibers greater than 16 pm in length increased significantly. It is likely that two phenom ena explain these results: (1) The clearance rate is inversely related to fiber length. (21 fibers undergo longitudinal splitting over time. Figure 6 shows that average fiber diameter decreases over time within each length class, consistent with longitudinal splitting of all length classes of fibers. In order to correct for longitudinal splitting, we calculated fiber mass within each length class and plotted clearance curves (Fig. 7). (If fibers split only longitudi nally. the mass within each length class will be conserved. This is an approxima tion. since transverse splitting of fibers probably occurs as well--see Discussion.) For each length class we calculated a clearance rate and half-life based on a one-compartment model. The clearance half-lives increased with increasing fiber length: --10 days for fibers 0.5-4 pm. increasing to 114 days for fibers 2*16 pm. Statistically, the clearance rate for fibers greater than 16 pm was not significantly different from zero (half-life infinity). These results are summarized in Fig. 8. DISCUSSION Deposition in Peripheral and Central Regions Bfhe lack of differentiaLdepositiop between peripheral and central regions of the lung was surprising. On the basis of two prevfous~experimental findingSTwe expected that there would be greater deposition in the central than in the periph eral region. First, deposition of chrysotile asbestos occurs preferentially at first alveolar duct bifurcations (Brody et ai., 1981). and we expected there to be a higher density of these bifurcations in central compared to peripheral tissues. Second, asbestos fiber deposition in a given region of the pulmonary parenchyma is inversely related to the pathlength (as measured from the carina) of the airway supplying that region (Pinkerton et al., 1986). and we expected the average airway pathlength to be greater in the peripheral than in the central region. In view of the present experimental findings, we examined histologic sections from rat lungs and found that first alveolar duct bifurcations can occur within 0.5 mm of the visceral pleura. Therefore, our dissection scheme is too crude to exclude many first duct bifurcations from "peripheral" tissue. A quantitative assessment of the density of first alveolar duct bifurcations in peripheral versus central regions would require morphometric analysis. Similar considerations may apply to airway pathlength. The dissection scheme used here does not reveal differences in pathlength between peripheral and central regions. Total (0 day postexposure) and pulmonary (1 day postexposure) deposition of chrysotile were similar (Table 3). indicating that little, if any. of the chrysotile burden measured represents fibers deposited in the ciliated airways. #anslocation DEPOSITION OF CHRYSOTILE IN RAT LL'NG 107 This study provides no evidence for the translocation of chrysotile fibers to ward the peripheral region or for preferential clearance of fibers from one region. Sensitivity analysis (see Results) suggests that, if translocation occurs, the rate can be no greater than 209? of the overall clearance rate. This finding does not agree with that of Morgan et at. (1977). who observed subpleural concentrations of asbestos in rats exposed by inhalation. However, there are significant differ ences between that study and the present one. Morgan et at. (1977) employed amphiboles (crocidolite and a synthetic fluoramphibole). and the observations of subpleural hot spots were made at 104 and 139 days postexposure, three to four times the follow-up of our study. Translocation may be a slow process, and our study may not have had sufficient follow-up time to detect it. Furthermore, some experimental work suggests subnleural-i reaction chrysoffle OOghisio et In addition, our dissection and digestion technique cannot differentiate between translocation from central to peripheral regions and slower clearance from the peripheral region relative to the central. In both cases, the ratio of peripheral to central fiber burden should increase over time. Likewise, autoradiographic tech niques such as those used by Morgan et at. (1977) suffer from the same limitations. An area of intense autoradiographic signal at one time point cannot be compared to the signal at another time point if the development times of the autoradiographs are different, as was the case in that study. Only a relative comparison of signal within a single autoradiograph is appropriate. Therefore the hot'spots'observed in that study may represent areas of slower clearance of deposited fibers and not ^^Kcentrations of fibers translocated from other regions. These hot spots may ^Wrespond to asbestos-induced inflammatory lesions observed by others (Oghiso et at.. 1984). Another limitation of the present work is the possibility that translocation of particles to the pleural regions occurs only at lung burdens much higher than those achieved here. Translocation of particles to the hilar lymph nodes of the rat increases when clearance mechanisms are overloaded at lung burdens of approx imately 1 mg (Ferin and Feldstein. 1978: Strom et at.. 1989). Particle translocation to the pleura may occur through the lymphatics (Hillerdal. 1980). so these findings may be relevant to the present work. Our study used a very modest exposure to chrysotile (10 mg/m3 for 3 hr), which gives an initial lung burden of -- 10 |ig in the left lung (Table 2) or --30 p.g for all lobes of the rat lung. This is well below the burden of asbestos (--1.5 mg) associated with clearance overload in the rat (Bol- Ftc. 3. Micrographs of lung digests. (A) Scanning electron micrograph (SEMi oflung digest from an animal sacrificed I day postexposurc. Many short (<5 pm) fibers are present. Average fiber diameter is -0.13 pm. Note the long, wide (length >20 pm. diameter -0.5 pm) fiber at the top of the micro graph. (Bl SEM of lung digest from an animal sacrificed 29 days postexposure. Fewer short (<5 pmi fibers are present (arrows) than at I day. Average fiber diameter is -0.09 pm. Note the long, narrow (length >20 pm. diameter -0.1 pm) fiber at the top of the micrograph. 108 COIN. ROGGLI. AND BRODY Fig. 4. Clearance in terms of mass. The points represent geometric means across animal: left lobe burden. Mass was calculated according to Eq. (21 in the text. The curve is a fit c compartment model to- the data: ' MU) = Mu[ae' f II - ale" Here .Wit) is fiber mass as a function of time, Ma is the initial burden, a is the fraction of mass di m compartment I. , is the clearance constant for compartment I. and ft, is the clearance concompartment 2. The best-fit model has values a = 0.45. k, => 0.24 (half life 3 days), = 0.C life 20 days). The parameters were derived by least-squares fit to the log-transformed mass b The value of ,W,, was forced to the geometric mean of the initial value (8.7 p.g). The geometn of fiber mass in the four control animals was 0.11 jxg. ton et /.. 1983). Note, however, that Morgan et al. (1977) observed "p drift" after modest inhalation exposures (a maximum of 50 mg/nr' for 30 giving initial lung burdens on the order of 10-20 (jcg, comparable to the bu obtained in our study. Clearance The clearance rate of chrysotile fibers is inversely related to fiber length, clearance rate of fibers greater than 16 p.m in length is very low and not si, cantly different from zero in this study. These observations would have been difficult without the use of the length-stratified counting technique. Asbi fibers have a lognormal length distribution. Proper description of such a dist tion requires that either a great number of fibers be measured or that a strat sampling scheme be used. For example, in typical lung digests, fibers of le greater than 16 p.m constitute 0.3% of the total number present. Our strat counting scheme ensures acceptable statistical accuracy by obtaining a count least 20 fibers in this class. Using this scheme, characterization of the le distribution between 0.5 and 16 p.m requires counting between 120 and 240 fi (six length classes x 20-40 fiber ends/class). In contrast, achieving this levt accuracy by measuring every fiber encountered in a random search would req DEPOSITION OF CHRYSOTILE IN RAT LUNG 109 2sL<4pm 4 s L < 8 pm & z3B 10 > 8 < L < 16 pm r 19 P 46 L 216 pm 10 = 0 10 20 30 days post-exposure Fig. 5. Fiber number by length class. The points plotted are geometric means of fiber number within a length class. The lines arc linear regressions of log-transformed fiber number for each animal. These can be interpreted as fits of a one-compartment clearance model for each length class: <V,U) = ,V,,,e ' * where N,U) is the fiber number in length class i as a function of time in. Sk, is the initial pulmonary deposition in lengthclass i. and k, is a clearance constant. Statistical significance ip I and correlation (n are shown with each graph. Fiber number is decreasing significantly over time for each length class under 8 pm. For the class 8-16 pm. fiber number is not changing significantly over time. For the class s> 16 pm. fiber number is increasing significantly over time. The geometric mean of total fiber number m control animals was 7 x |0\ For each length class the control value was well below that of the exposed animals. For example the mean control value for fibers s> 16 pm was 3 x |0\ less than `/'uxi the value in exposed animals. no COIN. ROGGL1. AND BRODY 0.S S L < 1 pm 02 1 L < 2 pm 02t 0 1 o.H 0.0 o 02 10 20 30 2 L < 4 pm 0.0 0 02 10 20 30 4 < L < 8 um 3E 01 w0) OJ EIQ 00 '5 0 <u ' Cw<4OUQ>) <> 10 20 30 8 < L < 16 um 01 00 0 10 20 30 L >16 pm 02 00 00 '0 20 30 0 10 20 30 days post-exposure Fig. ft. Average fiber duimeter by length class. Average fiber diameter was calculated aero mills for each length class according to a modification of Eq. (4). The plotted points are anti means across animals. measurement of approximately 7000 Fibers. (The total fibers counted to give the >16 p.m class is =20/0.003 = 6700.) In addition, the length-dependent clearance observed here is not a resu overload of the pulmonary clearance system. The lung burdens achieved in study are much lower than those associated with clearance overload (see dis sion above under Translocation). Interpretation of the clearance curves is complicated by the tendenc DEPOSITION OF CHRYSOTILE IN RAT LUNG 0.5 L < 1 um 1 L < 2 um 111 L >16 urn days post-exposure Fig. 7. Fiber mass by length class. The points plotted are geometric means of fiber mass within a length class. The lines are linear regressions of log-transformed fiber mass for each animal. These can be interpreted as fits of a one-compartment clearance model for each length class: M,ln = A/,,,r where M,U) is the fiber mass in length class i as a function of lime (r). .Vf,,, is the initial pulmonary deposition in length class i. and k, is a clearance constant. Statistical significance (pi and correlation (r) are shown with each graph. Fiber mass is decreasing significantly over lime for each length class under 16 pm. chrysotiie fibers to split. The length and diameter of a fiber may change during its residence in the lung, independent of any mechanical clearance to the tracheo bronchial region. Thus, splitting may alter the apparent clearance rate of fibers within a certain length class. We corrected for longitudinal splitting (which will 112 COIN, ROGGLI, AND BRODY Flo. 8. Summary of clearance rate by fiber length. The points plotted are values of tf compartment clearance model based on the mass curves of Fig. 7. The value of fiber length pl< the lower end of the length class. The error bar represents the standard error of the estimate bs the linear regression. The scale on the right shows the half life (h j coresponding to the cle constant (At on the left-hand scale (r,, = In2/Al. not change the length of a fiber) by calculating clearance rates based on fiber within each length class. This correction was successful for the fibers greater 16 p.m in length. Their clearance rate (based on fiber mass) is near zero, sugge that a great deal of transverse splitting did not occur. On the other hand clearance rate of the 0.5- to 1-p.m length class was somewhat lower than expt based on the trend of clearance rate versus fiber length (Fig. 8). This anom; probably due to splitting of larger fibers into fragments of length 0.5-1 |xm. Other experimental work concurs with our finding of length-dependent c aoce-. Many workers have noted that the average length of fibers retained ii lung increases over time in experimental animals (Kimizuka et al.. 1987; R and Brody. 1984: Roggli et al.. 1987). Such changes in size distribution are sistent with length-dependent clearance. Clearance of amosite from the luni hamsters shows a dependence on fiber length almost identical to that found ii present study (Coin et al.. 1990). Morgan et al. (1982) found that preparatiot glass fibers between 5 and 10 p.m in length were cleared from the rat lung half-lives of 60-90 days, while fibers of 30 p.m length were not cleared. Tim (1981) compared size distributions of anthophyliite fibers in aerosol samples that in the lungs of exposed humans and concluded that fibers greater than 1" in length were not cleared. Pulmonary clearance of isometric particles also depends on particle size experimental animals, isometric particles greater than 7-9 p.m in diameter cleared slowly, if at all. from the pulmonary region (Snipes and Clem. 1 Snipes et al., 1984). This is likely due to the inability of pulmonary alve macrophages (PAM) to phagocytize and/or transport these panicles. Mor (1988) suggests PAM may become immobilized after engulfing more than a crii DEPOSITION OF CHRYSOTILE IN RAT LUNG 113 lume (-60-600 pm3/PAM) of isometric particles. This corresponds to a single (article of between 5 and 11 pm diameter, similar to the clearance limit observed in vivo. On the basis of the present work, we suggest that immobilization of PAM (or failure of phagocytosis) may-occur upon attempted phagocytosis of a particle greater than a certain dimension. In our experiment, chrysotile fibers greater than 16 pm in length are not cleared at a significant rate. These fibers have an average diameter (at deposition) of -0.2 pm, giving a typical volume of only 0.3 pm3, well below the volumetric limit for clearance proposed for isometric particles (Mor row. 1988). Our proposed dimensional limit for the clearance of fibers correlates with the size of PAM. which are 10-20 pm in diameter (Warheit et al.. 1984b: Fels and Cohn. 1986). Possibly PAM cannot engulf fibers which are longer than their own cell diameter, and/or PAM become immobile after phagocytosis of such fibers. Slower clearance of longer fibers may explain the increased pathogenicity of long fiber preparations compared to short (Adamson and Bowden. 1987a, 1987b: Davis et al., 1986; Davis and Jones. 1988). Prolonged retention of asbestos fibers in the lung leads to development of asbestosis in an animal model (Begin and Sebastien, 1989). Thus, other factors being equal, a preparation of long fibers will be retained in the lung for a greater length of time and should cause more fibrosis than a preparation of short fibers. The length dependence of clearance needs to be considered in human studies correlating fiber burden with pathology. For example. Churg and co-workersitaye. found that average fiber length of asbestos.burden is negatjvety~cofrdated with grade of fibrosis (.Churg et al., 1989, 19905". i hey suggest several explanations for obsct vatiou; iiicludiiig-that shorf-fibeigi5fe^nipnrtipTT-geft(^a^iyTif~T7brri- K" or alternatively, that "preferential retention of short fibers would have to ur" inareas offibrosis. The results of our work and that of others suggest the latter explanation is plausible. We showed that fibers greater than 16 pm in length were cleared very slowly, if at all. This length dependence of clearance occurred in the absence of extensive pulmonary fibrosis and inflammation, which may inhibit clearance (Tryka et al., 1985; Slauson et al., 1989). The magnitude of asbestos exposure used here produces lesions only at the alveolar duct bifurca tions and does not cause generalized pulmonary inflammation (Brody et al.. 1981: Warheit et al., 1984a; Chang et al., 1988). Furthermore, in other animal models of asbestosis, generalized pulmonary fibrosis and suppression of clearance (over load) occur at much higher lung burdens (--1.5 mg) (Bolton et al., 1983) than those achieved here (--30 pg). Therefore, if any suppression of clearance occurs due to fibrosis or inflammation it will affect primarily the clearance of shorter fibers, since longer ones are not cleared, as discussed above. This means that suppres sion of fiber clearance for any reason will cause a decrease in the average length of retained fibers. Experimental results cannot rule out the possibility that, in humans, short fibers are important in causing fibrosis (Churget al., 19&>. tV9U). It is quite possible, as suggested by others (Chutg-ef u/r-I990TGoodglick and Kane, 1990), that short fibers contribute to fibrosis and other diseases once inflammation and/orTmpair- 114 COIN. ROGGLI. AND BRODY ment of clearance occurs. This hypothesis could be tested experiment pulmonary ribrosis, out Has not been addressed to date. For example, could be exposed to a short fiber preparation in combination with an agen produces inflammation and retards clearance of particles. CONCLUSIONS The results of the present study could have implications for the pathoge asbestos-related pleural disease. While we found no evidence of pleural d did find that substantial numbers of inhaled fibers are deposited within 1-2 the visceral pleura of the rat. Many are probably deposited within a fewli micrometers of the visceral pleura. These include fibers greater than 16 length, which are cleared slowly, if at all. Indeed their number increased ov due to longitudinal splitting (Fig. 5). These fibers may-be susceptible,! translocation processes not detectable in this st'udy.-Thus suETpleuraTfibei not'moveiuLorTa'st-in-order to reach the vicinity of tfilTvilcerSflpfeura. Pi thfTregion could cause inflammation in the subpleural parenchyma, thereb ing pleural disease, in concordance with this hypothesis. ongoing work laboratory" indicates that, in mice exposed for 5 hr to 10 mg/m3 of chr) subpleural peripheral mesenchymal cells show enhanced uptake of tritiau midine within 24 hr of inhalation exposure (Coin et al.. 1991). Studies are way to determine if this proliferative response is due to the presence of fil the subpleural layer and/or the diffusion of growth factors released by e: cells at first alveolar duct bifurcations (McGavran et al.. 1990: Bonner 1990). . 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