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Persistence of Long, Thin Chrysotile Asbestos Fibers in the Lungs of Rats Patrick G. Coin/-2 Victor L. Roggli,2 and Arnold R. Brody '3 Laboratory of Pulmonary Pathobiology, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina; department of Pathology, Duke University and Durham Veterans ' Administration Medical Centers, Durham, North Carolina; department of Pathology and Laboratory Medicine, ! Tuiane University Medical Center, New Orleans, Louisiana ! distribution of inhaled mineral fibers in the lung determines the site and severity of disease caused by the fibers. Some of our recent work has ascribed the fate of inhaled asbestos fibers in rodents. After a brief inhalation exposure, asbestos fibers are deposited primarily at the first alveolar | ;jct bifurcations, and fibrotic iesions are initiated. These sites of deposition occur as close to the visceral pleura as 220 pm. Several studies have ; rjggested that short fibers are cleared from the lung more efficiently than long ones, and our data support this view. Our laboratory has shown that I grosolized chrysotile fibers longer than 16 pm can be deposited in the peripheral lung parenchyma of rats, and the measured clearance rate of ; -ese fibers is not significantly different from zero. Chrysotile, but not amphibole, fibers spilt longitudinally, so that the number of retained chrysotile ' -xts 2:16 pm in length increases over time. We have not observed significant changes in chemical composition of chrysotile fibers up to 30 days ;ost-deposriion in the rat. Nor have we observed translocation of chrysotile fibers from the ''central" regions of the lung toward the subpleural ; sgions. However, 1 month after a single 3-hr exposure to chrysotile asbestos, the longest, most pathogenic fibers persist throughout the lung j .-aranchyma. These retained fibers have the potential to cause disease in both parenchyma and pleura. -- Environ Health Perspect 102{Suppl ] ;.:197-199 (1994) >.jy words: asbestos, chrysotile, pulmonary clearance, particle translocation, pleural disease ' Introduction Pulmonary deposition, clearance, alter. ;iion (leaching and splitting), and transloi ration of mineral fibers play important | rotes in determining the sites and severity j if disease caused by these fibers. In this j report, we review some of our recent find' mgs on the fate of inhaled chrysotile isbestos in the lungs of rats. . Deposition ) After brief inhalation exposures in rats and J mice, asbestos fibers are' deposited primarj ly at alveolar duct bifurcations, and J fibrotic lesions are confined to these sites 1,2). Recently our laboratory has been i investigating the mechanisms of asbestos' related pleural disease. We have sought to j :ti.dy the differential deposition of rhrysotile aerosols in peripheral (sub pleural) and central regions of the rat lung 3). This study showed no differential deposition of fibers in central versus , '*'s paper was presented at the Workshop on .- : operatefence of Respirable Synthetic Fibers and 1 '.'nerals held 7-8 September 1992 in Lyon, France. J This work was supported by the Department ot : -aeons Affaire Medical Research Funds. Address correspondence to Dr. Patrick G. Coin, oiroralofy of Pulmonary Pathobiology, National 'thtute of Environmental Health Sciences, Research 'angle Park. NC, 27709. Telephone 1919) 541-4410. !(919)5414133. peripheral regions. Indeed, even the longest fibers classified (16 pm) were deposited abundantly in the peripheral region within 1 mm of the visceral pleura. We attempted to reconcile this somewhat surprising finding with previous observa tions showing that deposition occurs largely at the first alveolar duct bifurca tions (/). Thus, we measured the distance from first alveolar duct bifurcations to the nearest visceral pleural surface in tissue sections from the caudal half of the left lung of rats (Figure 1). Roughly half of all first-duct bifurcations are found within 1 mm of the visceral pleural surface, which corresponds to the peripheral tissue resected and digested in our previous study (J). The minimum bifurcadon/pieural dis tance measured was just over 200 pm. These findings show that fibers of the putatively most carcinogenic sizes [10 pm length, <1.5 pm diameter (4)] can be deposited readily within a few hun dred microns of the visceral pleura of the rat. The significance of this finding is dis cussed below. Clearance Several laboratories have shown that, for modest exposures, a large proportion of the mass of inhaled asbestos is cleared from the lungs of rodents within 30 days (3-7). Given the well-known role of fiber dimensions in both pulmonary fibrosis and pleural carcinogenesis (4,3-10), description of pulmonary clearance kinet ics in terms of fiber mass is not adequate. There is now sufficient experimental data to describe the differential clearance of asbestos fibers in terms of fiber length. Several workers have noted that the aver age length of retained asbestos fibers increases after an inhalation exposure (5,6,9-11). These experiments suggest that, qualitatively, short asbestos fibers are cleared from the lung more effectively than long fibers. However, few workers have been able to quantify the dimensions of "short" versus "long" fibers. Morgan et al. (12), employing monodisperse fiber glass, showed that fibers greater than 30 pm in length are not cleared read ily from the lung. Recendy, a novel statis tical sampling scheme has been employed to show that asbestos fibers greater than 16-20 pm in length are not cleared effec tively from the lungs of racs or hamsters (3,13). These findings are interesting in light of work suggesting that longer mineral fibers are more fibrogenic than short fibers (9,10,14,15). Our findings show that the longest, most fibrogenic fibers are retained in the lung for extended periods. The slower clearance of the longer fibers may partly explain their enhanced fibrogenicity. t :nvonmental Health Perspectives HWBU10009151 GpIMETAL. I Ii distance to pleura (mm) Figure!. Distribution of alveolar duct bifurcation/pleural distances. Distances from first-duct bifurcations to the nearest visceral pleura! surface were measured on tissue sections of the caudal half of the left lung lobes of 11 adult rets. A total of 106 bifurcations was measured. Lungs were fixed intrafraeheally. giving comparable infla tion to that of the lungs used for peripheral/centra! digests (3). Approximately 50% of first-duct bifurcations are within 1 mm of the visceral pleura. The minimum bifurcatiWpteural distance measured was 0.22 mm. Therefore, resected 'peripheral' tissue 13) should contain substantial numbers of asbestos fibers deposited at first duct bifurcations. Alteration Asbestos fibers can undergo longitudinal and transverse splitting in the lung. The former causes a decrease in fiber diame' ter, while the latter causes a decrease in fiber length. Chrysotile fibers readily undergo both types of splitting in the rodent lung, while amphibole fibers do not {3,5,6,16, 17). We have found that long C>16 pm) chrysotile fibers are not cleared at a significant rate up to 30 days after deposition in the rat, but that they do split (3). The number of retained long fibers increased over time, and the aver age diameter decreased (Table 1). The mass of retained long fibers decreased slightly over time while the surface area increased slightly, though neither trend was statistically significant (Table I). These findings suggest that, in our model, long fibers undergo longitudinal splitting but little transverse splitting. In contrast. Churg et al. {17) found that chrysotile split both longitudinally and transversely within 30 days of instillation into the lungs of guinea pigs. Our find ings are not necessarily at odds with those. We did find evidence of transverse splitting of fibers shorter than 16 pm (3). In addition, Churg et al. classified fibers only up to a maximum >10 pm in length, and they did not compensate for longitu dinal splitting within a length category as we did (3). Our previous work has shown that there is a marked change in the fate of fibers >16 pm in length as compared to shorter fibers, probably due to the inability of pulmonary macrophages to phagocytize these fibers (3). This failure of phagocytosis may play a role in split ting of the longest fibers. Leaching of magnesium from chrysotile fibers has not been observed consistently in the lungs of experimental animals. We have found no significant leaching of long chrysotile fibers after 30 days residence in the rat lung (Table 1). Similarly, Churg et al. (17) did not observe leaching of chrysotile after 30 days residence in the guinea pig lung. Others have observed leaching of chrysotile fibers after 1 month to 2 years of residence in the lungs of hamsters and rats (11,16)- Bellmann et al. (16) found a decrease of 1 to 2% per day in the Mg/Si ratio of chrysotile fibers resident in the rat lung for 30 days. Calculations of sta tistical power show that our experiment had an 80% chance of detecting a decrease in Mg/Si of 1.3% per day. It is possible that the discrepancy arises from the restriction of our analysis to fibers >16 pm in length, which are not likely to be phagocytized completely by macrophages (3) and undergo leaching in the acidic environment of phagolyso somes (IS). Bellmann et al. (76) did not specify the lengths of the six fibers selected for chemical analysis in each sample, but 90% of the fibers in the instilled suspension were <3 pm in length. Translocation Postdepositional movement (translocation) from parenchyma to pleura is believed to be important in the genesis of asbestosinduced pleural disease (19), but there have been few experimental studies on the subject. Morgan et al. (20) exposed rats briefly to aerosols of radiolabeled amphi bole asbestos and found that the label was concentrated in "hot spots" adjacent to the visceral pleura, 100 or more days post exposure. They suggested that these con centrations were due to translocation of fibers from the central regions of the lung toward the peripheral (subpleurai) regions, a so-called "pleural drift." In contrast, we have found no evidence for pleural drift after inhalation of chrysotile asbestos in the tat (3). However, there were important differences between the two studies. Morgan et al. (20) employed amphibole asbestos, and the subpleurai accumulations of labeled fibers were observed more than 100 days postexposure, three rimes longer than the follow-up of our study, which may not have given time to detect a slow translocation process. In addition, neither our study (3), nor that of Morgan et al. (20) can distinguish between translocation from central to peripheral regions, and slower clearance from the peripheral rela tive to the central region (3). Extensive and rapid translocation ot asbestos may not be necessary for the devel opment of pleural disease. Our studies of deposition and clearance show that the longest, possibly most pathogenic fibers arc deposited near the visceral pleura and retained there for long periods of rime. These fibers could be translocated to the pleura by slow processes not detectable in our study (3). In addition, asbestos fibers in the pulmonary parenchyma cause the release of growth factors and other media tors (21), which could have an effect on the nearby pleura (22,23). Asbestos deposited at alveolar duct bifurcations has a mitogenic effect on endothelial and smooth muscle cells of small pulmonary vessels. )and Coricfi : In our n ! deposits I bifurcaci' few hunc The dept Bro inh eph 123 Ch; rcac to a 3. Coi trar regi 4. Scat Smi amp 67: 5. Rog chi) sure 6. Rog char '~wi 8. Jaur care. thei 797- 9. Dav Smis amo tone 10. Dav. and 69:7 11. Kim alter J To 12. Mor frorr 25:3 Table 1. Alteration of chrysotile fibers 216 pm in length. Pays postexposure 18 15 fiber number Average fiber diameter, pm fiber surface area, mm* fiber mass, pg Mg/Si mean 3.1x10s* 0.191 4.4 0.65 0.75 0.04 32 x10s 0.150 3.6 0.44 Not measured 6.6 x10s 0.133 6.6 0.69 Not measured 29 5.1x10s 0.125 4.8 0.47 0.67 0.08 Correlation, r +0.56 -0.82 +0.23 -0.17 Statistical sianrficance pcOJE* p<0.01* p-0.35 p=0.50 p>0.80(Mest) Values in the table are geometric means for fibers 216 pm length in digests of the entire left lung of four or more animals. See Coin et al. |3| for experimental details. For detemna tion of Mg/Si ten fibers 2 02 pm in diameterwere examinedfrom each of four animals at 1 and 29 days postexposure. Arithmetic mean and standard error are shorn in the table, ft* Mg/Si ratios were compared between time points with a f-tesl4 These parameters showa statistically significant (p 0.05) trend with time. 198 Environmental Health Perspectives volwne 10 HWBUI0009152 PERSISTENCE OF CHRYSOTtLE IN THE RAT LUNG os--- there on the osed rats i amphilabei was nt to the ys postese con ation of the lung regions, trast, we ral drift os in the iportanc studies, tphibole .ulations >re than s longer , which : a slow neither n et al. ocacton u, and al rela- -dies of rat the jers are ra and : time, to the able in : fibers ise the mediaect on jestos shasa mooth essels. and this effect presumably is mediated by cytokines or other diffusible factors (24). Conclusions In our model, inhaled asbestos fibers are deposited largely at first alveolar duct bifurcations, many of which are within a ` few hundred microns of the visceral pleura. The deposited fibers include many >16 pm in length and <1 pm in diameter, within the range considered most pathogenic. These fibers are cleared slowly, if at all. Long chrysotile fibers undergo longitudinal splitting in the lung, so that their number actually increases over time, possibly increasing their potential for biologic effects. Even though extensive splitting of chrysotile fibers occurs, we have not observed substantial leaching of magne sium from chrysotile fibers up to 30 days after deposition. Translocation of chry sotile from deep parenchymal regions toward the subpleurai regions of the lung does not occur in our model. Extensive translocation, however, may not be neces sary for the development of asbestos-related pleural disease. REFERENCES 1. Brody AR, Hill LH, Adkins B, O'Connor RW. Chtysotiie asbestos inhalation in rats: deposition pattern and reaction of alveolar epithelium and pulmonary macrophages. Am Rev Respir Dis j 123:670-679 (1981). I 2. 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