Pulmonary and Pleural Responses in Fischer 344 Rats Following Short-Term Inhalation of a Synthetic Vitreous Fiber

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1 FUNDAMENTAL AND APPLIED TOXICOLOGY 30, (1996) ARTICLE NO Pulmonary and Pleural Responses in Fischer 344 Rats Following Short-Term Inhalation of a Synthetic Vitreous Fiber I. Quantitation of Lung and Pleural Fiber Burdens THOMAS R. GELZLEICHTER, EDILBERTO BERMUDEZ, JAMES B. MANGUM, BRIAN A. WONG, JEFFREY I. EVERTTT, 1 AND OWEN R. MOSS Chemical Industry Institute of Toxicology, P.O. Box 12137, Research Triangle Park, North Carolina Pulmonary and Pleural Responses in Fischer 344 Rats Following Short-Term Inhalation of a Synthetic Vitreous Fiber. I. Quantitation of Lung and Pleural Fiber Burdens. GELZLEICHTER, T. R., BERMUDEZ, E., MANGUM, J. B., WONG, B. A., EVERTTT, J. I., AND Moss, O. R. (1996). Fundam. Appl. Toxicol. 30, Received March 14, 1995; accepted September 27, 1995 The pleura is an important target tissue of fiber-induced disease, although it is not known whether fibers must be in direct contact with pleural cells to exert pathologic effects. In the present study, we determined the kinetics of fiber movement into pleural tissues of rats following inhalation of RCF-1, a ceramic fiber previously shown to induce neoplasms in the lung and pleura of rats. Male Fischer 344 rats were exposed by nose-only inhalation to RCF-1 at 89 mg/m 3 (2645 WHOfibers/cc),6 hr/day for 5 consecutive days. On Days 5 and 32, thoracic tissues were analyzed to determine pulmonary and pleural fiber burdens. Mean fiber counts were 22 X 10*/lung (25 X lovpleura) at Day 5 and 18 X 10*/lung (16 X lovpleura) at Day 32. Similar geometric mean lengths (GML) and diameters (GMD) of pulmonary fiber burdens were observed at both time points. Values were 5 /xm for GML (geometric standard deviation GSD «2.3) and 0.3 /xm for GMD (GSD» 1.9), with correlations between length and diameter (r) of Size distributions of pleuralfiberburdens at both time points were approximately 1.5 ixm GML (GSD «2.0) and 0.09 fim GMD (GSD =» 1.5; r» ). Few fibers longer than 5 ixm were observed at either time point These findings demonstrate that fibers can rapidly translocate to pleural tissues. However, only short, thin (<5 itm in length)fiberscould be detected over the 32-day time course Of the experiment. C 1996 Society of Toxicology Concern over asbestos-related health issues has led to a gradual reduction of asbestos production and use in developed nations, with replacement by a variety of synthetic vitreous fibers such as fiberglass, refractory ceramic fibers, rock, and slag wool (Neisel and Verschoor, 1981). Since many of these new replacement fibrous materials share cer- 1 To whom correspondence should be addressed. Fax: (919) tain physical and chemical properties with asbestos, the need to understand the basic mechanisms involved in the development of fiber-induced diseases continues. Human exposure to certain natural mineral fibers such as amphibole asbestos results in a constellation of inflammatory, fibrogenic, and neoplastic diseases of the lung and pleura (Wagner et ai, 1960; Selikoff et ai, 1979). Mesothelioma, a highly fatal tumor of the serosal lining of the thoracic and peritoneal cavity, is one of the prominent asbestos-induced neoplasms found in the occupational and environmental setting, and its incidence is on the rise (Lilienfeld et ai, 1988; Walker et ai, 1983). Size, durability, and surface properties are all known to affect the carcinogenic potency of inhaled fibers. However, the underlying mechanisms of neoplastic transformation in the lung and pleura are still poorly understood (Walker et ai, 1992). Biopsy samples from both pleural plaques and mesotheliomas alike demonstrate the presence of asbestos fibers in patients with asbestos-related pleural disease. However, studies involving asbestos-exposed populations have demonstrated that incidence of pleural plaques and mesothelioma, while clearly linked to asbestos exposure, does not necessarily correlate with pulmonary fiber burdens or degree of asbestosis (Hirsch et ai, 1982; McDonald and Davis, 1988; Sebastien et ai, 1975). Thus, despite numerous epidemiological studies on asbestos-exposed workers, no clear relationships between exposure, dose, and development of pleural disease have been established. Since quantitative data on pleural fiber burdens are lacking from epidemiological studies, the relationships between pleural fiber burdens, incidence, and severity of pleural disease remain unknown. Many types of nonasbestos fibers and particles have been shown to induce mesothelioma in rodent species when instilled directly onto target mesothelial cells via intrapleural instillation (Stanton et ai, 1981; Pott, 1978; Wagner et ai, 1985). However, many of these materials (e.g., fiberglass) are not mesotheliomagenic when animals are exposed by the % $18.00 Copyright O 1996 by the Society of Toxicology. All rights of reproduction in any form reserved.

2 32 GELZLEICHTER ET AL inhalation route (World Health Organization, 1988; Hesterberg et ai, 1993). In light of these findings, the ability of fibers to induce pleural disease may be, in part, a function of their ability to migrate to and accumulate on mesothelial surfaces of the pleura. In the absence of dosimetric data on pleural fiber burdens, however, this relationship is unknown. The interpretation of laboratory animal data for the assessment of human health effects is difficult due to the fact that a paucity of information is available concerning how inhaled materials reach the pleural tissues of man and animals. Different types of mineral fibers have differing pleural carcinogenic potencies that do not necessarily correlate with their ability to induce neoplasms in the lung. This finding is exemplified by erionite, a natural zeolite that is extremely mesotheliomagenic, following inhalation in humans and laboratory rodents alike (Rohl et ai, 1982; Sebastien et ai, 1981; Wagner et ai, 1985). The reason for the potency of erionite is unknown. Amphibole types of asbestos such as crocidolite demonstrate a higher mesotheliomagenic potential than does chrysotile asbestos, a serpentine fiber with different physical properties. In analyses of pleural tissues in asbestos-exposed workers, the amphibole fraction tends to contain significant numbers of fibers longer than 5 /zm, while retained chrysotile fibers tend to be exclusively short (Dodson et ai, 1989; Sebastien et ai, 1980). Based on these findings, one might hypothesize that increased pleural migration of long amphibole fibers may be responsible for the enhanced mesotheliomagenic potential of this type of asbestos. Other studies, however, have failed to detect size differences in fibers recovered from pleural and pulmonary tissues (Kohyama and Suzuki, 1989; Churg et ai, 1984). Direct comparisons of experimental results from independent studies are confounded by the employment of different fiber recovery methods. Researchers have successfully isolated pleural plaques and tumors from humans for characterization of deposited fibers. However, surgical isolation of pleural and subpleural tissues is exceedingly difficult without also sampling parenchymal areas of the lung. These technical difficulties are most pronounced in rodents, where the pleura is extremely thin (Coin et ai, 1992). The goal of the present experiments was to study pleural tissues as a distinct pulmonary compartment, separate from the underlying lung parenchyma, for determination and characterization of fiber burdens. Since it is not known if fibers need to be in direct contact with pleural tissues to exert pathologic effects, a secondary goal was to determine the temporal relationship between the migration into and accumulation of fibers in the pleural compartment and the advent of pleural pathobiologic responses. We isolated and characterized fiber burdens from Fischer 344 rats exposed to refractory ceramic fibers (RCF-1), a kaolin-based material known to induce both pulmonary tumors and mesotheliomas in fiber-exposed rats following inhalation (Bunn et ai, 1993: Mast et ai, 1995). METHODS Animals. Male CDF (F344)/CrlBR Fischer rats (Charles River Breeding Laboratories Inc., Raleigh, NC) free of antibody to munne mycoplasmal and viral respiratory disease and weighing g were used for all experiments. Rats were held for a minimum of 10 days prior to use. Exposures. The RCF-1 fiber samples used in these studies were supplied by the Fiber Repository of the Thermal Insulation Manufacturer's Association (TIMA) Inc. (Stamford, CT) and consisted of a size-separated preparation enriched for respirable-sized fibers. The chemical composition has been characterized and previously reported (Mast et al., 1995). Rats were exposed to RCF-1 in closed-faced, nose-only tubes for 6 hr per day from 8 AM to 2 PM for 5 consecutive days on a Cannon exposure tower (Cannon et ai, 1983). The time-weighted average fiber aerosol concentration throughout the experiment was 89 ± 30 mg/m 3 and ranged from 57 to 164 mg/m 3. The aerosol contained 6206 total fibers/cc, of which 2645 fibers/ cc were classified as WHO respirable fibers (lid s* 3, / > 5 /*m, d < 3 ixm. World Health Organization, 1985). During the exposures, fiber concentrations were continuously monitored using light scatter (RAM, Monitoring Instruments for the Environment, Inc., Billerica, MA). Fiber mass was determined from samples captured on open-faced, 0.2-fim polycarbonate filters (Gelman Sciences, Ann Arbor, MI) and sampled directly from animal exposure ports. Aerosol size characteristics were determined from filters sampled at both the beginning and the end of the exposure. Filters were gold-coated (Technics, Hummer V, Alexandria, VA) and visualized with a scanning electron microscope (JEOL Model JSM 840A, Tokyo, Japan). Fibers were defined as particles with a length:diameter aspect ratio greater than 3:1. Fiber lengths and diameters were determined using imaging software (Image-I, Universal Imaging Corp., West Chester, PA) at magnifications of 500X or 2000X for fiber lengths and 2000X for fiber diameters using World Health Organization (WHO) guidelines for sizing fibers by scanning electron microscopy (WHO, 1985). For aerosol samples, size characteristics were also determined for nonfibrous particulates. Room temperature was maintained at F throughout the exposures and relative humidity at 40-60%. For fiber-exposed groups, six rats each were euthanized either immediately following the final exposure or following a 27-day postexposure recovery period and analyzed for the determination of tissue fiber burdens To determine background fiber concentrations, six unexposed rats were terminated and analyzed for determination of pulmonary and pleural fiber burdens. While not on exposure towers, rats were housed in polycarbonate cages with alpha cellulose bedding (ALPHA-dn, Shepherd Specialty Papers, Inc., Kalamazoo, MI) and supplied with both deionized water and NIH07 diet (Ziegler Bros., Gardner, PA) ad libitum Tissue sampling. Rats were killed by exsanguination following an intraperitoneal injection of sodium pentobarbital. Pleural tissues were immediately digested with an agarose/sodium dodecyl sulfate (SDS) mixture (Bermudez, 1994). Briefly, a 2% (w/v) low-melting-point agarose (BRL, Gaithersburg, MD) solution containing 0.1% SDS (Sigma, St. Louis, MO) was prepared and liquefied at 4I C. After the abdominal cavity was exposed, agarose/sds was injected into the pleural cavity with a 16-gauge needle through the diaphragm adjacent to the sternum. Following intrathoracic instillation, the lungs were expanded with 2-3 ml of a liquefied 2% agarose solution by intralracheal instillation, and the agarose/sds mixture was solidified by chilling the rats on ice for 30 min. After removal from the thoracic cavity, the lung and pleural cast were carefully separated and frozen at -20 C for subsequent recovery of fibers. Lung and pleural tissue specimens were freeze-dried, ashed in an oxygen plasma (Plasma Systems, Inc model LTA 504). resuspended in distilled water, and filtered onto QA-fim polycarbonate filters (Nuclepore, Pleasanton, CA). Lung samples were further chemically digested as previously described (Williams et al., 1982). Lung fiber size characteristics were determined as described above for aerosol samples. Pleural fiber size characteristics were determined at a magnification of I0,000x and included only those particles with a length:diameter aspect ratio greater than 5:1 with straight

3 FIBER DOSIMETRY FOLLOWING RCF-1 EXPOSURE parallel edges. For aerosol and lung samples, a minimum of 14 fields (at 2000X magnification) were examined. For pleural samples, a minimum of 1250 fields (at 10,000x magnification) were examined. Computational analysis. Fiber size characteristics were described based on the bivariate lognormal distribution of fiber lengths and diameters (Siegrist and Wylie, 1980; Cheng, 1986). Size distributions were described mathematically by the means and variances of the natural logarithm of the lengths and diameters and the correlation between ln(length) and ^(diameter) (Moss et ai. 1994). The size distributions of fibers were described by isobar plots. Fibers were categorized in a 12 x 12 block fashion as a bivariate function of (In)length and (In)diameter. The geometric mean sizes for each category were described on the x- and y-axes of isobar plots. The z-axes for these plots denote the relative abundance of fibers normalized to Day 5 data with total fiber yield, (*,>>, x.y,) = 100, on that day. a total of 6206 fibers/cc (89 mg/m3) or, when using World Health Organization guidelines (/ > 5, d < 3, lid > 3), 2645 fibers/cc (WHO 1985). Characteristics of Lung Fiber Burdens Physical characteristics of lung fiber burdens are described in Table 2. Fiber burdens were determined from lungs isolated immediately following the final 6-hr exposure on Day 5 and contained an average of 22 X 106 fibers/lung. When given a 27-day recovery period before sampling (Day 32), total lung burdens dropped by 17%, indicating a very slow rate of clearance from the pulmonary compartment. Approximately 103 fibrous particles were also detected in lungs from RESULTS Characteristics of RCF-1 Aerosol A sampling of the RCF-1 aerosol was captured on a polycarbonate filter and is shown in Fig. 1. Most nonfibrous particles were amorphous, while fibrous particles were cylindrical in shape. The size distribution of fibrous and nonfibrous particles from the exposure aerosol is shown in Table 1. The length of fibers ranged from 0.7 to 111 pm with a geometric mean length (GML) of 4.5 //m and a geometric mean diameter (GMD) of 0.56 fim. Nearly half of the particles by count (44%) were nonfibrous. The aerosol contained TABLE 1 Particle Size Characteristics of RCF-1 Aerosol Particle composition Percentage of total particles GML /jm (GSD) GMD /xm (GSD) Tau Fibers Nonfibrous particles (2.38) 2.12 (1.64) 0.56(1.92) 1.11 (1 68) Note. GML, geometric mean length; GSD, geometric standard deviation; GMD, geometric mean diameter, Tau, correlation coefficient for the relationship between In(length) and In(diameter). FIG. 1. Scanning electron micrograph of RCF-1, X2000. Sample was aerosolized and collected from one of the exposure tower delivery ports.

4 34 GELZLEICHTER ET AL. TABLE 2 Fiber Number and Dimensions of RCF-1 Recovered from Lung and Pleura! Samples Parameter Filtered air control RCF-1 Day 5 RCF-1 Day 32 Lung samples Total fibers/lung GML (fim) GSD (length) GMD (/xm) GSD (diameter) Tau 135,000 ± 117, ± ± ± ± ± ,200,000 ± 5,760, ± ± ± ± ± ,400,000 ; t 8,430, : t : t : t : t ± Pleural samples Total fibers/pleura GML (nm) GSD (length) GMD (/im) GSD (diameter) Tau 5070 ± ± ± ± ± ± ,000 ± 16,100* 1.5 ± ± ± ± ± ,700 ± : t t it : t ± 0.21 Note Results are expressed as arithmetic mean values (n = 6 per group) ± 1 standard deviation. * Group with n = 5. Tau values denote mean (± 1 standard deviation) correlation coefficients in ln(length) vs In(diameter). GML, geometric mean length; GDS, geometric standard deviation; GMD, geometric mean diameter. cage control rats; however, nearly all of these were elongated amorphous particles. Isobar plots indicating the relative abundance of fibers as a function of length and diameter are shown in Figs. 2A-2C. The bivariate lognormal size distributions for lung fibers at both the 5- and 32-day time points were nearly identical, suggesting that significant dissolution of fibers during the 27-day recovery period did not occur. Approximately half the fibers isolated from lung tissue were longer than 5 fim in length. The geometric mean fiber length on Day 5 was comparable to that found in the exposure aerosol, whereas the geometric mean fiber diameter was substantially thinner (39% decrease) than that found in the aerosol. Since fiber respirability is largely a function of aerodynamic diameter, thick fibers are more likely to be intercepted in the nose or upper airways. Characteristics of Pleural Fiber Burdens Fiber number and dimensions for pleural fiber burdens are presented in Table 2. The pleural compartment contained dramatically fewer fibers (three orders of magnitude less) than were found in the lung compartment following exposure to fibers. Pleural fiber yield was at its maximum immediately following the exposure on Day 5, whereas yields dropped by 37% by Day 32. The relative size distribution of pleural fibers are shown in Figs. 2D-2F. No significant shift in the bivariate lognormal size distribution was observed over time. However, fibers were much smaller than those found in the pulmonary compartment at both time points. There appeared to be a strict size limitation, whereby only those fibers shorter than 5 ^m in length were capable of migration to the pleural compartment within the time frame of these experiments. DISCUSSION The development of malignant mesothelioma has been presumed to develop following the accumulation of carcinogenic mineral fibers in the serosal tissues of the pleural and peritoneal cavities, although no information presently exists concerning how this might occur. Asbestos fibers are known to be transported widely throughout the body in lymphatics and blood following inhalation exposure and are found at autopsy in a wide variety of organs in individuals who have asbestosis (Auerbach et al., 1980). In the present study, size characteristics of pleural fiber burdens indicated that physical limits restricted the size of fibers that rapidly move from the lung to the pleural compartment. Only those fibers shorter than 5 fim in length and thinner than 0.35 ^xm in diameter were found in pleural tissues during the course of these experiments. This finding is in agreement with previous studies (Viallat et al., 1986; Viallat, personal communication) involving asbestos-exposed rats in which similar-sized fibers were recovered from pleural lavage fluid. Upon examination of the pulmonary fiber burdens, it is evident that fibers were excluded from the pleural compartment as a function of both length and diameter. In pulmonary tissues, significant numbers of fibers were thinner than 0.35 fj,m and longer than 5 ^m but did

5 FIBER DOSIMETRY FOLLOWING RCF-1 EXPOSURE 35 RCF Aerosol i 8.4- l Day 0 pleura Mean flber number per pleura - 5 x 10 3 d o d o " z «P * - ^ IN m o o o o B 8.4- I i Day 5 lung Mean fiber number per lung- 22 x 10 I I I I I I I I I I I CM CO Day 32 lung Mean f&or number per lung - 18 x 10,6 i i i i i i i i Day 5 pleura 0.34 x10 j) «t in n *- CJ a m *- cy *r r~,_: o <-> o o o d Day 32 pleura Mean tber number per pleura - 16 x 10 FIG. 2. Relative size distribution of fibers isolated from (A) aerosol, (B) Day 5 lung, (C) Day 32 lung, (D) Day 0 pleura, (E) Day 5 pleura, and (F) Day 32 pleura. Isobars were calculated based on histogram analysis of the aerosol cloud and fiber burden data (average of six rats). Median bin values are shown on the x- and v-axes. Pulmonary and pleural fiber burdens were normalized to Day 5 data with areas under the curve equal to 100 on Day 5. not appear to migrate to the pleura. Similarly, significant numbers of fibers were thicker than 0.35 /xm and shorter than 5 (im but were also not found in the pleura. It is of interest to note that these same size restrictions also appeared to apply to the very small number of fibers found in pleural samples from nonexposed rats. The average number of fibers recovered from pleural tissues immediately following the 5- day exposure was low with only 25,000 fibers/pleural sample. Given the small size of pleural fibers, only nanogram amounts were present in the pleural samples. Clearly, an extremely small fraction of the respired fiber dose translocated to the pleura at these early time points. The movement of fibers to the pleura also appears to be a dynamic process since pleural fiber burdens rapidly decrease following a 27- day recovery period. The mechanisms by which fibers are excluded from the pleura are most likely related to physical limitations imposed during migration. However, the pathways by which fibers migrate to the pleura are poorly understood. Oberdorster et al. (1988) showed that intrabronchially instilled amosite in dogs rapidly migrates to thoracic lymph nodes and was found in postnodal lymph fluid collected from the right lymph duct.

6 36 GELZLEICHTER ET AL. They noted a length cutoff of 16 fim in the nodes and 9 /xm in lymph fluid. Diameters appeared limited to <0.5 /xm in both lymph nodes and lymph fluid. These workers proposed that fibers may translocate through the pleural lymphatic plexus or be systemically distributed through the vasculature, although such pathways could only account for a small fraction of total pulmonary clearance. If lymphatic transport is involved, this would help to explain the size restrictions imposed on pleural translocation. However, it remains to be demonstrated that such pathways are involved in pleural migration of fibers. Lehnert et al. (1988) demonstrated that 2-fim polystyrene microspheres, while capable of rapid migration to tracheobronchial lymph nodes, did not reach the pleural space following intratracheal instillation in rats. Other routes (Hillerdal, 1980) involve movement across the visceral pleura and subsequent drainage by parietal lymphatics. Additional research is obviously needed to resolve these questions. The physical restriction of fiber movements to the pleural compartment may serve to protect against the advent of pleural disease. Using intrapleural implantation methods, Stanton and co-workers (1981) first demonstrated that fiber geometry greatly affects the mesotheliomagenic potential of fibers in rats, with potency being positively correlated with fiber length and negatively correlated with fiber diameter. They found that long, thin fibers (length =*8 ^m, diameter s 0.25) had the greatest mesotheliomagenic potential. Since then, similar results have been reported following intrapleural and intraperitoneal injection of fiber suspensions in rodents (Pott, 1978; Davis et al., 1991). Davis et al. (1986a,b) found that if fibers were restricted to sizes smaller than 5 fxm in length, the incidence of mesothelioma following intraperitoneal injection was dramatically reduced. The relevance of these studies is limited due to the nonphysiologic routes of exposure and the extremely high numbers of fibers injected. In the study cited by Davis (1986a), the "short-fiber" chrysotile rat intrathoracic injections contained 8.6 X 10 9 total fibers, 57 X 10 6 of which were greater than 8 /zm in length, and yet this treatment did not produce mesotheliomas. These fiber burdens represent six orders of magnitude more total fibers than we found in pleural samples under our experimental conditions. Furthermore, Kane and co-workers demonstrated that short crocidolite asbestos (99% ^5 ^m length), when injected intraperitoneally into mice, was highly mesotheliomagenic if lymphatic clearance mechanisms were either overloaded by multiple injections or blocked by agarose beads (Moalli et al., 1987; Kane, 1991; Kane, personal communication). In these studies, diaphragmatic serosal surfaces of fiber-treated mice revealed extensive fiber aggregation at sites of lymphatic drainage from the abdominal cavity. Based on these findings, Kane has hypothesized that the mesotheliomagenic potential of instilled long fibers may be due in part to the impairment of clearance from mesothelial surfaces. If these experiments can be used to model the disposition of inhaled fibers, the pleural carcinogenic potential of fibers may be dependent on both clearance from the pleural space as well as the ability of fibers to reach the target site. These findings raise the question of the relative contribution of dimension to the issue of carcinogenic potency, particularly for inhaled fibers. Some experimental evidence suggests that long fibers are more mesotheliomagenic than short fibers following inhalation. Davis and co-workers (1986a,b) exposed rats to sizefractionated preparations of amosite asbestos and found that preparations enriched for "long" fibers (20% >8 ^m in length) induced a higher incidence of mesothelioma (3/40 rats) than a preparation containing "short" amosite fibers (0.5% >8 [im in length, 1/40 rats). Neither control rats nor rats exposed to UICC amosite considered to be of "intermediate" size had any incidence of mesothelioma (0/61, 0/ 43 respectively). Similar results were obtained with sizefractionated chrysotile in aerosol-exposed rats, where "long" preparations produced a 5/80 incidence of mesothelioma vs 1/127 in animals that inhaled "short" fibers. However, the relative potency of these fiber preparations is difficult to quantitate since only a single concentration for each exposure was performed. This is true especially in light of subsequent studies demonstrating that macrophage-mediated clearance mechanisms are impaired at relatively high dust burdens, a factor that could potentially alter the kinetics of fiber movement to the pleura. Epidemiologic evidence indicates that fiber thickness is a critical factor in the prevalence of asbestos-induced mesothelioma. Northwestern Cape crocidolite accounts for most of the cases of mesothelioma in South Africa (Harington, 1981), although airborne fibers tend to be rather short and thin (99% <8 fim in length, 50% <0.1 //m in diameter). Harington contrasted these results with epidemiologic studies of anthophyllite asbestos fibers (60% >8 //m in length, 99% >0.1 ^m diameter) from mines in Paakkila, Finland, which are known to cause lung tumors in humans but no incidence of mesothelioma. In an analogous fashion, crocidolite from mines in northeastern Transvaal (99% >0.1 nm in diameter) caused predominantly lung cancer but few cases of mesothelioma. While containing large numbers of "Stanton-sized" fibers, neither Paakkila nor Transvaal asbestos, caused significant incidence of mesothelioma. In contrast, Cape crocidolite, thought to be one of the most mesotheliomagenic types of asbestos, contains relatively few Stantonsized fibers. Due to this data, it has been suggested that threshold limit values should be more on the order of 3-5 fim for fiber lengths and /zm for fiber diameters. Given that thin fibers are apparently more mesotheliomagenic in humans, these fibers may be more carcinogenic because they are able to penetrate to pleural locations. However, the types of fibers that can penetrate to pleural tissues

7 FIBER DOSIMETRY FOLLOWING RCF-I EXPOSURE 37 are not necessarily those that have the greatest mesotheliomagenic potential. Numerous studies have shown that chrysotile fibers are more commonly found in pleural plaques and pleural tumors in occupationally exposed workers than amphibole types of asbestos (LeBouffant et ai, 1976; Sebastien et ai, 1979; Dodson et al, 1989; Kohyama and Suzuki, 1989). This finding is surprising given that amphibole types of asbestos (e.g., crocidolite and amosite) rather than chrysotile are thought to have the greatest mesotheliomagenic potential. A study of North American insulation workers exposed to mixtures of South African amosite and Canadian chrysotile (Kohyama and Suzuki, 1989) demonstrated a 5:1 ratio of amositexhrysotile in pulmonary parenchyma but only a 0.07:1 ratio in either pleural plaques or pleural tumors, indicating enhanced translocation of chrysotile to pleural sites. However, these chrysotile fibers were found to be extremely short and thin (geometric mean lengths of 1-2 fim and geometric mean diameters of 0.03 ^tm). These findings may be partially due to the fact that amosite fibers, which predominated in the lung, were longer and thicker than chrysotile fibers, which predominated in pleural samples. Although relatively fewer amosite fibers were found in pleural tissues, those that were found tended to be longer and thicker than their chrysotile counterparts. However, fibers found in human pleural tissues do tend to be short and thin. The results of the present study are in agreement with data from humans and strongly suggest that translocation of fibers to the pleural compartment is restricted as a function of size, with only short, thin fibers capable of migration. It should be noted that the data derived from the present studies are limited to a 32-day time course. Due to the long latency of fiber-induced pleural diseases, longer studies are necessary to characterize chronic migration of fibers to the pleura. The present studies demonstrate the feasibility of separating the pleural fiber burden from that of the underlying pulmonary parenchyma in experimental fiber inhalation studies. Information gathered using this approach will allow dissection of the mechanism(s) underlying fiber-induced pleural disease, particularly the time course of cellular events and the role of direct versus indirect fiber-induced effects (McClellan and Hesterberg, 1994). ACKNOWLEDGMENTS This study has been supported in part by grants from the National Institute of Environmental Health Sciences (ESO ), the North American Insulation Manufacturers Association, and the Refractory Ceramic Fiber Coalition. The excellent technical assistance of Carolyn Shanley and Carl Parkinson is greatly appreciated. REFERENCES Auerbach, O., Conston, A. S., and Garfinkel, L. 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