Comparison of pleural responses of rats and hamsters to subchronic inhalation of refractory ceramic fibers.

In the present subchronic study, we compared pleural inflammation, visceral pleural collagen deposition, and visceral and parietal pleural mesothelial cell proliferation in rats and hamsters identically exposed to a kaolin-based refractory ceramic fiber, (RCF)-1 by nose-only inhalation exposure, and correlated the results to translocation of fibers to the pleural cavity. Fischer 344 rats and Syrian golden hamsters were exposed to 650 fibers/cc of RCF-1, for 4 hr/day, 5 days/week for 12 weeks. Following 4 and 12 weeks of exposure, and after a 12-week recovery period, pleural lavage fluid was analyzed for cytologic and biochemical evidence of inflammation. Visceral and parietal pleural mesothelial cell proliferation was assessed by immunocytochemical detection of bromodeoxyuridine incorporation. Pleural collagen was quantitated using morphometric analysis of lung sections stained with Sirius Red. Fiber-exposed rats and hamsters had qualitatively similar pleural inflammation at each time point. Mesothelial cell proliferation was more pronounced in hamsters than in rats at each time point and at each site. In both species, the mesothelial cell labeling index was highest in the parietal pleural mesothelial cells lining the surface of the diaphragm at each time point. Hamsters but not rats had significantly elevated collagen in the visceral pleura at the 12-week postexposure time point. Fibers were found in the pleural cavities of both species at each time point. These fibers were generally short and thin. These results suggest that mesothelial cell proliferation and fibroproliferative changes in the pleura of rodents following short-term inhalation exposure are associated with fiber translocation to the pleura and may be predictive of chronic pleural disease outcomes following long-term exposure.

Long-term fiber inhalation studies in rats and Syrian golden hamsters have demonstrated that there are significant interspecies differences in fiber-induced pleural disease (1). While both Fischer 344 rats and Syrian golden hamsters developed significant pulmonary inflammation and interstitial fibrosis following chronic inhalation of a kaolin-based refractory ceramic fiber (RCF)-1, there were significant interspecies differences in tumor induction and the development of pleural fibrosis. F344 rats developed lung tumors but relatively few malignant mesotheliomas. In contrast, Syrian golden hamsters exposed simultaneously to the same fibrous aerosols had no parenchymal lung tumors but developed a high incidence of pleural malignant mesothelioma and pleural fibrosis. The objectives of the present studies were 2-fold: a) to determine if there were differences in pleural responses to shortterm RCF-1 exposure in rats and hamsters that would correlate to long-term disease outcomes; and b) to determine if pleural responses were associated with fiber This paper is based on a presentation at The Sixth International Meeting on the Toxicology of Natural and Man-Made Fibrous and Non-Fibrous Particles held 15-18 September 1996 in Lake Placid, New York. Manuscript received at EHP 26 March 1997; accepted 1 1 April 1997. We acknowledge the helpful discussions of our colleagues P. Ferriola and F. Miller and the scientific editing assistance of B. Kuyper. This work was funded in part by grants from the North American Insulation Manufacturers Association  translocation. The purpose of these studies was to test the hypothesis that interspecies differences in pleural response to RCF-1 exposure were associated with differences in fiber translocation to the pleural space.

Animals
Male CDF (F344)/CrIBR Fischer rats, 13 weeks of age and weighing 250 to 275 g, were obtained from Charles River Breeding Laboratories (Raleigh, NC). Male Lak:LVG(SYR)BR Syrian golden hamsters, 13 weeks of age and weighing 140 to 150 g, were obtained from Charles River Breeding Laboratories (Montreal, Canada). All animals were quarantined for a minimum of 10 days prior to exposure and remained free from antibodies to common murine mycoplasmal and viral pathogens throughout the course of the experiment. While not on exposure towers, rats were housed in polycarbonate cages on direct-contact cellulose bedding (ALPHA-dri, Shepard Specialty Papers, Kalamazoo, MI) and supplied NIH-07 cereal-based diet (Ziegler Brothers, Gardner, PA) and water ad libitum. Room temperature was maintained at 60 to 65°C and humidity at 40 to 60% throughout the exposure and postexposure periods. Exposures Rats and hamsters were exposed to an RCF-1 aerosol by nose-only inhalation using previously described methods (2). Animals were exposed for 4 hr/day, 5 days/week. Various groups were removed from the exposure regimen at 0, 4, and 12 weeks or were held for an additional 12-week recovery period following the final exposure (week 12 exposures were for 5 consecutive days per week, Monday through Friday, animals were euthanatized immediately after the termination of Friday exposures. Characterization of pleural fiber burdens was as previously described (2) using an agarose casting method (4) to recover pleural fibers.

Celi Proliferation Studies
Animals used for pathobiology studies (six/group) were implanted with bromodeoxyuridine (BrdU)-filled miniosmotic pumps to quantify DNA synthesis. Three days after the final fiber exposure, miniosmotic pumps (Alza, Palo Alto, CA) were surgically implanted in the dorsal subdermal skin folds of fiber-exposed and control animals. Miniosmotic pumps were filled with a 2-ml sterile saline solution of BrdU at a concentration of 10 mg/ml, with a discharge rate of 5 pl/hr. After 3 days of labeling, animals were euthanatized and used for lavage and histopathology studies.

Pleurl Lavage Fluid
At each time point, six animals from each group were anesthetized with pentobarbital, exsanguinated, and both the lungs and pleural cavity were lavaged with sterile calcium-free, magnesium-free, and phenol red-free Hanks balanced salt solution (HBSS) (Gibco Laboratories, Grand Island, NY) as previously described (5). Rat and hamster pleural spaces were lavaged twice with 4 and 3 ml HBSS, respectively. Pleural lavage samples were pooled and centrifuged at 200xg for 10 min at 40C. The cell pellets were stored on ice while the lavage supernatant was retained for biochemical analyses. Cell pellets were resuspended in RPMI 1640 media (Gibco Laboratories) containing 10% fetal bovine serum (Hyclone Laboratories, Logan, UT). Cell numbers were determined with a Coulter Counter (ZM model, Coulter Electronics, Marietta, GA). Cell differentials were determined from cytocentrifuge slides stained with Wright Giemsa Diff-Quik (Leukostat, Fisher Diagnostics, Pittsburgh, PA) as described by Gelzleichter et al. (2). The cell-free lavage supernatants were immediately analyzed for lactate dehydrogenase (LDH) and N-acetylglucosaminidase (NAG) using a COBAS FARA II autoanalyzer (Roche Diagnostic Systems, Montclair, NJ). Protein content was assayed using a commercially available kit (Wako Pure Chemical Industries, Osaka, Japan). Fibronectin content was determined by enzyme-linked immunosorbent assay as previously described (6). For cellular and biochemical assays, all results are expressed as mean values ± 1 SD. Significant differences between groups were determined by an analysis of variance with Fisher protected least significant difference as a posthoc test (p< 0.05).

Pleural Collagen Determination
Morphometric collagen measurement in the visceral pleura was obtained from Sirius Red-stained paraffin sections [modified from Malkusch et al. (7)] viewed under polarized transmission illumination (20 x objective). Black and white video images were acquired and saved using an image analysis system (Image-i/AT, Universal Imaging, West Chester, PA). Regions evaluated were marked manually and the parameters of area and length were measured. The pleural collagen content was measured as the area of polarizable tissue per unit length of the visceral pleural surface, and the results from fiber-exposed animals were expressed as a percentage increase above control values.

Results
Qualitatively similar patterns of inflammatory cell infiltrate were found in the pleural spaces of both rats and hamsters, with increased numbers of pleural macrophages, eosinophils, neutrophils, and lymphocytes in the pleural lavage fluid (PLF) (Figure 1). In addition to cytologic changes, both hamsters and rats had alterations in PLF biochemical profiles indicative of inflammation (Table 2), including increased total protein, lactic dehydrogenase, N-acetyl glucosamine, and fibronectin. Hamsters had a greater inflammatory response than did rats, particularly at the 12-week postexposure time point. Time, weeks -Control rat -Fiber-exposed rat o Control hamster FRber-exposed hamster 4 12 24 e, weeks Time, weeks  At each time point, labeling indices in mesothelial cells were higher in fiberexposed hamsters than in rats in both parietal and visceral pleural sites (Figure 2). Mesothelial cell labeling was greater in control hamsters than in rats at all three time points, which indicates a higher basal level of mesothelial cell proliferation in this species. In both species, the labeling indices in parietal mesothelial cells lining the pleural diaphragmatic surface were greater than those of the visceral pleura. The highest labeling indices were found on the diaphragmatic surfaces in fiber-exposed hamsters ( Figure 2). Mesothelial cell proliferation remained significantly elevated at both sites in both species at the 12-week postexposure time point.
Visceral pleural collagen was not icantly increased over controls in ha or rats at the end of the 12th week of 1 exposure, nor were any pleural I noted. In contrast to the findings n( the end of the 12-week exposure E collagen was significantly increased visceral pleura in hamsters but not the postexposure group at the 24 time point (Figure 3). This fibros characterized by focal pleural thick with associated hypertrophy of v pleural mesothelial cells.
In both species, pleural agaroso had similar numbers of fibers pres each time point, although higher nu of fibers were found in the casts of each time point compared to hat B -Control rat lbar-axposed rat -_ Control hamster Fiber-exposed hamster  ( Table 3). Most of these fibers were very short and thin.

Discussion
isceral The presence of significant cytologic and biochemical changes present in the PLF of e casts subchronically exposed hamsters and rats sent at extends our observations made after acute imbers RCF-1 inhalation exposure. In those studies rats at inflammatory changes immediately followmsters ing a 1-week exposure in rats or hamsters were not noted, although we noted pleural inflammation 1-month postexposure (5,8). Inflammatory changes in the PLF have been reported in asbestos-exposed rodents with similar cytologic profiles (9,10). In this study, inflammatory changes persisted into the postexposure recovery period and became more marked for several of the cytologic and biochemical end points. The exacerbation of pleural inflammation after cessation of fiber exposure suggests a correlation with progressive injury and chronic fibroproliferative disease outcomes. The 24 finding of persistent pleural space inflammation in fiber toxicology studies underscores the value of PLF analysis whenever the pleura is a suspected target for toxicity. Study of PLF is likely to prove useful for examination of changes in the pleural space in a manner analogous to the way * bronchoalveolar lavage fluid analysis has advanced knowledge of pulmonary toxicity. At present, there is a limited study database that correlates pleural inflammatory changes with fiber-induced and nonfibrous particulate-induced pulmonary toxicity. For 24 this reason, there is little present understanding ofwhich, if any, pleural inflammatory parameters or changes are fiber-specific hamster or which correlate with long-term fibroprois. *, sig-liferative pleural disease. Complicating the picture is the finding that pulmonary  parenchymal inflammation itself causes cytologic and cytokine alterations in pleural cell populations (11).
An early mesothelial cell proliferative response has been reported in mice following inhalation or instillation of long crocidolite asbestos fibers under conditions that resulted in pulmonary fibrosis (12). Subsequent study revealed that this proliferation could result as a nonspecific pleural change following fibrogenic insult to the lung (13). Several authors have suggested that this asbestos-induced visceral pleural mesothelial cell proliferation does not represent a direct effect of fibers in contact with mesothelial cells but may be due to fiber-induced release of reactive oxygen species, cytokines, or growth factors that stimulate cell proliferation in these cells (13,14). These conclusions were made because light microscopic examination of lung and pleura sections found no fibers. The present study using RCF-1 strongly suggests that fiber-induced mesothelial cell proliferation is associated with fibers reaching pleural target sites.
At each of the time points in the present study, fibers were recovered in pleural casts, even though no fibers were found by examination of the visceral pleura using light microscopy. Lack of pleural fiber detection in some previous studies is probably due to the methodology employed. Many of the fibers detected in the present study are of a size that requires the use of electron microscopy for detection. In addition, the use of casts of the pleural space is believed to be a more efficient method of fiber recovery than examination of histologic sections. Relatively rapid translocation of short, thin fibers has been previously reported with chysotile asbestos and thus is not limited to the present study (15). The recovery of fibers from the pleural space, in conjunction with the finding of a high labeling index in the mesothelial lining of the central diaphragm, makes it highly unlikely that local cytokines or elaborated factors in the parenchymal lung were responsible for the mesothelial cell proliferation noted. The pathways through which fibers reach the pleura, and the populations and sizes of fibers that are responsible for this proliferation, are presently unknown. Our finding of site-specific mesothelial cell proliferation in the rodent parietal pleura strongly supports the recent observation of Boutin and colleagues (16), who found that asbestos fibers accumulate in specific sites in the human parietal pleura associated with lymphatic drainage.
Results of the present study show that the Syrian golden hamster develops more severe fibrotic change and mesothelial proliferation in the pleura than does the F344 rat following inhalation of RCF-1. This strongly suggests that hamster pleura is a more sensitive target organ for fiberinduced disease than is the pleura of the rat. Additional studies of the size distributions of pleural fiber burdens are needed to determine whether differences in retained fiber populations contribute to interspecies differences. There is some evidence, as noted by markedly higher mesothelial cell labeling indices in control and fiberexposed hamsters, that there are inherent tissue susceptibility differences that can explain interspecies differences in disease outcomes. This finding agrees with previous studies in our laboratory, where we used a fiber instillation model to demonstrate that hamsters respond with higher mesothelial cell proliferation to fiber exposure than do F344 rats (17,18). It is premature, however, to speculate on which rodent species, if any, is a more appropriate model for fiber-induced pleural disease in humans.
In summary, the present experiments demonstrate that pleural inflammation and fibroproliferative changes follow subchronic RCF-1 inhalation exposure in rats and hamsters, and correlate with pleural findings reported in long-term rodent inhalation bioassays. The more severe pleural changes noted in hamsters did not correlate with differences in the number of total fibers that translocated to the pleural space. The correlation of findings between subchronic rodent fiber inhalation exposures and long-term inhalation bioassays will allow the development of short-term rodent models useful for predicting fibrogenic and oncogenic pleural disease following chronic fiber exposure.