Comparison of pleural responses of rats and hamsters to subchronic ...

Comparison of Pleural Responsesof Rats and Hamsters to SubchronicInhalation of Refractory Ceramic FibersJeffrey 1. Everitt, Thomas R. Gelzleichter, EdilbertoBermudez, James B. Mangum, Brian A. Wong,Derek B. Janszen, and Owen R. MossChemical Industry Institute of Toxicology, Research Triangle Park,North Carolina

In the present subchronic study, we compared pleural inflammation, visceral pleural collagendeposition, and visceral and parietal pleural mesothelial cell proliferation in rats and hamstersidentically exposed to a kaolin-based refractory ceramic fiber, (RCF)-1 by nose-only inhalationexposure, and correlated the results to translocation of fibers to the pleural cavity. Fischer 344rats and Syrian golden hamsters were exposed to 650 fibers/cc of RCF-1, for 4 hr/day, 5days/week for 12 weeks. Following 4 and 12 weeks of exposure, and after a 12-week recoveryperiod, pleural lavage fluid was analyzed for cytologic and biochemical evidence of inflammation.Visceral and parietal pleural mesothelial cell proliferation was assessed by immunocytochemicaldetection of bromodeoxyuridine incorporation. Pleural collagen was quantitated usingmorphometric analysis of lung sections stained with Sirius Red. Fiber-exposed rats and hamstershad qualitatively similar pleural inflammation at each time point. Mesothelial cell proliferation wasmore 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 thesurface of the diaphragm at each time point. Hamsters but not rats had significantly elevatedcollagen in the visceral pleura at the 12-week postexposure time point. Fibers were found in thepleural 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 thepleura of rodents following short-term inhalation exposure are associated with fiber translocationto the pleura and may be predictive of chronic pleural disease outcomes following long-termexposure. Environ Health Perspect 105(Suppl 5):1209-1213 (1997)

Key words: fiber, inhalation, pleura, hamster, cell proliferation

Long-term fiber inhalation studies in ratsand Syrian golden hamsters have demon-strated that there are significant interspeciesdifferences in fiber-induced pleural disease(1). While both Fischer 344 rats and Syriangolden hamsters developed significant pul-monary inflammation and interstitial fibro-sis following chronic inhalation of akaolin-based refractory ceramic fiber(RCF)-1, there were significant interspeciesdifferences in tumor induction and thedevelopment of pleural fibrosis. F344 ratsdeveloped lung tumors but relatively few

malignant mesotheliomas. In contrast,Syrian golden hamsters exposed simultane-ously to the same fibrous aerosols had noparenchymal lung tumors but developed ahigh incidence of pleural malignantmesothelioma and pleural fibrosis.

The objectives of the present studieswere 2-fold: a) to determine if there weredifferences in pleural responses to short-term RCF-1 exposure in rats and hamstersthat would correlate to long-term diseaseoutcomes; and b) to determine if pleuralresponses 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. Manuscriptreceived 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 InsulationManufacturers Association and the Refractory Ceramic Fiber Coalition.

Address correspondence to: Dr. J.1. Everitt, Chemical Industry Institute of Toxicology, P.O. Box 12137, 6 DavisDrive, Research Triangle Park, NC 27709. Telephone: (919) 558-1267. Fax: (919) 558-1300. E-mail: [email protected]

Abbreviations used: BrdU, bromodeoxyuridine; HBSS, Hanks balanced salt solution; L/D, length/diameter; LDH,lactate dehydrogenase; NAG, N-acetylglucosaminidase; PLF, pleural lavage fluid; RCF, refractory ceramic fiber.

translocation. The purpose of these studieswas to test the hypothesis that interspeciesdifferences in pleural response to RCF-1exposure were associated with differencesin fiber translocation to the pleural space.

MethodsAnimalsMale CDF (F344)/CrIBR Fischer rats,13 weeks of age and weighing 250 to275 g, were obtained from Charles RiverBreeding Laboratories (Raleigh, NC). MaleLak:LVG(SYR)BR Syrian golden hamsters,13 weeks of age and weighing 140 to 150 g,

were obtained from Charles River BreedingLaboratories (Montreal, Canada). All ani-mals were quarantined for a minimum of10 days prior to exposure and remainedfree from antibodies to common murinemycoplasmal and viral pathogens through-out the course of the experiment. While noton exposure towers, rats were housed inpolycarbonate cages on direct-contact cellu-lose bedding (ALPHA-dri, Shepard SpecialtyPapers, Kalamazoo, MI) and suppliedNIH-07 cereal-based diet (Ziegler Brothers,Gardner, PA) and water ad libitum. Roomtemperature was maintained at 60 to 65°Cand humidity at 40 to 60% throughout theexposure and postexposure periods.

ExposuresRats and hamsters were exposed to an

RCF-1 aerosol by nose-only inhalationusing previously described methods (2).Animals were exposed for 4 hr/day, 5days/week. Various groups were removedfrom the exposure regimen at 0, 4, and 12weeks or were held for an additional 12-week recovery period following the finalexposure (week 12). During exposures, lightscatter (RAM, Monitoring Instruments forthe Environment, Billerica, MA) was usedto continuously monitor fiber concentra-

tions. Fiber mass (45.6 ± 10 mg/m3) was

determined from samples captured on open-faced, 0.2 pm polycarbonate filters (GelmanSciences, Ann Arbor, MI) and assayed as

previously described (2). The aerosol con-

tained 650 total fibers/cc (length/diamter[L/D] > 3), of which 300 fibers/cc were clas-sified as World Health Organization fibers(L/D > 3, L > 5 pm) (3) (Table 1).

Pleural Fiber BurdensGroups of six animals were sampled at0, 4, 12, and 24 weeks and used exclusivelyto determine pleural fiber burdens. As

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EVERITT ET AL.

Table 1. Bimodal, bivariate lognormal size distributionof fiber and particle aerosol.

Fibers, Particles,Aerosol parameters L/D03 L/D<3

Median length (pm) 5.3 2.41Median diameter (pm) 0.63 1.20GSD (length) 2.5 1.58GSD (diameter) 1.8 1.62Tau 0.54 0.80Percentage aerosol 65 35

Abbreviations: GSD, geometric SD; Tau, correlationbetween the natural logarithm length and diameter.

exposures were for 5 consecutive days perweek, Monday through Friday, animalswere euthanatized immediately afterthe termination of Friday exposures.Characterization of pleural fiber burdenswas as previously described (2) using anagarose casting method (4) to recoverpleural fibers.

Celi Proliferation StudiesAnimals used for pathobiology studies(six/group) were implanted with bromo-deoxyuridine (BrdU)-filled miniosmoticpumps to quantify DNA synthesis. Threedays after the final fiber exposure, minios-motic pumps (Alza, Palo Alto, CA) weresurgically implanted in the dorsal subder-mal skin folds of fiber-exposed and controlanimals. Miniosmotic pumps were filledwith a 2-ml sterile saline solution of BrdUat a concentration of 10 mg/ml, with a dis-charge rate of 5 pl/hr. After 3 days of label-ing, animals were euthanatized and usedfor lavage and histopathology studies.

Pleurl Lavage FluidAt each time point, six animals from eachgroup were anesthetized with pentobarbi-tal, exsanguinated, and both the lungs andpleural cavity were lavaged with sterile cal-cium-free, magnesium-free, and phenolred-free Hanks balanced salt solution(HBSS) (Gibco Laboratories, GrandIsland, NY) as previously described (5).Rat and hamster pleural spaces werelavaged twice with 4 and 3 ml HBSS,respectively. Pleural lavage samples werepooled and centrifuged at 200xg for 10min at 40C. The cell pellets were stored onice while the lavage supernatant wasretained for biochemical analyses. Cell pel-lets were resuspended in RPMI 1640media (Gibco Laboratories) containing10% fetal bovine serum (HycloneLaboratories, Logan, UT). Cell numberswere determined with a Coulter Counter(ZM model, Coulter Electronics, Marietta,

GA). Cell differentials were determinedfrom cytocentrifuge slides stained withWright Giemsa Diff-Quik (Leukostat,Fisher Diagnostics, Pittsburgh, PA) asdescribed by Gelzleichter et al. (2). Thecell-free lavage supernatants were immedi-ately analyzed for lactate dehydrogenase(LDH) and N-acetylglucosaminidase(NAG) using a COBAS FARA II auto-analyzer (Roche Diagnostic Systems,Montclair, NJ). Protein content wasassayed using a commercially available kit(Wako Pure Chemical Industries, Osaka,Japan). Fibronectin content was deter-mined by enzyme-linked immunosorbentassay as previously described (6). For cellu-lar and biochemical assays, all results areexpressed as mean values ± 1 SD. Significantdifferences between groups were deter-mined by an analysis of variance withFisher protected least significant differenceas a posthoc test (p< 0.05).

Pleural Collagen DeterminationMorphometric collagen measurement inthe visceral pleura was obtained from SiriusRed-stained paraffin sections [modifiedfrom Malkusch et al. (7)] viewed underpolarized transmission illumination

x0

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(20 x objective). Black and white videoimages were acquired and saved using animage analysis system (Image-i/AT,Universal Imaging, West Chester, PA).Regions evaluated were marked manuallyand the parameters of area and length weremeasured. The pleural collagen contentwas measured as the area of polarizable tis-sue per unit length of the visceral pleuralsurface, and the results from fiber-exposedanimals were expressed as a percentageincrease above control values.

ResultsQualitatively similar patterns of inflamma-tory cell infiltrate were found in the pleuralspaces of both rats and hamsters, withincreased numbers of pleural macrophages,eosinophils, neutrophils, and lymphocytesin the pleural lavage fluid (PLF) (Figure 1).In addition to cytologic changes, bothhamsters and rats had alterations in PLFbiochemical profiles indicative of inflam-mation (Table 2), including increased totalprotein, lactic dehydrogenase, N-acetyl glu-cosamine, and fibronectin. Hamsters had agreater inflammatory response than did rats,particularly at the 12-week postexposuretime point.

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Figure 1. Cell composition of the pleural lavage fluid (A) macrophages; (B) neutrophils; (C) eosinophils; (D) lym-phocytes in fiber-exposed and control rats and hamsters. Results are expressed as means ± SD. *, significantly dif-ferent from control values, p< 0.05 (analysis of variance [ANOVA] with Fisher protected least significant difference[PLSD] as a posthoc test).

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LUNG AND PLEURAL RESPONSES TO RCF-1

Table 2. Biochemical analysis of pleural lavage fluid. Data expressed as the percent increase over matchedcontrols.

Biochemical 4 weeks 12 weeks 24 weeksparameters Rat Hamster Rat Hamster Rat Hamster

LDH -5.5+50a 1.1±32 20±49 59±58* 28±61 112±39*NAG -10±30 87±33* 8±25 35±41 -12±35 78±48Total protein 19±16* 46±28* 78±28* 148±69* 44±10* 26±43Fibronectin 34±28 20±11* 153±57* 65±18* 86±44* 45±24*

"Each value represents the mean±SD for data obtained with pleural cell populations lavaged from five to sixanimals. *p. 0.05, as determined by the Fisher PLSD test.

At each time point, labeling indices inmesothelial cells were higher in fiber-exposed hamsters than in rats in both pari-etal and visceral pleural sites (Figure 2).Mesothelial cell labeling was greater incontrol hamsters than in rats at all threetime points, which indicates a higher basallevel of mesothelial cell proliferation in thisspecies. In both species, the labeling indicesin parietal mesothelial cells lining thepleural diaphragmatic surface were greaterthan those of the visceral pleura. The high-est labeling indices were found on thediaphragmatic surfaces in fiber-exposedhamsters (Figure 2). Mesothelial cell prolif-eration remained significantly elevated atboth sites in both species at the 12-weekpostexposure time point.

Visceral pleural collagen was noticantly increased over controls in haor rats at the end of the 12th week of1 exposure, nor were any pleural Inoted. In contrast to the findings n(the end of the 12-week exposure Ecollagen was significantly increasedvisceral pleura in hamsters but notthe postexposure group at the 24time point (Figure 3). This fibroscharacterized by focal pleural thickwith associated hypertrophy of vpleural mesothelial cells.

In both species, pleural agarosohad similar numbers of fibers preseach time point, although higher nuof fibers were found in the casts ofeach time point compared to hat

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Figure 2. Mesothelial cell proliferation in fiber-exposed and control rats and hamsters. (A) Rat and (B)visceral; (C) rat and (D) hamster diaphragmatic proliferation. Results are expressed as means with 95% Cl

nificantly different from control values, p<0.05 (ANOVA).

signif-imstersfRCF-lesionsoted atperiod,in therats in:-weekSis was-enings

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Figure 3. Morphometric quantitation of visceralpleural collagen 12 weeks after the end of the 12-weekexposure period. *, significantly different from controlvalues, p< 0.05 (ANOVA).

(Table 3). Most of these fibers were veryshort and thin.

Discussionisceral The presence of significant cytologic and

biochemical changes present in the PLF ofe casts subchronically exposed hamsters and ratssent at extends our observations made after acuteimbers RCF-1 inhalation exposure. In those studiesrats at inflammatory changes immediately follow-msters ing a 1-week exposure in rats or hamsters

were not noted, although we noted pleuralinflammation 1-month postexposure (5,8).Inflammatory changes in the PLF havebeen reported in asbestos-exposed rodentswith similar cytologic profiles (9,10). Inthis study, inflammatory changes persistedinto the postexposure recovery period andbecame more marked for several of thecytologic and biochemical end points. Theexacerbation of pleural inflammation aftercessation of fiber exposure suggests a corre-lation with progressive injury and chronicfibroproliferative disease outcomes. The

24 finding of persistent pleural space inflam-mation in fiber toxicology studies under-scores the value of PLF analysis wheneverthe pleura is a suspected target for toxicity.Study of PLF is likely to prove usefulfor examination of changes in the pleuralspace in a manner analogous to the way

* bronchoalveolar lavage fluid analysis hasadvanced knowledge ofpulmonary toxicity.

At present, there is a limited study data-base that correlates pleural inflammatorychanges with fiber-induced and nonfibrousparticulate-induced pulmonary toxicity. For

24 this reason, there is little present under-standing ofwhich, if any, pleural inflamma-tory parameters or changes are fiber-specific

hamster or which correlate with long-term fibropro-is. *, sig- liferative pleural disease. Complicating the

picture is the finding that pulmonary

Environmental Health Perspectives * Vol 105, Supplement 5 * September 1997

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EVERITT ET AL.

Table 3. Fiber size characteristics of pleural fiber burden.

Length DiameterWeek GML, pm GSD GMD, pm GSD Tau Fiber number, x 1000

Hamster4 1.4 1.7 0.10 1.6 0.42 17.5 ± 8.312 2.1 2.3 0.14 1.8 0.66 14.8 ± 1124 2.1 2.5 0.12 1.8 0.54 15.6 ± 5.4

Rat4 1.7 2.0 0.11 1.6 0.28 42.1 ±3512 1.5 1.7 0.09 1.5 -0.01 41.4 ± 9.424 1.6 1.8 0.10 1.6 0.21 40.3 ± 10

All GML, GSD, and Tau values are averages for animal groups. Abbreviations: GMD, geometric mean diameter;GML, geometric mean length.

parenchymal inflammation itself causescytologic and cytokine alterations in pleuralcell populations (11).

An early mesothelial cell proliferativeresponse has been reported in mice follow-ing inhalation or instillation of long croci-dolite asbestos fibers under conditions thatresulted in pulmonary fibrosis (12).Subsequent study revealed that this prolif-eration could result as a nonspecific pleuralchange following fibrogenic insult to thelung (13). Several authors have suggestedthat this asbestos-induced visceral pleuralmesothelial cell proliferation does not rep-resent a direct effect of fibers in contactwith mesothelial cells but may be due tofiber-induced release of reactive oxygenspecies, cytokines, or growth factors thatstimulate cell proliferation in these cells(13,14). These conclusions were madebecause light microscopic examination oflung and pleura sections found no fibers.The present study using RCF-1 stronglysuggests that fiber-induced mesothelialcell proliferation is associated with fibersreaching pleural target sites.

At each of the time points in thepresent study, fibers were recovered inpleural casts, even though no fibers werefound by examination of the visceral pleurausing light microscopy. Lack of pleural

fiber detection in some previous studiesis probably due to the methodologyemployed. Many of the fibers detected inthe present study are of a size that requiresthe use of electron microscopy for detec-tion. In addition, the use of casts of thepleural space is believed to be a more effi-cient method of fiber recovery than exami-nation of histologic sections. Relativelyrapid translocation of short, thin fibers hasbeen previously reported with chysotileasbestos and thus is not limited to the pre-sent study (15). The recovery of fibersfrom the pleural space, in conjunction withthe finding of a high labeling index inthe mesothelial lining of the centraldiaphragm, makes it highly unlikely thatlocal cytokines or elaborated factors in theparenchymal lung were responsible for themesothelial cell proliferation noted. Thepathways through which fibers reach thepleura, and the populations and sizes offibers that are responsible for this prolifera-tion, are presently unknown. Our findingof site-specific mesothelial cell proliferationin the rodent parietal pleura strongly sup-ports the recent observation of Boutin andcolleagues (16), who found that asbestosfibers accumulate in specific sites in thehuman parietal pleura associated withlymphatic drainage.

Results of the present study show thatthe Syrian golden hamster develops moresevere fibrotic change and mesothelial pro-liferation in the pleura than does the F344rat following inhalation of RCF-1. Thisstrongly suggests that hamster pleura is amore sensitive target organ for fiber-induced disease than is the pleura of therat. Additional studies of the size distribu-tions of pleural fiber burdens are needed todetermine whether differences in retainedfiber populations contribute to interspeciesdifferences. There is some evidence, asnoted by markedly higher mesothelial celllabeling indices in control and fiber-exposed hamsters, that there are inherenttissue susceptibility differences that canexplain interspecies differences in diseaseoutcomes. This finding agrees with previ-ous studies in our laboratory, where weused a fiber instillation model to demon-strate that hamsters respond with highermesothelial cell proliferation to fiber expo-sure than do F344 rats (17,18). It is pre-mature, however, to speculate on whichrodent species, if any, is a more appropri-ate model for fiber-induced pleural diseasein humans.

In summary, the present experimentsdemonstrate that pleural inflammation andfibroproliferative changes follow subchronicRCF-1 inhalation exposure in rats andhamsters, and correlate with pleural find-ings reported in long-term rodent inhala-tion bioassays. The more severe pleuralchanges noted in hamsters did not correlatewith differences in the number of totalfibers that translocated to the pleural space.The correlation of findings between sub-chronic rodent fiber inhalation exposuresand long-term inhalation bioassays willallow the development of short-term rodentmodels useful for predicting fibrogenic andoncogenic pleural disease following chronicfiber exposure.

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