- Email: [email protected]
Pulmonary response of hamsters to fibrous glass. — Clearance and morphology after a single intratracheal instillation
Exp. Pathol. 29, 197-209 (1986) VEll Gustav Fischer Verlag Jena 1) Depa rtment of Veterinary Pharmacology, Pharmacy and Toxicology, "Gniversity of "Gtrecht, The r-i et herlands : 2) 'rNO-CIVO To xicology and r-i utrition Institute, Zeist, The r\etherl ands
Pulmonary response of hamsters to fibrous glass. Clearance and morphology after a single intratracheal instillation
With 10 figures (Received June 24, 1985)
Address for cOl'respondence: Dr. V. J. FEROK, TNO-eIVa To xicology a.nd Nutrition Institute, P.O. Box 360, 3700 AJ Zeist, The Netherlands Key W 0 r ds: glass fibres; hamster lung; clearance; tracheal instillation; pneumonitis; alveolar macrophage
8ttmrlwry In a one-year serial sacrifice stud y 10 mg glass fibres (length, 95 % IX 20.um, 89 % IX
12.um, 58 % IX 5.um and 25 % IX 2.um; diameter, 88 % IX 1.0.um, 60 % IX 0.5.um and 31 %IX 0.25 .um) suspended in 0.2 ml saline solution were administered to Syrian golden hamsters by a. sing'le intratracheal instillation to determine the clearance of the glass fibres from the lungs and to examine their effects on the lungs using light and electron microscopy. The clearance was rather efficient with a half-time of about 3 months. Coating and corrosion of glass fibres were sporadic findings. A violent focal acute pneumonitis was evoked by the glass fibres and was followed by exeessive aecumulations of alveolar macro phages often loaden with glass fibres. Thereafter, "silieotie granulomas" developed which were seen as dusters of tightly packed iron-positive maerophages eontaining glass fibres. These granulomas were surrounded by a layer of alveolar epitheliulll, and wer e by the end of the study the predominant lesion in otherwise normal lungs.
I ntroduction The industrial production of glass wool and glass yarns started around 1930. Mineral wools are mainl y used for thermal and sonar insul ation ; glass yarns find wide applieation as reinforcement of rubber, plasti e, paper and cement (KOLK 1982). Fibrous glass (length/ diameter ratio> 3: 1) is used in th e manufacture of more than 33,000 products (MORISSET et al. 1979). One well-known adverse effect of fibrous glass is that it eauses itehing through meehaniral irritation of the skin (HEISEL and HUNT 1968; POSSICK et al. 1970). KUSCHNER and WRIGHT (1976) observed pulm onary fibrosis in guinea pigs aft er intratracheal instillation of fibrous glass. After intrapleural or intraperitoneal inoculation of fibrous glass sarcomas and mesotheliomas have been found in rats (STANTON and WRENCH 1972; POTT et aI. 1976; WAG NER et al. 1976 ; STANTON et al. 1977). Reeently, POTT (1981) and POTT et aI. (1983) showed that glass fibres admini stered intratraeheally to Syrian golden hamsters induced both pleural mesotheliomas and pulmonary careinomas. Both the carcinogenicity and the fibro g'enieity have been shown to be related to physical faCtors such as length and diameter of the fibres (STANTON and WRENCH 1972). Although most experimental evidence indicates the importanee of the geometry of minerai fibres for their biologieal aetivity including carcinogenieity, other factors such as 13
197
chemical composition (HARINGTON 1965) and polycyclic aromatic hydrocarbons and other compounds adsorbed on the fibres (DIXON eta!. 1970; CRALLEY and LAINHART 1973; SHABAD et a!. 1974) are thought to playa role in the carcinogenicity of fibrous dusts. Asbestos enhance the induction of lung cancer by cigarette smoke (SARACCI 1977; lVIEURMAN et al. 1979; SELIKOFF et a!. 1980). In mice respirable glass fibres have been found to enhance the adverse effect of styrene vapour on the bronchiolar epithelium (lVIORISSET et a!. 1979). Within the scope of a research programme concerning the significance of non-specifie physical injury to the respiratory tract for the formation of tumours at the site of damage, intratracheal instillation of glass fibres was used as one of the methods to inflict damage to the lungs. The short- and long-term effects on the lungs of single and repeated administration of fibrous glass with or without the careinogen benzo( a)pyrene have been studied in Syrian golden hamsters using light and electron mieroseopy. The present report describes the clearanee of glass fibres from the lungs and their effects on the lungs after a single intratracheal instillation.
Materials and Methods Animals The animals used in these studies were Syrian golden hamsters (111esocl'icetus auratus) obtained from the randomly-bred colony of the Central Institute for the Breeding of Laboratory Animals TNO, Zeist, The Netherlands. The hamsters were housed in groups of four or five in suspended stainless steel cages with wire-screen bottom in a room ventilated with 10 air changes/h, maintained at 23 ± 1°C, with a relative humidity of 45-GO % and a 12 h light/dark cyele (light from 6.00 a.m. till G.OO p.m.). The animals were offered a pelleted stock diet for hamsters (R13, Hope Farms, Woerden, The Netherlands) and bottled, unfluoridated, tap-water ad libitwil. The hamsters were 10 weeks old at the time of administration of the glass fibres. Glass fibres Glass microfibres (code 104, Johns-Manville, Denver, U.S.A.) were obtained commercially. The elemental composition of the glass fibres is: Si0 2 57.9 %, B2 0 3 10.7%, Na 2 0 10.1 %, Al 2 0 3 5.8 ')~, BaO 5.0 %, ZnO 3.9 %, CaO 3.0 %, K 2 0 2.9 %, F 2 0.6 % and Fe 2 0 3 0.1 %. The glass wool was milled in a ball mill with agate bowl and agate balls (Pulverisette 5, Alfred Fritsch and Co, Idar-Oberstein, F.R.G.). The following procedure was used to obtain a total of 43 g glass fibres: an 80 ml agate bowl was filled with 1 g glass wool and 5 agate balls of 20 mm diameter. The glass wool was milled lor 20 s at 70 % of the maximum rotation speed, followed by 10 min at 50 % of the maximum speed. This procedure was repeated 43 times. The different batehes of ground glass fibres were mixed by hand-stirring and stored in a glass eontainer at room temperature. The size distribution of the fibres was determined by seanning electron microscopy using a Philips EM 400 mieroscope fitted with a special computerized scanning equipment (Tracor N orthem TN 2000)1). Thirteen hundred and ten fibres were measured in a representative sample and the individual fibre dimensions were arranged in a length-diameter diagram (fig. 1). The following size distribution by number was found: length, 95 % c< 20 [tm, 89 % c< 12,um, 58 % ex 5 p.m and 25 % c< 2 [tm; diameter, 88 % ex 1.0 p.m, GO % ex 0.5,(tm and 31 % c< 0.25 p.m. Administration of glass fibres Samples of glass fibres were suspended in a sterile, non-pyrogenie 0.9 % NaCI solution. A suspension containing 5 % (w/v) glass fibres was prepared. During the treatment period of the hamsters the suspensions were magnetically stirred. Details of the instillation procedure have been described previously (JONG and FERON 1971). Under light ether anaesthesia each hamster reeeived a single intratracheal instillation of 10 mg glass fibres administered as 0.2 ml of the 5 % suspension. Determination of glass fibres in lungs 2 ) The elearance of glass fibres from the lungs was studied by determination of the amounts of glass in the lungs at 0 time (within 2 min after instillation) and further 14 days, 1, 3 and G months 1) The size distribution measurements were carried out by Mr. lVI. J. VAN N OORD and ]\1rs. Ing. C. J. G. BLOK-vAN HOEK of the Centre for Analytical Electron Microscopy TNO, Leiden, The Netherlands. 2) These measurements were carried out by Mr. K. KOOPMANS in the Department of Analytical Chemistry, TNO-Division of Teehnology for Society, Delft, The Netherlands.
198
Exp. PatllO!' 29 (1986) 4
~
fiJ@ 0.0
~
ffi6J
ri'tlj
tV
~
~ ~
~
Diameter in JIm
<>
r!I= 5%
of totaL
Length in JI m Fig. 1. Length-diameter distribution of the glass fibres used.
after intratracheal instillation of the suspension of glass fibres. The lungs were dissected out and the trachea and paratrachcal and tracheo-bronchial lymph nodes were removed; then the lungs were frozen with liquid nitrogen and stored in a freezer at -20 DC. The glass content of each lung was indirectly determined by spectrochemical analysis, measuring the aluminum content of the lung-ash obtained by the powder DC-arc technique. Ash samples were mixed with lithium carbonate-graphite mixtures and the obtained spectra were compared with spectra of commercial lithium carbonate standards. The aluminium contents of the ashes were measured in duplicate with an optical emission spectrograph at three different AI-spectral lines, viz. 265.25, 266.4 and 257.5 nm, respectively. The values of the duplicates and those obtained at the different spcctrallines showed a variation of 10-15%, which is somewhat larger than the usual accuracy of 10 % obtained with this technique. The average aluminum value of each sample was corrected by subtracting the average aluminum value of the control lungs collected at the corresponding time intervals. After converting the aluminum values into values for the amount of glass, the glass content of the lungs at the various time intervals was calculated and expressed in percentages of the amount of glass found at 0 time. Experimental design and conduct Forty four male and 44 female hamsters were treated with glass fibres. Groups of 4 males and 4 females were killed by cutting the abdominal aorta under ether anaesthesia at 0 time (within 2 min after instillation) and further after 4, S, and 24 h, after 3, 7 and 14 days, and after 1, 3, 6 and 12 months. After S h, after 14 days, and after 1, 3, and 6 months 4 male and 4 female hamsters treated with saline only were killed in the same way; they served as controls. At each point of time the groups of treated and control animals were divided into 3 subgroups viz. 1 male and 1 female for determination of the glass content of the lungs, 2 males and 2 females for light microscopy and 1 male and 1 female for electron mitroscopy. The lungs to be used for glass determinations were stored at -20 DC until the analyses could be carried out. The lungs to be studied by light microscopy were removed in toto with trachea and larynx, and preserved in a 4 % neutral, phosphatebuffered solution of formaldehyde by careful intratracheal infusion of the fixative under 10 cm water pressure. The paratracheal and tracheobronchial lymph nodes were also removed and fixed in formalin. The liver, kidneys, thymus and mesenteric lymph nodes were collected and preserved only of the hamsters killed after 6 months. All organs were processed through paraffin wax, sectioned at 5 pm (of the larynx, trachea and each pulmonary lobe sections were prepared at 3 different levels), stained with haematoxylin and eosin, and examined microscopically. For electron microscopical investigations the lungs were fixed by intratracheal instillation of the fixative consisting of 2.5 % glutaraldehyde buffered with 0.1 M sodium cacodylate to which 0.02 M CaCl 2 was added. The fixative had a pH of 7.3 and an osmolality of 500 m Osm/kg. The fixation was carried out while the hamsters were anaesthetized with Nembutal (50 mg/kg body weight). Then the lungs with the trachea were removed and stored overnight in the fixative. Thereafter, slices of the lungs were stored in the buffer solution at 4 DC. Finally small pieces of lung tissue were postfixed in 1 % OS04 buffered with 0.1 M sodium cacodylate, washed, dehydrated in acetone water mixtures and embedded in an araldite-glycidether mixture, followed by hardening at GO DC during 4S h. Semithin sections were prepared and stained with toluidine blue. Ultrathin sedions were cut with a diamond knife, stained with uranyl acetate and lead citrate, and viewed with a Philips 201 G electron microscope at GO kV.
1:3*
Exp. Pathol. 29 (1986) 4
199
Results
Clearance of glass fibres from the lungs The average amounts of glass fibres fo und in the lungs at different points of time after administration, are given in table 1. After 2 weeks there was no measurable reduction in the amount of glass present in the lungs. However, after 3 months the amount of glass in the lungs was decreased to about 50 ';6, and a further reduction to about 20 was found after 6 months. Gross pathology Apart from a few tiny greyish areas on the surface of the lungs of several hamsters treated with glass fibres, no gross lesions attributable to treatment were seen. Microscopic pathology Time period 0 The lungs of all animals killed immediately after instillation contained massive amounts of glass fibres. Glass fibres were nearly always found in each of the pulmonary lobes, but there was a considerable variation in the amount of fibres among the various lobes. The glass fibres occurred as large plugs, as small aggregates, and also as individual fibres (fig. 2). They were most frequently found in bronchioles, alveolar ducts and peribronchiolar alveoli, but were also present in larynx, trachea, bronchi and peripheral alveoli. 4 hours 4 hours after instillation, perivascular edema, haemorrhages, cellular debris, and neutrophilic leucocytes were visible in areas containing plugs of glass fibres, particularly in the peribronchiolar regions. Over large areas alveolar epithelial cells were destroyed and had disappeared leaving "naked" blood capillaries. Neutrophilic lencocytes having phagocytized glass fibres were already seen at this point of time (fig". 3). Free glass fibres seemed to be surrounded by a thin layer of electron-lucent material. Lamellar bodies of type II pneumocytes were empty. Capillaries and interstitium contained many neutrophilic leucocytes. The total of changes resulted in a chaotic picture of the pulmonary tissue in glass fibre-containing areas. S hours After 8 hours peribronchiolar regions containing glass fibres showed much cellular debris, extensive haemorrhages and massive infiltrates of neutrophils (fig. 4). As a consequence, Table 1. Clearance of glass fibres from the lungs of hamsters after intratracheal instillation of a single dose of 10 mg glass fibres suspended in 0.9 % NaCI solution Period of time between instillation of glass fibres and killing of the animals 1-2 min (= 0 time) 14 days 1 month el months G months 12 months
Amount of glass fibres retained in the lungs (expressed as % of the amount found at 0 time) Males Females
a) 100 Gel
47 28
NDc)
100b )
lOB
96
55 11
ND
a) No glass found probably due to faulty instillation. Therefore, the amount of glass found after 14 days was set at 100 %, which was deemed permissible in view of the lOB % found in females at this point of time. il) The average amonnt of glass fibres found in the lungs at 0 time was 9.2 mg. c) Xot determined by mistake.
200
Exp. Pathol. 29 (1986) 4
Fig. 2. Glass fibres in the lung of a male hamster immediately after treatment. I-Iaematoxylin and eosin. X 160.
Fig. 3. Neutrophilic leueocytes having phagocytized glass fibres in alveoli of a female hamster 4 h after treatment. Uranyl acetate and lead acetate. X 5,800. Exp.Eathol. 29 (1986) 4
201
Fig. 4. Cellula.r debris and massive infiltrates of neutrophilic lcucocytes in peri bronchiolar regions of the lung of a female hamster 8 h after treatment. Haematoxylin and eosin. x 40.
alveolar structures were hardly recognizable in these areas. Occasionally, small groups of alveolar macrophages could be detected. Bronchiolar epithelium in contact with plugs of glass fibres was flattened. 24 hours One day after instillation a violent inflammatory reaction accompanied by haemorrhages was observed in the lungs. An alveolar structure could no longer be distinguished in peribronchiolar regions containing glass fibres, neutrophilic leucoeytes and now also largo numbers of macrophages. Most of the glass fibres were ingested or surrounded by macrophages. Occasionally electron-dense granular material was attached to or surrounded the glass fibres (fig. 5). Neutrophils were seen to surround macrophagcs heavily laden with glass fibres. Perivascular edema and flattening of bronchiolar epithelium in contact with plugs of glass fibres were common findings. 3 and 7 days In affected areas the alveolar strueture was now better recognizable than after 24 hours. The alveolar septa were widened due to a thickened interstitium containing neutrophiIs and glass fibres-containing macrophages, and a lining of cuboidal epithelial cells. Alveoli were filled with macrophages containing glass fibres. Th e number of macrophages was invariable greatly increased, that of neutrophils in most animals clearly decreased as compared to 24 hours. After 7 days aggregates of glass fibres were seen to be surrounded by neutrophilic leueoeytes. Nuclear debris was found in varying amounts; macrophages with glass were seen to also contain phagocytized nuclear fragments. Individual eosinophils were frequently encountered. The alveolar knob endings (alveolar rings) protruding into the alveolar ducts were swollen as were the alveolar septa, which was due to thickened epithelium and the presence in the interstitium of neutrophilic leu eocytes and macrophages with glass fibres (fig. 6). T,h e alveolar epithelial cells frequently showed many short microvilli-like projections. Haemor-
202
Exp. Pathol. 29 (1986) 4
Fig. 5. Electron-dense granular material surrounding intracellular glass fibres in the lung of a female hamster 24 h after treatment. Uranyl acetate and lead citrate. X 40,000.
rhages and perivascular edema were less conspicuous than after 24 hours. Bronchioles often eo ntained plugs of glass fibres mingled with eellular debris and neutrophils. At these sites the bronchiolar epithelium was often hypertrophic. and oc.casionally stratified. In affected bronchioles Clara cells could not or hardly be recognized. 14 days Considerable numbers of neutrophils were still visible around plugs of glass fibres. Glass fibres in the alveolar lumen were either phagocytized or surrounded by macrophages, which often also contained nuclear remnants. Clusters of macrophages only containing phagorytized nuclear fragments were common findings; occasionally such clusters were surrounded hy neutrophilic leucocytes. Groups of macrophages with glass fibres and nuclear debris also contained iron-}Jositive material according to Perl's method for staining of iron. Individual Ill acrophages were not iron-positive. Areas not containing glass parti cles did not exhibit pathological changes. 1 month Peribronchiolar alveoli were often completely filled with macrophages containing glass fibres . In some animals the number of neutrophils was still considerable, whereas in others only a few of these cells were seen. The amount of cellular debri s was greatly reduced as (:ompared to 7 and 14 days. Haemorrhages were no longer observed. Multinucleated giant rells were occasionally found. Clusters of tightly packed macrophages laden with glass particles and covered with a continuous layer of simple epithelium were observed at this stage for the first time. The content of the macrophages in these clusters was yellowish-light brown and appeared to be moderately iron-positive. The clusters were vi sible as granulomas varying in diameter from 50 to 300 pm and protruding into'the lumen of alveoli and alveolar duets; they were attached to th e alveolar wall often only through a thin stalk. Thickened alveolar septa were often nearly exclusively lined by type II pneumocytes. Exp. Pathol. 29 (1986) 4
203
Fig. 6. Alveolus containing macrophages with glass fibr es. Thic.kened alveolar wall mainly due to the presence of neutrophils and ma,cropha.ges in a male hamst er 3 days after treatment. Uranyl acetate and lead citrate. x 1,700.
After 1 month a yellowi sh-brown, P erl-p ositive co ating around gl ass fibres was seen fo r the first time ; only a very few of such coat ed fibres were found. A striking feature was t he occurrenee of dark (electro n-dense) eyt oplasmic areas adj acent to glass fibres a,n d around phagosomes. 3 months Widely varying numbers of macrophages filled with glass fibres were still found in alveoli, mainly peribronehiolar alveoli. As a rule neutrophilic leueoeytes were present in limited numb ers, but in 1 animal considerable amounts of these inflammatory cells were still visible around group s of macrophages. An occasional multinur.Ieated giant cell laden with glass fibres was seen; the glass fibres were found all around th e nuclei and not in the central cytoplasm (fig. 7). Glass fibres lying freely in alveolar lumens were stilI a common finding. The number of coated glass fibres had increased in comparison with 1 month, but was stilI a very minor portion of the total number of glass fibres. An occasional glass fibre has a rough (" weathered" ) surface indicating corrosion (fig. 8). Nodular clusters of macrophages ("silieotic granulom as") with massive amounts of glass particl es were now conspi cuous findings (fi g. 9). These clusters never occurred in great numbers and in fact occupied only a very small portion of the lungs.
204
Exp. Pathol. 29 (1986) 4
Fig. 7. l'IIultinucleated giant cell with glass fibres in the lung of a femule hamster 3 months after treatment. Note the localisation of the glass fibres around the nueJei. Uranyl acetute and lead citrate. X 4,000.
6 and 12 months Nodul ar clusters of macrophages were the predominant lesion after 6 and 12 months. Electron microscopical examinations clearly showed that the epithelium covering the clusters consisted of type I and type II pneumocytes and was continuous with the alveolar epithelium (fig. 10). The dusters generally were smaller than after 1 and 3 months, occasionally contained small groups of inflammatory cells (mononuclear cells and polymorphonuclear leucocytes) and were seen to be interwoven with strands of a pink, homogeneo us material. Ultrastructurally, coll agen fibres were clearly reco~nizable. Some of the glass fibres appeared to be corroded. As opposed to the smooth interior, t he outermost rim of such a corroded fibre was seen as a relatively thick layer of very fine granu les most probably r epresenting leached glass consisting of the silica skeleton only. Varying numbers of glass fibres-containing macrophages, a, sporadic l11ultinueleated giant cell with glass fibres, and a few free glass fibres were still found in alveoli. Relatively few intra- or extracellular glass fibres were coated with a yellowish-brown, Perl-positive material. Bronchiolar epithelium looked normal; Clara cells were clearly recognizable. Exp. Patho!. 29 (1986) 4
205
Fig. 8. Corrosion of an intracellular glass fibre (arrows) in the lungs of a female hamster 3 months after treatment. Uranyl acetate and lead citrate. X 25,800.
Fig. 9. Nodular cluster of macrophages containing massive numbers of glass fibres in the lung of a male hamster 3 months after treatment. Haematoxylin and eosin. X 160.
206
Exp. Pathol. 29 (1986) 4
Fig. 10. "Silicotic granuloma" lin ed by alveolar epithelium in th e lungs of a female hamster 6 months after treatment. Uranyl acetate and lead citrate. X 1,700.
Discussion In the present study the half-time of clearance of the glass fibres (58 %
207
Dissoluti on of glass fibres in rat lun gs has been conv incingly demonstrated by MORGAN et al. (1982). The longer fibres (::2: 30 fim ) which are t oo long to be phagocytized by a macrophage app eared to dissol ve much faster than shorter fibres (::;; 10 fim ) which arc largely engulfed by macrophages. Since in th e presont study 89 % of the glass fib r es were 12,um or shorter, dissolution mORt probably pl ayed only a ver y mi nor role in the di sa,p p earance of glass fibres from the lungs. Nevertheless, corrosion of glass fibres present in maerophages was occasionall y observed, indicating tha,t leaching and dissolution of intracellul ar gla.ss fibres may occur. However, it ca.nnot be ruled out th at the corrosion of th e fibres had taken place prior t o their ingestion by macrophages . From the present study it also appeared that intratracheal admini stration of glass fib r es r esulted in sever e acute damage to th e bronchiolo-al veolar t issue accompanied by a neutrophilic inflammatory response of the ha mster lung. The numb er of neutrophils rapidl y decreased and massive amo unts of alveolar maerophag·es appeared , and phagocytized or surrounded glass fibres . In inflamed areas containing plugs of glass fibres bron chioli and alveoli were lined by hyper- and metaplas tic epithelium. Gradually the number of glass fibres diminished, more fibr es became coated, aDd the linin g epithelium of brollchi oli and alveoli became norma.! again. "Silicotic granulomas" seen as clusters of tightly packed, glass fibr es-containing, iron-positive macrophages surrounded by a layer of alveolar epithelium developed and were the predominant lesion ill animals kill ed after 6 or 12 month s. Areas not containing glass fibres or "silicotic nodules" were indistinguishable from comp arable pulmonary regioJls in sal in e-treated controls. The above pulmonary response was very similar t o changes seen in tIle lungs of hamsters after intratracheal injection of quartz du st (GROSS et al. 1967) or to lesio ns fo und in hamster s following inhalation of inorganic fibres or fibrou s glass (LEE et al. 1981). Moreover , t he acute and subaeute effects on the lungs were not essentially different from the pulmonary changes seen in rats and guinea pigs foJl owing intratracheal instillati on of silica particles (REISER et al. 1982; LUGANO et al. 1982). Th e " si li eotic granulomas" r esembl ed the foreign body granulom as observed by BERNSTEI N et a.J. (l980) in rats after intratracheal admini stration or inhalation of glass fibres. However, in contrast to our findings t hese granul omas in rats contained many multinucleated giant cells. iHORGAN et al. (1982) did not obs erve granulomatous lesions or fi brosis in the lungs of rats given glass fibres of differ ent sizes by intratracheal instillation. However, the do ses the y used (0.5 or ].0 mg) were much lower than those used by BERNSTEIN et al. (1980) or in our study viz. 20 or 10 mg, r espectively. Coating of glass fibres wa.s not uncommon in the present study, but t ypical glass fibrebodies with a beaded coating as found by BOTHAM and HOLT (1971) in lungs of guinea pigs exposed to glass fibre-dust by inhalation, were not observed. In conclusio n, the glass fi bres used were cleared from the lungs of hamsters rather efficiently, were coated onl y sporadically, occasionally showed slight eorrosio n, and evoked a violent ac ute pneumoniti s followed by a strong alveolar macrophage r ear;tion, and finally the formation of "silicotic gr anulom as" in the lungs.
Ac7cnowledgements The authors thank Mr. P. D. VAN DEN HEUVEL for biotcehnical assistance, Mrs. E. C. M. VAN OOSTRml, Mr. W. G. ROVERTS and Thorr. M. C. A. HOFSTEE for invaluable histotechnical assistance, Mr. A. VAN WELlE for the excellent photographic work, iVlr. H . J. DE VETTER fo r typing the man uscript, and Dr. A. P. DE GROOT for critical reading of the man uscript. The studies were sponsored by th e Netherlands Cancer foundation ("Koninging Wilhelmina Fonds"), Amsterdam, The Netherlands.
Literature BERNSTEIN, D. M. , R. T. nREW and M. KUSCHNER, Experim en tal approaches for exposurc to sizcd glass fibr es. Environm. Health Perspect. 34, 47 ~57 (1980). BOTHAM, S. 1(., and P. F. HOLT, The development of glass fi bre bodies in th e lun gs of guinra pigs . J. Pathol. 103, 149 ~156 (1971).
208
Exp. Pathol. 29 (1986) 4
CRALLEY, L. J., and W. S. LAINHART, Are trace metals associated with asbestos responsible for the biologic effects attributed to asbestos? J. Oceup. l\Ied. Vi, 262-266 (1973). DIXON, J. R., D. B. LOWE, D. E. RICHARDS, L. J. CRALLEY and H. E. STOCKINGER, The role of trace metals in ehemical careinogenesis: asbestos cancer. Cancer Res. 30, 1068-1074 (1970). GROSS, P., J. DE VILLIERS and R. T. P. DE TREVILLE, Experimental silicosis. The "atypical" reaction in the Syrian hamster. Arch. Path. 84, 87-94 (1967). HARINGTON, J. S., Chemical studies of asbestos. Ann. NY Acad. Sci. 132, 31-47 (1965). HEISEL, E. B., and F. E. HUNT, Further studies ill cutaneous reactions to glass fibres. Arch. Ellyironm. Health 17, 705-711 (1968). J ONG, D. DE, and V. J. FERON, Intratracheale intubatie bij hamsters. Biotechniek 10, 66-74 (1971). KOLK, J. J., Biologische effeeten van man made mineral fibres. T. Soc. Geneesk. 60, 770-773 (1982). KUSCIINEH, 1\1., and G. M. WmGHT, The effects of intratracheal instillation of glass fiber of varying size in guinea pigs. In: Occupational Exposure to Fibrous Glass. HEW Pub!. N°. (NIOSH) 76-151, US Department of Health, Education and Welfare, Washington DC 1976, pp. 151-168. LEE, K. P., C. E. BAHHAS, F. D. GHIFFITH, R. S. WARITZ and C. A. LAPIN, Comparative pulmonary responses to inhaled inorganic fibres with asbestos and fiberglass. Environm. Res. 24, 167-191 (1981). LUGANO, E. M., J. H. DAUBER and R. P. DANIELE, Acute experimental silicosis. Lung morphology, histology and macrophage chemotaxin secretion. Am. J. Patho!. 109, 27-36 (1982). MEUH}IAN, L. 0., R. KIVILNOTO and M. HAKAl\IA, Combined effect of asbestos exposure and tobacco smoking on Finnish anthrophyUite miners and millers. Ann. NY Acad. Sci. 330, 491-496 (1979). i\IOHGAN, A., A. HOLMES and W. DAVISON, Clearance of sized glass fibres from the rat lung and their solubility in vivo. Ann. Occup. Hyg. 2il, 317 -331 (1982). lVloHISSET, Y., A. P'AN and Z. JEGIEH, Effeet of styrene and fiber glass on small airways of mice. J. Toxico!. Environm. Health 5, 943-956 (1979). POSSICK, P. A., A. GELLING and M. lVI. KEY, Fibrous glass dermatitis. Am. Ind. Hyg. Assoc. J. 31, 12-15 (1970). POTT, F., Tumorinduktion durch Asbest und andere Fasern - Tierexperimentelle Ergebnisse und ihre Bedeutung fiir die Praventivmedizin. Gmwelthygiene, Girardot, Diisseldorf - Essen 1981, pp.31-66. K. H. FRIEDRICHS and F. HUTI!, Ergebnisse aus Tierversuehen zur kanzerogenen Wirkung faserfiinniger Staube und ihre Deutung im Hinbliek auf die Tumorentstehung beim lVIenschen. Zb!. T:htkt. Hyg. I, Abt. Orig. B 162, 467-505 (1976). - U. ZIEM and U. lVIolm, Lung carcinomas and mesotheliomas following intratracheal instillation of glass fibres and asbestos. In: Proc. VIth Int. Pneumoconiosis Coni., Bochum 1983. REISER, K. lVI., T. W. HESTEHBERG, W. lVI. HASCHEK and J. A. LAST, Experimental silicosis 1. Acute effects of intratracheally instilled quartz on collagen metabolism and morphologic characteristics of rat lungs. Am. J. Patho!. 107, 176-185 (1982). SAR,\CCI, R., Asbestos and lung cancer: an analysis of the epidemiological evidence on the asbestossmoking interaction. Int. J. Cancer 20, 323-331 (1977). SELIKOFF, 1. J., H. SEIDMAN and E. C. HAMMOND, lVIortality effects of cigarette smoking among amosite asbestos factory workers. J. Nat!. Cancer lnst. 6il, 507-513 (1980). SHABAD, L. lVI., L. N. PYLEV, L. V. KRIVOSHEEVA, T. F. KULA GINA and B. A. NElIIENKO, Experimental studies on asbestos carcinogenicity. J. Nat!. Cancer lnst. il2, 1175-1187 (1974). STANTON, lVI. F., lVI. LAYARD, A. TEGERIS, E. MILLER, lVI. lVIAY and E. KENT, Carcinogenicity of fibrous glass: Pleural response in the rat in relation to fiber dimension. J. Nat!. Cancer lnst. il8, 587-603 (1977). - and C. WRENCH, Mechanisms of mesothelioma induction with asbestos and fibrous glass. J. Nat. Cancer Inst. 48, 797-821 (1972). Wc\GNER, J. C., G. BERRY and J. W. SKIDMOHE, Studies of the carcinogenic effects of fiber glass of different diameters following intrapleural inoculation in experimental animals. In: Occupational Exposure to Fibrous Glass. Proc. Symp., HEW Publications N° (NIOSH) 76-151, US Department of Health, Education and Welfare, Washington DC 1976, pp. 193-197.
Exp. Pathol. 29 (1986) 4
209
Pulmonary response of hamsters to fibrous glass. Clearance and morphology after a single intratracheal instillation
With 10 figures (Received June 24, 1985)
Address for cOl'respondence: Dr. V. J. FEROK, TNO-eIVa To xicology a.nd Nutrition Institute, P.O. Box 360, 3700 AJ Zeist, The Netherlands Key W 0 r ds: glass fibres; hamster lung; clearance; tracheal instillation; pneumonitis; alveolar macrophage
8ttmrlwry In a one-year serial sacrifice stud y 10 mg glass fibres (length, 95 % IX 20.um, 89 % IX
12.um, 58 % IX 5.um and 25 % IX 2.um; diameter, 88 % IX 1.0.um, 60 % IX 0.5.um and 31 %IX 0.25 .um) suspended in 0.2 ml saline solution were administered to Syrian golden hamsters by a. sing'le intratracheal instillation to determine the clearance of the glass fibres from the lungs and to examine their effects on the lungs using light and electron microscopy. The clearance was rather efficient with a half-time of about 3 months. Coating and corrosion of glass fibres were sporadic findings. A violent focal acute pneumonitis was evoked by the glass fibres and was followed by exeessive aecumulations of alveolar macro phages often loaden with glass fibres. Thereafter, "silieotie granulomas" developed which were seen as dusters of tightly packed iron-positive maerophages eontaining glass fibres. These granulomas were surrounded by a layer of alveolar epitheliulll, and wer e by the end of the study the predominant lesion in otherwise normal lungs.
I ntroduction The industrial production of glass wool and glass yarns started around 1930. Mineral wools are mainl y used for thermal and sonar insul ation ; glass yarns find wide applieation as reinforcement of rubber, plasti e, paper and cement (KOLK 1982). Fibrous glass (length/ diameter ratio> 3: 1) is used in th e manufacture of more than 33,000 products (MORISSET et al. 1979). One well-known adverse effect of fibrous glass is that it eauses itehing through meehaniral irritation of the skin (HEISEL and HUNT 1968; POSSICK et al. 1970). KUSCHNER and WRIGHT (1976) observed pulm onary fibrosis in guinea pigs aft er intratracheal instillation of fibrous glass. After intrapleural or intraperitoneal inoculation of fibrous glass sarcomas and mesotheliomas have been found in rats (STANTON and WRENCH 1972; POTT et aI. 1976; WAG NER et al. 1976 ; STANTON et al. 1977). Reeently, POTT (1981) and POTT et aI. (1983) showed that glass fibres admini stered intratraeheally to Syrian golden hamsters induced both pleural mesotheliomas and pulmonary careinomas. Both the carcinogenicity and the fibro g'enieity have been shown to be related to physical faCtors such as length and diameter of the fibres (STANTON and WRENCH 1972). Although most experimental evidence indicates the importanee of the geometry of minerai fibres for their biologieal aetivity including carcinogenieity, other factors such as 13
197
chemical composition (HARINGTON 1965) and polycyclic aromatic hydrocarbons and other compounds adsorbed on the fibres (DIXON eta!. 1970; CRALLEY and LAINHART 1973; SHABAD et a!. 1974) are thought to playa role in the carcinogenicity of fibrous dusts. Asbestos enhance the induction of lung cancer by cigarette smoke (SARACCI 1977; lVIEURMAN et al. 1979; SELIKOFF et a!. 1980). In mice respirable glass fibres have been found to enhance the adverse effect of styrene vapour on the bronchiolar epithelium (lVIORISSET et a!. 1979). Within the scope of a research programme concerning the significance of non-specifie physical injury to the respiratory tract for the formation of tumours at the site of damage, intratracheal instillation of glass fibres was used as one of the methods to inflict damage to the lungs. The short- and long-term effects on the lungs of single and repeated administration of fibrous glass with or without the careinogen benzo( a)pyrene have been studied in Syrian golden hamsters using light and electron mieroseopy. The present report describes the clearanee of glass fibres from the lungs and their effects on the lungs after a single intratracheal instillation.
Materials and Methods Animals The animals used in these studies were Syrian golden hamsters (111esocl'icetus auratus) obtained from the randomly-bred colony of the Central Institute for the Breeding of Laboratory Animals TNO, Zeist, The Netherlands. The hamsters were housed in groups of four or five in suspended stainless steel cages with wire-screen bottom in a room ventilated with 10 air changes/h, maintained at 23 ± 1°C, with a relative humidity of 45-GO % and a 12 h light/dark cyele (light from 6.00 a.m. till G.OO p.m.). The animals were offered a pelleted stock diet for hamsters (R13, Hope Farms, Woerden, The Netherlands) and bottled, unfluoridated, tap-water ad libitwil. The hamsters were 10 weeks old at the time of administration of the glass fibres. Glass fibres Glass microfibres (code 104, Johns-Manville, Denver, U.S.A.) were obtained commercially. The elemental composition of the glass fibres is: Si0 2 57.9 %, B2 0 3 10.7%, Na 2 0 10.1 %, Al 2 0 3 5.8 ')~, BaO 5.0 %, ZnO 3.9 %, CaO 3.0 %, K 2 0 2.9 %, F 2 0.6 % and Fe 2 0 3 0.1 %. The glass wool was milled in a ball mill with agate bowl and agate balls (Pulverisette 5, Alfred Fritsch and Co, Idar-Oberstein, F.R.G.). The following procedure was used to obtain a total of 43 g glass fibres: an 80 ml agate bowl was filled with 1 g glass wool and 5 agate balls of 20 mm diameter. The glass wool was milled lor 20 s at 70 % of the maximum rotation speed, followed by 10 min at 50 % of the maximum speed. This procedure was repeated 43 times. The different batehes of ground glass fibres were mixed by hand-stirring and stored in a glass eontainer at room temperature. The size distribution of the fibres was determined by seanning electron microscopy using a Philips EM 400 mieroscope fitted with a special computerized scanning equipment (Tracor N orthem TN 2000)1). Thirteen hundred and ten fibres were measured in a representative sample and the individual fibre dimensions were arranged in a length-diameter diagram (fig. 1). The following size distribution by number was found: length, 95 % c< 20 [tm, 89 % c< 12,um, 58 % ex 5 p.m and 25 % c< 2 [tm; diameter, 88 % ex 1.0 p.m, GO % ex 0.5,(tm and 31 % c< 0.25 p.m. Administration of glass fibres Samples of glass fibres were suspended in a sterile, non-pyrogenie 0.9 % NaCI solution. A suspension containing 5 % (w/v) glass fibres was prepared. During the treatment period of the hamsters the suspensions were magnetically stirred. Details of the instillation procedure have been described previously (JONG and FERON 1971). Under light ether anaesthesia each hamster reeeived a single intratracheal instillation of 10 mg glass fibres administered as 0.2 ml of the 5 % suspension. Determination of glass fibres in lungs 2 ) The elearance of glass fibres from the lungs was studied by determination of the amounts of glass in the lungs at 0 time (within 2 min after instillation) and further 14 days, 1, 3 and G months 1) The size distribution measurements were carried out by Mr. lVI. J. VAN N OORD and ]\1rs. Ing. C. J. G. BLOK-vAN HOEK of the Centre for Analytical Electron Microscopy TNO, Leiden, The Netherlands. 2) These measurements were carried out by Mr. K. KOOPMANS in the Department of Analytical Chemistry, TNO-Division of Teehnology for Society, Delft, The Netherlands.
198
Exp. PatllO!' 29 (1986) 4
~
fiJ@ 0.0
~
ffi6J
ri'tlj
tV
~
~ ~
~
Diameter in JIm
<>
r!I= 5%
of totaL
Length in JI m Fig. 1. Length-diameter distribution of the glass fibres used.
after intratracheal instillation of the suspension of glass fibres. The lungs were dissected out and the trachea and paratrachcal and tracheo-bronchial lymph nodes were removed; then the lungs were frozen with liquid nitrogen and stored in a freezer at -20 DC. The glass content of each lung was indirectly determined by spectrochemical analysis, measuring the aluminum content of the lung-ash obtained by the powder DC-arc technique. Ash samples were mixed with lithium carbonate-graphite mixtures and the obtained spectra were compared with spectra of commercial lithium carbonate standards. The aluminium contents of the ashes were measured in duplicate with an optical emission spectrograph at three different AI-spectral lines, viz. 265.25, 266.4 and 257.5 nm, respectively. The values of the duplicates and those obtained at the different spcctrallines showed a variation of 10-15%, which is somewhat larger than the usual accuracy of 10 % obtained with this technique. The average aluminum value of each sample was corrected by subtracting the average aluminum value of the control lungs collected at the corresponding time intervals. After converting the aluminum values into values for the amount of glass, the glass content of the lungs at the various time intervals was calculated and expressed in percentages of the amount of glass found at 0 time. Experimental design and conduct Forty four male and 44 female hamsters were treated with glass fibres. Groups of 4 males and 4 females were killed by cutting the abdominal aorta under ether anaesthesia at 0 time (within 2 min after instillation) and further after 4, S, and 24 h, after 3, 7 and 14 days, and after 1, 3, 6 and 12 months. After S h, after 14 days, and after 1, 3, and 6 months 4 male and 4 female hamsters treated with saline only were killed in the same way; they served as controls. At each point of time the groups of treated and control animals were divided into 3 subgroups viz. 1 male and 1 female for determination of the glass content of the lungs, 2 males and 2 females for light microscopy and 1 male and 1 female for electron mitroscopy. The lungs to be used for glass determinations were stored at -20 DC until the analyses could be carried out. The lungs to be studied by light microscopy were removed in toto with trachea and larynx, and preserved in a 4 % neutral, phosphatebuffered solution of formaldehyde by careful intratracheal infusion of the fixative under 10 cm water pressure. The paratracheal and tracheobronchial lymph nodes were also removed and fixed in formalin. The liver, kidneys, thymus and mesenteric lymph nodes were collected and preserved only of the hamsters killed after 6 months. All organs were processed through paraffin wax, sectioned at 5 pm (of the larynx, trachea and each pulmonary lobe sections were prepared at 3 different levels), stained with haematoxylin and eosin, and examined microscopically. For electron microscopical investigations the lungs were fixed by intratracheal instillation of the fixative consisting of 2.5 % glutaraldehyde buffered with 0.1 M sodium cacodylate to which 0.02 M CaCl 2 was added. The fixative had a pH of 7.3 and an osmolality of 500 m Osm/kg. The fixation was carried out while the hamsters were anaesthetized with Nembutal (50 mg/kg body weight). Then the lungs with the trachea were removed and stored overnight in the fixative. Thereafter, slices of the lungs were stored in the buffer solution at 4 DC. Finally small pieces of lung tissue were postfixed in 1 % OS04 buffered with 0.1 M sodium cacodylate, washed, dehydrated in acetone water mixtures and embedded in an araldite-glycidether mixture, followed by hardening at GO DC during 4S h. Semithin sections were prepared and stained with toluidine blue. Ultrathin sedions were cut with a diamond knife, stained with uranyl acetate and lead citrate, and viewed with a Philips 201 G electron microscope at GO kV.
1:3*
Exp. Pathol. 29 (1986) 4
199
Results
Clearance of glass fibres from the lungs The average amounts of glass fibres fo und in the lungs at different points of time after administration, are given in table 1. After 2 weeks there was no measurable reduction in the amount of glass present in the lungs. However, after 3 months the amount of glass in the lungs was decreased to about 50 ';6, and a further reduction to about 20 was found after 6 months. Gross pathology Apart from a few tiny greyish areas on the surface of the lungs of several hamsters treated with glass fibres, no gross lesions attributable to treatment were seen. Microscopic pathology Time period 0 The lungs of all animals killed immediately after instillation contained massive amounts of glass fibres. Glass fibres were nearly always found in each of the pulmonary lobes, but there was a considerable variation in the amount of fibres among the various lobes. The glass fibres occurred as large plugs, as small aggregates, and also as individual fibres (fig. 2). They were most frequently found in bronchioles, alveolar ducts and peribronchiolar alveoli, but were also present in larynx, trachea, bronchi and peripheral alveoli. 4 hours 4 hours after instillation, perivascular edema, haemorrhages, cellular debris, and neutrophilic leucocytes were visible in areas containing plugs of glass fibres, particularly in the peribronchiolar regions. Over large areas alveolar epithelial cells were destroyed and had disappeared leaving "naked" blood capillaries. Neutrophilic lencocytes having phagocytized glass fibres were already seen at this point of time (fig". 3). Free glass fibres seemed to be surrounded by a thin layer of electron-lucent material. Lamellar bodies of type II pneumocytes were empty. Capillaries and interstitium contained many neutrophilic leucocytes. The total of changes resulted in a chaotic picture of the pulmonary tissue in glass fibre-containing areas. S hours After 8 hours peribronchiolar regions containing glass fibres showed much cellular debris, extensive haemorrhages and massive infiltrates of neutrophils (fig. 4). As a consequence, Table 1. Clearance of glass fibres from the lungs of hamsters after intratracheal instillation of a single dose of 10 mg glass fibres suspended in 0.9 % NaCI solution Period of time between instillation of glass fibres and killing of the animals 1-2 min (= 0 time) 14 days 1 month el months G months 12 months
Amount of glass fibres retained in the lungs (expressed as % of the amount found at 0 time) Males Females
a) 100 Gel
47 28
NDc)
100b )
lOB
96
55 11
ND
a) No glass found probably due to faulty instillation. Therefore, the amount of glass found after 14 days was set at 100 %, which was deemed permissible in view of the lOB % found in females at this point of time. il) The average amonnt of glass fibres found in the lungs at 0 time was 9.2 mg. c) Xot determined by mistake.
200
Exp. Pathol. 29 (1986) 4
Fig. 2. Glass fibres in the lung of a male hamster immediately after treatment. I-Iaematoxylin and eosin. X 160.
Fig. 3. Neutrophilic leueocytes having phagocytized glass fibres in alveoli of a female hamster 4 h after treatment. Uranyl acetate and lead acetate. X 5,800. Exp.Eathol. 29 (1986) 4
201
Fig. 4. Cellula.r debris and massive infiltrates of neutrophilic lcucocytes in peri bronchiolar regions of the lung of a female hamster 8 h after treatment. Haematoxylin and eosin. x 40.
alveolar structures were hardly recognizable in these areas. Occasionally, small groups of alveolar macrophages could be detected. Bronchiolar epithelium in contact with plugs of glass fibres was flattened. 24 hours One day after instillation a violent inflammatory reaction accompanied by haemorrhages was observed in the lungs. An alveolar structure could no longer be distinguished in peribronchiolar regions containing glass fibres, neutrophilic leucoeytes and now also largo numbers of macrophages. Most of the glass fibres were ingested or surrounded by macrophages. Occasionally electron-dense granular material was attached to or surrounded the glass fibres (fig. 5). Neutrophils were seen to surround macrophagcs heavily laden with glass fibres. Perivascular edema and flattening of bronchiolar epithelium in contact with plugs of glass fibres were common findings. 3 and 7 days In affected areas the alveolar strueture was now better recognizable than after 24 hours. The alveolar septa were widened due to a thickened interstitium containing neutrophiIs and glass fibres-containing macrophages, and a lining of cuboidal epithelial cells. Alveoli were filled with macrophages containing glass fibres. Th e number of macrophages was invariable greatly increased, that of neutrophils in most animals clearly decreased as compared to 24 hours. After 7 days aggregates of glass fibres were seen to be surrounded by neutrophilic leueoeytes. Nuclear debris was found in varying amounts; macrophages with glass were seen to also contain phagocytized nuclear fragments. Individual eosinophils were frequently encountered. The alveolar knob endings (alveolar rings) protruding into the alveolar ducts were swollen as were the alveolar septa, which was due to thickened epithelium and the presence in the interstitium of neutrophilic leu eocytes and macrophages with glass fibres (fig. 6). T,h e alveolar epithelial cells frequently showed many short microvilli-like projections. Haemor-
202
Exp. Pathol. 29 (1986) 4
Fig. 5. Electron-dense granular material surrounding intracellular glass fibres in the lung of a female hamster 24 h after treatment. Uranyl acetate and lead citrate. X 40,000.
rhages and perivascular edema were less conspicuous than after 24 hours. Bronchioles often eo ntained plugs of glass fibres mingled with eellular debris and neutrophils. At these sites the bronchiolar epithelium was often hypertrophic. and oc.casionally stratified. In affected bronchioles Clara cells could not or hardly be recognized. 14 days Considerable numbers of neutrophils were still visible around plugs of glass fibres. Glass fibres in the alveolar lumen were either phagocytized or surrounded by macrophages, which often also contained nuclear remnants. Clusters of macrophages only containing phagorytized nuclear fragments were common findings; occasionally such clusters were surrounded hy neutrophilic leucocytes. Groups of macrophages with glass fibres and nuclear debris also contained iron-}Jositive material according to Perl's method for staining of iron. Individual Ill acrophages were not iron-positive. Areas not containing glass parti cles did not exhibit pathological changes. 1 month Peribronchiolar alveoli were often completely filled with macrophages containing glass fibres . In some animals the number of neutrophils was still considerable, whereas in others only a few of these cells were seen. The amount of cellular debri s was greatly reduced as (:ompared to 7 and 14 days. Haemorrhages were no longer observed. Multinucleated giant rells were occasionally found. Clusters of tightly packed macrophages laden with glass particles and covered with a continuous layer of simple epithelium were observed at this stage for the first time. The content of the macrophages in these clusters was yellowish-light brown and appeared to be moderately iron-positive. The clusters were vi sible as granulomas varying in diameter from 50 to 300 pm and protruding into'the lumen of alveoli and alveolar duets; they were attached to th e alveolar wall often only through a thin stalk. Thickened alveolar septa were often nearly exclusively lined by type II pneumocytes. Exp. Pathol. 29 (1986) 4
203
Fig. 6. Alveolus containing macrophages with glass fibr es. Thic.kened alveolar wall mainly due to the presence of neutrophils and ma,cropha.ges in a male hamst er 3 days after treatment. Uranyl acetate and lead citrate. x 1,700.
After 1 month a yellowi sh-brown, P erl-p ositive co ating around gl ass fibres was seen fo r the first time ; only a very few of such coat ed fibres were found. A striking feature was t he occurrenee of dark (electro n-dense) eyt oplasmic areas adj acent to glass fibres a,n d around phagosomes. 3 months Widely varying numbers of macrophages filled with glass fibres were still found in alveoli, mainly peribronehiolar alveoli. As a rule neutrophilic leueoeytes were present in limited numb ers, but in 1 animal considerable amounts of these inflammatory cells were still visible around group s of macrophages. An occasional multinur.Ieated giant cell laden with glass fibres was seen; the glass fibres were found all around th e nuclei and not in the central cytoplasm (fig. 7). Glass fibres lying freely in alveolar lumens were stilI a common finding. The number of coated glass fibres had increased in comparison with 1 month, but was stilI a very minor portion of the total number of glass fibres. An occasional glass fibre has a rough (" weathered" ) surface indicating corrosion (fig. 8). Nodular clusters of macrophages ("silieotic granulom as") with massive amounts of glass particl es were now conspi cuous findings (fi g. 9). These clusters never occurred in great numbers and in fact occupied only a very small portion of the lungs.
204
Exp. Pathol. 29 (1986) 4
Fig. 7. l'IIultinucleated giant cell with glass fibres in the lung of a femule hamster 3 months after treatment. Note the localisation of the glass fibres around the nueJei. Uranyl acetute and lead citrate. X 4,000.
6 and 12 months Nodul ar clusters of macrophages were the predominant lesion after 6 and 12 months. Electron microscopical examinations clearly showed that the epithelium covering the clusters consisted of type I and type II pneumocytes and was continuous with the alveolar epithelium (fig. 10). The dusters generally were smaller than after 1 and 3 months, occasionally contained small groups of inflammatory cells (mononuclear cells and polymorphonuclear leucocytes) and were seen to be interwoven with strands of a pink, homogeneo us material. Ultrastructurally, coll agen fibres were clearly reco~nizable. Some of the glass fibres appeared to be corroded. As opposed to the smooth interior, t he outermost rim of such a corroded fibre was seen as a relatively thick layer of very fine granu les most probably r epresenting leached glass consisting of the silica skeleton only. Varying numbers of glass fibres-containing macrophages, a, sporadic l11ultinueleated giant cell with glass fibres, and a few free glass fibres were still found in alveoli. Relatively few intra- or extracellular glass fibres were coated with a yellowish-brown, Perl-positive material. Bronchiolar epithelium looked normal; Clara cells were clearly recognizable. Exp. Patho!. 29 (1986) 4
205
Fig. 8. Corrosion of an intracellular glass fibre (arrows) in the lungs of a female hamster 3 months after treatment. Uranyl acetate and lead citrate. X 25,800.
Fig. 9. Nodular cluster of macrophages containing massive numbers of glass fibres in the lung of a male hamster 3 months after treatment. Haematoxylin and eosin. X 160.
206
Exp. Pathol. 29 (1986) 4
Fig. 10. "Silicotic granuloma" lin ed by alveolar epithelium in th e lungs of a female hamster 6 months after treatment. Uranyl acetate and lead citrate. X 1,700.
Discussion In the present study the half-time of clearance of the glass fibres (58 %
207
Dissoluti on of glass fibres in rat lun gs has been conv incingly demonstrated by MORGAN et al. (1982). The longer fibres (::2: 30 fim ) which are t oo long to be phagocytized by a macrophage app eared to dissol ve much faster than shorter fibres (::;; 10 fim ) which arc largely engulfed by macrophages. Since in th e presont study 89 % of the glass fib r es were 12,um or shorter, dissolution mORt probably pl ayed only a ver y mi nor role in the di sa,p p earance of glass fibres from the lungs. Nevertheless, corrosion of glass fibres present in maerophages was occasionall y observed, indicating tha,t leaching and dissolution of intracellul ar gla.ss fibres may occur. However, it ca.nnot be ruled out th at the corrosion of th e fibres had taken place prior t o their ingestion by macrophages . From the present study it also appeared that intratracheal admini stration of glass fib r es r esulted in sever e acute damage to th e bronchiolo-al veolar t issue accompanied by a neutrophilic inflammatory response of the ha mster lung. The numb er of neutrophils rapidl y decreased and massive amo unts of alveolar maerophag·es appeared , and phagocytized or surrounded glass fibres . In inflamed areas containing plugs of glass fibres bron chioli and alveoli were lined by hyper- and metaplas tic epithelium. Gradually the number of glass fibres diminished, more fibr es became coated, aDd the linin g epithelium of brollchi oli and alveoli became norma.! again. "Silicotic granulomas" seen as clusters of tightly packed, glass fibr es-containing, iron-positive macrophages surrounded by a layer of alveolar epithelium developed and were the predominant lesion ill animals kill ed after 6 or 12 month s. Areas not containing glass fibres or "silicotic nodules" were indistinguishable from comp arable pulmonary regioJls in sal in e-treated controls. The above pulmonary response was very similar t o changes seen in tIle lungs of hamsters after intratracheal injection of quartz du st (GROSS et al. 1967) or to lesio ns fo und in hamster s following inhalation of inorganic fibres or fibrou s glass (LEE et al. 1981). Moreover , t he acute and subaeute effects on the lungs were not essentially different from the pulmonary changes seen in rats and guinea pigs foJl owing intratracheal instillati on of silica particles (REISER et al. 1982; LUGANO et al. 1982). Th e " si li eotic granulomas" r esembl ed the foreign body granulom as observed by BERNSTEI N et a.J. (l980) in rats after intratracheal admini stration or inhalation of glass fibres. However, in contrast to our findings t hese granul omas in rats contained many multinucleated giant cells. iHORGAN et al. (1982) did not obs erve granulomatous lesions or fi brosis in the lungs of rats given glass fibres of differ ent sizes by intratracheal instillation. However, the do ses the y used (0.5 or ].0 mg) were much lower than those used by BERNSTEIN et al. (1980) or in our study viz. 20 or 10 mg, r espectively. Coating of glass fibres wa.s not uncommon in the present study, but t ypical glass fibrebodies with a beaded coating as found by BOTHAM and HOLT (1971) in lungs of guinea pigs exposed to glass fibre-dust by inhalation, were not observed. In conclusio n, the glass fi bres used were cleared from the lungs of hamsters rather efficiently, were coated onl y sporadically, occasionally showed slight eorrosio n, and evoked a violent ac ute pneumoniti s followed by a strong alveolar macrophage r ear;tion, and finally the formation of "silicotic gr anulom as" in the lungs.
Ac7cnowledgements The authors thank Mr. P. D. VAN DEN HEUVEL for biotcehnical assistance, Mrs. E. C. M. VAN OOSTRml, Mr. W. G. ROVERTS and Thorr. M. C. A. HOFSTEE for invaluable histotechnical assistance, Mr. A. VAN WELlE for the excellent photographic work, iVlr. H . J. DE VETTER fo r typing the man uscript, and Dr. A. P. DE GROOT for critical reading of the man uscript. The studies were sponsored by th e Netherlands Cancer foundation ("Koninging Wilhelmina Fonds"), Amsterdam, The Netherlands.
Literature BERNSTEIN, D. M. , R. T. nREW and M. KUSCHNER, Experim en tal approaches for exposurc to sizcd glass fibr es. Environm. Health Perspect. 34, 47 ~57 (1980). BOTHAM, S. 1(., and P. F. HOLT, The development of glass fi bre bodies in th e lun gs of guinra pigs . J. Pathol. 103, 149 ~156 (1971).
208
Exp. Pathol. 29 (1986) 4
CRALLEY, L. J., and W. S. LAINHART, Are trace metals associated with asbestos responsible for the biologic effects attributed to asbestos? J. Oceup. l\Ied. Vi, 262-266 (1973). DIXON, J. R., D. B. LOWE, D. E. RICHARDS, L. J. CRALLEY and H. E. STOCKINGER, The role of trace metals in ehemical careinogenesis: asbestos cancer. Cancer Res. 30, 1068-1074 (1970). GROSS, P., J. DE VILLIERS and R. T. P. DE TREVILLE, Experimental silicosis. The "atypical" reaction in the Syrian hamster. Arch. Path. 84, 87-94 (1967). HARINGTON, J. S., Chemical studies of asbestos. Ann. NY Acad. Sci. 132, 31-47 (1965). HEISEL, E. B., and F. E. HUNT, Further studies ill cutaneous reactions to glass fibres. Arch. Ellyironm. Health 17, 705-711 (1968). J ONG, D. DE, and V. J. FERON, Intratracheale intubatie bij hamsters. Biotechniek 10, 66-74 (1971). KOLK, J. J., Biologische effeeten van man made mineral fibres. T. Soc. Geneesk. 60, 770-773 (1982). KUSCIINEH, 1\1., and G. M. WmGHT, The effects of intratracheal instillation of glass fiber of varying size in guinea pigs. In: Occupational Exposure to Fibrous Glass. HEW Pub!. N°. (NIOSH) 76-151, US Department of Health, Education and Welfare, Washington DC 1976, pp. 151-168. LEE, K. P., C. E. BAHHAS, F. D. GHIFFITH, R. S. WARITZ and C. A. LAPIN, Comparative pulmonary responses to inhaled inorganic fibres with asbestos and fiberglass. Environm. Res. 24, 167-191 (1981). LUGANO, E. M., J. H. DAUBER and R. P. DANIELE, Acute experimental silicosis. Lung morphology, histology and macrophage chemotaxin secretion. Am. J. Patho!. 109, 27-36 (1982). MEUH}IAN, L. 0., R. KIVILNOTO and M. HAKAl\IA, Combined effect of asbestos exposure and tobacco smoking on Finnish anthrophyUite miners and millers. Ann. NY Acad. Sci. 330, 491-496 (1979). i\IOHGAN, A., A. HOLMES and W. DAVISON, Clearance of sized glass fibres from the rat lung and their solubility in vivo. Ann. Occup. Hyg. 2il, 317 -331 (1982). lVloHISSET, Y., A. P'AN and Z. JEGIEH, Effeet of styrene and fiber glass on small airways of mice. J. Toxico!. Environm. Health 5, 943-956 (1979). POSSICK, P. A., A. GELLING and M. lVI. KEY, Fibrous glass dermatitis. Am. Ind. Hyg. Assoc. J. 31, 12-15 (1970). POTT, F., Tumorinduktion durch Asbest und andere Fasern - Tierexperimentelle Ergebnisse und ihre Bedeutung fiir die Praventivmedizin. Gmwelthygiene, Girardot, Diisseldorf - Essen 1981, pp.31-66. K. H. FRIEDRICHS and F. HUTI!, Ergebnisse aus Tierversuehen zur kanzerogenen Wirkung faserfiinniger Staube und ihre Deutung im Hinbliek auf die Tumorentstehung beim lVIenschen. Zb!. T:htkt. Hyg. I, Abt. Orig. B 162, 467-505 (1976). - U. ZIEM and U. lVIolm, Lung carcinomas and mesotheliomas following intratracheal instillation of glass fibres and asbestos. In: Proc. VIth Int. Pneumoconiosis Coni., Bochum 1983. REISER, K. lVI., T. W. HESTEHBERG, W. lVI. HASCHEK and J. A. LAST, Experimental silicosis 1. Acute effects of intratracheally instilled quartz on collagen metabolism and morphologic characteristics of rat lungs. Am. J. Patho!. 107, 176-185 (1982). SAR,\CCI, R., Asbestos and lung cancer: an analysis of the epidemiological evidence on the asbestossmoking interaction. Int. J. Cancer 20, 323-331 (1977). SELIKOFF, 1. J., H. SEIDMAN and E. C. HAMMOND, lVIortality effects of cigarette smoking among amosite asbestos factory workers. J. Nat!. Cancer lnst. 6il, 507-513 (1980). SHABAD, L. lVI., L. N. PYLEV, L. V. KRIVOSHEEVA, T. F. KULA GINA and B. A. NElIIENKO, Experimental studies on asbestos carcinogenicity. J. Nat!. Cancer lnst. il2, 1175-1187 (1974). STANTON, lVI. F., lVI. LAYARD, A. TEGERIS, E. MILLER, lVI. lVIAY and E. KENT, Carcinogenicity of fibrous glass: Pleural response in the rat in relation to fiber dimension. J. Nat!. Cancer lnst. il8, 587-603 (1977). - and C. WRENCH, Mechanisms of mesothelioma induction with asbestos and fibrous glass. J. Nat. Cancer Inst. 48, 797-821 (1972). Wc\GNER, J. C., G. BERRY and J. W. SKIDMOHE, Studies of the carcinogenic effects of fiber glass of different diameters following intrapleural inoculation in experimental animals. In: Occupational Exposure to Fibrous Glass. Proc. Symp., HEW Publications N° (NIOSH) 76-151, US Department of Health, Education and Welfare, Washington DC 1976, pp. 193-197.
Exp. Pathol. 29 (1986) 4
209