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Animal Models ofPneumoconiosis B ACOUSTIC IMPEDANCE METHOD FOR DETECTING LUNG DYSFUNCTION JOHN SNECKENBERGER, Professor Timothy Whitmoyer, Doctoral Student Mechanical and Aerospace Engineering, West Virginia University Morgantown, WV 26506, USA ABSTRACT The acoustic impedances of seven rat lungs were measured at frequencies between 100 and 6400 Hz. Rats were divided into two groups: a silica exposed group (N=3) and a control group (N=4). The silica exposed group was injected intratracheally with silica solution. Three of die control group were intratracheally in jected with saline. Between four and six weeks after die injections, all lungs were excised and degassed. Lungs were suspended in a pressure chamber, with the trachea canula attached to the end of a tapered im pedance tube. The lungs were subjected to transpulmonary pressures between --30 cm H20 and 6 cm H20 to simulate deflation and inflation. With transpulmonary pressure being held constant, die impedance tube was excited with random noise. A dual channel analyzer calculated H12(f), die transfer function between the two microphones. This function was used to calculate the lung's impedance at that pressure. The im pedance magnitude spectra of both groups typically had peals at 2000, 3500, and 5500 Hz. Statistically significant differences {90% confidence level or greater) between die two groups occur at die 3500 Hz peak at transpulmonary pressures of 20, 8, 6,4, and 2 cm of H20. This feet seems to confirm that this method can detect lung disease. Further research will indicate whether this method will be able to detect the onset of coal worker's pneumoconiosis. INTRODUCTION One ofthe first studies ofimpedance of the human lung were conducted by DuBois et al.2 using die forced oscillation technique. This technique, however, liras limited to frequen cies below 30 Hz. Further studies by Van Den Berg* re vealed that the lung reflected higher frequency sounds (100-10,000 Hz), instead of behaving as an anechoic ter mination. This discovery has lead to several studies of die acoustical properties ofboth human and animal lungs at high frequencies.3'4*5 Tshizalra et al.4 measured the input impedances of laryngectomized human subjects using a two microphone technique. This study reported peaks in the impedance magnitude at 640, 1400, and 2100 Hz. Fredberg et al.3 used a transient forced oscillation technique to measure the input impedance of ex cised canine lungs for frequencies up to 10,000 Hz. Jayaraman and Frazer5 used a two microphone technique in combination with transmission matrix theory to study changes in die acoustic impedance of excised rat hings during defla tion and inflation. This study's focus is to determine the differences in die acoustic impedance ofexcised silicotic and healthy rat hings. Seven Long Evans Hooded rats, weighing between 200 and 250 g, were divided into two groups. The silica-exposed group (N=3) were intratracheally injected with a silica-saline solution to induce silicosis. Three rats of die control group (N=4) were given a sham exposure of saline. During a 312 period four to six weeks after injection, all lungs were ex cised and degassed. METHODS Figure 1 displays a block diagram of die impedance tube facility used in this study. An excised lung is attached to die end of a tapered tube within a plexiglass pressure chamber. Random noise, produced by a Bmel and Kjaer 2032 dual channel analyzer, is amplified and introduced into the tube via a side-mounted speaker driver (University, type ID-30C-8). The standing waves thus formed in the impedance tube are measured by two Bruel and Kjaer 4136 pressure microphones mounted 2.3 cm apart in a plexiglass cylinder. The signals of these microphones are die inputs to die dual channel analyzer, which calculates die transfer function be tween die two microphones, Hj2, and its inverse Fourier transform , h(t). Following Jayaraman and Frazer's exam ple,5 exponential weighting is applied to h(t) and transmis sion matrix theory applied to die resulting transfer function to yield the input impedance of the excised lung. The plexiglass chamber's pressure is controlled by a variable speed pump to produce transpulmonary pressures between 30 and --6 cm of H20. The difference between chamber pressure and atmospheric pressure is monitored by a water manometer. A lung is first inflated to 30 cm of H20, then deflated to --6 cm of H20, pausing at several pressures for impedance measurements. Once fully deflated, the lung is mfiatftd to 30 cm of H20, again stopping at various pressures for measurements. Animal Models ofPneumoconiosis JZ Figure 1. Block diagram of impedance measurement facility. RESULTS The average magnitude spectra of the silica and control groups are presented in Figures 2, 3, and 4 for transpulmonary pressures of 30,8, and 2 cm of H2O, respectively. DISCUSSION The impedance magnitude spectra ofall rats have been com puted for transpulmonary pressures of 30,20,10, 8, 6, 4, 2, 0, and -2 cm of H20. Typical rats in both groups had 313 Animal Models ofPneumoconiosis 27 Comparison of Normal and Silica Rate Deflation JO cm 03 4 0J 4 Ir 1 rj o Mrjrj ?dOO n siiiCrj rats 2*00 3200 4tJOQ *300 . 'roquonc.-, rfz normal fiafir 3dOO 0*00 Figure 2. Comparison of average impedance spectra of silica and control groups. Deflation 30 cm H20. peaks at 2000,3300, and 3500 Hz. Hie placement of these peaks compare favorably with the study of Jayaraman and Frazer,5 with die exception that in this study, no peak oc curred at 600 Hz. Significant differences between the silica and control groups occurred at the peak at 3300 Hz. T-tests performed on the peak magnitudes at this frequency showed that the silica group had significantly higher impedance (90% confidence 314 Animal Models ofPneumoconiosis II domoarioon of Normal and Nilica Rato ~<^t-3tfon * cm *,20 * I c0r. 1 t od *00 .'000 .J.200 -iWO 4*00 3000 0400 q ^w/c^ rirs rraoijenr.Mr -- orrr h.hs Figure 3. Comparison of average impedance spectra of silica and control groups. Deflation 8 cm H2Q. level) at transpulmonary pressures of 20 and 8 cm of H20 during deflation. The silica group also had significantly higher impedance (98% confidence level) at 3500 Hz at pressures of 6 and 4 cm of H20 during deflation. These findings show that changes in the mechanical properties of lung tissue and die closure of airways occurred at higher pressures in the silica group than with the control group. This finding agrees with the work of Chvalova et al.1 which found that the pressure-volume curve of silicotic rats was shifted to higher pressures compared to normal rat lungs. CONCLUSIONS The above findings indicate that silicosis in rat lungs can be detected by measuring the lung input impedance. The key indicator thus far is the impedance magnitude of the peak at about 3500 Hz. Further studies will determine the effec tiveness of the method in detecting die development of lung diseases and if the measurement of acoustic impedance can be an effective clinical tool for the treatment oflung diseases. 315 Animal Models ofPneumoconiosis U Comparison of Normal and Silica Rats Otjfl-y/on J cm HCO torn 3*00 3300 4000 0400 Silica rats frequency. Hz ------- normal Rats Figure 4. Comparison of average impedance spectra of silica and control groups. Deflation 2 on H2O. REFERENCES 1. Chval0va,M.,Kiincova, J.,Havrankova,J., andPakcek,F.: Regula tion of Respiration in Experimental Silicosis. Physiol. Booenoslov. 23:539-547 (1974). 2. DuBois, A3., Brody, DU., Lewis, DA, and Burgess, B.F.: Oscilla tion Mechanics ofLungs and Chest in Man. J. Appl. PbyskJ. 8:587-594 (1956). 3. Fredbcrg, JJ., Sided, R.S., Wohl, M.E., andDeJong, R.G.: Canine Pulmonary Input Impedance Measured by Transient Forced Oscilla tions. /. Bkmech. Bog. 100:67-71 (1978). 4. lahimira, K., Matsodaira, M., and Kaneko, T.: Input Acoustic Im pedance Measurement of (be Subglottal System. /. Accost. Soc. Am. 60:190-197 (1976). 5. Jayaraman, K. and Frazer, D.: Broadband Acoustical Impedance of Excised Rat Lungs at Ttanspulmonaiy Pressures between --5 cm and 30 cm ofH2O. PaB Conference ofthe American Physiological Socie ty. New Orieans, LA (1986). 6. Van Den Bog, J.W.: An Electrical Analogue ofthe Trachea, Lungs, and Tissues. Acta. Physiol. Pharmacol. Need. 9:361-385 (1960). ACKNOWLEDGMENTS: The authors wish to thank the Division of Respiratory Disease Studies, NIQSH, Morgantown, WV for the use oftheir facilities during this study. This research has been supported by the Depart ment ofthe Interior's Mineral Institute Program administered by the Bureau of Mines through the Generic Mineral Technology Center for Respirable Dust mvfer grant number GU35142. 316 Animal Models of Pneumoconiosis II CONNECTIVE TISSUE COMPONENTS AS STRUCTURAL BASIS IN LUNG RESEARCH a VOSS A. FIsseler-Eckhoff K.-M. Muller Silikose-Forschungsinstitut der Bergbau-Berufsgenossenschaft and Institut fur Pathologie, Berufsgenossenschaftliche Krankenanstalten ``BergmannsheU Bochum", Universitatsklinik Gilsingstrasse 14, 4630 Bochum 1/FRG ABSTRACT The connective tissue of the lung is a complex structure influencing its functional properties in health and disease. Under normal circumstances, the connective tissue helps to evenly distribute mechanical forces over the entire surface ofthe lung, contributes to the overall elastic properties ofthe parenchyma and mediates intercellular communications as well as cell-matrix-interactions. Components of the connective tissue are collagen molecules, proteoglycans, glycoproteins and elastin. In lung fibrosis an accelerated accumulation of interstitial matrix molecules, predominantly collagen, has been reported. To achieve a morphological analysis of the composition of the lung connective tissue, collagen types I, m, IV and V, the glycoproteins fibronectin and laminin, and proteodermatan-sulfate were isolated. Polyclonal antibodies directed against these antigens were used in indirect immunofluorescence studies of the lung tissue. The results revealed that under normal conditions the matrix molecules participated in the development of specific structural properties ofthe lung. However, irritation ofdie lung resulted in a disorder ofconnective tissue components. The present results indicate that the morphological analysis of matrix molecules may be able to explain functional properties of die lung. INTRODUCTION A fundamental prerequisite for understanding convulsive pro cedures at the alveolar wall is die knowledge about its struc tural composition in correlation to its functions. The func tional structures of the alveolar-capillary region of die lung are die epithelial cells of the alveolar space, the surfactant, the alveolar macrophages, the components of the interstitial connective tissue and the endothelial cells of the capillaries. The distortion of the genetically defined functional structure by exogeneous or endogeneous noxes obviously induces a metabolic disorder of lung collagens and other components of the extracellular matrix. The changes to matrix com ponents may result in an enhanced accumulation ofcollagen and fibrous material like elastin leading to lung fibrosis.1 Since collagen (60-70%), elastin (25-30%), proteoglycans (1 %) and fibronectin (0.5%) are major components of the interstitial structure, their inappropriate distribution is of im portant interest. Moreover, die development of basement membranes, which can be studied by the distribution of laminin, might be a key feature of fibrotic lung disorders.1 The distribution of different collagen types has been made with immuno-histochemical markers.2,3 However, these studies showed different results concerning die ratio of col lagen type I and type m. In our study we focused on die distribution of these collagen types and of fibronectin and laminin. MATERIAL AND METHODS Tissue samples of human lungs were obtained after autopsy and stored immediately at --70C. IMMUNOLOGICAL REAGENTS Antibodies directed against collagen types, fibronectin or laminin were of die same origin as previously described.4 Antibodies against the core protein ofproteodennatan sulfate were kindly provided by Dr. H. Kresse, PhysiologischChemiscbes Institut of die University of Munster/FRG. The specificity' of these antibodies was shown in an earlier publication.5 IMMUNOFLUORESCENCE Tissue section 6-8 tun were cut with a SLEE (Mainz,FRG) cryomicrotome, mounted on glass slides, and fixed by air drying for up to 12 hrs. The fixed tissue sections underwent two different staining procedures: for collagen types, fibronectin and laminin they were stained directly with die first antibody, for proteoglycan they were pie-incubated with chondroitin ABC lyase (Sigma). For pre-incubation, sections were treated with 5 mU chondroitin ABC lyase for 5 minutes at room temperature. The enzyme was inactivatedby washing with distilled water for 1 minute. Pre-incubated and untreated tissue sections were allowed to react with antibodies either 317 Animal Models of Pneumoconiosis n directed against die core protein of proteodermatan sulfate or against collagen type I, type ID, fibronecdn or laminin for 30 minutes of room temperature. Excess antibody was removed by washing five times with phosphate buffered saline, pH 7.5. Bound antibodies were labelled for visualiza tion in a fluorescence microscope (Orthoplan, Leitz, FRG) with anti-rabbit IgG conjugated with fhiorescin-isotihocyanate or anti-goat IgG in case ofcollagen types, also isolated with FTTC (Behringwerke, Marburg, FRG). After washing again five times with phosphate buffered saline, die stained tissue sections were preserved by embedding in Entelan (MERCK, Darmstadt, FRG). For all tissues, controls were performed by treatment with whole pre-immune serum or chromatographically purified IgG. RESULTS The structural components of the normal alveolar septa are deposited in a thin filamentous backbone. Collagen types I and IH were found to be co-distributed. Light microscopically elastin was observed in similar structures as revealed by col lagen staining. Collagen type I and type m obviously were associated with elastin fibres. The glycoprotein fibronecdn was additionally visible in laminar structures representing either the epithelial or the subendothelial basement mem branes. Proteodermatan sulfate was present not only in the interstitial spaces but was also observed close to basement membranes and all surfaces. In hmg fibrosis the alveolar sep ta become enlarged due to higher amounts of connective tissue components. Collagen type I and type m now enveloped foe larger elastic fibres. These fibres were addi tionally covered with fibronecdn. The denser connective tissue matrix reduced die capillaries in the alveolar walls visuable by the reduction of basement membrane material laminus. In this stage of fibrosis, sometimes fibronecdn was accumulated in alveolar spaces, opsoniering carbon or other dust particles, fit further development of lung fibrosis especially in pneumoconiosis granuloma formation is visi ble. Although the connective tissue exhibited a dense struc ture, collagen type I and type m were decreased in pericen tral areas while elastin seemed to be increased. Also fibronectin or laminin were either diminished or not further recognizable in central parts of granulomas. Discussion The present results show the participation of different con nective tissue components, especially collagen type I, type HI, elastin, fibronecdn and laminin in the formation offibrous material in alveolar walls. Collagen types IV and V were not separately shown. Both collagen types are either in base ment membranes (type IV) or co-distributed with collagen type I and type m or with fibronecdn. In further develop ment offibrosis collagen type I and type III depositions were increased. However, in granuloma formation their central parts contermined predominantly elastin. These results in dicate that analysis of collagen content of a tissue sample depends partially on foe stage of disease.6 The distribution of foe glycoprotein laminin was mainly visible in either epithelial or subendothelial basement membranes. The specific fluorescence for laminin was diminished in later stages offibrosis indicating a reduced microcirculation. Thus, foe functional properties of the lung are totally disturbed. The increase and decrease of connective tissue components reflect their regulation by different cells. In recent years foe in vitro investigations elaborated different cytokines which in cooperation with proteases may influence the formation of fibrous material.7*8 New therapeutic approaches should include these cellular factors. REFERENCES 1. Murray, J.C., Laurent, GJ.: What is pulmonary fibrosis? Thorax 43:9-11 (1988). 2. Madri, J.A., Furthmayr, H.: Collagen polymorphism in the hmg. An nrnnuDohistocheinical study of pulmonary fibrosis. Hum. Path. 11:355-363 (1980). 3. Bateman, E.D., Tbmer-Warwick, M., Halmt P., Adelmann-Grill, B.C.: Cryptogenic fibrosing alveolitis: prediction of fibrogenic activi ty from immunohistocbemical studies ofcollagen types in hmg biopsy specimens. Thorax 38:93-101 (1983). 4. Voss, B., Rauteiberg, 1.: Localization ofcollagen types L HI, IV and V, fibroocctin *H laminin in human arteries by the indirect im munofluorescence method. Path. Res. Pact. 181:568-575 (1986). 5. Voss, B., Glossl, J., Cully, Z., Kiesse, H.: Immunocytochemical in vestigation on the distribution of small chandroitin sulfate--dermatan sulfate proteoglycan in the human. J. Histocbem. Cytocbem. 34:1013-1019 (1986). 6. Kirk, J.M.E., Bateman, E.D., Haslam, P.L., Laurent, G.J., TumerWarwkk, M.: Serum type m procollagen peptide concentration in cryp togenic fibrosing alveolitis and its clinical relevance. Thorax 39:726-732 (1984). 7. Crystal, R.G., Bittennan, P.B., Rennard, SJ., Hance, AJ., Keogh, B.A.: Interstitial lung disease of unknown cause. L Eag. J. Med. 310:154-166 (1984). 8. Crystal, R.G., Bittennan, P.B., Rennard, SJ., Hance, AJ., Keogh, B.A.: Interstitial hmg disease of unknown cause, n. Engl. J. Med. 310:235-244 (1984). 318 Animal Models ofPneumoconiosis B STUDY OF FIBROGENIC EFFECTS OF POLYPROPYLENE AND POLYTHENE ON RAT LUNGS L. ZHANYGN F. Yuxiang Y. Fengting Liaoning Institute of Labour Hygiene Shenyang, P.R. China ABSTRACT Polypropylene and polythene are macromolecular compounds and typical synthetic organisms. In order to research their fibrogenic effects, 12$ rats (half males and half females) were selected and 50 mg of the polypropylene or polythene dusts was injected intrachacheally into each rat. The observation was made for 18 months. The results showed that in the early stage the prominent histopathological changes in the lungs were foci of dust-granuloma (polythene group showed polynucleo-macrophage granuloma) and hyperplasia of reticular fibers. At die 18th month after injecting die dust, in experiment groups pronounced hyperplasia of reticular fibers as well as collagen fibers were seen in these foci and around bronchi. Collagen content of the lungs in experiment groups was higher than that in control groups (treated with normal saline). The author in dicated that the slight fibrogenic effect on the rat lung was caused by both polypropylene and polythene dust. See Table of Contents, Part II, for Paper. Animal Models ofPneumoconiosis U CHEMOTACTIC RESPONSES OF LEUKOCYTES FROM THE BRONCHOALVEOLAR SPACE OF RATS EXPOSED TO AIRBORNE QUARTZ, COALMINE DUSTS OR TITANIUM DIOXIDE KENNETH DONALDSON, Ph.D. Joan Slight Geraldine M. Brown David M. Brown, BA. Maura Dl Robertson, Ph.D. John M. G. Davis, ScD. Institute of Occupational Medicine, 8 Roxburgh Place Edinburgh EH8 9SU INTRODUCTION Studies on humans and in laboratory animals have revealed that bronchoalveolar deposition ofdusts commonly associated with pneumoconiosis, results in recruitment of leukocytes to the lung parenchyma.1>2 In view of the important role of the leukocytes in mediating both injury and mesenchymal cell proliferation, die resulting alveolitis is considered to be an important factor in determining the progress of disease.3*4 During inflammation leukocytes are known to marginate and then migrate from the capillaries to die interstitium and alveolar space under die influence of chemotacdc factors generated in this region. As part of a study on leukocyte recruitment into the lungs of rats exposed, by inhalation, to pneumoconiosis-producing dusts,5 we examined the chemotacdc activity of bronchoalveolar leukocytes lavaged from these animals. We report here on die chemotacdc ac tivity of bronchoalveolar leukocytes from die lungs of rats exposed to 10 mg/m3 or SO mg/m3 airborne mass concen tration of; (a) the pathogenic particulate quartz, which causes silicosis; (b) dusts collected from the air of coalmines min ing anthracite, high rank coking coal and low rank bituminous coal; (c) as a negative control, titanium dioxide, a fine par ticulate of respirable size which is not associated with pneumoconiosis. MATERIALS AND METHODS Rats Syngeneic, PVG rats, SPF maintained and fifteen weeks of age at commencement of exposure, were used. Minerals The dusts used in die study were (a) titanium dioxide (Rutile), obtained from Tioxide Limited, Stockton-on-Tees; (b) the quartz standard DQt2; (c) coalmine dusts collected from die air of British collieries mining anthracite, high rank (cok ing) coal and low rank (bituminous) coal. Airborne coalmine dust samples were collected on dry Bondina socks mounted in the return roadway of a single face at each of die three collieries; full details of this procedure are given elsewhere.5 Details of die mineralogical composition of die samples used are shown in Table I. Inhalation Exposure Groups of 48 rats were exposed to airborne dust for 5 days per week, seven hours per day in exposure chambers de scribed by Beckett.6 The dusts were dispensed using either Wright or die dust dispensers. The concentration of dust in the chambers was monitored as the mass concentration of respirable dust defined by the BMR.C Johannesburg sam pling criterion7 using a Casella MRE 113A dust sampler. Full details of die exposure system are described in full elsewhere.5 Bronchoalveolar Lavage At 8,32 and 75 days into exposure, groups of four rats, and two control rats maintained in room air, were removed from die chamber and subjected to bronchoalveolar lavage. The method is described in detail elsewhere5 but involved removal of the lungs, exsanguination, followed by lavage ofdie bronchoalveolar space with 4 x 8 ml volumes ofsaline at 37C. The bronchoalveolar leukocytes, so obtained, were concentrated by centrifugation, counted and the proportions ofthe different leukocyte types assessed by differential count ing of May-Gnmwald Giemsa stained cytospin preparations. Assay of Bronchoalveolar Leukocyte Chemotaxis Cbemotaxis was assessed using Blindwell chambers. Three hundred microlitres of 10% zymosan-activated serum (ZAS) (high in the chemotactic complement component C5a), were placed in the lower compartment and a filter (Nuclepore, Pleasanton, California) placed on top. The top compartment was screwed down and 6 x 10s alveolar macrophages in 400 pi of RPMI medium (Gibco, Paisley) were placed in the upper compartment. The filters used were 5 pm pore size and incubation was for 3.5 hours at 37C in 5% CO2 to allow migration of cells through the filter towards die chemotactic material in the lower compartment. At the end of the incubation period the filter was removed from the diamber, washed, stained and allowed to dry before being mounted on a slide in plastic mountant. Two chambers were set up for each condition and die number of migrated cells in 5 high power fields (xlOOO) were assessed for each filter. 320 Animal Models of Pneumoconiosis n Table I Mineralogical Composition of Dusts Used in the Study DUST QUARTZ Coalmine dust A Coalmine dust H Coalmine dust L Quartz Titanium dioxide (TiQ2) CLASSIFICATION % ASH % IN DUST KAOLIN MICA Anthracite High rank Low rank DQj2 Standard Rutile 10.6 0.8 4.3 1.4 13.2 0.6 0.5 1.0 53.2 18.1 0.0 6.7 NOT APPLICABLE - PURE QUARTZ NOT APPLICABLE - PURE Ti02 Statistical Analysis of Results Results were obtained from four experimental and two con trol rats at each time point. Data were analysed by analysis of variance using die Genstat computer package and com parisons made using a *1* test.3 RESULTS Chemotaxis versus Chemoklnesls in Leukocyte Migration To ensure that chemotaxis was the dominant activity being measured in each sample, and not chemokinesis, we used a modified "checkerboard" method:--(mean standard deviation migrated cells/high power field); spontaneous migration 0.0 0.0; chemokinesis (measured as migration with 5% ZAS in both the upper and lower compartments) 14.8 6.6; chemotaxis (measured with 5% ZAS in die lower compartment) 46.4 4.8. These results confirm that the majority ofthe migration was in fret chemotaxis and migra tion in the Blindwell Chambers will henceforth be referred to as chemotaxis. Effect of Dust inhalation on Chemotaxis of Bronchoalveolar Leukocytes Figure 1 shows typical data obtained for chemotaxis ex periments with leukocytes from rats exposed, by inhalation, to 10 mg/m3 of the five dusts. This data clearly shows that inhalation exposure to Ti02 had very litde effect whereas exposure to quartz and the coalmine dust was associated with a marked reduction in the ability of the bronchoalveolar leukocytes to chemotact. Figure 2 shows the data, from all experiments at 10 mg/m3 airborne mass concentration, expressed as percentage inhibi tion of chemotaxis compared to the controls on that day, to more clearly highlight the effect ofdust exposure. It is clear that, although inhibition of 30% was present with Ti02 at 8 days, thereafter the inhibitory effect of TiC>2 did not ex ceed 17%. In the case of quartz, however, this was as great as 89.4% by day 75. All three coalmine dusts traded to show a gradual rise in the impairment ofchemotaxis shown ty the bronchoalveolar leukocytes as time of exposure progressed, reaching 50-70% inhibition by day 75. Figure 3 documents the effects of increasing airborne mass concentration of coalmine dust, on the inhibition of chemotatic activity. The increase from 10-50 mg/m3 air borne mass concentration was associated with a marked in crease in the impairment of chemotaxis, observable in the coalmine dust-exposed bronchoalveolar leukocytes, reaching 70-90% at 50 mg/m3. Attempts to Elucidate the Mechanism of DustRelated Impairment of Leukocyte Chemotaxis Limited experiments were carried out to try and elucidate the mechanism whereby dust deposition in the lungs of rats, as described above, caused loss of ability to chemotact. (a) Effect of ingested dust on macrophage chemotaxis. Control rat alveolar macrophages were allowed to adhere to filters and then incubated with quartz or Ti02 for 1 hour to allow phagocytosis. A chemotaxis gradient was then set up by plac ing the filters in a chamber with ZAS in the bottom com partment. We then allowed chemotaxis to proceed:--all data given as migrated cells/high power field mean standard deviation; untreated macrophages, with no phagocytic burden 54.4 11.3, TiOrexposed 51.8 6.2, quartz-exposed 59.8 6.0. Clearly merely having a phagocytic burden in side the macrophages was not sufficiently detrimental to cause impairment of chemotaxis. (b) Effect of incubation for 4 hours on chemotaxis. Allowing dust-exposed macrophages with inquired chemotaxis (obtained after 75 days ofexposure to coalmine dust L) to incubate for 4 hours in medium to allow recycling ofchemotaxin receptors had no effect on die impaired ability of the cells to chemotact:--control alveolar macrophages, freshly derived 55.0 (7.0)--incubated for 4 hours 48.2 (11.0); dust-exposed bronchoalveolar leukocytes, freshly derived 12.6 (3.6>--incubated for 4 hours 9.1 (2.4). (c) Relationship between % neutrophils in die lavage and % inhibition of chemotaxis. Since neutrophils were present to substantial proportion in some samples of bronchoalveolar leukocytes we assessed-whether the presence of neutrophils was related to impairment ofchemotaxis. There was no clear relationship between the proportion of neutrophils present in any bronchoalveolar leukocyte sample and impairment of chemotaxis--10-60% inhibition was caused with <10% neutrophils while increasing the percentage of neutrophils 321 Animal Models of Pneumoconiosis n to between 10 and 50%, only caused a maximum further 20% inhibition. CCALMNE DUST Figure 1. Chemotactic response of bronchoalveolar from rats exposed to die indicated dusts at 10 mg/m3. Data derived as mean + sd of pooled results ob tained for days 8,32 and 75 (6-12 rats per group). Significant differences dust-exposed v control for all except TiCV 0 D 20 30 40 50 AIRBORNE MASS CONC (rng^n*) Figure 3. Airborne mass concentration dependence of the chemotaxis inhibition present in bronchoalveolar leukocytes from rats exposed for 32 days to die indicated dusts; no 50 mg/m3 data available for quartz or TiOj. Data derived as described in die legend to Figure 2. DAYS Figure 2. Mean percentage inhibition ofchemotaxis shown by bronchoalveolar leukocytes from dust-exposed compared to control rats exposed to 10 mg/m3 of the indicated dusts. Mean percentage inhibition ob tained as: 1Q0 _ mean migration ^ dusted broochoalveolar leukocytes x jqq mean migration control bronchoalveolar leukocytes Raw data obtained from 2 control and 4 dust-exposed rats. DISCUSSION Ti02 is a fine particulate used extensively in industry and is not associated with pneumoconiosis in exposed popula tions.8 It causes minimal response in rats when given by in halation or intraperitoneal injection.2*9 Coalmine dust and quartz both cause pneumoconiosis and 3 coalmine dusts of different mineralogical composition, including quartz con tent, were included in order to test whether such differences would contribute to differences in leukocyte recruitment. These studies are reported in detail elsewhere5 but revealed alveolitis in rats exposed to quartz and all 3 coalmine dusts and failure of TiC>2 to elicit any substantial leukocyte response except at high dose following a long period of exposure. The studies on die chemotactic activity of bronchoalveolar leukocytes reported here show impairment ofchemotaxis in line with die ability of die dust to cause inflammation, i.e., (a) titanium dioxide which caused minimal inflammation caused least impairment of leukocyte chemotaxis; (b) quartz, caused large scale inflammation and the bronchoalveolar leukocytes ofdie alveolitis had impaired chemotactic activi ty; (c) coalmine dusts were intermediate in response between TiC>2 and quartz in ability to cause inflammation and impair chemotactic responses. There were no well defined dif ferences between die three coalmine dusts with different mineralogical composition, in terms of their ability to im pair chemotaxis. The results described here do show that chronic deposition of titanium dioxide, a dust not associated with pneumoconiosis did cause a measure of loss of impairment of chemotactic activity. In die cases where quartz and coalmine dusts caused impairment of chemotaxis there was 322 clear dose dependency in terms of die airborne mass con centration to which die rats were exposed. The data described here was obtained as counts of all migrated leukocytes which included both macrophages and neutrophils in inflammatory populations. However die decreased number of migrated leukocytes present in dust-exposed populations could not be explained on die basis of the neutrophils present, either as different migration characteristics compared to macrophages or effects of neutrophils on macrophage ability to migrate. This was evident since (a) profound inhibition was present even with low percentages of neutrophils;3 (b) in a limited number of cases differential count ofthe migrated cells were carried out (data not included) revealing, in some cases, similar proportions of macrophages and neutrophils in the migrated cells to those in the cells as lavaged; in some cases die proportion of neutrophils was decreased but this was never sufficient to explain die overall reduction in migra tion shown by the inflammatory population and impairment of macrophage chemotaxis must have been present. From this it is clear that macrophages from dust-exposed lung have impaired chemotactic activity and that neutrophils from dustexposed lung have less chemotactic activity than control alveolar macrophages, at least under the conditions of the assay. The net effect ofthis is that the ability ofthe leukocytes to clear dust from dust-inflammed alveoli is likely to be severely impaired. We have shown that the biological mechanisms underlying the loss of ability to chemotact do not include mere difficul ty encountered by dust-loaded cells in dying to pass through die pores ofdie filter towards the source ofchemotaxin. Since die leukocytes lavaged from the bronchoalveolar space have exudated in response to a chemotactic stimulus, it seemed possible that chemotaxin receptors might already be oc cupied. However, experiments allowing chemotaxin recep tors to regenerate, by incubation for 4 hours, produced no effect and impairment was maintained. Other studies from our Institute have suggested that neutrophils could cause sane inhibition of the chemotactic activity of macrophages.10 However plotting % inhibition against % neutrophils in die lavage failed to show any clear relationship between numbers of neutrophils and loss of chemotaxis.5 We believe that leukocytes from dust-exposed lung have im paired expression of chemotaxin receptors or inhibition of die cytoskeletal proteins involved in cell movement, or their energy supply. Myrvik11 reported inhibition of migration of rabbit alveolar macrophages which had phagocytosed asbestos in vitro; whilst die impairment ofchemotaxis could not be attributed to toxicity in this study, unfortunately inert control dusts were not included. Following exposure in vivo and bronchoalveolar lavage, Warheit et a!.12 reported im pairment of chemotaxis with asbestos whilst Dauber et al.,13 and Martin et al.,14 both described impairment of leukocyte chemotaxis following inhalation exposure to silica. The pre sent response is the first, to our knowledge, showing that coalmine dusts also cause profound impairment of bron choalveolar macrophage chemotaxis. It was noteable that in hibition of chemotaxis was present following chronic inhala tion exposure at an airborne mass concentration of 10 mg/m3 Animal Models ofPneumoconiosis U approximating to die maximum allowable level in British col lieries (7 mg/m3). The fact that impairment of migration in bronchoalveolar leukocytes was much less with die inert dust Ti02 than with the two pneumoconiosis-producing dusts suggests that this phenomenon may be important in contributing to lung damage and pathological change in pneumoconiosis. This could be brought about by die pneumoconiosis-producing dusts being allowed to persist in the bronchoalveolar region within alveolar macrophages chronically stimulated by the ingested pathogenic dust. Such stimulated macrophages, refractory to die normal chemotactic gradients which govern their movements, could persist in die alveolar region, releas ing injurious agents such as proteases and oxidants and growth factors such as interleukin 1 and tumour necrosis fac tor, leading to fibrosis. REFERENCES 1. Begin, R., Bison, G., Boileau, R., Masse, S.: Assessment of disease activity by Gallium 67, scan and lung lavage in the pneumoconiosis. Sew. Resp. Med. 7:275-280 (1986). 2. Donaldson, K., Bolton, R.E., Jones, A., Brown, G.M., Robertson, M.D., Slight, J., Cowie, A.H., Davis, J.M.G.: Kinetics of die bron choalveolar leukocyte response in rats following exposure to equal air borne mass concentrations of quartz, chrysotile asbestos or titanium dioxide. Thorax. 43:525-533 3. Keogh, B.A., Crystal, R.G.: Alveolitis--the key to the interstitial lung disorders. Thorax. 37:1-10 (1982). 4. Crystal, R.G., Reynolds, H.Y., Kalica, A.R.: Bronchoalveolar lavage: the report of an international conference. Chest. 90:122-131 (1986). 5. Donaldson, K., Bolton, R.E., Brown, D.M., Brown, G.M., Cowie, H.A., Jones, A.D., Robertson, M.D., Slight, J., Davis, J.M.G.: Studies on the cellular response in hmg tissue to the inhalation of mineral dust. Institute of Occupational Medicine report TM/88/01. (1988). 6. Beckett, S.T.: The generation and evaluation of UICC asbestos clouds in animal exposure chambers. Ana. Occup. Hyg. 18:187-198 (1975). 7. Orenstein, AJ.: Proceedings of the pneumoconiosis conference Johan nesburg 1959. London: Churchill Press 610-621. (1960). 8. Ophus, E.M., Rode, L., Gylseth, B., Nicholson, D.G., Saeed, K.: Analysis of titanium dioxide pigment in human lung tissue. Scand. J. of Work Environment and Health. 5:290-296 (1979). 9. Donaldson, K., Bolton, R.E., Brown, D.M.: InhaledParticles, VI. Pro ceedings of the symposium. Inflammatory cell recruitment as a measure ofmineral dust toxicity. J. Dodgson and R.I. McCallum, Eds. Pergammoo Press, (in (moss) 1988. 10. Donaldson, K., Slight J., Biown, D.M.: The effect of products from inflammatory pulmonary neutrophils on alveolar macrophage chemotaxis, spreading and thymidine incorporation. Thorax. (Submit ted for publication) (1988) 11. Myrvik, Q.N., Knox, E.A., Gordon, M., Shirley, P.S.: Effects of asbestos on the random migration ofrabbit alveolar macrophages. Env. Health Peispect. 60:387-393 (1985). 12. Warheit, D.B., Chang, L.Y., Hill, L.H., Hook, G.E.R., Crapo, J.D., Brody, A.R.: Pulmonary macrophage accumulation and asbestos-induced lesions at sites of fiber deposition. Am. Rev. Resp. Dis. 129:301-310 (1984). 13. Dauber, J.H., Rossman, M.D., Daniele, R.P.: Pulmonary fibrosis: bronchoalveolar cell types and inquired function of alveolar macrophages in experimental silicosis. Env. Res. 27:226-236 (1982). 14. Martin, T.R., Chi, E.Y., Covert, D.S., Hodson, W.A., Kessler, D.E., Moore, W.E., Altman, L.C., Butler, J.: Comparative effects ofinhal ed volcanic ash and quartz in rats. Am. Rev. Resp. Dis. 128:144-152 (1983). ACKNOWLEDGEMENT: This research was funded by the Council ofthe European Communities. 323 Animal Models of Pneumoconiosis n PATHOPHYSIOLOGICAL EVIDENCE IN MODIFICATION OF COAL-INDQCED LESIONS BY JAGGERY IN RATS ANAND P. SAHQ, Ph.EX Scientific Commission for Continuing Studies on Effects of Bhopal Gas Leakage on life Systems, Sardar Pate! Bhavan New Delhi-110 001, INDIA INTRODUCTION Inspite of rapid technological advancements made during the twentieth century, occupational lung diseases due to inhala tion ofairborne particulate or fibrous matters continue to con stitute exceptionally high incidence among industrial workers. Pulmonary dust diseases, in general, are not curable in true sense because die usual therapy of destroying or removing foe morbific agent from lung has not proved efficaceous as is possible with bacterial or viral infections of die lungs. Apart from protective devices, as designed by engineers and put into actual use, various experimental ap proaches for tiie treatment of pulmonary dust diseases were also extensively made in the past with partial success using different types ofaerosol therapy, hormonal therapy, vitamin therapy and other substances including dietetic factors.17 The discovery of antisilicotic drug-PVNO and its efficacy both under in vitro and in vivo situations demonstrated its usefulness not only against experimental silicosis but also against the pathogenic effects of a mixture ofcoal and quartz dust in foe lungs of rats.11 In 1975, Chinese investigators demonstrated the therapeutic and preventive effect of Tetrandrine--an alkaloid ofbisbenzyl isoquinoline (Stephania tetrandra S.) on experimental silicosis in rats and monkeys as well as marked improvement in symptoms and chest Xray of human silicotics seen with tetrandrine treatment.3 Beletsky and Coworkers1 have reported some success with alkali inhalation (aerosol of potassium carbonate) by in dustrial workers before and after work shift, indicating reduc tion of silicosis incidence up to 28%. Hydrolysed dextran or glutamate have also helped in bringing about significant prevention of silicosis.**13 However, all these agents have also shown to be associated with various harmful side effects. Many therapeutic properties have been attributed tojaggery in foe Ayurvedic system of medicine. Regular consumption ofjaggery conferred great symptomatic relief of indukrial mine workers in mining and industrial establishments of India. Our earlier work9,10 and this report indicates foe beneficial effects of jaggery, a nutritive substance of con siderable potential and without any harmful side effects, on coal mine dust induced lesions in rats. MATERIALS AND METHODS Jaggery A solidified form offoe product obtained on boiling in open pan and concentrating sugarcane juice (Saccharum sinense Roxb.) is an indigenous edible item which is produced in all parts of India and is consumed as such or as confec tionaries. The production ofgur involves extraction ofjuice from cane, purification by straining, boiling and treating with vegetable darificant followed by further boiling and concen tration into a thick semi-solid mass which solidifies on cool ing. Usually jaggery contains all the soluble constituents of sugarcane but the exact composition depends upon the variety and age of canes and nature of foe soil on which they are cultivated. The chemical composition of jaggery6*7*12 is given in Table I. Coal Mine Dust The respirable size (5 pm diameter) with fairly well defined chemical composition was obtained from Environmental Monitoring Section of Industrial Toxicology Research Cen tre, Lucknow. To increase fibrogenic potential of coal mine dust, it was supplemented with quartz up to 10% ofdie total mixture. Experimental Male albino rats (108) were procured from ITRC animal breeding facility and maintained cm normal laboratory con ditions, on standard pellet diet (Gold Mohur, Upton India Ltd.) and water ad libitum. Animals were divided into 5 groups as shown in Experimental Protocol (Table II). The dose ofjaggery in rat was based on its average daily con sumption of mine workers in India. Following treatment from first day, 6 animals from each group were weighed in dividually and sacrificed at foe end of 30, 60 and 90 days. Hematology Blood was collected from jugular vein in heparinized tubes for RBC and WBC counts and estimation of hemoglobin. RBC and WBC were counted on Cell Counter 2041 Labora Manheim GmBH, Germany. Haemoglobin was estimated ac cording to foe method of Dacie and Lewis.4 Fresh blood 324 Animal Models of Pneumoconiosis II Table I General Composition of Indian Jaggery (Gur) Compiled from Reference 6, 7 and 12 Content Moisture ( % ) Protein ( % ) (a) Non-protein nitrogen (mg/100 g) (b) Protein nitrogen (mg/100 g) Carbohydrate ( % ) (a) Sucrose ( % ) (b) Reducing sugar ( % ) Minerals ( % ) (a) Calcium ( % ) (b) Chloride ( % ) (c) Phosphorus ( % ) (d) Potassium ( % ) (e) Sodium ( % ) (f) Iron ( % ) (g) Magnesium ( % ) (h) Copper ( % ) (i) Cobalt, Nickel and Molybdenum ( % ) Vitamins (a) Thiamine (mg/100 g) (b) Riboflavin (mg/100 g) (c) Nicotinic acid (mg/100 g) (d) Vitamin C (mg/100 g) Carotene (jug/100 g) Phenolics (mg/100 g) Fat, Wax, Pectin and Organic acids ( % ) Value (ranee) 3.9-7.2 0.35-0-40 19.6-42.9 13.7-17.6 83.5-95.0 72.8-80.3 6.8-14.2 0.6-2.6 0.2-0.36 0.2-0.34 0.03-0.22 0.10-0.16 0.006-0.025 0.005-0.020 0.008-0.105 0.007-0.010 0.001-0.008 0.018-0.030 0.042-0.046 3.92-4.50 5.20-30.00 155.0-168.0 280.0-320.0 0.10-0.60 325 Animal Models ofPneumoconiosis II Group I U III IVC vd Table n Experimental Protocol Number of rats used 22 22 22 22 20 Treatment Coala + - + + Jaggery - + + + _ aCoal dust (50 mg/1 ml of 0.15 M NaCl solution, sterile suspension) was injected intratracheally. ^Jaggery (500 mg/1 ml of sterile distilled water) given orally (po) 5 days/week for 90 days (termination of the study). CAnimals were first treated with gur (po) for 2 weeks followed by coal dust (intratracheally) and gur (po) treatment for 90 days (termination of the study). dThe control animals were treated orally (po) 1 ml of sterile waters 5 days/ week for 90 days (termination of the study). was used for making blood films to enmurates differential leukocytes alter Leishman's staining. Relative Organ Weights and Lymphoid Cell Counts At 30,60, and 90 days post treatment, body weight, weights ofthymus, spleen, peripheral (axial a popliteal lymph nodes) and tracheobronchial lymph nodes, adrenal glands, kidney, liver and lung were determined from individual rats and relative organ weights calculated. Thymus, spleen, peripheral and tracheobronchial lymph nodes cell counts were counted as number of nucleated cell per organ in Counter 2041 alter making cell suspension. Viability of cells was determined by Trypan Blue exclusion method. Histological Lungs were inflated in situ with 10% buffered formalin while other organs were used for estimation of collagen content. Alter preliminary fixation, the blocks were selected on fire fixed positions along with foe long axes of both foe lung at the level of foe hilum m include foe maximum area of foe lung. Tracheobronchial lymph nodes were excised careful ly and fixed in Bouin's solution. Other visceral organs were 326 alsn fixed in formalin. The paraffin section of5 |im thickness were prepared and stained with hematoxylin and eosin, silver impregnation for reticulin and Van Gieson's stain for collagen. Collagen and Sulphydrol Content Lung Collagen content was estimated by measuring hydroxyproline levels. In brief, dry lung tissues were hydrolysed with 6 N HC1 in sealed glass tubes at 110C for 16hrs. The hydrolyate was titrated to pH 7 and diluted with distilled water. Hydroxyproline content was then assayed according to Woessner.15 Concentration of total and free Sulphydryls in hing homogenates prepared in 0.25 M sucrose containing 0.005 M EDTA and free Sulphydryls in blood was deter mined using the oilman procedure.3 RESULTS Gross Observation The visceral organs ofrats of different groups did not reveal any significant macroscopic changes up to 90 except in lungs and tracheobronchial lymph nodes of rats receiving coal dust The lungs of group 1 at 60 days showed black pin point pat ches of coal dust on the lobes and by 90 days these patches became more prominent. Black patches of die coal dust were also seen in group in and IV at 90 days but they were less prominent than in group I judged from visual observation. In the early phase in group I, in and IV, the tracheobron chial lymph nodes became prominent and black in colour. However, in group m and IV at 90 days, a marked lymph- adenopathy was observed. Tracheobronchial lymph nodes of Group I animals showed some macroscopic changes. Relative Weight The relative weight of lungs at 30 days showed marked in crease in groups I, QI and IV. At 60 and 90 days relative weight of lungs in group I increased significantly (51% and 41 % respectively) in comparison to controls while relative lung weight of lungs in group m and IV were almost similar to that of control. The relative weight of tracheobronchial lymph nodes (TLN) of coal dust exposed group (I) showed 61-74% increase. In general die relative weight of lung, TLN, liver and spleen showed an increase at 30 days and a decline at 60 and 90 days. Hematological Changes RBC counts increased significantly in all the experimental groups. Concomitantly jaggery treatment in group III and IV significantly increased the RBC counts at different periods. At 90 days the values were higher than those of group I. At 30 days in coal exposed and jaggery treated group (HI and IV) die increase in WBC count was over 50%. A time dependent increase was further observed in these groups with maximum increase of76% (P <0.001) at the end of90 days. The haemoglobin content in group I was more or less the same at all periods but slightly higher than those of controls. However, die percent haemoglobin increased (45-50%; P <0.05) in all the threejaggery receiving groups (II, HI and IV) at 30, 60 and 90 days. Cellularity of Lymphoid Organs Animals treated with coal and jaggery group m and IV in dicated 18-20% (P 0.05) increase in thymocyte counts at all periods. At the termination ofexperiment (90 days), a decline in splenocyte counts (16% and 33%; P 0.05 respectively) was observed in group I and II animals. The cell counts of tracheobronchial lymph node indicated an increase in group I, m and IV in a time dependent manner at the end of 90 days. Peripheral lymph nodes cell counts indicated signifi cant increase in group in and IV at 60 and 90 days (25-45%; P <0.05). Histological findings In group I at 90 days typical coal-induced focal areas of fibrosis was seen which upon silver impregnation showed presence of thick reticulin fibres enclosing coal dust mass. Lung of animals treated orally with jaggery did not reveal any significant histopathological alteration at 30, 60 or 90 days. In group m (coal and jaggery simultaneously) at the termination of study (90 days), the alveolar parenchyma, in general, showed widely scattered and partly unphagocytosed coal particles with minimal cellular reaction and less Animal Models of Pneumoconiosis n fibrogenic response upon silver impregnation in comparison to group I. In group IV (pretreatment withjaggery followed by coal plus jaggery) at 90 days there was hardly any indication of the development of characteristic coal-induced lesions except there was mild thickening of alveolar Septa (Figure 1). Moreover, minimal cellular proliferation around coal deposits in the parenchyma as well as small aggregates of coal par ticles in the lumen of alveolar duct respiratory and terminal bronchioles as was seen in group m. The changes in tracheobronchial lymph nodes: the nodes were slightly enlarged with diffuse distribution of coal particles in the paracortical regions. Silver impregnation revealed presence of dense fibres along with thick branches of reticulin enclos ing coal aggregates, hi group IU at 90 days there was minimal reaction provoked by coal aggregates which were rather focally distributed and not diffusely as in group I. Focal areas ofcoal aggregates did not reveal any significant fibroblastic reaction. In group IV and 90 days, the enlarged lymph nodes demonstrated many scattered focal areas of coal aggregates with minimal reaction (Figure 1). In spleen die fibroblastic reaction was seen in red pulp in group I at 90 days while very mild reaction was observed in group m and IV at same period. Histological examination ofother organs did not show any significant alterations in their structures in various treat ment groups at 90 days. Lung Collagen Lung collagen as measured by hydroxyproline content in various treatment groups (I to V) up to 90 days is shown in Figure 2. A time dependent increase was seen in hydrox yproline content of group I animals. Animals of group n, however, did not show any alteration in die lung hydrox yproline content up to 90 days. Group m animals also did not exhibit any initial increase in hydroxyproline content at 30 days but at later periods (60 and 90 days) 10% increase (P 0.001) did occur. Interestingly, hydroxyproline content oflung from animal ofgroup IV remained unaltered and were within the limits of normal variation up to 90 days. Total and Free Sulphydryl Content of Lung The jaggery treatment group m and IV showed an elevated level of their lung-SH content. Free SH-contents of the jag gery treated animals (group m and IV) were exceptionally high at 90 days. Free Sulphydryl Content of Blood Coal dust instillation and oral treatment of jaggery (group I and II) did not change the free SH-content of blood. In group m and IV the SH-content increased in initial stage (30 days) but an increase became evident at 60 and 90 days in group IV. DISCUSSION The result of the present experiments showed significant in hibition of fibrotic changes in the lungs at 90 days in coal treated rats which received prior treatment of jaggery. Moreover the initial reaction of the coal induced cytotoxici ty, phagocytosis as well as fibroblastic reaction in lungs re mained less prominent and did not damage the lung archi- 327 Animal Models ofPneumoconiosis B Figure 1A. Rat hmg after treatment of Coal alone (group I) at 90 days, thick redculin fibrosis upon silver impregnation, X 128. Figure IB. Rat lung, pretreatment with Jaggery and Coal + Jaggery (group IV) at 90 days, upon silver impregnation showing mild thickening of alveolar septa, X 104. Figure 1C. Tracheobronchial lymph node after treatment of Coal alone (group I) at 90 days upon silver im pregnation showing dense reticulin fibres and coal particles, X 104. Figure ID. Tracheobronchial lymph node, pretreated with Jaggery and Coal + Jaggery (group IV) upon silver impregnation, few reticulin fibres along with coal particles, X 104. 328 Animal Models of Pneumoconiosis B Although it has been reported that protein is essential for fibrogenesis,2,14 multideficient or protein deficient diets did not modulate silicotic fibrogenesis.17 In the pre-fibrogenesis stage, there is likely to be proliferation of endoplasmic reticulum leading to enhanced-SH levels. If fibrosis is re tarded, as in the jaggery treated groups as evident histopathologically, hydroxylation is initiated with affecting the en doplasmic reticulum. In that case SH is likely to increase. The enhancement of sulphydryl content of coal exposed rat lung following jaggery treatment suggest that jaggery or its microingredient(s) play some protective role in the release of these toxic biologically active substances. 30 60 Days 90 Figure 2. Changes in Hydroxyproline content of rat lung of different groups at 30, 60 and 90 days. I I -- Control; I I -- Coal alone (group I); BSS -- Jag gery alone (group II); S3 -- Coal and Jaggery simultaneous treatment (group HI); mil| -- Pre treatment with Jaggery and Coal + Jaggery (group IV). tecture. In addition to histologic evidence in lungs, pretreat ment ofjaggery prevented the increase in die hydroxyproline content (collagen) of lungs. Significant changes in the relative weights of lungs, liver, TLN and spleen ofcoal exposed rats were observed followingjaggery treatment. The WBC counts and haemoglobin contents were significantly higher in all die jaggery treated groups. Concomitantly, die cell counts of lymphoid organs were also elevated. These findings indicate that jaggery treatment presumably increased the physiological status of almost every important organ and cells of the body suggesting that dust clearance from lung could be by die enhanced physiological pathway. Our earlier studies9 following jaggery treatment have shown that pathological pathway of clearance is also effectively operative in these groups as dust can be observed in TLNs. REFERENCES 1. Beletsky, V.S. etal: Gig. Tiud. Prof. Zabol. 11:29-34(1982). Abstract from Indsutrial Hygiene Digest 126/83. 2. Bhuyan, U.N., Nayak, N.C., Deo, M.G., Ramalingaswami, V.: Ef fect ofdietary protein fibrogenesis in albino rats. Lab. Invest. 14:184-190 (1965). 3. Change-qi, Z., Xi-Rong, L., Yu-Rui, L.: VI International Pneumoconiosis Conference, 1:467-478 Bochum (1983). 4. Dacie, J.V., Lewis, S.M.: Modified Drabkin and Austin Colorimeteric assay ofhemoglobin (cyanomethoglobin method). PracticalHematology 5th Ed., p. 30. ELBS, Churchill Livingston (1975). 5. EUaman, G.L.: Tissue sulphydryl groups. Arch. Biocbem. Biopbys. 82:70-77 (1959). 6. Gut: Tbe wealth ofIndia. Industrial Product Part IV: F-H., pp 182-186. Council of Scientific and Industrial Research, New Delhi (1957). 7. Monograph on the Gur Industry ofIndia, p. 285. S.C. Roy, Ed. In dian Central Sugarcane Committee, Indian Institute ofSugar Technology, Kanpur (1951). 8. Morosova, K.I., Katsnelson, B.A., Rotenberg, Yu, Belobragina, G.V.: A further experimental study of the antisilicotic effect of Glutamate. Brit. J: Ind. Med. 41:518-525 (1984). 9. Sahu, A.P., Upreti, R.K., Singh, K.P.: Pathomorphologic evidence ofmodification ofcoal-induced lesions by jaggery in rats. Indian J. Med. Res. 87:308-317 (1988). 10. Sahu, A.P., Upreti, R.K., Saxena, A.K., Shanker, R.: Modification of coal-induced lesions by jaggery (Gur): Part n-Patbophysiological evidence in rats. Indian J. Exp. Biol. 26:112-117 (1988). 11. Schlipkoeter H.-W., Brockhaus, A.: Die Hemmung der experimentellen silikose druch sabcutane verabeichung von polyvinylpridine-N-Oxid. Klin. Wscb: 1182-1189 (1961). 12. Scientific Monograph on Technical aspects relating the Improvement of die Gur Industry, p. 14. K.L. Khanna and A.S. Charavarti, Eds. Bangalore Press, Bangalore (1955). 13. Slincbenko, N.Z. et al.: Hydrolysed dextran as an antisilicotic. Gig. Trud. Prof. Zabol. 1:25-28 (1984). Abstract from Industrial Hygiene Digest 430/1984. 14. Stary, H.C., McMillan, G. C., Weigenberg, B.I.: Wound healing in lysine deficiency. Arch. Pathol. 82:280-286 (1966). 15. Woessner, J.F.: The determination ofhydroxyproline in tissue and pro tein samples containing small proportions of this amino acid. Arch. Biocbem. Biophys.: 93:440447 (1961). 16. Zaidi, S.H.: Experimental Pneumoconiosis, pp. 121-143. Johns Hopkins Press, Baltimore (1969). 17. Zaidi, S.H., Kaw, J.L.: Protein malnutritions and silicotic fibrogenesis. XVI International Congress on Occupational Health, pp. 193-196(1969). 329 Animal Models ofPneumoconiosis n IMMUNOLOGIC FEATURES OF THE BRONCHOALVEOLAR LAVAGE FLUID OF RATS WITH SILICO-PROTEINOSIS a E. BANKS, M.ZX J. E. Morgan, Ph.U Y. Y. Hammad, DlSc. Sections of Pulmonary Diseases & Allergy and Clinical Immunology Tulane University School of Medicine, New Orleans, LA, 70112, USA ABSTRACT We provoked silico-proteinosis in specific pathogen free Fisher 344 rats by exposing diem to 10 mg/m3 respirable free crystalline silica for 3 months (6 hrs./day, 5 days/wk) and compared the immunologic features to a group of non-exposed rats. Bronchoalveolar lavage fluid (BALF) return, total cell count, differential cell count, and BALF and serum IgG, albumin, and IgA levels were measured and expressed as mean S.E.M. Percent fluid recovered was do different in the silica exposed and non-exposed rats (51.66 v. 648). Total cells recovered (xl0fy(64 17 v. 3.11.0), % macrophages (67.43.0v. 94.80.5), % neutrophils (5.91.4 v. 1.30.8), and % lymphocytes (262 v. 4.50.6) were significantly different in the silica versus non-exposed rats, respectively. IgG (g/dl) was significantly increased in the serum (2.00.2 v. 1.20.3) and BALF (0.70.01 V. 0.020.01) of die exposed rats. Serum albumin (g/dl) was not significantly different in the 2 groups but BALF albumin (g/dl) was significantly increased in the silico-proteinosis group (0.200.02 v. 0.03 0.01). IgA levels did not significantly differ in the serum and were below detectable limits in both of the groups. A dramatic influx of humoral and cellular components occurs into die bronchoalveolar lavage fluid of rats with silicoproteinosis, reflecting the extensive inflammatory response associated with this disease. No Paper provided. 330