Document zN3zzgkLapmrnBkG32agLr2a

Poster Session II STUDY OM SUPPRESSION OF SOLUBLE ALUMINUM AEROSOLS ON QUARTZ-INDUCED CYTOTOXICITY --Combined Effects of Both Aerosols in an Artificial Dust Atmosphere CHEN JIN, M.S. Shljie Liu, M.D. Department of Occupational Health, Beijing Medical University School of Public Health, Beijing, P.R. China INTRODUCTION Since 1930's it has been noted that aluminum (Al) and its compounds could prevent and treat silicosis. A number of studies have shown that Al could resist the cytotoxicity of SK>2 particles on macrophages and erythrocytes, and sup press or lessen quartz-induced pulmonary fibrosis.1-4,6.9.12,18 More recently, it has been found that the mechanism of the effects of Al was the combination of it with Si02 particles, which altered some properties of the particle surface, so leading to the decrease of pathogenic activity of SiO*.418 And the charges on Si02 particles were greatly reduced after the mineral particles were combined with Al, which was quite valuable to aggregation and dropping SK^ dusts. Consequently, if Al is used in dust workplace, such as spray ing aqueous solution of aluminum compound or adding solu ble aluminum into the water for dropping dusts from which the soluble aluminum aerosols are generated and probably combined with the Si02 dusts in air and/or inside lungs before or after the dusts are inspired into the worker's respiratory passage, it will give play to the dual effects on both resistance against the pathogenicity of Si02 and drip ping the SiO 2 dusts in working atmosphere. It is possible to explore a new way to treat dusts in our industries and mines to prevent the development of silicosis. The present study made an attempt to demonstrate whether or not the soluble aluminum aerosols could combine with Si02 particles in the atmosphere, and if they could suppress or lessen the cytotoxicity of the minerals on guinea pig alveolar macrophages and rat erythrocytes. MATERIALS AND METHODS Development of a Dynamic Inhalation Exposure System and Collection of Si02 Particles Combine with AlAs or DWAs A dynamic inhalation exposure system was designed and developed to simulate an atmosphere interacting of both aerosols--SiO* particles and soluble aluminum aerosols (AlAs) or deionized water aerosols (DWAs). SiO^ dusts generated by a dust generator (modified F-710 Elec tromagnetic feeder made in China Jiujian) were conveyed into the exposure chamber (made of polymethyl methacrylate, 0.8M3 in volume) of the system to form the average concentration of 200mg SKtyM3. AlAs or DWAs, on the other hand, nebulized by an ultrasonic nebulizer (JWC-2A Transistor Ultrasonic Nebulizer made in China Anshan, 0.5-10pm in aerosol diameter) were sprayed into the chamber from another inlet. In the exposure chamber both aerosols interacted on each other, and Si02 particles combined with and/or without AlAs or DWAs were sam pled on a microfilter (0.4S pm in pore size, made in China Beijing) by WY-1 cascade impactor (7 catch stages, made in Chinese Academy of Preventive Medicine),11 on the basis of which following experiments were done. Measurement of Aluminum Combined on Si02 Particles The contents of Al and SiC>2 in the sample on the microfilter were directly analyzed and measured by thin film X-ray fluorescence (XRF), based on modification ofmethod of Cui et al. ,7 with PW-1400 X-Ray Fluorescence Meter (made in Philip Inc.) from which the amounts of Al combined on Si(>2 particles were calculated and expressed in pg Al per cm2 specific area of SiC^, i.e., pgAl/cm2 SiO*. The specific area was calculated from the diameter and density of Si02 particles.16 Cytotoxicity Examination of the Si02 Particles Combined with AlAs Using hemolysis assay and macrophage viability test monitored in vitro die cytotoxicity of AlAs--or DWAscombined Si02 particle samples collected from die 5th stage microfilter ofthe Impactor, represented by antihemolysis rate (AHR) and macrophage viability index (MVI). The former was based on the hemolysis assay system previously de scribed by Hefner et al.,10 Briefly, whole blood was taken from rat aorta abdominalis, washed 3 times in normal salt (NS) by centrifugation at 1500 rpm for 10 min., and the pellets, all of which are almost erythrocytes (RBC), were resuspended in NS to 20% (v/v). 1 mg SiC>2 or SiCVAl sample was added to 3 ml or the 2% RBC suspension, and the mixture incubated in 37C water bath for 30 min and gently shook every 5 min. At the end of incubation, with centrifugating the mixtures the supernatants was measured for optical density (OD) at 420nm wavelength. Antihemolysis rate (AHR) was calculated from below formula: (HR of SiQ2-H2Q) - (HR of SKyAI) x 100% HR of SiC>2-H20 1299 Poster Session U where HR (hemolysis rate) = -------O---D----o--f--S---iO--r---A---l-g2_r-o--u-pL------ x 100% OD of whole hemolysis group MVI is complex indicators of damage effects of SK>2 par ticles ou macrophages, including rate ofmacrophage viability (RMV), intracellular K+ contents and lactate dehydrogenase (LDH) activity in die conditioned medium of macrophages cultured with the Si02 samples. Alveolar macrophages (TxlOtyml) harvested from male guinea pig lungs with bronchopulmonary lavage as described earlier9 were in cubated with ISOpg/mg Si02 (Si02-Al or SiOj-HjO samples) or normal salt (NS, as negative control) in medium RPMI1640 at 37C in 5% COz-95% air for 4 hours, RMV(%) was assayed by trypan blue exclusion test, in tracellular K+ contents of macrophages by Flame Atomic Absorption Spectroscopy and LDH activity in die conditioned medium by a colorimetric process.14,18 According to these parameters above, MVI was obtained from the formula: and die rate of AlAs-suppressed toxicity of SiOj dusts to macrophages (RAST) was calculated from: (MVI in SiOz-Al) - (MVI in SiprHjO) xl0Q% (MVI in NS) - (MVI in SiOr-H20) * RESULTS AND DISCUSSIONS Combination of Soluble Aluminum Aerosols with SiOz Particles 1) The amounts of A1 combined on SiOz particles at nebulization with various aluminum compounds. Four solu ble aluminum compounds, Al-I, Al-II, Al-m and Al-IV (syn thesized and supplied by the Beijing Medical University School of Pharmacy) were prepared to O.SmgAl/mg solu tion in deionized water. Experiments were respectively done with each of diem under die consistent conditions such as nebulizing, sampling and measuring described above. As shown in Figure 1, all but Al-I aqueous solution were found to significantly increase die amounts of A1 combined on /RMV in SiCVAl group ' RMV in NS group K+ in NS group LDH in Si<V x 300% id 13- Figure 1. The amounts of A1 combined on Si02 particles at nebulization using four soluble aluminum compounds (Al-I, Al-H, Al-m and Al-IV aqueous solution) and deionized water (H20). Mass Mean Aerodynamic Diameter (MMAD) of SiC^ particles in these samples was 5.14pm. *P<0.01; **P<0.05t-test withH20 group; expresses die net amounts of A1 combined on SiO^ particles. 1300 SiC>2 particles as compared with the DWAs-sprayed Si02 samples. The net amounts of A1 on SiC>2 particles ranged from 0.09 to 0.15pgAl/cm2 SiC>2. Although the amounts of A1 at the Al-IH-nebulized aerosols group were situated be tween Al-H and Al-IV, considering that Al-m was in great resource and easy to be produced in our country, our in vestigations were mainly done on the interaction of Al-m aqueous solution aerosols with SKD2 dusts as follows. 2) Combination of S1O2 particles with the AlAs nebulized with the various concentrations ofAl-m aqueous solution. Al-m aqueous solution was diluted into 0.1, 0.3, 0.5 and 1.0 mgAl/ml with deionized water, and respectively nebu lized into the exposure chamber with Si(>2 dusts at same conditions. Significant linearity between die Al-m aqueous soludon concentrations and the amounts of A1 combined on SiC>2 particles was observed in Figure 2, i.e., the amounts of A1 on Si02 particles increased with increasing the con centrations ofAl-m solution. It was noted that in the presence of nebulization by the lower concentrations of Al-m solu tion the amounts of A1 on Si02 particles was 0.160.03 pgAl/cm2 SiOj and reached an effective level reducing pathogenicity of the mineral dusts, as the previous experi ment in which the interaction of A1 with SiC>2 was in test tube had suggested that while the amounts of A1 on Si02 were 0.15pgAl/cm2 or so, the cytotoxicity of Si02 was ob viously suppressed. When die SiC>2 samples combined with AlAs or DWAs were washed 4 to 5 times in deionized water by centrifugation at 4000 rpm for 20 min., respectively resuspended and filtered on microfilter by suction for XRF Poster Session II analysis, it was found that amounts of A1 on the post-washed Si02 particles were yet more enough (data not shown) to lessen the SiO^induced cytotoxicity on the basis 4 of preliminary experiments in test tube.4 This suggested that the combination of AlAs with Si02 particles was quite firm and relatively stable. 3) Combination of various SiOj dusts in diameter with AlAs. With nebulizing 0.5mgAl/ml of Al-m aqueous solu tion into die Si02 dust atmosphere of the exposure chamber, the Si(>2 samples were collected on four stage microfilters from 2.54 to 6.97 pm MMAD (2.54, 3.68, 5.14 and 6.97 pm MMAD, respectively) by the cascade impactor (lOL/min sampling flow rate), and measured by XRF to calculate the amounts of A1 combined on various Si02 particles in diameter. The results shown that the higher the SiC>2 particle-size distribution was, i.e. the smaller SiOz particles, die more AlAs could be combined with diem (Figure 3). The net amounts of A1 on the 2.54 pm Si02 MMAD were 9 times as many as that on the 6.97 pm SiC>2. In accordance with the deposition curve of SiC>2 particles in lungs, the 2 to 3 pm MMAD SiC>2 particles were of maximum deposi tion in lungs.17 Therefore, no doubt will the AlAs in dusty workplace be much more significant as a preventive measure to silicosis. Anti-cytotoxic Effect of Soluble Aluminum Aerosols on Si02 Dusts To estimate the preventive effects of AlAs on silicosis, the changes in cytotoxic effects (AHR and MVI) of the SiC>2 D concentrations of Al-III Solution (mgAl/ml) Figure 2. The combination of Si02 dusts with AlAs nebulized by various concentrations (0.1, 0.3, 0.5 and 1.0mgAl/ml) of Al-m aqueous solution. NAALs represents die net amounts of A1 combined on SiC>2 par ticles, i.e., die amounts of A1 on AlAs-treated S1O2 minus the ones on DWAs-treated Si02. The number of samples was 4 per group. 1301 Poster Session U particles combined with aerosols of Al-XII aqueous solution ofvarious concentrations on guinea pig alveolar macrophages and rat erythrocytes were examined, which is rapid, sensitive and common marks to test cytotoxicity of mineral dusts and also an important evidence to reflect pathogenicity of them.5 The greater AHR and MVI are, the lower die cytotoxicity of SiOj particles is and the more effectively the AlAs could resist SiO^induced pulmonary damage. As seen in Figure 4, die AlAs-combined SiC^ particles greatly increased die AHR and MVI with statistical significance as compared to control group. To some extent the increases were correlated with die amounts of A1 on SiOj. When the amount of A1 was 0.180.06pgAl/cm2 (in die nebulizing group of0.3mgAl/ml Al-m aqueous solution), AHR and MVI reached maximum level, 66.23% and 64.3%, respectively. At nebulization with the lower concentration NAALs (ugAl/cn2 SiC>2) Figure 3. The net amounts of A1 (NAALs) combined on the SiOj particles of various diameter (MMAD 2.54 to 6.97pm) sampled by cascade impactor (lOL/min sampling flow rate). Figure 4. The suppressing effects of soluble aluminum aerosols on SiCVinduced cytotoxicity to macrophages and erythrocytes. NAALs: net amounts of A1 combined on S1O2 particles; AHR: antihemolysis rate; RAST: rate of AlAs-suppressed toxicity of Si02 dusts to macrophages. 1302 of A1-TTT aqueous solution (0. lmgAl), the cytotoxicity of the Si02 particles was also suppressed by more than 40% (AHR by 48.32% and RAST by 52.1%). CONCLUSIONS Hie experimental results indicated that AlAs could not only stably combine with Si02 dusts in an atmosphere, but also effectively suppress the cytotoxicity of the minerals of macrophages and erythrocytes, which is consistent with the previous investigations in water system.4*18 Although the present study did not examine the change of surface charges of Si02 particles, according to the preliminary experiments in which the surface charges of SiC>2 particles significantly decreased once the Si02 particles were combined with Al, it may be speculated that AlAs could reduce the charges of SiC>2 surface and promote aggregating and dropping of the SiOj particles in a dusty atmosphere. It must be pointed out that in this study die AlAs combined with SiC>2 dusts were only involved, but the other AlAs combined without SiC>2 in the air can be inspired into lungs together with Si02 dusts and interact with the minerals in respiratory passage, by which the AlAs could in the same way make resistance against die cytotoxicity of Si02 par ticles. Therefore, though in the nebulizing group with 0. ImgAl/ml Al-m aqueous solution the amounts of Al com bined on SiC>2 particles and the suppression of Cytotoxicity were not maximum, the effective amounts of Al will greatly increase if it is considered that the AlAs can combine with SiC>2 dusts in respiratory passage. It is suggested that 0. ImgAl/ml Al-m aqueous solution or lower may be suitable for further experiments in lab and dust working sites. However, the optimal dosage of soluble aluminum com pounds used in workplace remain to be further experimented. REFERENCES 1. Begin, R., Masse, S., Rola-Pteszczgnsld, M., Martel, M., Desmarais, Y., Geoffrpy, M., LeBouffant, L., Danniel.H., Martin, J.: Aluminum lactate treatment alters the lung biological activity ofquarts. Exp. Lung Res. 10(4):385-399 (1986). Poster Session n 2. Begin, R., Masse, S., Sebastien, P., Martel, M., Geoffrey, M., Labbe, J.: I-ate aluminum therapy reduces the cellular activities of simple silicosis in the sheep model. J. Leukocyte Bio. 41:400-406 (1987). 3. Bouffant, L.L., Daniel, H., Martin, J.C.: Inhaled Particles IV (pert I), pp. 389-401. Pergamon Press Ltd. (1977). 4. Cao, C.J., Liu, S.J., 1-in, K.C.: The injurious effect ofsilica and the anti-injurious effect ofaluminum citrate on biological membranes. The Graduate Thesis of M.D. in BMU (1985). (in Chinese) 5. Chamberlain, M., Davies, R., Brown, R.C., Griffiths, D.M.: In vitro tests for tbe pathogenicity of mineral dusts. Ann. Occup. Hyg. 26(1-4:583-592 (1982). 6. Crombie, D.W., Blaisdell, J.L., MacPherson, G.: Tbe treatment of silicosis by aluminum powder. Canad. Med. Assoc. J. 50:318-328 (1944). 7. Cui, F.H., Wang, S.L., Pu, S.B.: Analysis of suspended particles in atmosphere with X-ray fluorescence. Environ, Qtem. 2(3):52-58 (1983). (in Chinese) 8. Dauber, J.H., Daniele, K.R.: Chemotactic activity ofguinea pig alveolar macrophages. Am. Rev. Respir. Dis. 117:673-684 (1978). 9. Dix, W.B.: Aluminum Powder and silicosis prophylaxis. Canad. Min. J. 92(10):35-42 (1971). 10. Hefner, R.E., Jr., Gehring, P.J.: A comparison of the relative rates of hemolytic induced by various fibrogenic and non-fibrogenic parti cle with washed rat erythrocytes in vitro. Am. Ind. Hyg. Assoc. J. 36:734-740 (1975). 11. Liu, G.Q., Wang, Y.S., Chen, M.N., Liu, J., Ma, L.X.: The develop ment of WY-1 cascade impactor for flue pipe. Environ. Engineering 5:17-24 (1985). (in Chinese) 12. Liu, S.J., Zou, S.Q.: Symposium ofStudies on Occupational Medicine Vol.ni, pp. 86-97. Dept, of Occup. Med. of BMU, Beijing (1979). (in Chinese) 13. Macnab, G., Harington, J.S.: Heamolytic activity ofasbestos and other mineral (hist. Nature 214:522-523 (1967). 14. MftriTihrarfQlfy, F , Marh, Ml: Measures <ifviahility in isolated cells. Biocbem. Med. 13:164-177 (1975). 15. Skaug, V., Davies, R., Gylseth, B.: In vitro macrophage cytotoxicity of five calcium silicates. Brit. J. Ind. Med. 41:116-121 (1984). 16. Tan, T.Y., Liang, F.Z.: Technology forIndustrial Ventilation and Trap ping Dusts, pp. 33-35. Architectural Industry Press of China, Beijing (1984). (in Chinese) 17. Watson, H.H.: Dust sampling to simulate the human lung. Brit. J. Ind. Med. 10:93-100 (1953). 18. Zou, T.T., Liu, S.J.: Studies forprevention ofaluminum citrate from cytotoxicity ofsilica in vitro. The Graduate Thesis of M.M. in BMU, (1982). (in Chinese) ACKNOWLEDGEMENT: This work was supported by grant from the Ministry of Labour and Personnel, P.R. China. 1303 Poster Sessioa II PSEUDO-TUMORAL LUNG FORMATIONS FROM SILICA FREE DUSTS LUCIANO ROMEO L_ Perbellini P. Apostoli F. Malesani Institute of Occupational Medicine, University of Verona, Italy INTRODUCTION Li our experience we have encountered some workers who exhibited round shaped opacities in their lung suspected as being neoplastic in nature. In certain cases it was possible to exclude malignancy of such lesions, identifying diem on die contrary, as the results of prolonged inhalation of silica free dusts at work. In this work we report four cases exposed to calcium and magnesium salts (as previously stated) and one case of talc exposure, recently observed, who exhibited these types of lesions.5 SUBJECTS AND METHODS Our study was carried out on five patients: four were magnesium and barium hydroxide production workers and one was a natural rubber processing operator. The first four subjects were allocated in the same industry and had done various tasks in two departments: 1. magnesium production employing heat processing (Pidgeon system), using dolomite, ferrosflicon and calcium fluoride as raw materials; 2. barium hydroxide production employing "Sodabesgher" furnaces, by use of barium carbonate. In die first department the enviromental dust level was be tween 1.6 and 71.3 mg/m3 and in die second one from 0.8 to 128 mg/m3. The four workers between the ages of43 and 58, had worked in the two departments for long periods (from 17 to 34 years). Their chest X-ray films were very similar and presented coin shaped lesions measuring 1-2 cm in diameter, single in three cases and multiple in one. Only one case was also a pneumoconiosis suspect with reticular shadows. In two workers the coin shaped lesions were found 3-4 years before. We had suspected the presence of secondary neoplasm of die lung but as it proved impossible to locate die primary site of neoplasm, in spite of accurate investigations, chest X-ray film follow-ups were suggested. In three of the four subjects die coin lesions remained un changed at every follow-up for five years. In the fourth sub ject, after three years, die two shadows originally identified had increased about 30% in diameter reaching a dimension of 3 and 1.5 cm respectively. We therefore proceeded with a toracotomy which permitted removal ofa sub-pleural well encapsulated node. 1304 On cryoscopy it was described as: "fibrous wall node with signs ofchronic inflammation and a central area ofnecrosis." The hystological examination revealed:'* well defined node oflung parenchyma consisting essentially of interwoven and whirl-pool-like collagen fibers, free of identifiable cells, and minute calcified particles. The surface area of the node is rich in fibrocytes, newly formed vessels, clusters of lymphoplasmocytic cells and occasional foreign body multinucleated giant cells." Mineralogical studies on the node employing X-ray diffrac tion, neutronic activation, and X-ray fluorescence showed scarse presence of silica and no significant concentrations of any specific metals. The Debye X-ray diffraction reveal ed this node as being rich in calcium and magnesium diphosphate. The subjects maintained good health and showed no further lesions ofthe hmg whatsoever on radiological investigations. The fifth worker, 45 years old, had worked in a department for the processing of natural rubber where according to die requirements for production, dust of talc, mica and fecula were alternatively used. The results ofenviromental investigation carried out to deter mine the type and qualify of corpuscolar pollution showed a variation of 0.6 and 1.4 mg/m3 of dust concentration. Microscopic examination of the particles removed revealed that die dust was rich of fibers 1-2 pm in diameter, 80% 10-20 pm in length and 20% less than 10 pm long. Fecula consisted ofparticles less that 5 pm in diameter which under polarized light had the typical "malta cross" appearance. Lastly, mica consisted oflamellae ranging from 20 to 60 pm in size. No asbestos fibers or silica was found. The patient gave no history of respiratory or other diseases. All at once, a few days before admission, die subject had slight fever, cough and exertional dyspnoea. The chest X-ray film showed presence ofan irregular digitated shadow, 3.5 cm in diameter, at die base of die left lung. All the various investigations carried out were negative. Be tween thirty and ninety days after, other coin shaped shadows appeared bilaterally, becoming larger and more numerous, tending to be confluent. The rapid growth of die lesions in die lung compared to the satisfactory clinical state ofthe patient, led us to proceed with a toracotomy. The cryoscopic examination ofthe heaviest mass which was first localized at the base of die left lung, ruled out any presence of neoplasm. The histological examination reported: "Lung parenchyma greatly altered by wide-spread granulomatose inflammation consisting of epithelioid cells and numerous foreign body giant cells, containing birefrangent needle-like fibers 1-2 pm in length and asteroid bodies.'* In conclusion the histological diagnosis was "giant cell talc granulomatosis of the lung." The results of die study made on the material used by die patient at work confirm that the needle-like formations seen in cytoplasm of the giant cells were talc fibers and one may assume that die asteroid bodies were, cm the contrary, fecula granules. The postoperative course was normal and his overall condi tion improved, hi addition, within the following months a slow but constant regression of die shadows was observed and seven months later only rare radiological irregular opacities were seen. Two years later the chest X-ray film was normal apart from the signs of die toracotomy. Discussion The description of pseudo-tumoral lung formations from silica free dusts is rare in literature.2'4 Although coin lesions ofthe lung due to inhalation ofcalcium and magnesium salts have not been reported, scientific studies have described talc granulomas of the lung and other organs.3'5*6'7'8 Poster Session U With regards to the first four cases we have described among the raw materials used at work, dolomite results the most suspected responsible for round shaped opacities of the lung.1 The rapid development and progression of talc granulomatosis found in our rubber worker and its disap pearance after he stopped work was probably due to revers ible flogistic-proliferative tissue reaction, without fibrosis, from talc free of silica or asbestos fibers. Hence, in the presence of lung lesions, in particular those we have described, it seems beneficial to add to routine clinical and instrumental practice suitably deeper investiga tion into work history of subjects, possibly including analysis of work environment and raw materials in use. REFERENCES 1. Anttila, S., Sutinen, S., Paako, P., Finell, B.: Rheumatoid Pneumo coniosis in a Dolomite Worker A Light and Electron Microscopic, and X-ray Microanalytical Study. Br. J. Dis. Chest 78/2:195-200 (1984). 2. Alivisatos, G.P., Poutikalds, A.E., Terzis, B.: Talcosis of Unusually Rapid Development. Br. J. lad. Med 12:43-49 (1955). 3. Brody, A.R., Dwyer, D.M., Vallyathan, N.V., Craidied, J.E.: Elemen tal Contenent of Granulomata in Biopsied and Autopsy Lung Tissue. Am. J. Path. 86:72 (1977). 4. Cavjgnaux, A., Fuchs, C.A., Tara, S.: Un Cas de Talcose a Forme Pseudo Tnmorale. Arch. Mai. Prof. 11:34-39 (1950). 5. D'Andrea, F., Apostoli, P.,Chiesa, A., Menestrina, F., Pucchetti, V.: Opacita' Rotonde Polmonaii in Lavoratori Addetti alia Produzione di Magnesio: Considerazioni Eziologiche. Med. Lav. 2:161-166 (1980). 6. Eisemen B., Seeling M.C.: Talcum Powder Granuloma: a Frequent and Serious Postoperative Complication. Ann.Surg. 126:820 (1974). 7. Gordon, B. Me Donald, D.F.: Tile Granuloma Presenting as a Testicular Mass. /. Urol. 118:122 (1977). 8. Tukiainen, P., Nicbels, I., Tasldnen, E., Nyberg, M.: Pulmonary Granulomatous Reaction: Talc Pneumoconiosis or Chronic Sarcoidosis? Br. J. Iod. Med. 41:84-89 (1984). 1305 nPoster Session MINERAL FIBERS AND DUSTS IN THE LUNGS OF SUBJECTS LIVING IN AN URBAN ENVIRONMENT F. MALCHIODI ALBEDI L. Paoletti M. Falchi M. P. Carrieri* A. M. Cassano G. Donelli Laboratorio di Ultrastrutture and Laboratorio di Epidemiologia e Biostatistica, Istituto Superiore di Sanita' Viale Regina Elena, 299--00161 Rome, Italy INTRODUCTION It is well-known that toxic and carcinogenic substances can be present in die breathable airborne particulate of urban areas and the higher incidence oftumors and chronic obstruc tive lung diseases, documented in these areas as compared with rural ones, is generally considered to be a consequence of the environmental conditions.3 Recently, the need to be ttor define die carcinogenetic role of atmospheric pollution in comparison with that played by other factors, such as cigarette smoking, has been emphasized.4 Hoe, we have studied mineral particulate from autoptic hmgs in a sample ofpopulation having lived in Rome area and not occupationally exposed to dusts. Their life-style, in particular their smoke habits, and die quantity and quality offibers and mineral dusts found in their hmgs were correlated. MATERIALS AND METHODS Sixty subjects, who had lived in Rome, aging from 15 to 65 years, were selected for this study. Subjects with a histoiy of occupational exposition to mineral dusts or with serious pathologic conditions and drug addicts were excluded. Dur ing post-mortem examination, fragments oflung tissue were taken from die upper lobe of right lung.1*2 They were mineralized in atomic oxygen plasma. Dry weight was also estimated. Inorganic component was resuspended in deioniz ed water and filtered on 0.45 *m cellulose membrane filters. Mineral particles were then transferred on copper grids, which had been coated with carbon films. They were observ ed under a 430 Philips transmission electron microscope, equipped with an energy-dispersive spectrometer for X-ray. Adjacent fragments ofhmg tissue were fixed in formalin and embedded in paraffin for light microscope observation. RESULTS Concentration and Type of Mineral Particulate in the Lungs Mineral particle concentrations in the hmg parenchima rang ed from 0.7 x 10s to 1.7 x 10s particle/mg of dry tissue (Figure 1). Two principal components were found--Silicates and crystalline silica (52%)--Heavy metal oxides and sulfates (48%). The relative percentage of components, however, showed significative differences, since in nearly 12% of sub 1306 jects the ratio between silicates and metal compounds dif fered for more than 50% of the average value. Fibrous par ticles were detected in 16% ofsubjects. They were general ly represented by asbestos fibers (chrysotile and amphiboles), but small amounts oftalc, rutile (titanium oxide) and calcium sulfate fibers were also found. Asbestos fiber concentration ranged from 200 to 300 fi/mg ofdry tissue and represented 0.5-1% of total particulate. Seven groups of silicates were detected: micas, clays, talc, chlorites, serpentine and amphiboles. The majority of par ticles had a diameter ranging from 1 to 5 pm. No particles more than 30 pm in diameter were observed. Asbestos fibers were represented for more than two thirds tty chrysotile and ranged in length from 1 to 8 pm, with a length/width ratio higher than 10 (Figure 2). Up to sixteen different metallic elements, in the form of ox ides and sulfates, were found (Figure 3). Nearly 80% ofpar ticles ranged in size from 0.1 to 1 pm and no particles larger than 2 pm were found. Six elements (Al, Ca, Ti, Cr, Fe, Ni) could be identified in more than two thirds of subjects. Dependence between Mineral Particulate, Age and Life-style of Subjects Tbe dependence between particle concentrations and the age of subjects is shown in Figure 4. In general, concentration appeared to increase with age. Moreover, observation in light microscopy showed that anthracosis, which was scored from 0 (absent) to 3 (severe) also tended to increase with age (Figure 5). Smoking habit seemed to influence the quantity ofmineral particulate deposited in the hmgs, since in the same age-groups, a greater amount ofparticulate was found in the hmg parenchyma of smokers than in non-smokers (Figure 6). CONCLUSIONS Our results confirm the high degree ofdependence between tile concentration of mineral particulate in die lung paren chyma and foe environmental situation. Particularly, it ap pears foal subjects living in an urban area are exposed to toxic and carcinogenic substances released by motor vehicles (heavy metals, asbestos fibers). Finally, our results confirm the effect of smoking on foe quality and quantity offoe par ticulate in the hmgs. y, o f subjects MINERAL PARTICULATE IN LUN6 TISSUE Poster Session U p*rt.icu1Ate concentrAtion <pp/'m9>*X00000 Figure 1. Particulate concentration in the lung parenchyma of sixty subjects. T< OF ASBESTOS GUALIITIES detected in of subjects Figure 2. Percentage of asbestos types detected in the lung tissue. 1307 Poster Session II DETECTION FREQUENCY OF METALLIC ELEMENTS Figure 3. Frequency of the metal elements observed. AGE - RELATED DISIRIBUTION OF PARTICULATE CONCENTRATIONS PP/mg 1308 Figure 4. Particulate concentration in relation to the age-groups. 69 59 m 45 f U 35 3 25 SUBJECTS FREOUENCT nPoster Session REFERENCES 1. Paoletd, L.: Electron microscopy techniques in the control of mineral dust environmental pollution. Ultramicroscopy 12:166 (1983). 2. Paoletti, L., Batisti, D., Caiazza, S., Petrelli, M.G., Taggi, F., De Zorzi, L., Dina, M.A. and Donelli, G.: Mineral particles in the lungs of subjects resident in the Rome area and not occupationally exposed to mineral dust. Environ. Res. 4:18-28 (1987). 3. Vena, J.I.: Air pollution as a risk factor in hmg cancer. Am. J. Epidem. 116:42-56 (1982). 4. Saracci, R.: The interactions of tobacco smoking and other agents in cancer etiology. Epidem. Rev. 9:175-193 (1987). Figure 5. Distribution of anthracosis (scored from 0-absentto 3-severe) in relation to age-groups in sixty subjects. MINKFmL pfikTlCULrtTE IN LUNG TISSUE p p /ir19 * 1 0 0 0 6 0 20-40 40-60 60-S cl Assess (ye-Ars) no smohers smoher-i Figure 6. Influence of smoking in particulate concentration in the lung tissue. 1309 Poster Session B PREVALENCE OF PNEUMOCONIOSES AMONG PHOSPHATE ROCK WORKERS IN BRAZIL EDUARDO MELLO DE CAPITANI Department of Preventive and Social Medicine UNICAMP, Campinas, Sao Paulo, Brazil INTRODUCTION The phosphate rock as a pure chemical compound is defined as a calcium phosphate, Cas<F, Cl) (P04)3. Its use in the fertilizer industry is well known all over the world and has increased since the 2nd world war end period. The industrial manipulation of that compound has brought concern about fluorose hazard by die treatment of die phosphate rock by H2SO4 or other strong acids to liberate phosphate rich com pounds. Some respiratory Illnesses mainly ofupper airways were described related to this manipulation.3'4,6,7*8*9*10 A very poor literature is available concerning pneumoconioses hazard linked with inhalation of die powdered rock.1*2'6*6-16 In die same way, good reviews about pneumoconioses don't quote any information cm phosphate rock pathogenicity.16 CRETEANU et al.,2 in 1969 and PISLARU et al.,15 in 1969 described 7 (seven) and 6 (six) cases ofpneumoconioses in phosphate rock mill and transportation workers. Both of the papers did not tell us anything about die level of free silica in die samples ofdie inhaled rock. On die other hand, EL GAWABI & IBRAHIM3 in 1975 described some cases of pneumoconioses in phosphate rock workers, but die analysis ofdie inhaled dust showed a high percentage offree silica and the lung disease was characterized as silicosis. MATERIAL AND METHODS The workers studied in this investigation were exposed to phosphate rock extracted in the states of Goias and Minas Gerais, Brazil, where die material is crushed and then transported by train reaching Paulinia, state of Sao Paulo where the compound is stored in underground mills. The underground work and a twelve hours work shift every day, with only one day of rest each fortnight created a condition of very high risk to lung diseases. Eighty one workers that had some kind of exposition to die rock dust were asked to participate ofthe study. During the investigation, eight (8) of them were put out of die study because they did not conclude all the proposed examinations. All die remanent 73 were submitted to a) occupational anamnesis; b) detailed respiratory questionnaire; c) physical examination emphasizing respiratory apparatus; d) pulmonary function tests using Collins Maxi Survey Com puter Systems analyzing. The parameters analyzed were the CVF%; VEF,/CVF and MMEF FEF^;12-13-1417 e) thorax X-ray which were read by three readers in a blind 1310 schedule using die HjO Classification of Radiographs of pneumoconiosis 1980;11 f) two workers were submitted to lung biopsies through thoracotomy. Tissue samples were stained by H.E., van Giemsa, Masson and argentic dye to found out fibrosis or even reticulin fibres; g) die quantity of free silica in die airborne samples was measured by col orimetric methods using Physical and Chemical Analysis Branch; h) a semiquantitative analysis was done with die air borne sample using an X-ray spectrometry EG 86 ORTEC. RESULTS AND DISCUSSION Fran the 73 examined workers we found 2024-7 with pneumoconioses by X-rays characterization. Trying to determine an average time of exposition for these 20 cases it was found a mean time of46 months with a range from 12 to 73 months. CRETEANU et al.,2 and PISLARU et al.,13 found a mean time for their workers of 24 and 36 months respectively. The differences between ill and not ill workers concerning the smoke habit were not found signifi cant. Relating to past inhalatoiy risk conditions, only one worker had previously worked in a fertilizer plant for a short period of time. The majority of the 20 cases didn't suffer from any respiratory symptoms (85%). Fourteen cases (70%) showed MEFF FEFjsns alterations; two cases (10%) mild restrictive patterns; three cases (15%) a pure obstructive pat tern and five cases (25%) with normal patterns. Data from the study of CRETEANU et al. obstructive pattern in six of die seven cases. No correlation between time of exposi tion and lung function alterations was found. Opacities in thorax X-rays were classified as small and round in 17 cases (85%) and small and irregular in 3 cases (15%). No pleura disease or mediastinum alterations were found (see Table I and Figure 1). Lung biopsies were examined through optical microscopy. What appeared was a very extense deposit ofbrownish crystal material with focal refringence to polarized light in the perivascular, peribronquic and septa tissues, also occupy ing intra alveolar spaces. A mild histiocytary inflammatory reaction with alveolar collapses were also seen. Despite the use for special dyes to find out fibrosis or increased reticulin fibres, no gjgnifirant fibrosis was seen (see Figures 2 and 3). Diffraction analysis by EG 86 ORTEC showed relative amounts ofCa, P, Fe, Mn, Si, Ti, Ba, Nb and S. Small quan tities of Fluoride are not detectable by this method but it is Table I Thorax X-rays Alterations in the 20 Cases of Pneumoconioses radiologic alterations n? small opacities round irreyular total P 10 q7 s1 t2 20 Poster Session U % 50 35 5 10 100 clear this element exists in the composition of that rock. Despite the good sensitivity of the method for free silica, not even traces were found. Colorimetric methods showed free silica in less than 1% of the total inhaled dust. CONCLUSIONS From the results one can conclude about the non fibergenicity of this pneumoconioses, at least with the available data. A prospective study must be carried out to assess the former statement. A regular follow-up using lung function tests and thorax X-rays must be included in the routine examinations of the phosphate rock workers. The actual etiology of that pneumoconioses should be studied using electronic microscopy linked with microanalysis by X-rays diffrac tion.18 The high prevalence of pneucomonioses found in this study must derive from the specially bad conditions for work ing people were submitted at this plant. REFERENCES 1. CHAUDERON, I.--Phosphates, superphosphates. Id:. Encyclopedia ofOccupationalHealth andSafety. 3rd. ed. (revised) ILO--GENEVA, 1983, p. 1679-80. 2. CRETEANU, G. H. et al.--Coosiieratii aspura udos cezuri ie poeumocooioze la apatita la munciotorii feroviari. Rev. Medkochirlasi, 73:837-42, 1969. 3. DERRYBERRY, O.M. et al.--Fluoride exposure and worker health. Arch. Envir. Health, 6:303-11, 1963. 4. D'ONOFRIO, V. et al.--Osservazkmi sulla patologia professionale di operari addetd alia produzione di superfosfati. Rass. Med. Iodustr. 6:369-78, 1954. 5. EL GHAWABI, S. H. A IBRAHIM, S. M.--Pnwimnconjoses in the superphosphate industry. Egpt. J. Occ. Med. 3:55-62, 1976. 6. FABBRI, L. et al.--Broncopoeumopatia cronka e pneumoconiosi in operai adidetti alia produziooe ii acido fosforico. Lav. Um. 23:50-57, 1977. 7. FABBRI, L. et al.--Patologia respiratoria del ciclo di proiuzione de fertilizzand fosfati: 2. Bronchite cronies ed alterazione funzionale respiratorie. Med. Lav. 69:723-27, 1978 8. GANDEVIA, B. & RITCHIE, B.--Relevance of respiratory symptoms and signs to ventilatory capacity changes after exposure to grain dust and phosphate rock dust. Brit. J. Iod. Med. 23:181-87, 1966. 9. HODGE, C. H. A SMITH, F. A.--Occupational fluoride exposure. J. Occ. Med. 19:12-39, 1977. 10. HUGHES, J. P.--Fluorides, a new critical review. J. Occ. Med. 19:11-12, 1977. 11. INTERNATIONAL LABOR OFFICE--ILO--Guidelines for the use ofILO-- International Classification ofradiographs ofpneumoconioses. Revised edition, ILO--Geneva, 1980. 12. KNUDSEN, R. J. et al.--The maximal expiratory flow volume curve: its use in the detection ofventilatory abnormalities in a population study. Amer. Rev. Resp. Dis. 114:871-79, 1976. 13. KORY, R. C. et al.--The veterans Adm-Army Cooperative study of pulmonary function: I. Clinical spirometry in normal man Amer. J. Med. 30:243-58, 1961. 14. MORRIS, I. F. et al.--Spirocnetric standards for the non-smoking adults. Am. Rev. Resp. Dis. 103.57-67, 1971. 15. PISLARU, V. et al.--Riscul de imbolnavire la apatita' in trasportul feroviar. Rev. Medicochir. Iasi. 73:961-66, 1969. 16. ROM, W. N. et al.--Other occupational lung diseases. In: ROM, W. N. Ed.--Environmental and occupational medicine. Boston, Little, Brown and Co. 1983, p. 251-266. 17. SOBOL, B. J. et al.--Relative value of various a spirometric tests in the early detection ofchronic obstructive disease. Am. Rev. Resp. Dis. 107:753-62, 1973. 18. VALLYATHAN, F.H. Y. et al.--Recent advances in the study of mineral pneumoconiosis. Pathol. Ann. 15:77-104, 1980. Figures not provided. 1311 Poster Session H SIZE CHARACTERIZATION OF INDUSTRIAL PRODUCTS (MMMF) USED IN BUILDINGS AND STRUCTURES AS SUBSTITUTES OF SPRAYED ASBESTOS-CONTAINING MATERIAL CORRADO CICCARELLI Istituto Superiore PrevenzioDe e Sicurezza Lavoro, Rome, Italy INTRODUCTION It is known die fire risk prevention problem has determined a very wide use of insulation materials and mainly of asbestos, as a consequence of its desiderable physical and chemical properties. For these purposes it has been used in many different industrial sectors, and particularly in con siderable quantity in the building industry. In this last field insulation products are mainly used as surfacing materials and partitions to provide both protection from fire spread and acoustic insulation. In the past, asbestos was sprayed extensively on structural steel structures and decks as fireproofing to enhance their fire resistance. Such use was required in Italy by national regulations on fire prevention. The health hazard due to die inhalation of airborne fibres arises from dust release in air caused by an aging process, bad maintenance or vandalism actions. All those facts have induced many governments to regulate the use of asbestos products and to ban asbestos-sprayed techniques. Like in some other countries die Italian Health Ministry has taken urgent measures for asbestos-control in schools and hospitals (circ.n.45 on 10.7.86). Such measures aim at iden tifying the presence of asbestos and to check if it is in good condition or if there is a potential release of asbestos dust into the air. In this case there will be a health risk and asbestos should be removed. These measures have determined in directly a lot of work in order to remove asbestos materials from buildings, even if they were not in bad conditions. Fibrous alternative materials (with die same heat and fire resistance) are generally used as substitutes for asbestos. At present, alternative products are available on the market with a silicate like chemical composition and various mor phologies, either fibrous or not-fibrous. Most of them have amorphous structures. As a rule, fibres of alternative prod ucts have a diameter coarser than asbestos-fibres, although some of them have size similar to asbestos. Generally such man-made mineral fibres (MMMF) are manufactured with different diameters relating to die specific use they are des tined. The most important MMMF are grouped in: slag wools, rock wools, glass wools and continuous filament with diameters varying from 1 up to 20 pm, averaging about 8 pm for filament glass fibres 1-5 pm for insulation wools. Often a considerable percentage of these fibres shows diameters in die range from less than 3 pm to 0 2 pm. In this paper, special attention is given to representative prod ucts used in public buildings as banks or offices, as substitutes for asbestos. These materials have been sent by U.S.L. (Local Sanitary Unit) to our Institute to obtain all information about their chemical composition and size characterization, in order to decide their use as asbestos substitutes. In a recent summary report of WHO-IARC international symposium,1 an increased lung cancer risk has been reported among workers exposed to small-diameter fibres since the early days of man-made mineral fibres production. The risk has been greater in die rock or slag wool sector than in the glass wool one. Moreover the IARC has revised of carcinogenicity and it has classified MMMF as possibly carcinogenic to humans (Group 2B). The cancerogenic activity is generally attributed to the fibre dimensions and to their durability in biological tissues. To day die opinion of the scientific community based on die epidemiological, toxicological and mineralogical studies, in dicates that any mineral fibre with specific dimensions and sufficient biological durability must be regarded as possibly cancerogenic. MATERIALS AND METHODS All materials studied appear composed of fibrous elements and a low density matrix of rounded aggregates. The color is usually brown to white. Inspection of the bulk materials by a stereomicroscope at about 40 X show a very friable con sistency and a largely empty structure. Fibres show a wide range ofdiameters and often they are in association with unfiberized particles mostly in the form of solidified droplets. Few milligrams ofthe bulk materials, taken at random were shaken into a beaker with a "policeman" for a few minutes using H202 in order to help particle disgregation and dispersion. Because of the mechanical treatment, fibre length distribu tion was not determined in the subsequent analysis. Material was then dispersed in prefiltered distilled water. Aliquots of die suspension were filtered through a polycarbonate filter (NPF), 25 mm diameter, 0.2 pm, by a small funnel (Nuclepore Corp.). 1312 A quarter of each filter was mounted on a carbon stub, 12 mm diameter and coated by a gold layer of approximately 100 nm of thickness. The samples were analyzed by scanning electron microscope, Cambridge model 200, at 0 tilt angle, 25 KV accelerating voltage. Fibre size was measured directly on die screen, separately recording diameters of 100 fibres. The airborne samples were collected on 25 mm 0.8 microns cellulosic filters. Half ofthe filters were cleared and exam ined by phase contrast optical microscope (PCOM) method. The remaining portion of the filters were mounted on a car bon stub in the same way, but examined by SEM at 15 KV acceleration voltage in order to prevent local overheat. Poster Session n RESULTS Table I shows size distributions from bulk samples of materials used as sprayed insulations in buildings. From diameters distributions it results that in samples 2 and 5 the respirable fraction (diameters less than 3pm) is about 30%, while in the other samples the same fraction is about 60%. During the removal operation from building structures coated with the material of sample n. 1, personal air samples were collected according to the standard method.2 Table II shows a consistent size distribution by diameter and by length of airborne respirable fibres from 4 different samples. Sample Table I Frequency of Diameter in the Bulk Materials D=1 1D3 3 D=5 D5 1 29 26 3 20 4 25 5 10 39 22 10 27 33 34 41 26 13 35 25 15 19 34 47 1313 Poster Session II Table n Size Distribution of Airborne Respirable Fraction A) Distribution by diameter pm D=1 1D2 2D3 D5 % 39 44 15 2 B) Distribution by length pm 5 L 10 10 L 15 15 L 20 20 L 30 30 L 100 L 100 % 41 18 1? 15 11 5 In this case die respirable fraction is about 98% showing a clear preferential loss ofcoarser fibres. About 40% ofthese fibres are between 5 and 10 microns in length, and the rest is equally distributed among die subsequent length classes. Chemically fibres consist principally of silicon, calcium and aluminium. Other elements such as iron, magnesium, potassium are also present. CONCLQSIOMS Recently there has been an increasing demand for insulation products and especially of man-made mineral fibrous materials to replace asbestos-products. Owing to die possible fibre release from MMMF insulation materials in bad conditions, sometimes they have been removed from structures. The results from this study, even iflimited, indicate that die physical characteristics of MMM products show 50% diameters less than 3 microns, while during die removal about 98% of airborne fibres have diameters less than 3 microns. Although, at present, investigation3 carried out in different countries unnecessary exposures to these types of fibres. FmaHy, it seems important that clear information be reported on appropriate labels in which should appear not only technical instructions, but also chemical data as well as die diameter range offibres in order to evaluate the possible risk of people exposed. 1314 Fi(an 1 NlfMf MtUitatlw * j Poster Session H Figure 1. Distribution of fibre diameters of bulk materials. REFERENCES 1. WORLD HEALTH ORGANIZATION, REGIONAL OFFICE FOR EUROPE: Les fibres minerales artificielles sur les beux de travail Sym posium international. Rapport sommaire, Copenhague (1986). 2. WORLD HEALTH ORGANIZATION,REGIONAL OFFICE FOR EUROPE: Reference Methods fix- Measuring Airborne Man-Made mineral Fibres, 4, Copenhagen (1985). 3. Doll, R.: Symposium on MMMF, Copenhagen, October 1986: Over view and Conclusions. Ann. Occup. Hyg. 31:805-819(1987). 1315 Poster Session B SILICOSIS IN PIT DIGGERS IN SERRA DA IBIAPABA, CEARA, BRAZIL EDSON JOSE DE BARROS HATEM* Fernando Marcos Torres Bandeira Cavalcanti! Occupational Physician {Occupational Safety Engineer/ Fundacentro/Brazil A survey carried out by technicians from FUNDACENTRO, Pernambuco, showed that out of 134 pit excavators, 38 presented silicosis after clinical and laboratorial examina tions. Out of these 38, 5 died during this survey. En vironmental evaluations in workplaces were also made through dust measurements inside pits being excavated. A conventional water supply is here considered a definite solu tion to these problems. Specific measures are also proposed as transient solutions. We participated in the research on silicosis in Serra da Ibiapaba, approx. 350 km from Fortaleza, because of the professional concern and particularly because of die great social vision of die INAMPS pneumologist. Dr. Marcia Alcantara Holanda, who in her ordinary work as the coordi nator of the apprenticeship in pneumology at the INAMPS hospital in Messejana, remembered Ramazzini and asked two patients: "What's your occupation?" Today, only in Tiangua', a town with 40,355 inh. (IBGE 1980), 138 pit diggers have already been clinically and radiologically examined, and a very large number of workers suffering from the disease was found. FUNDACENTRO participated in this work through its Divisions of Occupa tional Medicine and Hygiene, its Regional Center in Per nambuco, supported by its National Technical Center, mak ing clinical examinations, radiological comparisons based on OIT standards (International Labor Organization) en vironmental dust analyses and other risk analyses, general guidance in relation to occupational safety, hygiene and medicine, and chiefly in the proposition of solutions com patible with the serious nature of the situation, in order to solve definitely this crucial problem that affects seven towns in Serra da Ibiapaba, comprising approximately 200,000 peo ple. None of these towns has a conventional water supply, and there is no natural water reservoir in the surroundings. The population has to dig water holes manually, one per fami ly, keeping a large number of workers busy. HAZARDS OF THE ACTIVITY--BACKGROUND A pit is generally dug by two persons who substitute each other alternatively. While one worker is inside the pit, the other stays on the surface, helping with whatever is necessary. The pits usually have a diameter of 1,50m and they are around 12m deep. The geologic profile of die soil shows a stratum containing lOm of sandstone, which is easily excavated using tools. There is a hard silicified stratum below it that may only be removed with the use of explosives or 1316 mechanized tools. A sample of this silicified stratum was taken to be analyzed and it was found to contain 97.44% of SiOj. After this stratum we reach the saturated zone, where the walls of the pit are then covered with premolded concrete rings. A bucket lifted with ropes through a drum fixed to a wooden girder set at the pit entry is used to remove the material. It can also be used by die worker to go up to die surface or down to die bottom. In February 1987, they used to charge the amount of US $10.00 for each "palm" (approx. 25cm) excavated or US $385.00 for a whole pit of any size. Operational Hazards Due to die poor conditions where the work is performed, it is easy to foresee die numerous hazards involved: Falls caused tty rope rupture, drum break, rupture of drum fastening, rupture of drum supporting girder, slipping. Being buried in the earth by caving in of pit walls. Accidents caused by tools in the confined workplace. Ergonomic risks due to worker's posture because of con fined workplace. Misuse of explosives. Here we wish to focus on a number of irregular proceedings, starting with equipment and material purchase, which is trad ed freely, with no observance ofcurrent laws. The transport, storage and handling are also totally inappropriate. Ex plosives have been seen in worker's bouses in places within children's reach. The preparation of explosives for detona tion is done in a very primitive way, even die teeth are employed to fix the detonator to the fuse and it is done in the presence of ordinary people (even children) in residen tial areas, with no measure taken to isolate the area. Thus, besides die danger of an accidental explosion, there is the danger ofstones being thrown at considerable distances, often causing physical and material damage. Another circumstance which deserves attention is that die worker has only 2 minutes and a half to get out of the pit, after lighting die fuse of the explosive charge at die bottom of die pit. It is easy to imagine what may occur in case be foils because of any reason already mentioned, hi die survey we found that at least one fatal accident had been caused by this reason. Environmental Hazards Undoubtedly, the biggest hazard of this type of work is the dust produced by the excavation. In rainy weather, the aerosol is present almost exclusively in the operation of removing die silicified sandstone stratum through mechanical means or explosions (the usual manner). The situation is ag gravated in dry weather when the dust is also present in the strata prior to the silicified sandstone. Therefore, die risk generating factors are: Low humidity of the soil above die saturated zone. Pit depth, making the dust reduction by means of natural ventilation impossible. Lack of any exhaustion device. The concentration of dust is higher just after die silicified sandstone stratum is exploded and it remains like that for hours. The worker generally goes back to the bottom of the pit to remove fragments 3 hours after the detonation which is not enough time for the dust to have dissipated. MATERIAL AND PROCEDURE The measurements of these concentrations were carried out since then. The adopted procedures are described on Table I. The threshold limit values represent dust concentrations in workplaces under which it is believed that most of the workers may be repeatedly exposed to during their worklife with no harm to their health. Poster Session H Procedures for Evaluation and Analysis The dust sampling was collected by means of 37mm PVC filters, with 5pm porosity, where low flow rate suction pumps were used (pump BENDDC model BDx 44). A oneinch cyclone was used to select respiratory particles. It allows 90% of particles having a diameter smaller than or equal to 2pm to go through and it detains particles with diameters bigger than 10pm. The determination of dust concentration was made by gravimetry and an analytical balance of 0.000010 precision was used. The percentage determination of crystallized free silica in the dust samples was carried out through X-Ray diffractometry. Note. The BENDIX sampling kit is an equipment for in dividual use, fitted out in the worker. Hie samples collected represent the circumstances of exposure to dust when per forming any analyzed activity. The flow rate used in the collection oftotal dust is 1.5L/min and in the collection of respirable dust it is 1.7L/min. The period for sampling varied according to environmental characteristics. Procedure for Assistance The procedure for assisting exposed workers has been the following: THRESHOLD LIMIT 8.5 MPPCD* %$i02+10 Table I Threshold Limit for Crystallized Free Silica Dust COLLECTED DUST SAMPLING METHOD Total Impinger PROCEDURE FOR ANALYSIS Field Count 8.0 Mg/m3** tSiO,+Z Respirable Gravimetric with Cyclone Gravimetric (weighing) 24 Mg/m3** ISi027T Total Gravimetric Gravimetric (weighing) (*) MPPCD = Millions of particles per cubic decimeter (**) Mg/m3= Milligrammes per cubic meter 1317 Poster Session n 1. Simple spirometry, measuring die forced vital capaci ty, measuring the forced expiratory volume in die first second oftime and forced respiratory flow between 25% and 75% of forced vital capacity. 2. Effort tests (with arterial blood gases, exhausted gas sample and realization of ECG). 3. Fiberoptic bronchopy and collection of bronchiolealveolar lavage (BAL). Patients with changes in effort tests and normal radiological examinations are subject to a transbronchial biopsy of the pulmonary paren chyma. 4. Determination ofthe immunological profile ofeach pa tient, including: Hemogram, plasmatic protein elec trophoresis, serum immunogloblins, cutaneous tests for cellular immunity evaluation, rheumatoid factor. 5. Outdoor pulmonary biopsy, where transbronchial biop sy is not enough for a diagnosis. 6. Diagnosed patients are registered in their hometowns for attendance and especially for treatment of the associated diseases mentioned above. These stages are carried out in Tiangua', by die team of Dr. Marcia' Holanda. MEASUREMENTS The evaluations were based on Tiangua' The measurements were done in pits located in 3 different regions of the town. Pit No.l (urban area). Pit No.2 (suburban area) and Pit No. 3 (rural area). The situation ofthis hard working population is very serious, as can be seen in Table II, The threshold Table n Concentration Measurement of Environmental Dust Inside Pits in the Town of Tiangua', Brazil PLACE SAMPLE VOLUME OF XSi02 CONCENTRATION THRESHOLD EXCESS' WEIGHT AIR SAMPLE (mg/m3) LIMIT (Mg) (in*) (mg/m3) Pit no-1 Bairro Sto. Antonio 0,33 Pit no- 1 Bairro Sto. Antonio 0,96 Pit no. 1 Bairro Sto. Antonio 0.25 Pit no. 2 Rua das Almas 0,40 Pit no. 2 Rua das Almas 0,19 Pit no. 3 Health Center 0,11 Pit no. 3 Health Center 0,27 0,0066 0,0068 0,0034 0,0045 0,0075 0,0051 0,0085 18,2 55,0 TD 18,8 141,2 TD 16,0 73,5 RD 10,0 88,9 TD 10,5 25,3 TD 9,1 21,6 RD 3,7 31,8 RD 1,13 0,41 0,44 1,85 1,78 0,72 1,40 48 344 167 48 14 30 23 According to NR 15, Governmental Decree 3214/78 of the Ministry of Labor TD = Total Dust RD = Respirable Dust * = Number of times the Threshold limit was exceeded 1318 limit for silica dust occupational exposure was exceeded up to 344 times. The legislation considers these cases as max imum degree insalubrity. Dust concentration at these levels, inevitably causes silicosis. This was confirmed by a number of diagnosed cases in the region. In Tiangua, out of the 200 pit diggers estimated, 134 were examined. Out ofthese, silicosis was confirmed in 38 through clinical and laboratorial examinations, and 3 workers, out of these 38, died within 60 days. CONCLUSIONS AND RECOMMENDATIONS As far as the workers already affected by the disease are con cerned, consequently with no chance of cure, what could be done to help diem is give them a monthly life annuity. Therefore, it is necessary that the workers be examined and considered disabled by means of a medical examination. The disease is known to exist and to be extremely serious, however, because it occurs in a deep poverty region, distant from die big cities, prevention is laid aside by die supervis ing, educational and medical assistance structures in charge of the matter. It is common knowledge that silicosis prevention in general includes environmental control measures, such as: Product substitution Process alteration Dust suppression in the source by means of moistening Process insulation Dust removal through a general scattered ventilation, on/site exhausting ventilation or electrostatic precipi tation v Use of personal protective equipment We consider a conventional water supply system a definite solution to die problem. A dam has already been built 18km from Tiangua', which is just waiting for a governmental deci sion so that supplementary works can be built and put in operation (wafer mains, water-treating plant and water supply system). In the rural area the solution would be die machine sinking of wells, with no direct participation of die worker in die process. In addition to die benefits that these solutions could bring, they would also create jobs for the local population. However, these are not short term measures, thus while water-holes still need to be built as the unique source of potable water for die people, some specific measures could be carried out: For die use of explosives--Careful attention to safety instructions about transportation, storage and handling of these materials. Provide appropriate safety conditions in order to avoid fells when going down and up die pit. Provide ap propriate dimensions for ropes, drums.sheaves and sup port beam. Paster Session U Keep the excavation site moist. Wait for at least 24 hrs after the explosion before enter ing the pit. Standardize a diameter for the water-hole of at least 2,50m so that die worker may have better working conditions. Limit the use of personal protective equipment, which should only be used as a transient solution for its several deficiencies and above all, its low efficiency and discom fort for the person who uses it. Cover die pit walls with pre-molded concrete rings as the excavation gets deeper. Make regularly die previously mentioned examinations in accordance with the current labor legislation at the expense of die Ministry of Social Welfare. However, a question is to be asked: How are these pieces of information to get to the workers, and principally, how to make them understand how relevant they arc? Dr. Marcia Alcantara has taken the first steps in relation to this. In Tiangua', on a daily radio program she talks about the general aspects of the problem attacking the pit diggers ofdie region. The workers themselves, after the first deaths, have mobilized to fight for better working conditions, but due to the feet that most workers are not educated, and need to support themselves and their family, they do not give at tention to the matter. While definitive solutions are not adopted, it is necessary to make die transient measures known to mitigate the disease. For this to occur, we suggest die following measures be taken: 1. Campaign about silicosis prevention in the Tiangua'daily radio program. Fundacentro may cooperate recording cassette tapes with this information. 2. Fundacentro can prepare posters and booklets to be distributed to health centers, town halls, church com munities and labor unions. 3. Educative lectures held at community centers, health centers, schools and churches, given by technicians from die Regional labor office. Welfare and State Health office. 4. Intensification of supervision by die Regional Labor Of fice of preventive aspects and of die work entailment between employees and employers. Orientation about the use of explosives by die Army. 3. Facilities for purchase of personal respirators or even their distribution free of charge to workers, by die Ministry of Social Welfare together with die State Health office. Nevertheless, we stand upon the point that the eradication of the problem will only occur completely, when there is the engagement of the federal, state and municipal govern ments in order to provide these populations with what they do not have so far: a potable water supply system. 1319 Poster Session II DNA FLOW CYTOMOETRIC ANALYSIS OF MESOTHELIAL CELLS EXPOSED IN VIVO TO ASBESTOS FRANCIS H.Y. GREEN Amba Balu University of Calgary, Calgary, Alberta T2N 4N1, Canada ABSTRACT Epidemiologic and experimental studies have established a causal relationship between malignant mesothelioma and exposure to asbestos and other fibrous minerals (e.g., erionite) which have similar structural but not chemical characteristics. Predicting whether a given fibrous mineral will be a human carcinogen is very difficult in view of the long latency interval (usually in excess of 30 years) seen in human cases of mesothelioma. It has been shown by others that mesothelial cells exposed to fine chrysotile fibres in vitro exhibit chromosomal damage associated with fibre penetration of die nucleus. In view of the likelihood of DNA damage induced by asbestos, we studied DNA profiles in mesothelial cells exposed to asbestos in vivo. Fischer 344 rats given a single intraperitoneal injection of 50 mg crocidolite are being followed for periods up to 18 months. Cell cycle analysis using DNA flow cytometry showed a rapid increase in DNA synthesis (S-Phase) by mesothelial cells, which peaked at 3 days and remained relatively stable until 28 days. Observations at later times have not yet been completed. The proportion of mesothelial cells in S-phase following asbestos stimulation was approximately 3 times greater than that seen in normal mesothelium. The mesothelial cell proliferative response was accompanied by an inflammatory reaction which peaked at 2 weeks. Light and electron microscopy showed asbestos fibres within mesothelial cells. The evidence so for indicates dial asbestos stimulates an early and intense proliferative response in mesothelial cells. Based on DNA flow cytometric analysis of2 human cases ofmesothelioma showimg marked aneuploidy, we expect to observe die development of chromosomal abnormalities in our experimental lesion. We con sider that quantitation of these changes using different types of fibrous minerals and fibrous minerals of different dimensions, will ultimately lead to a short-term bioassay of high predictive value. INTRODUCTION The association between asbestos exposure and diffuse malig nant mesothelioma (DMM) has now been well established by experimental and epidemiological methods.2*9'13*14 Moreover, other fibrous minerals (e.g., erionite) have also been causally implicated.15 The latency interval of this disease is long (usually 15-40 years from initial exposure) and death generally occurs within 12-14 months of diagnosis. DMM is exceedingly difficult to diagnose, no effective treatment exists, and currently there are no screening techniques for high-risk individuals such as asbestos workers. In recent years, cell proliferation characteristics of normal and malignant cells have gained increasing attention and have been the focus of studies of various human and animal turnon 6.7,io,i Cytokineticists have relied heavily on the fre quency of DNA synthesizing cells to determine die cell cy cle traverse characteristics of normal and malignant cells. Structural chromosomal damage and changes in cellular DNA content have been shown in association with several tumors including mesotheliomas.3-6-12 Analysis of two cases of human mesothelioma in our laboratory revealed DNA ploidy changes. (Figure 1) Although the mechanism(s) of these karyotypic changes are not entirely understood, it may involve a direct interaction between asbestos fibres and metaphase chromosomes.16 Flow cytometry is the latest and in many ways the most prac tical of die current adjunctive techniques for studying the cytokinetic properties of malignant tumours. However, the cell dynamics of malignant, or indeed of normal mesothelial cells are virtually unknown. To determine the possible value of DNA quantitation by flow cytometry in the evolution of DMM, we studied the cytokinetic properties, specifically die DNA synthesizing phase (S-phase) changes of mesothelial cells exposed to asbestos in vivo. Our main purpose was to correlate phase distribution changes of cycling cells with the early histological and cytological alterations induced by asbestos. MATERIALS AND METHODS Dust Suspensions Crocidolite samples were obtained from NIOSH (courtesy Dr. V. Vallyathan). Details of fibre dimensions are shown in Figure 2. 1320 200 Figure 1. DNA histogram of human mesothelioma show ing an aneuploid population at 3.5 N. Animals Male Fischer 344 rats were intraperitonially injected with 50 mg crocidolite asbestos in 10 ml of saline or with 10 ml saline alone. Uninoculated animals served as additional baseline controls. Four animals from each treatment group were sacrificed, under halothane anesthetic, at 1, 3, 7, 14 and 28 days post-inoculation. Cell Isolation and Separation The peritoneal cavity was lavaged thrice with normal saline to remove the majority of free-floating cells, mostly macrophages. EDTA (20 ml) was then instilled in the peritoneal cavity, which was lavaged 30 minutes later to ex tract mesothelial cells from the parietal peritomeum and serosal surfaces (Figure 3). The cell sheets were separated by gentle mechanical agitation. In crocidolite treated animals, firm nodules surrounding asbestos fibres were present throughout the omental and serosal surfaces. These were surgically excised. Cells were extracted by mechanical dissociation, and suspended in RPMI 1640 media. Poster Session U Cytology Cytological smears from the peritoneal lavage fluids and from single cell suspensions of the reactive modules, were sprayfixed with Cytospray, an alcohol-based fixative, and stained with Papanicalaou. Differential cell counts were performed on 100 cells for each specimen. A hemocytometer was used to obtain total cell counts. Histology Tissue blocks obtained from the reactive peritoneal nodules and the parietal and visceral peritoneal surfaces, were fixed in Camoy's solution (60% ethanol, 30% acetic acid and 10% chloroform), and processed in paraffin. 5 |im tissue sections were stained with hematoxylin and eosin and examined by light microscopy. Scanning Electron Microscopy Samples of parietal and visceral peritoneum were immediate ly excised from animals at termination and immersed in Karnovsky's fixative. Following complete fixation, samples were dehydrated in graded concentrations of ethyl alcohol, critical point dried and coated with gold/palladium. Specimens were examined in a Hitachi S-450 scanning electron microscope. Flow Cytometry Single cell suspensions from the above tissues were prepared in RPMI media and fixed in 50% ethanol. The cells were washed twice in PBS and resuspended in 1 ml of PBS, con taining 5 pg of Propidium iodide (Sigma 0P-417O, Lot 107F-08201). (Figure 4) This suspension was then filtered through a 44 p nylon mesh to exclude cell clumps. Cell cy cle analysis was performed with a FACS Ortho flow cytometer. During flow cytometry, cells were excited at 488 nm. Red fluorescence from Propidium iodide (PI) was collected through a 600 nm wavelength long pass filter and recorded as a measure of total DNA. The resulting electrical pulses were stored in the memory unit of a pulse height analyzer and displayed as a histogram. The phase distribution ofeach cell population was ascertained from the DNA histogram.8 RESULTS Cytology Analysis of cells isolated from the abdominal cavity by saline wash (free floatimg cells) from all groups, showed that more 1321 Poster Session n CROCDOLTTE: Ffcre Length FREQUENCY FREQUENCY CROODOLTTE: Fibre Wkfth WIDTH (Nm) Figure 2. Crocidolite asbestos--fibre dimensions: a) fibre length; b) fibre diameter. 1322 Poster Session U Figure 3. Sheet of normal mesothelial cells obtained by EDTA extraction (Papanicalaou stain X 400). than 90% were macrophages and polymorphonuclear cells. Rats injected intraperitoneally with saline showed a small increase in inflammatory cells at 24 hrs which returned to normal values by 3 days. (Figure 5) Asbestos injection, on the other hand, produced an intense and sustained inflam matory reaction which peaked at 14 days. Lavageates from asbestos exposed animals; also contained large numbers of red blood cells, at 1, 3 and 7 days. Cytological examination of cells extracted from the mesothelial surfaces of saline injected rats by EDTA, showed that over 90% were of mesothelial type. ETDA extraction of asbestos treated rats produced a more mixed cellular pro file, with mesothelial cells constituting 70-90% of the total population. The number ofcells retrieved from saline injected rats remained relatively constant at all time periods. (Figure 5) Maximal retrieval of mesothelial cells in the crocidolite treated group occurred at 7 days, reflecting die increased pro liferative response (see below). Histology and Scanning Electron Microscopy The normal mesothelium consisted of a single layer of flat tened mesothelial cells on a basal lamina overlying a thin connective tissue stroma. A distinct submesothelial cell population was not apparent by light microscopy. Asbestos injection produced an early and intense acute in flammatory reaction in the mesothelial and submesothelial tissues, consisting of capillary dilatation, edema and macrophage and polymorphonuclear cell infiltration. The serosal surfaces were covered with a fibrinous exudate. (Figure 6) This inflammatory response was observed at all time periods, but by 28 days macrophages predominated in the inflammatory infiltrate. Initially, the asbestos fibres lay on the peritoneal surfaces and were concentrated on the omentum, upper small in testine, and around tire liver, spleen and stomach. Fewer fibres were noted on the parietal surfaces of the peritoneal cavity. At subsequent time intervals, the majority of fibres were actively phagocytosed by reactive mesothelial cells and incorporated into the submesothelial tissues. Fibres also became trapped within the tissues through infolding of omen tal surfaces (Figure 6). Incorporation of fibres into the peritoneum was associated with the proliferation ofboth sur face and submesothelial cells (Figure 7). The former became multilayered and developed hyperchromatic and irregularly shaped nuclei. Many assumed bizarre multinucleate forms (Figure 6). Scanning electron micrographs revealed these cells to be highly abnormal and many had fibres penetrating their cytoplasm. (Figure 8) Submesothelial cell proliferation was associated with diffuse thickening of the peritoneum and nodule formation. (Figures 9,10) At 28 days post-injection, collagen was also found within the nodules. 1323 Poster Session II Figure 4. Fluorescent micrograph of normal mesotbelial cells stained with Propidium iodide (X 1000). i am ic mm*- ancmnc wr omxmks) *a>i effMCTHK- omcidm.lit waracsoneJM. ceus> 3.ii m*- axna. air wwim > an* oomcnat- coma, amp o*sonj*i i&i IDE CdlS ftSt iMClUtiN) Figure 5. Total and differential cell counts in lavageates from abdominal cavities of rats exposed to crocidolite asbestos. 1324 Flow Cytometry Cell cycle analysis of inflammatory cells retrieved from the abdomimal cavities, showed similar phase distribution for control and asbestos treated rats. Hie S-phase values were low and ranged from 0.5-3%. Values pertaining to mesothelial cells differed markedly between the two groups (Figure 11). Approximately 7% of normal mesothelial cells were in the S-phase ofthe cell cycle at any given time. Saline injection produced a small increase in DNA synthesizing cells (S-phase) at 24 hours, which peaked at 3 days and returned to normal values at 7 days. Crocidolite asbestos, by contrast, produced a rapid increase in the proportion of DNA syn thesizing mesothelial cells which reached maximal values at 3 days and was sustained up to 28 days. Discussion The inflammatory and proliferative changes associated with intraperitoneal injection of asbestos, have been well documented by others.3'4,14 In this study, saline injection alone produced a mild inflammatory response and increased proliferation of mesothelial cells. This response was short lived and normal values returned by 7 days. Cell cycle analysis of inflammatory cells retrieved from the abdominal cavities of both saline and asbestos treated animals, showed similar distribution of cells in S-phase. These values were typically low (1-2%) and indicate that Poster Session U Figure 6. Section of visceral peritoneum (omentum) from rat dosed with 50 mg crocidolite IP 7 days previously. There is an intense proliferation of sur face and subepithelial mesothelial cells associated with a fibrinous and in flammatory response. The surface mesothelial cells also show giant cell for mation and nuclear atypia (X 100 hematoxylin and eosin). inflammatory cells migrating into the abdominal cavity, are termimally differentiated and do not proliferate in response to asbestos stimulation. Mesothelial cells, on the other hand, developed an early and sustained proliferative response to asbestos with S-phase populations constituting 20-30% of the total mesothelial cell population. The relatively high S-phase value (7%) for nor mal mesothelial cells indicates that these cells have a fairly rapid turnover rate. Unfortunately, there is very little recent information on cell cycle characteristics of normal mesothelial cells. Experiments have been initiated in our laboratory to determine these parameters, using DNA flow cytometry combined with bromodeoxyuridine (BuDr) incorporation.5 At this stage, it is not possible to say whether the changes observed in mesothelial cell populations exposed to asbestos, are specific for this mineral type or whether they progress to neoplasia. Preliminary experimental studies with Fischer rats suggest that intraperitoneal injection of other dusts (silica, iron oxide and wollastonite) induce less proliferative activi ty of mesothelial cells than crocidolite asbestos. It should be noted that wollastonite is a fibrous silicate mineral that has potential for substitution in asbestos products. If these preliminary experiments are confirmed, they would indicate the potential utility for this approach as a short term bioassay for assessing the carcinogenic potential of fibrous minerals. Analysis of two human mesothelioma cases showed a definite second population of cells in both instances with DNA con- 1325 Poster Session D Figure 7. Proliferating mesothelial cells in juxtaposition to asbestos fibres (X 400 hematoxylin and eosin). tents of approximately 3.5 N (Figure 1). Craighead et al., have reported similar ploidy changes in experimentally in duced malignant mesotheliomas in rats.3 We therefore ex pect to see die development ofmalignant change and changes in DNA ploidy in our experimental animals. We are par ticularly concerned to determine die time of onset of these changes, the clonality ofthe resulting tumors and die presence or absence of specific cytogenetic abnormalities which might serve as markers of exposure to asbestos or other car cinogenic fibrous minerals. 1326 Foster Session U Figure 8. A. Scanning electron micrograph of peritoneal surface of rat two weeks after receiving 50 mg crocidolite asbestos I.P. The mesothelium is hyperplastic and shows incorporation of asbestos fibres into the mesothelial cells. B. Normal mesothelium (X 2000). 1327 Paster Session JJ Figure 9. Serosal aspect of abdominal cavity from rat exposed to crocidolite asbestos two weeks previously. There is a marked proliferation of spindle-shaped submesothelial cells (X 250 hematoxylin and eosin). REFERENCES 1. Ballantyoe, K.C. ct al.: Flow cytometric analysis of the DNA con tent of gastric cancer. Br. J. Cancer. 56:52-M (1987). 2. Becldake, MJL: Asbestos related diseases ofthe lung and other organs: their epidemiology and implications for clinical practice. Am. Rev. Resp. Dis. 114:187-237 (1976). 3. Craighead, J.E., Akley, NJ., Gould, L.B., Libbus, B.L.: Characteristics oftumors and tumor cells cultured from experimental asbestos-induced mesotheliomas in rats. Am. J. Path. 129:448-462 (1987). 4. Davis, I.M.G.: The histopatbology and ultrastructure of pleural mesotheliomas produced in the rat by injections ofcrocidolite asbestos. Br. J. Pathology. 60:642-652 (1979). 5. Dolbeare, F. et al.: Flow cytometric measurement of total DNA con tent and incorporated brotnodeoxyuridine. Proceedings of National Academy ofScience, U.SA. Vci. 80, pp. 5575-5577 (1983). 6. Gibas, Z, Li, F.P., Amman, K.H., Bernal, S., Stahel, R., Sandberg. A.A.: Chromosome changes in malignant mesothelioma. Cancer Genet. Cytogenet. 20:191-201 (1986). 7. Gray, J.W., Dolbeare. F., Pallavicini. M.G., Beisker, W., Waldman, F.: Cell cycle analysis using flow-cytometry. Int. J. ofRadiation Bio. Vol. 49a), PP 237-255 (1986). 8. Krishan, A: Rapid flow cytofluorometric analysis of mammalian cell cycle by Propidium iodide staining. /. Ceil Biology. Vol. 66, pp. 188-193 (1975). 9. McDonald. J.C., McDonald A.D.: Epidemiology of mesothelioma from estimated incidence. Prev. Med. 6:426-446 (1977). 10. Murphy, W. et al.: Flow cytometry of deparaffinized nuclei compared to histological grading for the pathological evaluation of transitional cell carcinomas. J. of Urology. 135:694-697 (1986). 11. Shin, M.C., Fbnunger, H.: Acute and chronic effects ofnXiapetiluneal injection of two types of asbestos in rats with a study of the histopathogenesis and ultrastructure of resulting mesotheliomas. Am. J. Pathology. 70(3):291-306 (1973). 12. Sincock, A, Seabright, N.: Induction of chromosome changes in Chinese hamster cells by exposure to asbestos fibres. Nature. Vol. 257, pp. 56-58 (1975). 1328 Poster Session II Figure 10. Nodular lesion on parietal aspect of peritoneum of a rat 7 days after receiving 50 mg crocidolite I.P. Giant cells are common in this lesion (X 100 hematoxylin and eosin). 13. Solomons K.: Malignant mesothelioma clincial and epidemiological features. South African Med. J. 66:407-412 (1984). 14. Wagner. J.C. et al.: Experimental production of mesothelial tumors of the pleura by inplantation of dusts in laboratory animals. Nature. Vol. 196, pp. 180-181 (1962). 15. Wagner, J.C., Skidmore, J.W., Hill, R.J., Griffiths, D.M.: Eronite exposure and mesotheliomas in rats. fir. J. Cancer. 51:727-730(1985). 16. Wang, M.D. et al.: The interactions between asbestos fibers and metaphase chromosomes of rat pleural mesothelial cells in culture. Am. J. Path. pp. 343-349 (February 1987). Figure 11. Percentage change in mesothelial cells in S-phase with time in response to intraperitoneal injection of asbestos or normal saline. ACKNOWLEDGEMENTS: Technical expertise was provided by M. Skromeda. Secretarial assistance was provided by Marguerite J. Schultz. This work was supported by the Alberta Heritage Foundation for Medical Research, Grant #71-8168. 1329