Document Ddy7q3KNXwjbOD1JqLppNEGdn

Poster Session n ALTERATIONS IN PULMONARY XENOBIOTIC METABOLIZING ENZYME SYSTEMS IN ASBESTOTIC ANIMALS QAMAR RAHMAN, Ph.IX S. G. Khan, M.Phil. S. Ali, Ph.D Industrial Toxicology Research Centre, Post Box No. 80, Mahatma Gandhi Marg Lucknow 226001, India INTRODUCTION The synergistic interaction of asbestos and tobacco smoke in die genesis ofbronchogenic carcinoma has been reported extensively by both epidemiological and experimental studies.1*30*42'18'26'31'49 Insphe of several efforts in this direc tion, the precise biochemical mechanisms involved in the potentiating effects of asbestos in die development of bron chogenic carcinoma remains obscure. It is conceivable that die fibres may adsorb die known carcinogens of cigarette smoke on their surface and thus facilitate their entry and retention in the system.27*28 Experimental studies have shown die adsorption of benzo(a)pyiene, a major compo nent of cigarette smoke cm the surface of asbestos and their poor excretion from experimental animals.41*44 Alternative ly, asbestos fibre may directly modify carcinogen metaboliz ing enzyme system, viz. activation arid conjugation reactions. It has been reported that asbestos fibres partially inactivate microsomal mixed function oxidase (MFO) system.25*40 The present paper is concerned with the alterations in die xenobiodc metabolizing enzyme Systran, lipid peroxidation, antioxidant levels in die lung of rats at progressive stages of dust exposure. MATERIALS AND METHODS Dust Chrysotile UICC standard reference sample, particle size <30 p, was obtained as a gift from Dr. J.B. Leinweber, John-Manville Mills, U.S.A. Chemicals Benzo(a)pyrene, 3-hydroxy benzo(a)pyrene, styrene epox ide and bovine serum albumin were procured from Sigma Chemical Co., U.S.A. All die other chemicals and reagents were either purchased from V.P. Chest Institute, New Delhi or Sisco Research Laboratories (SRL), Bombay, India, and were of analytical grade. Treatment of Animals Male albino rats from the 1TKC colony, weighing 150-180 gm were used. The dry dust and 0.15 M NaCl were separate ly autoclaved at 15 ltxs pressure for 15 min, suspended and mixed thoroughly just before inoculation. The animals were intratracheally instilled with 5 mg of dust suspended in 0.5 ml of normal saline, according to the procedure, described by Zaidi.54 Corresponding controls received 0.5 ml of 1330 normal saline only. The animals were maintained on a com mercial pellet diet, supplied by Hindustan Lever limited, Bombay, India and tap water ad libitum. Six animals from each group were sacrificed at 1,4, 8, 16,90 and 290 days after inoculation. Isolation of Microsomes The rat lung microsomal fraction was isolated by the pro cedure of Johannesen et al.22 Enzyme Assays Benzo(a)pyTene hydroxylase was assayed by the fluorimetric techniques as described by Dehnen et al.8 The quantitation of phenolic metabolites was based on comparison of fluorescence to a standard solution of 3-hydroxy benzo(a)pyrene. Epoxide hydratase activity was measured by die fluorimetric technique according to the method of Dansette et al.7 by using styrene epoxide as substrate. Glutathione-S-transferase activity was determined tty the pro cedure described by Habig et al.,15 using l-chloro-2, 4-dinitrobenzene (CDNB) as substrate. Chemical Estimation Microsomal cytochrome P-450 was quantitated from carbon monoxide plus dithionite-reduced difference spectra as described by Omura and Sato.38 An extinction coefficient of 91,000 cm-1, M_1 was used for absorbance change be tween 450 and 490 nm. Glutathione content was measured in rat lung cytosolic fraction, according to die method of EHmann.11 Ascorbic acid content was estimated in lung cytosol accord ing to the procedure of Schaffert and Kingsley.43 Enzymatic and non-enzymatic lipid peroxidation was deter mined by the procedure of Ottolenghi39 as modified by Hunter et al.17 Protein was estimated by the method of Lowry et al.,29 with crystalline bovine serum albumin as standard. Statistics The values presented mean standard error of six animals, statistical significance was determined by Students* `t' test. RESULTS Effect of Chrysotile on Lung Weight There was significant increase in lung weight of experimen tal animals at 90 and 290 days after treatment (Figure 1). At 290 days of exposure, the increase was 95 % in fresh lung weight over the untreated group. Poster Session U At 90 and 290 days, the decrease in the activity was 21 % and 39% which is statistically quite significant. Effect of Chrysotile on Water Soluble Antioxidants As recorded in Figure 6, at 90 and 290 days after exposure, a significant decrease in the content of ascorbic acid was Lung Weight Cytochrome P-450 content days Figure 1. Fresh lung weight ofcontrol and chrysotile treated rats. The values are expressed as mean SEM of six animals. *p >0.001. Effect of Chrysotile on Rat Lung Microsomal and Cytosolic Fractions The biochemical changes related to drug metabolizing en zyme system induced by chrysotile at different time inter vals are given in Figures 2, 3,4,5. As Figure 2 shows there was decrease in lung microsomal cytochrome P-450 content from 1 to 16 days but at 90 and 290 days there was signifi cant increase in the content of P-450. At 290 days, the in crease was recorded (45%). Same pattern was obtained with the activity of benzo(a)pyrene hydroxylase as shown in Figure 3. At 90 and 290 days, 49% and 48% increase were obtained over their controls, respectively. In case of epox ide hydratase as shown in Figure 4, till 16 days of exposure there was a decrease in the activity but at 90 and 290 days after treatment 90% and 96% increase were recorded respec tively, over their controls. However, in cytosolic fraction there was a continuous decrease in die activity ofglutathioneS-transferase in experimental animals as shown in Figure 5. Figure 2. Lung cytochrome P-450 content of control and chrysotile treated rats. The values are expressed as mean SEM of six animals. *p<0.02; bp<0.01; ^<0.05; dp<0.001. observed in the lung cytosolic fraction of the experimental animals. In case of reduced glutathione content as given in Figure 7, there was continuous decrease in experimental animals at all the stages of exposure but at 90 and 290 days after treatment the decrease is very significant. Effect of Chrysotile on Lipid Peroxidation As shown in Figure 8, at all the stages of exposure there was significant induction in microsomal lipid peroxidation in asbestotic animals. DISCUSSION It is revealed from the study that asbestos fibre alters mixed function oxidase system of rat lung at all the stages of the exposure. At the initial stages of exposure, the content of cytochrome P-450 and the activity ofbenzo(a)pyrene hydrox- 1331 Poster Session n Benzo(o) Pyrene Hydroxylase Activity Epoxide Hyretose Activity days Figure 3. Benzo(a)pyrene hydroxylase activity in lung microsomes isolated from control and chrysotile treated rats. The values are expressed as mean SEM of six animals. *p<0.05; ^<0.02; *{><0.001; ^<0.01. ylase and epoxide hydratase were reduced as compared to their respective controls, but at later stages a reversed pat tern with a progressive increase was observed. The decrease in the content of cytochrome P-450 at initial stages of ex posure may be due to destabilization of heme proteins.10'34'52 Partial inactivation of microsomal mixed function oxidase system in vivo and in vitro at the initial stages of the disease have been reported.25'40'24'36 The inhibition in the activity ofphase I reaction suggests that at the early stages chrysotile prolongs the tissue retention of carcinogens. However, the content of cytochrome P-450 and die activity of benzo(a)pyrene hydroxylase and epoxide hydratase increased at 90 days and thereafter, thus indicating that chrysotile fibre participates actively in the activation of phase I reaction at the advanced stages of the disease when fibrosis developed. At this stage, our results are in agreement with Naseem et al.35 and Dzugaj et al.,19 who have reported high activity of benzo(a)pyrene hydroxylase in lymphocytes isolated from asbestos workers and liver of asbestos exposed mice. The increase in the activity of aryl hydrocarbon hydroxylase system is very important because they play the major role in die regulation of the microsomal biotransformation of Figure 4. Epoxide hydratase activity in lung microsomes, isolated from control and chrysotile treated rats. The isolated from control and chrysotile treated rats. The values are expressed as mean SEM of six animals. *p<0.02; ^<0.05; cp-NS; ^><0.01; <fc<0.001. polycyclic aromatic hydrocarbons (PAHs), the major car cinogen of cigarette smoke.13 It is associated with die ac tivation of PAHs in chemical carcinogenesis and also die epoxide hydratase catalyzed formation of dihydrodiols.37 Therefore, the higher activities of microsomal benzo(a)pyrene hydroxylase and epoxide hydratase on prolonged period of asbestos exposure may produce more reactive metabolites from die known carcinogens present in die cigarette smoke in die target tissue thereby increasing the possibility of higher DNA adduct formation.40 A linear decrease in the activity of glutathione-S-transferase in the case of chrysotile treated animals was observed. The max imum inhibition for the activity was observed at 290 days of treatment, registering a 39% inhibition. This is in agree ment with the findings of Brown et al.3 Glutathione-Stransferase is involved in the detoxification of metabolically modified carcinogens by conjugation with reduced glutathione. The decrease in activity of this enzyme as observed in this study may, in turn, result in the accumula tion ofunscavenged reactive metabolites which may find ac cess to other sites and exert deleterious effects like the ad duct formation with DNA. The hydrolytic enzymes which released from lysosomes have been reported in asbestotic 1332 6Mothon-S-Tronsfrot octivrty Poster Session II Ascorbic ocid content n Control QU Experimental 27 c o & u 22 o (OA >> c_> 17 H Ii * 1 Figure 5. Glutathione-S-transferase activity in lung cytosol, fractionated from control and chrysotile treated rats. The values are expressed as mean SEM of six animals. ^<0.05; *^<0.02; *^<0.001. animals at the advanced stages of the disease50 may further negate the clearance by the hydrolysis of preformed con jugates releasing reactive metabolites in the cells. A higher rate of both enzymatic and non-enzymatic lipid peroxidation have been recorded in pulmonary microsomal fractions isolated from chrysotile treated rats after 1,4, 8, 16, 90 and 290 days of exposure. Recently, other in vestigators have also reported similar findings.12'21 The lipid peroxides generated due to the peroxidative damage of polyunsaturated fatty acids of die biological membrane have tremendous toxic potential in the biological systems.9'14'33*53 These include alterations in membrane fluidity, initiation of free radical chain reactions, and effects on intermediatory metabolism. The lipid peroxide also stimulates the metabolism of benzo(a)pyrene.14 There are several evidences to prove that hydroxyl and superoxide radicals are involved in asbestos induced lipid peroxidation5'16*20'32*51 Therefore, the enhanced lipid peroxidation of the lung microsomes in asbestotic rat may contribute to the delayed toxic and carcinogenic effect of these mineral fibres. A remarkable decrease in the contents of water soluble an tioxidants like ascorbic acid and reduced glutathione have been recorded in chrysotile treated rats after 90 and 290 days of exposure. However, insignificant depletion in the levels 12 4 8 IS 90 290 days Figure 6. Ascorbic acid content in the lung of control and chrysotile treated rats. The values are expressed as mean SEM of six animals. *p<0.001; **p<0.001. of these antioxidants was observed at the initial stages of ex posure. Several antioxidants are known to inhibit tumors in duced by a variety of carcinogens, including PAHs.2'4'6*23'45-48 The low contents of antioxidants in the lung after chrysotile inhalation may hamper the defense of the tissue against other environmental and occupational con taminants. It may be concluded from the above study that the events like quick generation of active carcinogens, their poor elimination from the tissue, hydrolysis of preformed conjugates, generation of free radicals and low antioxidants level in the lung following exposure to asbestos dust may be initiating, favouring and stimulating the process of bron chogenic carcinoma. REFERENCES 1. Berry, G., Newhouse, M.L., Turok, M.: Combined effect ofasbestos exposure and smoking on mortality from lung cancer in factory workers. Lancet, 2:476-479 (1972). 2. Benedict, W.F., Wheatley, W.L., Jones, P.A.: Inhibition of chemically induced morphological transformation and reversion ofthe transformed phenotype by ascorbic acid in C3H/10T1/2 cells. Cancer Res. 40:2796-2802 (1980). 3. Brown, R.C., Fleming, G.T.A., Knight, A.J.C.: Asbestos effects on the in vitro uptake and detoxification of aromatic compounds. Environ. Hlth. Perspect. 51:315-318 (1983). 4. Carcinogens: Identification and mechanism of action, 1st ed., pp. 299-316, A.C. Criffin and C.R. Shane eds. Raven Press, New York (1979). 1333 Poster Session B OSH Content Control 04 QD Experimental Upid Peroxidation ("I Control > o E o E Figure 7. GSH content in the lung ofcontrol and chrysotile treated rats. The values are expressed as mean SEM of six animals. *p<0.01; **p<0.001. 5. Case, B.W., Clip, M.P., Padilla, M., Kkinennan, J.: Asbestos effects on superoxide production: An in vitro study of hamster alveolar macrophages. Environ. Res. 39:299*305 (1986). 6. Collacchio, T.A., Memoli, V.A., Hanover, N.H.: Cfcemoprevention of colorectal neoplasm: ascorbic acid and fi-carotene. Arch. Stirg. 121:1421-1427(1986). 7. Dansette, P.M., DuBois, G.C., Jenna, D.M.: Continuous fluorometric assay ofepoxide bydratase activity. Anal. Biocbem. 97:340-345 (1979). 8. Dehnen, W., Tomings, R., Roos, J.: A modified method for the assay of benzo(a)pyrene hydroxylase. Anal. Biocbem. 53:373-380 (1973). 9. Dix,TA.,Mamett,J.L.: Metabolism ofpolynuclear aromatic hydrocar bon derivatives to ultimate carcinogens during lipid peroxidation. Science, 221:77-79 (1983). 10. Dixit, R., Mukhtar, H., Bickers, D.R.: Destruction of microsomal cytochrome P-450 by reactive oxygen species generated during pbotosensitization of bemropoiphyria derivative. Pbotocbem. Pbotobiol. 37:173-176 (1983). 11. EUmann, G.L.: Tissue sulfhydiyl groups. Arcb. Biocbem. Biopbys. 82:70-77 (1959). 12. Fboteeaus, M., Mansay, D., Jaquen, M., Fezerat, H.: The stimulatory effects ofasbestos on NADPH-dependent lipid peroxidation in rat liver microsomes. Biocbem. J. 241:561-565 (1987). 13- Gelboin, H.V.: Benzo(a)pyrene metabolism. Activation and car cinogenesis: Role and reguktionofmixed function oxidases and related enzymes. Physiol. Rev. 60:1107-1166 (1980). 14. Gower, J.D., Willis, E.D.: The oxidation ofbenzo(a)pyrene mediated by lipid peroxidation irradiated synthetic dust Radiat. Biol. 49:471-478 (1986). 15. Habig, W.H., Pabst, MJ.. Jakoby, WJ.: Glutathione-Stransferase: The first enzymatic step m the mercapturic acid formation. J. Biol. Chem. 249:7130-7139 (1974). 16.Hansen, K., Mossman, B.T.: Generation of superoxide (Oj-) from alvtolar macrophages exposed to asbestiform and nonfibrous particles. Cancer Res. 47:1681-1687 (1987). 1334 Figure 8. Lipid peroxidation in lung microsomes isolated from control and chrysotile treated rats. The values are expressed as mean SEM of six animals. *p<0.001; **p>0.001. 17. Hunter, F.E., Gebicld, J.M., Hoffstein, P.E., Einstein. J., Scott, A.: Swelling and lysis of rat liver mitochondria induced by ferrous ions. /. Biol. Chem. 238:828-835 (1963). 18. Inhaled Particles, Isted. pp. 553-567. A Critchlow, ed. Ifergamoo Press, London (1982). 19. io vitro Effects ofmineral dusts, 1st ed. pp. 467-474, E.G. Beck and J. Bignon eds. Springer-Verlag, New York, Tokyo (1985). 20. In vitro Effects ofmineral dust, 1st ed. pp. 483-488, E.G. Beck A J. Bignon, eds. Springer-Verlag, New York, Tokyo (1985). 21. Jazte, J., Leo, I., Wishiewsnaknypt, J.M.: Enhanced lipid peroxida tion and lysosomal enzyme activity in the tnngs of rat with prolonged pulmonary deposition of crociddite asbestos. Brit. J. Indust. Med. 44:180-186 (1987). 22. Johanneses, K., Depierre, J.W., Beigstrand, A., Dallner, G., Eiester, L.: Preparation and characterization of total rough and smooth microsomes from the lung of control and methylcholanthrene-treated rats. Biochim. Biopbys. Act 496:115-135 (1977). 23. KallistraKB, G., Fasske, E.: Inhibition ofbenzo(a)pyrene carcinogenesis in rat with vitamin C. J. Cancer Res. din. Oncol. 97:91-96 (1980). 24. Kandaswami, C., O'Brien, PJ.: The effect ofchrysotile asbestos and silica on the microsomal metabolism ofbenzo(a)pyiene. Environ. Hhb. Ptrspcct. 51:311-314 (1983). 25. Kandaswami, C., Rahimtula, M. and O'Brien, JJ.: Effects ofasbestos fibres on arylhydrocaibon hydroxylase and ammopyrene-N-demetfaylase activities of rat liver microsomes. Toxicology 38:119-132 (1986). 26. Kimizuka, G., Ohwada, H., Hayashi, Y.: Co-carcinogenic effect of asbestos and benzo(a)pyrene in the lung of hamster. Acta Pathol. 37:465-474 (1987). 27. Lakowicz, J.R., England, F., Hidmark, A.: Particle-enhanced mem brane uptake of a polynuclear aromatic hydrocarbon: A possible role in co-carcinogenesis. J. Nat/. Cancer Inst. 61:1155-1139 (1978). 28. Lakowicz, J.R., Bevan, D.R.: Effects of asbestos, iron oxide, silica and caibon Mack on tbe microsomal availability of benzo(a)pyreoe. Biochemistry 18:5170-5176 (1979). 29. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J.: Col orimetric determination of protein with Folin phenol reagent. /. Biol. Cbem. 198:265-275 (1951). 30. Meunnan, L.O., Livilnoto, R., Hakama, M.: Combined effects of asbestos exposure and tobacco smoking on Finnish antbophyllite miners and millers. Ann. N.Y. Acad Sci. 330:419496 (1979). 31. Mossman, B.T., light, W., Wei, E.: Asbestos: Mechanism oftoxici ty and carcinogenicity in tbe respiratory tract. Ann. Rev. Pharmacol. Toxicol. 23:595-615 (1983). 32. Mossman, B.T.,Landesman,J.M.: Importance ofoxygen free radicals in asbestos jnrfnowrf injury to airway epithelial cells. Chest, 835:509-515 (1983). 33. Mukai, F.H., Goldstein, B.: Mutagenicity ofmalonaldehyde, a decom position products of peroxidized polyunsaturated fatty acids. Science 191:868-869 (1976). 34. Murakami, K., Mason, H.S.: An electron spin resonance study of microsomal Fe x. J. Biol. Cbem. 242:1102-1110 (1967). 35. Naseem, S.M., Tishler, P.V., Anderson, H.A., Selikoff, I.J.: Arylhydrocarbon hydroxylase in asbestos workers. Am. Rev. Respir. Dis. 118:693-700 (1978). 36. O'Brien, PJ., Rahimtula, M., Kandaswami, C.: Asbestos enhanced metabolic activation of carcinogenic polycyclic aromatic hydrocarbons and arylamines. Proc. Am. Cancer Res. 25:11-17 (1984). 37. Oesch, F.: Mammalian epoxide hydrases: Inducible enzymes, catalyz ing tbe inactivation of carcinogenic and cytotoxic metabolites derived from aromatic and olefinic compounds. Xeoobiotiea 3:305-340 (1972). 38. Omura, T., Sato, R.: Tbe carbon-monoxide-binding pigment of liver microsomes. J. Biol. Cbem. 239:2379-2385 (1964). 39. Ottolenghi, A.: Interacdon of ascorbic acid and mitochondrial lipids. Arch. Biocbem. Biopbys. 79:355-360 (1959). 40. PdyiHJClear Aromatic Hydrocarbons: Physical and Biological Chemistry. 1st ed. pp. 384403, M. Cooke, AJ.Dennis and G.L. Fisher, eds. Battella Press, Ohio (1982). 41. Pylev, L.N., Roe, F.J.C., Warwick, G.P.: Elimination ofradioactivi ty after intratracheal instillation of tritiated 3,4-benzo(a)pyrene in hamsters. Brit. J. Cancer 23:103-115 (1969). 42. Saracchi, R.: Asbestos and lung cancer: An analysis of the epidemiological evidence on the asbestos-smoking interaction. Int. J. Cancer 20:323-331 (1977). 43. Schaffert, R.R., Kingsley, G.R.: A rapid, simple method for tbe deter mination of reduced, dehydro- and total ascorbic acid in biological material. /. Biol. Cbem. 212:59-68 (1955). Poster Session B 44. Shabad, L.M., Pylev, L.N., Krivasheeva, L.V., Kulagina, T.F., Nemenki, B.A.: Experimental studies on asbestos carcinogenicity. /. Nad. Cancer Inst. 52:1175-1182 (1974). 45. Shah, G.M., Bhattacharya, R.K.: In vivo effect of L-ascorbic acid on benzo(a)pyiene metabolite--DNA adduct formation in rat liver. J. Biosci. 4:263-270 (1982). 46. Shatos, M.A., Doherty, J.M., Marsh, J.P., Mossman, B.T.: Preven tion of asbestos-induced cell death in rat lung fibroblasts and alveolar macmphages by scavengers of active oxygen species. Environ. Res. 44:103-116(1987). 47. Slaga, I.J., Bracken, W.M.: Tbe effects of antioxidants on skin tumor initiation and aiylhydrocarbon hydroxylase. CancerRes. 37:1631-1636 (1977). 48. Speicr, J.L., Lam, L.K., Wattenberg, L.W.: Effects ofadministration to mice ofbutylated hydroxyanisde by oral intubation on benzo(a)pyrene induced pulmonary adenoma formation and metabolism of benzo(a)pyrene. /. Nad. Cancer Inst. 60:605-609 (1978). 49. Topping, D.C., Nettesheim, P., Martini, D.H.: Toxic and tumorigenic effects of asbestos on tracheal mucosa. J. Environ. Pathol. Toxicol. 3:261-267 (1980). 50. Viswanathan, P.N., Rahman, Q., Beg, M.U., Zaidi, S.H.: Pulmonary lysosomal enzymes in experimental asbestosis in guinea pigs. Environ. Physiol. Biochem. 3:120-126 (1973). 51. Weitzman, S.A., Gracqja, P.: Asbestos catalyzes hydroxyl and superox ide radical generation from hydrogen peroxide. Arch. Biodtem. Biophys. 228:373-376 (1984). 52. Yamano, J., Ichikawa, Y.: Cytochrome P450-JV. Stability of cytochrome P450 and conversion to cytochrome P420: Isolation and properties ofcytochrome P420. Pharmacol. Tber. A2:673-692 (1978). 53. Yonei, S., Furei, H.: Lethal and mutagenic effects of malonaldehyde, a decomposition product ofperoxidized lipids on E. cdi with different DNA repair capacities. Mutat. Res. 88:23-32 (1981). 54. Zaidi, S.H.: Experimental Pneumoconiosis, 1st ed.,pp. 94-106. Johns Hopkin's Press, Baltimore (1969). ACKNOWLEDGEMENTS: The authors are thankful to Dr. P.K. Ray, Director, Industrial Toxicology Research Centre, Lucknow, India, for his interest in the work. One of die authors (S.G. Khan) is grateful to State Department of Environment, Lucknow, U.P., for the award of Junior Research Fellowship. 1335 Poster Session O THE ASSOCIATION OF SMALL IRREGULAR OPACITIES ON CHEST RADIOGRAPH WITH AGING IN A NONSMOKING POPULATION WITHOUT OCCUPATIONAL DUST EXPOSURE ANDREW J. GHIO, M.D. Attilio EX Renzetti, Jr., M.Dl Robert O. Crapo, M.D. Pulmonary Division, Department of Internal Medicine, LDS Hospital, and Division of Respiratory, Critical Care, and Occupational (Pulmonary) Medicine Department of Internal Medicine, University of Utah, Salt Lake City, UT, USA ABSTRACT Small opacities on chest radiograph have been found to increase with age in several studies which have been confounded by dust exposure and/or cigarette smoking. To analyze the association of small opacities with age, we used the ILO 1980 Classification to categorize 159 radiographs of asymptomatic, lifetime nonsmokers without occupational exposure to dusts. The study population included 84 males and 75 females. Age ranged from 15 to 85 years with a mean of 51.2 years and a standard deviation of 19.9 years. Chest radiographs with ages concealed were classified independently by two B readers. Reader 1 found 133 (83.6%) to have profusion category 0/0 and 26 (16.4%) to have category 0/1. Reader 2 found 125 (78.6%) to have category 0/0 and 34 (21.4%) to have category 0/1. No subject had a profusion category greater than 0/1. Significant point biserial correlation coefficients (r,*) were found between profusion category and age (^=.1659 and .1611 for readers 1 and 2 respectively; both p<.05). Analysis by gender demonstrated an association of small opacities with age only in females (^=.2761 and .3091 for readers 1 and 2 respec tively; both p^ .01). Changes of the breasts which take place with aging may account for this association. The International Labor Office (ILO) International Classifica tion of Radiographs of Pneumoconioses is used for epidemiologic research and surveillance of workers in dus ty occupations.1 It may also contribute to the evaluation of a worker for compensation. A variety of normal and abnor mal structures produce radiographic patterns similar to those ofthe pneumoconioses complicating interpretation of the ILO 1980 Classification.2,3 Studies have described an increase in small opacities on chest radiograph associated with age.4*9 These investigations have been confounded by dust exposure and/or cigarette smoking which also increase small opacities on chest radiographs. ,0'15 To test the hypothesis that small opacities increase with age independent ofdust exposure and cigarette smoking, we used tiie ILO 1980 Classification to categorize chest radiographs of asymptomatic, lifetime nonsmokers without occupational exposure to dusts. METHOD Subjects were volunteers, predominantly from the Church of Jesus Christ Latter-Day Saints (Mormons). A modified version of the Medical Research Council questionnaire for respiratory symptoms was administered to each in dividual.16 A detailed occupational history was also ob tained. Height (in meters) and weight (in kilograms) were measured with the subject wearing light outdoor clothing without shoes. A pulmonary physician examined all subjects. A 14" x 17" posteroanterior (PA) chest radiograph was 1336 taken at six feet on full inspiration with the patient in the standing position. PA radiographs with ages concealed were classified independently by the National Institute for Occupa tional Safety and Heatlh (NIOSH) certified B readers (readers 1 and 2). ILO 1980 Classification standard films were used. Results were reported with OMB Form No. 68-5 1322 pro vided by NIOSH for the complete classification. Subjects were included in the study population if they met tiie following criteria: 1) a lifetime nonsmoker (total smok ing of less than 0.5 pack-year and no smoking within six months of the study); 2) no symptoms of chest wall, lung, or heart disease; 3) no history of work in a mine, quarry, foundry, or pottery; 4) no occupational exposure to asbestos, irritating gases, or chemical fumes; 5) a normal physical ex amination of the chest wall, lungs, and heart; and 6) a PA radiograph of technical quality acceptable to both readers. The Chi-square goodness of fit test was used to examine the relationship of profusion category with gender.17 To analyze associations of small opacities with age and an obesity in dex (weight/height2), the point biserial correlation coeffi cient (rpb) was applied.18 The r^ allows correlation of a continuous variable (age and obesity index) with a categorical variable which has two values (all chest radiographs were classified into two profusion categories). RESULTS Eight volunteers were excluded from the study as a result of work in mines or exposure to asbestos. Technical quality prevented classification of six radiographs. The study popula tion included the remaining 159 subjects. There were 84 males and 75 females. Ages of males and females were com parable and were uniformly distributed from 15 to 85 years (Table I). Males were, as expected, taller and heavier. Table I Ages and Anthropometric Measures n Age in years Range Mean Stand. Dev. Males 84 15-85 52.1 19.7 Height in meters Range Mean Stand. Dev. 1.490-1.940 1.733 0.073 Weight in kilograms Range 59.6-110.9 Mean 78.9 Stand. Dev. 11.4 Females 75 17-84 50.2 20.0 1.460-1.780 1.611 0.068 43.8-104.7 67.7 12.2 Reader 1 categorized 133 (83.6%) radiographs as profusion category 0/0 and 26 (16.4%) as category 0/1. Reader 2 categorized 125 (78.6%) radiographs as category 0/0 and 34 (21.4%) as category 0/1 (Table II). No chest radiograph was found to have a profusion category greater than 0/1. Agreement between the two readers was 80.5%. Poster Session II In subjects with radiographs categorized 0/1, small opacities were found only in the lower zones. The predominant shapes and sizes were s and t varieties. There were no large opacities. Males had a higher prevalence of radiographs categorized as 0/1 but this difference between genders reached statistical significance with reader 2 only (Chi square = 4.08, p=.04). Correlation of profusion category with age, with males and females included, provided values of 0.1659 and 0.1611 (readers 1 and 2 respectively). Both were statistically signifi cant (Table HI). This association was then analyzed separately for each gender. Males were found to have no statistically significant correlation of profusion category with age while females had significant r^ values of 0.2761 and 0.3091 (Table HI). There was an association of profusion category with the obesity index in females with reader 1 only (Table ID). However when age and obesity index were simultaneously regressed against profusion category, the association of the obesity index with the profusion category was not found to be significant. DISCUSSION No subject in our group of asymptomatic, lifetime nonsmokers without occupational exposure to dusts had a profusion category greater than 0/1 and only 16 to 21 per cent were classified as category 0/1. Small opacities were predominandy s and t in shape and size and were located in the lower zones. Males were found to have category 0/1 radiographs more ffequendy than females. We found small opacities on chest radiograph to increase with age. However, when analyzed by gender, this association was statistically significant for females only. A possible explanation of the association of profusion category and age in females is that the small opacities result Table U ILO 1980 Classification of Profusion Category Reader 1 Male Female 0/0 67 66 0/1 17 9 Reader 2 Male Female 59 66 25 9 1337 Poster Session II Table in Correlation of Profusion Category with Age and Obesity Index AGE All Subjects Males Females Reader 1 Ipb* P Value 0.1650 0.0732 0.2761 .03 .51 .01 Reader 2 rpb* P Value 0.1611 0.0562 0.3091 .04 .61 .00 OBESITY INDEX (WEIGHT/HEIGHT^ All Subjects 0.0225 .77 0.1478 .06 Males 0.0904 .41 0.0978 .38 FA emales V0..A1334 24 0.2246 ,.V0^4 *rpt, is the point biserial correlation coefficient between profusion category and either age or obesity index from changes in breast tissue. The lobules ofglandular paren chyma and its stroma are hormonally dependent. With age, involution ofthese tissues occurs with replacement by adipose tissue.19'20 Fat absorbs relatively few X-rays and is therefore less radiopaque than the other tissues ofthe breast. As a result ofthis differential absorption, fat would provide sharp contrast on a radiograph to other tissues including per sistent strands of fibrous connective tissue, veins, and calcified arteries. These structures may be seen as small opacities in older females and would explain the location of the small opacities and their association with aging in females. Age in females explained less than 10% (coefficient ofdeter mination) ofthe variance ofprofusion category in this popula tion without occupational exposure to dusts. Ten percent is an underestimate since the maximal coefficient ofdetermina tion obtained using the point biserial correlation coefficient is approximately 0.80.18 Li addition, as the proportion of the study population in each ofthe two categories varies from 0.50, both rpb and the coefficient of determination will be underestimated.18 In our study, the inequality of subjects in categories 0/0 and 0/1 leads to an error in our determina tion of tiie true variance ofprofusion category explained by age. Although the exact value cannot be determined, it can be concluded that the majority of the variance of profusion category with both genders is unexplained. Soft tissues overlying the chest wall are thought to account for small opacities8 but could not be demonstrated to explain any variance ofprofusion category in our group. Radiographs of females should be categorized 0/1 more frequently than males as a result of overlying breasts if soft tissues explained a significant portion of the variance of profusion category. There was an effect of gender in our study but both readers categorized more radiographs of males as 0/1. Our study also showed that an obesity index (weight/height2) had no association with small opacities when a multiple regression was done with age as another independent variable. Subject 1338 characteristics not investigated in our study, technical quality of the radiograph, or error in classification may explain the majority of variance of profusion category in nonsmoking, unexposed populations. Two other investigations have categorized unexposed popula tions using the ILO Classification. Castellan et al. studied 1422 blue collar workers without exposure to known occupa tional respiratory hazards.8 Only ten workers had profusion category 0/1 and three had categories 1/0 and 1/1. Small opacities were irregular in shape. A statistically significant difference in ages of workers with profusion category ^ 0/1 was detected when compared to those with category 0/0. Almost all workers with small opacities were smokers. Ep stein et al. found 35 of 200 radiographs of hospitalized pa tients had a profusion category of 0/1 and 22 had category I/O.21 Small opacities were predominantly irregular in shape and located in either the lower zones or all zones of the lungs. The higher prevalence (11%) of radiographs with categories ^0/1 may have been the result of classifying a hospitalized population. An association of profusion category with age was not described. Some studies in exposed populations have also shown pro fusion category to increase with age.5*7'9 These findings may have resulted from incomplete accounting for foe ef fect ofdust exposure, decreased clearance ofthe dust by foe respiratory tract with aging, confounding factors (e.g., other occupational exposures, environmental exposures, cigarette smoking), or an age associated increase in small opacities independent of dust exposure and confounding factors. We conclude there is an association of small opacities on chest radiograph with age independent ofdust exposure and cigarette smoking in females only. Changes in breast tissue occurring with age may account for this finding. REFERENCES 1. International Labor Office. Guidelines for the Use ofILO International Classification ofRadiographs of Pneumoconioses. Revised editioa 1980. Geneva, Switzerland: EX), 1980 (ILO Occupational Safety and Health Series No. 22. Revised 80). 2. Van Ordstand, H.S.: Pneumoconioses and their masqueraders. /. Occup. Med. 1977; 19:747-753. 3. Pendergrass, E.P., Lainhart, W.S., Bristol, LJ., Felson, B., Jacob son, G.: Roentgenological patterns in hmg changes that simulate those found in coal workers' pneumoconiosis. Annals New York Academy Sciences 1972; 200:494-502. 4. Amandus, H.E., Lapp, N.L., Jacobson, G., Reger, R.B.: Significance of irregular small opacities in radiographs in coalminers in the USA. Brit. J. Indus. Med. 1976; 33:13-17. 5. Cockcroft, A., Lyons, J.P., Andersson, N., Saunders, MJ.: Prevalence and relation to underground exposure of radiological irregular opacities in South Wales coal workers with pneumoconiosis. Brit. J. Indus. Medicine 1983; 40:169-172. 6. Dick, J.A., Jacobsen, M., Gauld, S., Pern, P.O.:. The significance of irregular opacities in the chest radiographs of British coal miners. In: Proceedings of VI International Pneumoconiosis Conference. Bochum 1983. Geneva International Labour Office, 1984; 283-299. 7. Castellan, R.M., Sanderson, W.T., Petersen, M.R.: Prevalence of radiographic appearance ofpneumoconiosis in an unexposed blue col lar population. Am Rev Respir Dis 1985; 131:684-686. 8. Amandus, H.E., Ahhouse, R., Morgan, W.K.C., Sargent, E.N., Jones, R.: The morbidity and mortality of vermiculite miners and millers ex posed to remolite-actinolite: Part m. Radiographic findings. Am. J. Indus. Med. 1987; 11:27-37. 9. Collins, H.P.R., Dick, J.A., Bennett, J.G., Pern, P.O., Rickards, M.A., Thomas, DJ., Washington, J.S., Jacobsen, M.: Irregularly shaped email Poster Session H shadows on chest radiographs, dust exposure, and hmg function in coalworkers' pneumoconiosis. Brit. J. Indus. Med. 1988; 45:41-55. 10. Weiss, W.: Cigarette smoking and diffuse pulmonary fibrosis. A preliminary report. Arch. Environ. Health 1967; 14:564-568. 11. Weiss, W.: Cigarette smoking and diffuse pulmonary fibrosis. Am. Rev. Respir. Dis. 1969; 99:67-72. 12.Weiss, W.: Cigarette smoking, asbestos, and pulmonary fibrosis. Am. Rev. Respir. Dis. 1971; 104:223-227. 13. Carilli, A.D., Kotzen, L.M., Fischer, M.J.: The chest roentgenogram in smoking females. Am. Rev. Respir. Dis. 1973; 107:133-136. 14. TGheriault, G.P., Peters, J.M., Johnson, W.M.: Pulmonary function and roentgenographic changes in granite dust exposure, Arch. Environ. Health 1974; 28:23-27. 15. Weiss, W.: Pleuropulmonary disease among asbestos workers in rela tion to smoking and type ofexposure. S. Occup. Med. 1978; 20:341-345. 16. Medical Research Council 1966. Medical Research Council's enmmrtt on research into chronic bronchitis: Instructions for the use ofthe ques tionnaire on respiratory symptoms. Medical Research Council, London. 17. Zar, J.H.: Biostatistical Analysis. Prentice-Hall, Inc., 1974. 18. McNemar, Q.: Psychologic Statistics. Fourth edition. New York: Wiley, 1969. 19. Witten, D.M.: The Breast. First edition. Chicago: Year Book Medical Publishers Inc., 1969. 20. Gersbon-Coben, J.: Atlas of Mammography. Fust edition. New York: Springer-Verlag, 1970. 21. Epstein, D.M., Miller, W.T., Bresnitz, E.A., Levine, M.S., Gefter, W.B.: Application of ILO Classification to a population without in dustrial exposure: Findings to be differentiated from pneumoconiosis. Am. /. Radiol. 1984; 142:53-58. 1339 Poster Session n PULMONARY EFFECTS OF ACUTE EXPOSURE TO SULFUR TETRAFLUORIDE DURING ELECTRICAL CABLE REPAIR WORK ALLEN KRAUT, M.U Ruth Lilis, M.D. Division of Environmental and Occupational Medicine Mount Sinai Medical Center, New York, NY, USA ABSTRACT Six electrical workers were accidentally exposed to sulfur tetrafluoride (SF4) while repairing an electrical cable in an underground confined space. Repairs began 4 days after a burnout at a nearby substation. Symp toms noted approximately 1 hour after beginning work were shortness ofbreath, chest tightness, productive cough, nose and eye irritation, and headache. Some workers also experienced fatigue, nausea, and vomiting. Partial resolution of symptoms occurred when exposure was interrupted while attempts to identify the cause ofthe problem were made. Although exposure ended after several hours, 4 workers remained symptomatic for over one week. Chest radiographic abnormalities included, several discrete areas of transitory platelike atelectasis in 1 worker, and soft hazy infiltrates in another. Pulmonary function changes included reversible decrements in FVC and FEV). Sulfur hexafluoride (SF6), an inert gas, used in circuit breakers as an electrical arc-interrupting medium, decomposes to SF4 and other compounds when subjected to intense beat. SF4, an irritant gas, with toxic effects similar to phosgene, was eventually identified by mass spectrometry of worksite air samples, and is die likely cause of the illness developed by these workers. Effects of SF4 exposure on humans have not been reported in die medical literature, although available information indicates that it is a highly irritant compound. Occupational health personnel should be aware that exposure to SF4 is an important health hazard for workers repairing damaged electrical systems containing SF6. INTRODUCTION Unpredicted exposures to industrial chemicals place workers at serious ride, as they are both unprepared for die event and may not know the compound's toxic effects. We wish to report the consequences ofan unexpected, unpredicted, and unrecognized exposure to die irritant gas, sulfur tetrafluoride (SF4). The exposure occurred after initiation of repairs following a burnout (small explosion) in an electrical substa tion. During die burnout circuit breakers using sulfur hex afluoride (SF6) as an insulating gas were damaged. When SF6 is subjected to intense heat it decomposes to SF4 and other compounds.* Although SF4 has been reported to have toxicity similar to that of phosgene,2 we could find no published documentation of illnesses in humans caused by this compound. CIRCUMSTANCES OF EXPOSURE AND ACUTE SYMPTOMS. On January 12th 1988 approximately 15,000 gallons of palybutene insulating oil was lost from an electrical transmis sion cable. Concurrently, a burnout occurred in circuit breakers at a substation several miles further down die line. Four days later a team of six gas operators (workers #1-6) began repair work approximately 100 yards from the substa tion by cutting the surrounding pipe to gain access to die 1340 enclosed electrical cable. The worksite was an underground space, 10' X4' X 8', with two three foot diameter openings. Four members of the crew (#1-4) worked underground, while the safety officer, (#5), remained on the surface. The foreman, (#6), worked above or below ground as needed. All workers were previously healthy, except for (#5) who had a history of emphysema. Prior to entering the worksite, routine measurements for natural gas and oxygen concentrations were found to be satisfactory. At approximately 9:00 AM die team began us ing compressed air powered smith cutters to open the pipe. About one hour later, five underground workers began ex periencing burning eyes, tearing, dry and burning throat, and chest tightness. An odor similar to a "burning car battery'* was noticed. The crew stopped working and went above ground; symptoms decreased fifteen minutes later. A fan was obtained to improve air circulation at the worksite. The previously mentioned symptoms, however, recurred shortly after work was resumed. In addition, some workers started experiencing headache, fatigue and cough produc tive of clear sputum. Concern was raised that die air com pressor was the cause ofthe problems. Work was again halted and a different compressor ordered. Symptoms subsided when the men stopped working underground and went into the fresh air. Nevertheless, die same symptoms recurred one hour after work was resumed with die new compressor. Due to the persistence and worsening of symptoms a worksite investigation was performed by the company chemist at ap proximately 3:00 PM. No abnormalities were detected on routine air monitoring and samples from the partially opened pipe were taken for analysis. Although no problems were identified, a second fan was brought to improve the air cir culation. Two hours later workers again reported chest tightness and/or shortness of breath. Two complained of headache, fatigue and nose bleeds; two felt nauseous and one worker vomited. Work was again halted and the entire crew waited above ground for die chemist's final report. At 10:00 PM workers were sent home as die report had not yet arrived. At approximately 1:00 AM all six workers were notified by telephone that SF4, a potentially hazardous material, was identified in the air samples taken from the partially opened pipe at the worksite. In addition they were instructed to im mediately go to die nearest hospital emergency room. Five ofthe workers went to one hospital and die sixth to another. Oxygen was administered by mask in the emergency room of the first hospital. Chest radiographs were not taken until approximately 11:00 AM, approximately 26 hours after the onset of exposure. Five of the workers were discharged a few hours later. Poster Session II Worker #1, complained of headache, cough productive of blood streaked sputum, and wheezing. Three discrete areas of atelectasis were observed on chest radiograph. He was admitted to hospital and treated with bronchodilators and an tibiotics. Pulmonary function testing (PFTs) performed January 19th were normal. While in hospital he became febrile. The headache and productive cough persisted for over one week. All six workers elected to come to die Occupational Medicine Center at the Mount Sinai Hospital for evaluation between January 26 and February 1,1988. Initial and persistent symp toms are summarized in Table 1. Three workers, (#2-4), com plained of fatigue at the time of evaluation. Physical examina tion did not reveal any pertinent abnormalities. On reviewing the initial radiographs, worker #3 had hazy infiltrates in the lower lung fields, while worker #6 had a slight infiltrate in his left lower lung field. All follow-up radiographs taken between 10-21 days after the accident were normal. Pulmonary function testing was not performed during the initial emergency room evaluation. PFTs were ordered by the company physician between three and ten days after the event for five of die six workers, the sixth a few days later. Three of these were normal. Workers #5 and 6 had slight Table I Symptoms of Workers Exposed to Sulfur Tetrafluoride Worker Symptom Burning/ Tearing eves Nasal irritation/ F.nistJiTiR Throat irritation Chest tightness/ Wheezing/ S. O. B. Cough Nausea/Vomitting Fatigue Headache #1 0 * 6 * #2 #3 #4 * 4c 4c oo 4c 4c o o 4c 4c 4c o 0o o * Symptoms following exposure Symptoms lasting longer than one week #5 4c #6 4c o 4c 1341 Poster Session n decreases in FVC, 75% and 77% percent ofpredicted, which normalized to 89 and 98% on follow-up testing a few days later. PFT results are summarized in Table n. Interpreta tion of these findings is limited by the fact that different equipment was used at each location. Worker #3 had three sets of PFTs, the first set performed on January 19,1988 was normal. PFTs taken prior to resum ing work cme week after the event, revealed an obstructive pattern, FVC 109% and FEVj 67% of predicted. He did not have a history of asthma, but did complain of chest tightness and shortness of breath cm exposure to cold air for approx imately one week following the exposure. Repeat testing when be was asymptomatic was normal. DLCOs were nor mal in all workers except for #5 who had a history ofasbestos exposure and emphysema. DISCUSSION SF6 was first synthesized by Moissen and Lebeau in 1902 by burning sulfur in a fluorine atmosphere.3 SF6 has been used in electrical equipment in die United States since 1953.4 ft is a heavy, colorless, odorless gas of high chemical stability. By being an effective electron scavenger SF6 can efficiently retard electrical conduction. These prop erties have led to its use as an electrical insulating material in circuit breakers, cables, capacitors, and transformers.5 SF6 containing equipment has allowed the creation of com pact electrical substations requiring one twentieth the land of previous designs.6 The use of SF6 has increased markedly in recent years. The National Occupational Hazard survey initiated in 1971, ap- Table H Pulmonary Function Results Worker A #3 #5 #6 Date FVC FEV1 FVC/FEV1 FEF25-75 DLOO Jan. 19* 4.15 3.7 89% Date FVC FEV1 FVC/FEV1 FEF25-75 DLOO Jan. 19 ' 3.58 (77%) 2.17 (60%) 60% 1.08 (34%) Date FVC FEV1 FVC/FEV1 FEF25-75 DLOO Jan. 21 ' 3.64 (75%) 3.28 (87%) 90% 5-96 (160%) Jan. 26 ' 4.40 (109%) 2.08 (67%) 47% 2.00 (62%) Jan. 28 " 4.52 (98%) 2.71 (78%) 64% 1.50 (33%) 16.2 (60%) Jan. 26 " 4.23 (89%) 3.97 (107%) 94% 7.66 (153%) 24.3 (82%) * Private Physician's office ' Company medical facility " Mount Sinai Medical Center A Workers #1,2,4 all had unchanged results on repeat testing Percentages in parenthesis are % perdicted Feb. 1" 4.87 (116%) 3.92 (120%) 80% 2.69 (81%) 38.1 (141%) 1342 proximated that 177 American workers were potentially ex posed to this compound.7 Preliminary information from the early 1980's estimates over 9,000 potentially exposed workers, over half repairers of electrical and electronic equipment.8 SF6 is an inert gas; in experimental studies no ill effects were found in mice breathing a mixture of 80% SF6 and 20% O2 for 12-16 hours.2 SF6 will break down to toxic sulfur oxyfluorides during electrical arcing in the presence of ox ygen.1*9 Worker exposure to these gases can be significantly reduced by the presence of properly maintained absorptive filters. In experiments specifically designed to identify die decomposition products of SF6, SF4 was only generated by higher energy arcs after the consumption of available ox ygen.1 Temperatures above 150C have been reported to lead to the decomposition of SF6 to SF4 and other compounds.10 SF4, a highly reactive, colorless gas which fumes in moist air, has an irritating odor similar to sulfur dioxide.11 No comprehensive studies of this compound's toxicity could be found in die medical literature. The material safety data sheet on this compound reports it to be extremely irritating and corrosive to the upper and lower respiratory tracts, skin, and eyes.9 SF4 hydrolyses in air to form hydrofluoric acid. Thus skin and mucous membranes lesions similar to those caused by this acid can be expected in workers exposed to SF4. SF4 may cause chemical pneumonitis and pulmonary edema.4 Animals exposed to 10 ppm SF4 for one hour developed rapid labored breathing, weakness, and cyanosis.12 The manufacturer has reported that animals ex posed to 50 ppm for 4 hours died from pulmonary edema.13 Ten repeated exposures of4 ppm for 4 hours produced signs of respiratory effects in rats. Pulmonary damage was ob served in rats sacrificed immediately after the tenth exposure. Those subsequently unexposed for 14 days recovered clinical ly and showed no anatomical lesions.14 In 1959 in vestigators for E.I. Du Pont de Nemours & Company recom mended that SF4 should be treated with extreme caution as it has an inhalation toxicity comparable to phosgene.2 Con sistent with this high level of toxicity the ACGIH has set a ceiling exposure limit of 0.01 ppm for this compound.15 Electrical substations contain switches, circuit breakers, con ductors, and transformers to switch power circuits and transform power from one voltage to another or from one system to another. At the station in question three circuit breakers were connected to the damaged cable. Each ofthese were approximately the size of a 55 gallon drum and filled with SF6. Although the exact sequence of events leading to the SF4 exposure has not been determined, a likely sequence is as follows. Due to damage at a distant site insulating oil was lost from the cable. This, or the following burnout led to a disruption of the valve separating the circuit breakers from the cable. Due to die intense beat of the burnout SF6 decom posed and all oxygen in the system was consumed. Further breakdown of SF6 occurred leading to the production of SF4 and possibly other compounds. As the circuit breakers were not externally damaged foe SF4 was forced into foe pipe con taining the cable and was released when the pipe was cut Poster Session n at foe worksite. Although other breakdown products may have been present, SF4 was foe only one qualitatively iden tified. The level of exposure was not quantified. Repeat testing foe following day revealed barely detectable levels. Workers were exposed to SF4 for about 6 hours over a 12 hour period while repairing foe cable. Chest radiographs were not taken until 26 hours after foe start of exposure. In addition any early PFT changes may have been missed as testing was not performed until a few days after foe event when foe majority of acute symptoms had already subsided. Radiographic evidence ofmultilobar atelectasis was present in one worker. In addition a second worker, who did not have a previous history of asthma, complained of chest tightness on exposure to cold air and developed a transitory obstructive pattern on pulmonary function testing. His chest radiograph revealed hazy infiltrates in his lower lung fields. These findings are consistent with known toxic effects of irri tant gas exposure. All five underground workers had respiratory tract symp toms, foe sixth worker who remained above ground, ex perienced only eye irritation. The intermittent nature of foe exposure most probably prevented the development of more severe effects such as chemical pneumonitis or toxic pulmonary edema. These workers were unaware that theirjob could lead to ex posure to SF4. Although they had worked for many, some for over twenty years in this field, none had heard of SF4 before or were aware that their job may lead to exposure to irritating chemicals in general and to SF4 in particular. Had they, or foe company management, physician, chemist, or industrial hygienists been aware of foe potential for this exposure, it is likely that foe exposure would have been of much shorter duration. The potential for toxic exposures, however, is documented in foe material safety data sheet describing SF6.9 Proper education may have prevented foe adverse health effects suffered by these workers. Although foe presence or absence of odor should not in general be relied upon to identify toxic exposures, odors pres ent in areas containing heated SF6 must be considered to be coming from decomposition products and be a signal for foe use of proper safety procedures.1,4 An odor similar to a "burning car battery" was identified by workers, but no one involved in foe initial investigation recognized this to be a warning signal. Fortunately, no worker developed severe complications and all have been able to return to work. In order to limit foe potential adverse effects of a similar event in the future, foe following recommendations were given to both workers and management: 1. Comprehensive air tests be conducted before work is begun after accidents. 2. Knowledgeable individuals should be available for im mediate on sit consultation if needed. 3. If a problem is presumed to exist work should not be resumed until the evaluation has been completed. 4. Proper respiratory protective equipment should be available at the worksite. . 1343 Poster Session U Due to the expanding role ofSF6 in the electrical transmis sion industry16 it is likely that exposures to its decomposi tion products will occur in die future. Occupational health personnel should thus be aware that exposure to SF4 is an important health hazard for workers repairing damaged elec trical systems containing SF6. REFERENCES 1. Boudene, C., Chiet, J.L., Kteb, G., and Wind, G.: Identification and study of some properties ofcompounds resulting from the decomposi tion ofSF6 under die effect ofelectrical arcing in circuit breakers. Revue Generate de I'ekctricite. 185:45-78 (1974). 2. Smith. W.C.. ToUocfc, C.W., Muedertics, EJ_, Hoeek, WJL, Fawcett, F.S., Engelhard!, V.A., and Coffinann, D.D.: American CbcmicaJ Society Journal. 81:3165-66(1959). 3. Moissen, H. and Lebeau, P.: Etude des Fluomires et Oxyfluorides de Soufre. Ann. dim. Phys. 26:145-178 (1902). 4. Amalric, J., FaDou, M., Mazingarbe, E., Tellier, R., and Vigreux, J. Practical consequences of research on the decomposition by arc of SF6. Revue Generate de 1'etectricite. 185:78-85 (1974). 5. Kirk-OthmerEncyclopedia ofChemical Technology, 3rd Ed., Vol. 10 pp. 799-811. M. Grayson, Ed. John Wiley and Sons, New York 1980. 6. Standard Handbook forElectrical Engineers, 12th Ed., pp. 17.1-17.51. D.G. Fink and H.W. Beaty, Eds. McGiaw, New York (1987). 7. Natrona/ Occupational ExposureSurvey, National Institute for Occupa tional Safety and Health DHEW (NIOSH) Publication No. 78-114. Cin cinnati (1978). 8. National Occupational Hazard Survey. National Institute for Occupa tional Safety and Health DHEW (NIOSH) (pending). 9. AirProducts Specialty Gas Material Safety--Safety SheetSulfurHexaBouride. Air Products and Chemicals, Inc. Allentown, Pa. (1986). 10. Kiric-Othmer Encyclopedia ofChemical Technology, 3rd Ed.** Vol. 13 pp. 534-564. M. Grayson, Ed. John Wiley and Sons, New York (1980). 11. Information Profiles on Potential Occupational Hazards: Sulfur Tetrafluoride, Center for Chemical Hazard Assessment, Syracuse Research Corporation Report No. SRC TR 81-549. Syracuse N.Y. (1981). 12. Documentation of the Threshold Limit Values for Substance in Workroom Air. 3rd Ed., American Conference ofGovernmentIndustrial Hygienists. Cincinnati, OH (1979). 13. Sulfur Tetrafluoride, Bulletin JC174BUB-0. Air Products and Chemical, Inc. Allentown, PA. (n.d.) 14. Clayton, J.W. The toxicity of fluorocarbons with special reference to chemical constitution.: /. Occ. Med. 4:(5)262-273. (1962). 15. Threshold Limit Values and Biologic Exposure Indices for 1987-1988. American Conference ofGovernmental Industrial Hygienists. Cincin nati, OH. (1987). 16. The State ofInternational Development and Experience with SF6 In sulated High Voltage Switchgear, Session 23.01. International con ference on large high voltage electrical systems (CIGRE), Paris, (1982). 1344 Paster Session II EXPERIMENTAL STUDIES ON THE EFFECT ON THE IMMUNE SYSTEM OF EXPOSURE TO COALMINE DUST AND QUARTZ. Y. KUSAKA, M.D. R. T. Cullen, Ph.D K. Donaldson, Ph.D Institute of Occupational Medicine 8 Roxburgh Place, Edinburgh EH8 9SU INTRODUCTION . Immune effects ofmineral dusts may influence the develop ment and progression of pneumoconiosis. In coalworkers' pneumoconiosis, progressive massive fibrosis and Kaplan's Syndrome are said to be related to immunologic abnor malities.1 In addition, experimental studies have revealed that fibrogenic mineral dust alter immune responses.2'3 As part of a programme to examine the effect of silica and coalmine dust on die immune system, two approaches were taken: firstly immunocompetent cells from the rat spleen were exposed to dust in vitro and their mitogenic responses were assessed; secondly, dusts were intratracheally administered to rats and die effects of elicited bronchoalveolar leukocytes on splenocyte mitogenesis were studied. MATERIALS AND METHODS Animals Twelve to fifteen-week old, female, SPF-maintained, inbred PVG rats were supplied by the Institute of Occupational Medicine breeding unit. Dusts Four kinds ofdusts were used in the experiments: two were coalmine dusts collected from the air ofBritish collieries min ing 1) anthracite coalmine dust (A) and 2) low rank coalmine dust (L), 3) titanium dioxide (Ti02; rutile, Tioxide Ltd. Stockton on Tees), a dust oflow biological activity, 4) quartz dust (DQ12 standard). Splenocyte Mitogenesis Rats were killed by intraperitoneal injection with Nembutal and spleens were aseptically removed and disaggregated with a glass homogenizer. After lysing the erythrocytes, splenocytes were suspended in Hepes-buffered RPMI1640 medium supplemented with 50mM 2-Mercaptoethanol, 2mM glutamine, 100mg/l kanamycin and 10% fetal calf serum (cRPMl). Finally 2x10s splenocytes, in medium were delivered to each well of 96-well microtiter plates. The splenocytes, with or without dust suspensions, super natant or bronchoalveolar cells, were cultured in the presence or absence of a suboptimal dose of phytohemagglutinin (PHA, 10 |ig/ml) for three days at 37C in 5% COj. The cultures were then pulsed with 0.25uCi tritiated thymidine. incubated overnight, and the uptake of 3H Thymidine was determined by liquid scintillation counting. Effect of Dusts on Splenocyte Mitogenesis The four kinds of dusts were autoclaved and suspended in cRPMl. Each aliquot was added to splenocytes to obtain a final concentration in the well of 10,50 or 100 pg/ml. They were then co-cultured at 37C in 5 % CO2 for 24 hours and stimulated with suboptimal PHA for a further three days in culture. A preliminary study showed that 24 hours of co culture of the splenocytes with dusts led to the optimal response to PHA. The cultures were assessed for mitogenesis as described above. Effect of Supernatants on Mitogenesis Splenocytes were adjusted to 1 x 106 cells/ml and aliquots of 5ml were delivered to plastic flasks. The splenocytes were allowed to adhere for six hours (adherence efficiency 27 10%, x sd) and non-adherent cells were removed by washing. The adherent splenocytes were cultured with dusts at a final concentration of 100 pg/ml for 24 hours and super natants were collected which were spun, filtered and frozen until use. The supernatants, at various dilutions were delivered to wells containing 2 x 10s splenocytes and these were cultured and harvested as described above. Interleukin-1 Activity in Spleen Cell Supernatants Three-fold dilutions of supernatants from cultures of adherent spleen cells exposed to dust at 100 pg/ml, were incubated with C3H mouse thymocytes at 6 x 10s per well in microtiter plates. PHA was added at a final concentration of 4 |ig/ml and the plates cultured for 72 hours. Thymocyte proliferation was determined by die incorporation of tritiated thymidine added during die final 16 hours of culture. Super natant from unfractionated spleen cells cultured with 10 pg/ml Concanavalin A (Con A) served as a positive con trol. Con A activity was neutralized with methylmannoside before use in the thymocyte assay. Effect of Bronchoalveolar Leukocytes from DustExposed Rats on Splenocyte Mitogenesis Rats were intratracheally instilled with lmg of the four dif ferent kinds of dusts suspended in 0.5ml Phosphate Buffered Saline (PBS). PBS alone was injected into rats as a control. Bronchoalveolar cells (BAC) were obtained by lavage seven days later. BAC were washed with RPMI1640 and sus 1345 Poster Session U pended in cRPML BACs from quartz-treated rat were separated into a macrophage and neutrophil-enriched popula tions by density gradient centrifugation through Sepra-cell medium. Total and differential counting was done on Diffquick stained cytospin preparations and viability was assessed by trypan blue exclusion. Total or separated BACs were add ed to splenocytes at final ratios of from 1:4 to 1:128. The cultures were incubated and assessed for mitogenesis as above. Statistical Analysis Since variation between experiments was large, the data were expressed as mitogenic indices for each condition: the mitogenic indices were obtained by dividing the suboptimal PHA-driven splenocyte mitogenesis with dust, supernatant or bronchoalveolar leukocytes, by the mitogenesis without these treatments. The differences in mean values ofmitogenic indices between treated and untreated were tested by paired t-test. The differences were considered as significant ifvalues were less than 0.05. In the IL-1 assay, the 3H uptake by die cultures with various supernatants were compared to those with control supernatant (no dust treatment). RESULTS Effect of Dusts on Splenocyte Mitogenesis In Vitro Both quartz at 10, SO or 100 pg/ml and low rank coalmine dust L at 50 and 100 pg/ml significantly enhanced mitogenesis. Quartz especially augmented splenocyte pro liferation even without mitogen (data not shown). On the con trary, both T1O2 and coalmine dust A suppressed splenocyte proliferation in a dose-dependent manner (Figure 1). Effect of Supernatant from Dust-Exposed Adherent Splenocytes on Mitogenesis The supernatant, tested at various dilutions did not cause enhancement of mitogenesis and, in fact, supernatant from splenocytes treated with quartz at a high dose were inhibitory to mitogenesis (typical results of 1:16 dilution shown in Figure 2). Interleukin-1 Activity in Supernatants Despite the lack ofenhancement in the spleen cell mitogenesis assay, die thymocyte assay did show interleukin 1-like ac tivity in die quartz supernatant diluted at 1:7.5 (Figure 3). 6r 5h 10 50100 10 50 100 10 50 100 10 50 100jug/ml QUARTZ Ti02 L A Figure 1. Mitogenic indices (means with standard errors) of splenocytes cultured with four kinds ofdusts. Mitogenic index derived as the ratio of mitogenesis with dustrmitogenesis without dust. An asterisk denotes a significant (p<0.05) difference from the control. 1346 Poster Session II MITOGENIC INDEX QUART Z Ti 02 L A I-J Figure 2. Effects of supernatants from dust-exposed adherent splenocytes on mitogenesis. Data are shown as mitogenic indices (means with standard deviations). Mitogenic index derived as the ratio of mitogenesis with supernatant from dusted or not-dusted adherent splenocytes:mitogenesis without supernatant. INTERLEUKIN 1 ACTIVITY (cpm xIO3) --* K> Lu -C- LT1 -t--------------------------r------------------------- 1---------------- ----------1-------------------------- 1 control}* i QUART7 "T-i EHH I CONA POSITIVE CONTROL ~ [---------------------- 1 Figure 3. Interleukin 1 activity in supernatants from dust-exposed adherent splenocytes. An asterisk denotes a significant (p<0.05) difference from the control supernatant. 1347 Poster Session U Higher concentrations of supernatant were inhibitory and supernatant from spleen adherent cells treated with other dust had no detectable 11*1 activity. Effect of Bronchoalveolar Leukocytes from DustExposed Lungs on Mitogenesis Control, PBS-elicited bronchoalveolar leukocytes (total 4.16 0.33 x 106 (x sd) cells per rat, macrophages >98%, viability >95%) showed an inhibitory effect on splenocyte mitogenesis which was effectorrindicator cell ratiodependent. The bronchoalveolar leukocytes from coal dust L (total 3.08 0.76 x 106 cells per rat, macrophages 96 2%, neutrophils 3 2%, viability >91%), coalmine dust (A) (total 2.58 0.33 x 106 cells per rat, macrophages >99%, viability >92%) or TiOj (total 4.61 0.33 x 106 macrophages >99%, viability >93%) did not affect mitogenesis. Figure 4 shows die results for BAC from coalmine dust A. The BAC from quartz-treated rats (total 16.83 4.64 x 106 cells per rat, macrophages 42 4%, neutrophils 57 4 %) was significantly less inhibitory to splenocyte prolifera tion, at ratios of 1:64,1:32 and 1:16, than the control. After the separation into macrophage- and neutrophil-enriched frac tions (recovery rate 60 1%), die macrophage-enriched population (macrophages 89 5%) also showed less inhibi tion at ratios of 1:64 and 1:32. In contrast to the inhibitory effect of the total leukocytes or separated macrophages, the neutrophil-enriched population (neutrophils 82 2%) markedly enhanced mitogenesis compared to control BAC (Figure 5). DISCUSSION In our rat model system, we have examined die effects of exposure to mineral dusts on the immune system. The splenic lymphocytes were taken as indicator cells for the direct ef fect of dust on die immunomodulatory role of leukocytes within the lung. Both quartz and coalmine dust with a high (>5%) quartz component, enhanced splenocyte mitogenesis in vitro. Super natant from adherent splenocytes, presumed to be mostly macrophages, treated with quartz showed increased IL-1 ac tivity, whilst supernatant from coalmine dust or TiOz-treated macrophages had no such activity. None of these supernatants caused enhanced mitogenesis. These ap parently conflicting findings may be explained as follows. Adherent macrophages secrete, in addition to IL-1, a varie ty of substances including prostaglandins and hydrogen peroxide, which are inhibitory to lymphocyte prolifera tion.4'5 Subsequently die ability of any supernatant to in fluence mitogenesis is likely to be die product of both the inhibitory and enhancing activity present in it. Evidence that inhibitory factors were present, and could be diluted out was X LU O 1348 1 2345 Number of 3ronchoalveolar Leukocytes (xlO4/well) Figure 4. Bronchoalveolar leukocytes from coalmine dust L--instilled rats inhibited splenocyte mitogenesis in a dose-dependent manner. No significant differences from control leukocytes were present. Mitogenic index derived as the ratio of mitogenesis with bron choalveolar leukocytes:without leukocytes. 1-5 Poster Session II .Control ;o. Whole Bronchoalveolar Leukocytes; .Macrophage-enriched Population: . Neutrophil-enriched Population. Nunber of Brcnchoalveolar Leukocyte (xlO^/wdl) Figure 5. Effects of bronchoalveolar leukocytes from quartz-injected rats on splenocyte mitogenesis. Data are shown as mitogenic indices (means with standard deviations). An asterisk denotes a significant difference (p<0.05) from controls treated with PBS. shown by the fact that IL-1 activity in the supernatant from quartz-exposed splenocytes was expressed only at higher dilutions. Further studies are needed to elucidate the mechanism of enhanced mitogenesis by quartz and low rank coalmine dust in vitro including further characterization of the secreted product present in supernatant form dust-treated macrophages. Alveolar macrophages are situated at the air-tissue interface, strategically located for initial contact with inhaled par ticulates. They also play a crucial role in pulmonary immune responses. Alveolar macrophages in some species including rats are said to be poor accessory cells for mitogen or antigenderived lymphocyte proliferation.6 In our study normal bronchoalveolar macrophages inhibited splenocyte mitogenesis in a dose-dependent manner. TiC>2 and two kinds of coalmine dusts did not affect this down-regulatory function of alveolar macrophages. However die whole BAC and the alveolar macrophage-enriched population from quartz-treated rats inhibited lymphocyte response to a lesser extent than control BAC although this may be due to con taminating neutrophils as described below. Further studies are needed to confirm whether alveolar macrophages elicited by exposure to quartz have altered immunomodulatory prop erties, as suggested by this study. The neutrophils separated from quartz BAC strikingly enhanced mitogenesis and this could be mediated through protease7 or an 11-1 analogue which has been described in secretions from peritoneal neutrophils.8 Inhalation exposure to asbestos fiber, another type of fibrogenic dust, causes recruitment of la-positive alveolar macrophages and secretion of II-1 by alveolar macrophages.9'10 Additionally, alveolar macrohpages from asbestos-exposed rat enhanced T lymphocyte proliferation in vivo.11 In vitro fibrogenic dust such as asbestos and silica stimulated alveolar macrophages to secrete IL-1.12 Inhala tion exposure to silica also causes secretion of IL-1 by alveolar macrophages when stimulated with endotoxin.13 These studies suggest that fibrogenic dusts have immunostimulatory effects on alveolar macrophages and our results partially support these findings. However the com plex effect of recruitment of newly exudated, monocytederived populations with altered cytokine production and the role of neutrophils, which are also found in dust exposed lung,14 remain to be resolved. This study suggests that, in the lungs of individuals inhaling quartz or quartz-containing coalmine dusts, the alveolar macrophages may be affected by phagocytosed dust to release a range of mediators which could modulate lymphocyte responses in the local environment of the lung. Additional ly, neutrophils which are recruited into dust-exposed lung could also enhance immune responses leading to localized immunodulation. Any "adjuvant-type" effect on the immune system could contribute directly to heightened responses within the lung both to dust itself and to infectious agents, both of which could contribute to die tissue injury and fibrosis found in pneumoconiosis. 1349 Poster Session U REFERENCES 1. Morgan, W.K.C., Seaton, A.: Occupational LungDiseases, 2nd Ed., pp. 377-448. W.B. Saunders Co., Philadelphia (1984). 2. UnnalHsnn, 1C , Davis. James. K AAeaos-acrivated Peritoneal Macrophages Release a Factors) which Inhibit Lymphocyte Mitogenesis- Environ. Res. 104-113 (1983). 3. Hannan!, D., Donaldson, K., Bolton, R.E.: Immunomodulatory Ef fect of Mineral Dust. I. Effects ofIntraperitooeal Dust Inoculation on Splenic Lymphocyte Function and Humoral Immune Responses in vivo. /. din. Lab. Immunol. 16:81-85 (1985). 4. Bonney.RJ., Humes, J.L.: Physiological and RiannacoJogjcal Regula tion of Prostglandin and Leukotriene Production by Macrophages. J. Leukocyte Biol. 35:1-10 (1984). 5. Davis, L,Lipsky,P.E.: Signals Involved in T cell Activation. LPhofbol Esters Enhance Responsiveness but Cannot Replace Intact Accessory Cells in the Induction ofMitogen-stimulated T cell Proliferation. J. Im munol. 135:2946-2952 (1985). 6. Holt, P.G.: Alveolar Macrophages. IV. Inlerspecies Differences in Ac tivity in Proliferating Lymphocyte Cultures, dll. Immunol. 50:210-215 (1980). 7. Nakamura, S., Yosbinaga, M., Hayashi, H.: Interaction between Lym phocytes and inflammatory Exudate Cells, n. A Proteolytic Enzyme Released by PMNL as a Possible Mediator for Enhancement of Thymocyte Response. /. Immunol. 117:1-6 (1976). 8. Goto, F., Nakamura, S., Goto, K., Yosbinaga, M.: Production of a Lymphocyte Proliferation Potentiating Factor by Purified Folymorpbonudear Leukocytes from Mice and Rabbit. Immunol. 53:683-692 (1984). 9. Hartmann, D.P., Georgian, M.M., Oghiso, Y., Kagan, E.: Enhanced Interleukin Activity following Asbestos Inhalation. CSin. Exp. Immunol. 55:643-650 (1984). 10. Hartman, D.P., Georgian, M.M., Kagan, E.: FnhanenH Alveolar Macrophage la Atigen Expression after Asbestos inhalation. J. Immunol. 132:2693-2695 (1984). 11. Miller, K., Kagan, E.: Manifestation nf (Vlhilar Immunity in the Rat after Prolonged Asbestos Inhalation. Environ. Res. 26:182-194 (1981). 12. Oghiso, Y.,, Kubota, Y.: Interleukin l production and Accessory Cell Function of Rat Alveolar Macrophages Exposed to Mineral Dust Par ticles. Microbial. Immunol. 31:275-287 (1987). 13. Oghiso, Y., Kubota, Y.: Enhanced Interleukin 1 Production by Alveolar Macrophages and Increase in Ia-Positive Lung Cells in Silica-Exposed Rats. Microbiol. Immunol. 30:1189-1198 (1986). 14. Donaldson, K., Bolton, R.E., Jones, A., Brown, G.M., Robertson, M.D., Slight, J., Cowie, H., Davis, I.M.G.: Kinetics of the Bronchoalveolar Leukocyte Response in Rats during Exposure to Equal Air borne Mass Concentrations ofQuartz, CfcrysocBe Asbestos, orTitanium Dioxide. Thorn. 43:525-533 (1988). ACKNOWLEDGEMENT: The research was supported by the Commis sion ofthe European Communities. Dr. Y. Kusaka would like to gratefully acknowledge a kind contribution by the Colt Foundation which allowed this work to be completed. 1350 Poster Session U BRONCHOALVEOLAR LAVAGE IN SUBJECTS EXPOSED TO OCCUPATIONAL DUSTS GEORGE GOODMAN, M.D* f Norman L. Lapp, M.Dt* William H. Pailes, M.S4 Daniel Lewis, Ph.D.$* Vincent Castranova, Ph.D4 West Virginia University Hospital, Morgantown, West Virginia tAllegheny General Hospital, Pittsburgh, Pennsylvania ^Division of Respiratory Disease Studies, NIOSH, Morgantown, West Virginia INTRODUCTION Alveolar macrophages are free lung cells located on die sur face of small airways and alveoli. These phagocytes play an important role in die protection of the lung against airborne bacteria and particles.1 However, hyperactivation of pul monary phagocytes can lead to excessive secretion of en zymes and reactive oxygen species which could result in lung injury, emphysema, or fibrosis.2*3 Analysis of bronchoalveolar lavage effluents for cell types and cellular activity has yielded information concerning the etiology of various pneumoconioses. For example, em physema associated with inhalation of coal dust or cigarette smoke has been related to enhanced secretion of reactive ox ygen species from alveolar macrophages.4-7 In contrast, hypoactivation of alveolar macrophages has been associated with inhalation of diesel particulates4 and has been related to increased susceptibility to pulmonary infection.8 Inhala tion of cotton dust has been associated with dramatic in creases in lavagable polymorphonuclear leukocytes9 while pulmonary sarcoidosis and silicosis have been related with high numbers of lymphocytes in lavage effluents1011 The objective of the present study was to obtain lavage ef fluents from 8 control subjects, 8 healthy power plant workers exposed to fly ash, 1 healthy coal miner, and 1 rock driller with acute silicosis. These effluents were analyzed for total numbers of alveolar macrophages, lymphocytes and neutrophils and for secretory activity of alveolar macrophages. Data were then analyzed to determine what response patterns were characteristic for given dust exposures. METHODS Selection of Subjects The 8 control subjects were all adult males from the area of Morgantown, WV. Two subjects among the controls had smoked cigarettes for approximately three pack-years and had stopped more than ten years ago. The 8 power plant workers were all healthy adult male employees of a power facility in Hatfield, PA, approximately thirty miles from Morgantown. One of the power plant personnel was a com parable ex-smoker. The remaining subjects were all lifelong non-smokers. The coal miner was a healthy non-smoking male from Morgantown, WV. The rock driller was a pa tient under treatment with corticosteroids for acute silicosis. He had shown considerable clinical improvement with this treatment but still manifested significant radiologic and pulmonary functional abnormalities at the time of lavage. The mean age of the 8 control subjects was 36 years (range: 31 to 49 years); the mean age of the remaining subjects studied was 38 years (range: 29 to 53 years). Each volunteer was interviewed prior to participation in the study. Each completed the British Medical Research Coun cil standardized questionnaire concerning respiratory and oc cupational history. One individual among the controls occa sionally used a metered-dose inhaler for the management of mild asthma; otherwise there was no history of significant concurrent respiratory illness among the subjects except for the one with acute silicosis. Other than these two individuals, none of our volunteers received any medications regularly. None of the control subjects had a history of significant ex posure to occupational dusts. The coal miner had worked in underground mines for approximately 20 years and rare ly wore a respirator. The power plant employees had variable histories of exposure to dusts at their plant, ranging from 4 to 15 years of employment. Although some of their work brought them into contact with both asbestos and coal dust, the primary dust exposure was to fly ash. None of them wore respirators consistently. The rock driller was exposed to significant levels of sandstone, coal and rock dust over a 12 year period during which time he never wore a respirator. Experimental Procedures Protocols were approved by HSRB at West Virginia Univer sity. In each subject, pulmonary function tests were obtained (spirometry and diffusion capacity) as well as a 12-lead elec trocardiogram, PA and lateral chest x-ray films, and screen ing blood tests. All of these studies were normal except for the presence of mild obstruction in our one asthmatic con trol and moderate restriction and reduced diffusion capacity in the patient under treatment for acute silicosis. Flexible bronchoscopy and bronchoalveolar lavage was per formed in a consistent fashion in all subjects.5*7 After the tip of the bronchoscope had been wedged into a distal subseg ment in the right middle or right lower lobe, this area was 1351 Poster Session n lavaged with 10 aliquots of 20 cc of 0.9 saline. The pooled return from die lavage was passed through nylon mesh (150 mesh) to remove mucus from die specimen. The specimen was then centrifuged at 500 g for 5 min. at 2C, die supernate decanted, and die pellet ofcells resuspended in HEPES buffered medium (145 mM NaCl, 5mM KC1, 10 mM HEPES, 1 mM CaCl2 5mM glucose; pH:7.4). These cells were then washed twice by alternate centrifugation and resuspension in HEPES-buffered medium. Total cell counts in die lavage effluent were determined us ing an electronic cell counter. Lymphocytes, neutrophils, and alveolar macrophages were identified by their distinctive cellular volumes using an electronic cell sizing attachment as described previously.12'13 Chemiluminescence was measured in die presence of 1.7 mg luminol and 8 serum using a Berthold 9505 Luminometer. Chemiluminescence was monitored at rest (Rest CL) and after stimulation with either 3 x 10-6M phorbol-12-myristate acetate (PMA-CL) or 2 mg/ml zymosan (Zym-CL). Chemiluminescence was expressed as total counts per sec ond/lO min/1.63 x 106 alveolar macrophages. RESULTS The data are shown in Table I. Fly ash exposure resulted in significant increases in lavagable alveolar macrophages, lymphocytes, and neutrophils compared to controls. In ad dition, zymosan-stimulated chemiluminescence was significantly elevated while resting CL and PMA-CL ex hibited a trend toward elevation. Coal dust exposure resulted in only a slight increase in resting chemiluminescence. In contrast, neither PMA-CL or Zym-CL was enhanced. The most striking changes were observed in acute silicosis where very large increases in lymphocytes, neutrophils, resting CL, PMA-CL, and Zym-CL were noted. DISCUSSION Our data indicate significant differences in alveolar cell populations and phagocytotic activity when asymptomatic occupationally-exposed individuals are compared with con trols. Subjects exposed to fly ash exhibited significant pulmonary inflammation, i.e., their bronchoalveolar lavage contained approximately twice the number of macrophages, lymphocytes, and neutrophils as controls. In addition, phagocytotic activities as measured by chemiluminescence were all increased, with the activity after zymosan stimula tion being significantly enhanced. No significant activation of alveolar macrophages was noted after coal dust exposure. This contrasts with coal-induced activation noted in animal studies.4 Clearly more coal miners are needed before definitive conclusions can be drawn. The acute silicotic exhibited die most striking changes. Lavagable cells were elevated by 32% for alveolar macrophages, 14.6 fold for lymphocytes, and 10.5 fold for neutrophils. Similar increases in lymphocytes and neutrophils due to silica exposure have been reported previously in both rats and humans.14*12 These observations are more dramatic when one considers dial this subject had been on cor ticosteroid therapy prior to lavage. Indeed, such treatment would tend to decrease the yield of lavagable cells.15 The high levels of chemiluminescence seen in the silicotic pa tient (i.e.. Rest CL increased 5.3 fold, PMA-CL increased 3.7 fold, and Zym-CL increased 12.4 fold) suggest that a substantial oxidant burden exists in the lungs of this subject. Oxidant injury could explain die restrictive lung disease and diminished diffusion capacity observed in this patient. CONCLUSION We present data on bronchoalveolar lavage in normal con trol subjects and individuals occupationally exposed to in- Table I Characterization of Bronchoalveolar Lavage Parameter^ Controls (mean + SEM) Fly Ash (mean + SEM) Coal Dust Acute Silicosis # Alveolar macrophages 7.4 + 1.2 # Lymphocytes 4.5 + 0.8 # Reutrophiis 2.9 + 0.7 Rest CL 27.0 6.0 PMA CL 68.2 + 22.6 ZYM CL 41.4 + 12.4 14.1 + 2.1* 8.4 + 1.1* 7.3 + 2.5* 38.9 + 6.0 83.0 10.7 84.0 8.4* 4.0 4.6 3.1 65.8 64.5 47.4 9.8 65.6* 30.3* 144.6* 250.2* 509.0* *Cell counts are given as millions/lavage. CL values are millions of counts per second/10 minutes/1.63 X 106 alveolar macrophages. ^Significantly greater than control at p < 0.05 using a Student's T test. 1352 dustrial dusts. Asymptomatic exposed subjects who were nor mal by clinical, radiologic and pulmonary function criteria nevertheless showed significant changes in cell populations and phagocytotic activity when compared with unexposed individuals. Much more extreme changes were observed in one subject suffering acute silicosis. REFERENCES 1. Green, G.M.: Ambersoo Lecture: In Defense of die Lung. Am. Rev. Respir. Dis. 102:691-700 (1970). 2. Weiss, S.J., LoBuglio, A.F.: Biology of Disease: Phagocyte Generated Oxygen Metabolites and Cellular Injury. Lab. Invest. 47:5-18 (1982). 3. Heppleston, A.G.: Silicotic Fibrogenesis: A Concept of Pulmonary Fibrosis. Ann.Occup. Hyg. 26:449-462 (1982). 4. Castranova, V., Bowman, L., Reasor, M.J., Lewis, T., Tucker, J., Miles, P.R.: The Response of Rat Alveolar Macrophages to Chronic Inhalation ofCoal Dust and/or Diesel Exhaust, Environ. Res. 36:405-419 (1985). 5. Hoidal, J.R., Fox, R.B., LeMarbe, P.A., Perri, R., Repine, J.E.: Altered Oxidative Metabolic Response In Vitro ofAlveolar Macrophages from Asymptomatic Cigarette Smokers. Am. Rev. Respir. Dis. 123:85-89 (1981). 6. Greening, A.P., Lowric, D.B.: Extracellular Release of Hydrogen Peroxide by Human Alveolar Macrophages: The Relationship to Cigarette Smoking and Lower Respiratory Tract Infections. Clin. Sci. 65:661-664 (1983). 7. Harris, J.O., Swenson, E.W. Johnson HI, J.E.: Human Alveolar Macrophages: Comparison of Phagocytic Ability, Glucose Utilization, Poster Session U and infrastructure in Smokers and Nonsmokers. J. Clin. Invest. 49:2086-2096 (1970). 8. Hahon, N., Booth, J.A., Green, F., Lewis, T.R.: Influenza Virus In fection in Mice After Exposure to Coal Dust and Diesel Engine Emis sions. Environ. Res. 37:44-60 (1985). 9. Castranova, V., Robinson, V.A., Tucker, J., Schwegler, D., Rose, D.A., DeLong, D.S., Frazer, D.G.: Time Course of Pulmonary Response to Inhalation of Cotton Dust in Guinea Pigs and Rats. Iu: Proceedings ofthe Eleventh Cotton Dust Research Conference. R.R. Jacobs and P.J. Wakelyn (eds). National Cotton Council, Memphis, TN, pp. 79-83 (1987). 10. Peterson, M.W., Nugent, K.M., Jolles, H., Monick, M., Hunninghake, G.W.: Uniformity of Bronchoalveolar Lavage in Patients with Pulmonary Sarcoidosis. Am. Rev. Respir. Dis. 137:79-84 (1988). 11. DeAraujo, A.T., Mendes, A.C., Monteiro, J., Duarte, G., Costa, M.F.E.: Bronchoalveolar Lavage and Silicosis Pathogenesis. VUth In ternational Pneumoconioses Conference (Abs) pp. 48 (1988). 12. Jones, G.S., Van Dyke, K., Castranova, V.: Purification of Human Granulocytes by Centrifugal Ehitriatkm and Measurement ofTransmem brane Potential. /. Cell. Physiol. 104:425-431 (1980). 13. Castranova, V., Bowman, L., Miles, P.R.: Ionic Content and Transmembrane Potential ofRat Alveolar Macrophages. J. Cell. Physiol. 101:471-480 (1979). 14. Banks, D.E., Morgan, J.E., Hammad, Y.Y.: Immunologic Features of the Bronchoalveolar Lavage Fluid of Rats with Silica-proteinosis. VUth International Pneumoconioses Conference (Abs) pp. 35 (1988). 15. Goncalves, J.R., Correia. I., Raymundo, E., DeAraujo, A.T., Costa. M.F.E.: Inhaled Corticosteroids in the Treatment of Occupational Respiratory Diseases (ORD). VUth International Pneumoconioses Con ference (Abs) pp. 48 (1988). 1353 Poster Session II EXPOSURES OF PRODUCTION EMPLOYEES TO AIRBORNE CONCENTRATIONS OF FIBROUS GLASS DURING THE MANUFACTURING PROCESS C. W. AXTEN* J. R. Bender* P. F. Aubourgf T. R. Jacobst M. R. Kalinowskif E. J. Klotzf *Owens-Coming Fiberglas Corp., Fiberglas Tower, T/ll, Toledo, OH, 45659, USA tOwens-Coming Fiberglas Corp., Technical Center, P.0. Box 415, Granville, OH, 43023, USA INTRODUCTION Airborne concentrations of fibrous glass can be evaluated either gravimetrically or by optical fiber counting methodologies. However, numerous studies1'2 have demonstrated that there is little correlation between gravimetric results and concentrations of fibrous glass pres ent. For this reason, optical fiber counting methodologies rather than gravimetric analysis have become die methods of choice for fibrous glass analysis. Prior to the publication of the Occupational Safety and Health Administration's Revised Asbestos Standard (29CFR 1910.1001) in June of 1986,3 the generally accepted pro cedure for the determination of airborne concentrations of asbestos, fibrous glass, and other man-made mineral fibers was the NIOSH P&CAM 239 method.4 However, with the promulgation of this Standard, a new methodology for the evaluation of airborne concentrations of fibrous materials was introduced, the NIOSH 7400 method.5 This method introduced a new sampling train for fiber col lection (i.e. 25 mm cassette with 50mm extension cowl) as well as alternative methods for fiber counting (Rules "A" and "B"). Though similar in other respects to the NIOSH P&CAM 239 method, die new NIOSH 7400 method guickly began to receive increased attention from the industrial hygiene community as it was utilized to evaluate individuals* exposures to not only asbestos, but other man-made mineral fibers as well. Of particular concern was die notable adherence of fibers to the sampling cowl and the differing results obtained when fibers were counted via the "A** ver sus die "B" rules.6 With a considerable body of data on employees' exposures to fibrous glass obtained through use of the NIOSH P&CAM 239 method, it became imperative for Owens-Coming Fiberglas to evaluate the correlation between die two methods in terms of the sample results produced and to determine if the NIOSH 7400 method should be adopted for future ex posure evaluations. Furthermore, since most of the infor mation concerning use of die NIOSH 7400 method had been generated as a result of asbestos monitoring, it was felt that additional information could be gleaned through the use of the method to evaluate airborne concentrations of a man made mineral fiber such as fibrous glass. Thus, the follow ing study was designed and implemented. MATERIALS AND METHODS Seventy-five paired personal and area samples were collected in parallel on 0.8 micron pore size mixed cellulose ester filters mounted in 37 mm diameter polystyrene plastic cas settes with 16 mm non-electrically conductive extension cowls (i.e. NIOSH P&CAM 239 sampling method) or in 25 mm diameter polystyrene plastic cassettes with 50 mm elec trically conductive extension cowls (i.e. NIOSH 7400 sampl ing method). During the initial phase ofdie study, additional samples were collected using 0.45 polycarbonate filters mounted in 37 mm diameter cassettes with 16mm extensions cowls for analysis by scanning electron microscopy. However, this approach was guickly discontinued due to the poor fiber retention (i.e. fibers were collected but were easily dislodged during transportation). All samples were collected at a flow rate of two liters per minute (i.e. 2.01/m) using constant flow sampling pumps. The pumps were calibrated, with the filter and sampling train in line, before and after sampling using a precision rotameter calibrated against a primary standard (i.e. soap bubble meter for volumetric rate of air flow). Samples were collected at specific sites along plant manufacturing lines during the pro duction of a variety of fibrous glass insulating products (e.g. batts, blankets, and loose fill). Samples were collected over significant portions of the work shift and are believed to be representative of full shift exposures. All sample filters were mounted using die acetone/triacetin clearing method and analyzed via phase contrast optical microscopy (PCOM) at a magnification of 400X. Fiber counts for all sample filters were derived utilizing the pro cedures specified in both die NIOSH P&CAM 239 method as well as the NIOSH 7400 "A" method (i.e. all fibers >5 microns in length with aspect ratios egual to or greater than 3:1 were counted). Glass fibers were differentiated from other fibers by shape recognition using polarized light microscopy. Additionally, fiber length and diameter measurements were determined for a fraction ofdie samples. To address fiber adherence to die sampling cowls, after filter removal, all cowls were rinsed with 25% isopropanol in distilled water. Rinse solutions were then filtered through 0.4 micron polycarbonate filters, and analyzed using the counting procedures described above. 1354 After all sample results had been obtained, matched pair results were analyzed statistically to determine differences between the 37 and 25 mm diameter filters and correspond ing cowls. Natural log transformed data were used to deter mine statistical difference at the 0.05 significance level. RESULTS AND DISCUSSION The sample results obtained from this study are indicated in Table I. The mean total fiber exposure and the lower and upper 95 % confidence limits are shown for the forehearth, line, packer, bagger, rollup, repack cubed, and repack milled operators. The overall mean total fiber (both glass fiber and all other fiber) exposures of employees in OCF production facilities involved in the manufacture of fibrous glass insula tion products were 0.024 f/cc for filters only and 0.03 f/cc for filters and cowls combined (NIOSH P&CAM 239 and 7400 methods combined). Additional analyses revealed that 70 to 75% were glass fibers and that 60% of the glass fibers were of a respirable size (i.e. diameters <3.5 microns, lengths of 5 to 250 microns, and length to diameter ratios of 3:1 or greater). Furthermore, these sample results were consistent irrespective of the type of product produced (i.e. faced vs. unfaced insulation) or the physical parameters of the product produced (i.e. R 30 vs. R19 or 24" width vs. 18" width). Poster Session B Because a significant concentration of fibers were found adhering to the sidewalls of the cassettes (i.e. NIOSH P&CAM 239 Procedure) and to die sampling cowls (NIOSH 7400 Procedure), these fibers were also counted. Results are reported on Table I and Figure I as filter only and as filter and cowl combined. Figure I also includes results of samples collected in end-user applications. The data indicate that there was no statistically significant difference in sample results obtained from the NIOSH P&CAM 239 and 7400 methods when the "A" counting rules were used (see Figure 1). Fur thermore, this result was consistent irrespective of the fiber type or size analyzed (i.e. total fiber, total glass fiber, or respirable glass fiber). Statistical analysis also indicated that there was no difference between the total fiber results obtained from the NIOSH P&CAM 239 and 7400 methods using the "A" counting rules when the fibers on the filters and cowls were combined, Table n. Table II also includes results of samples collected in end-user applications. As indicated in Table II, the ratio, R, of (fibers deposited on cowls + fibers deposited on filters)/ fibers deposited on filters, was 1.7 for die NIOSH P&CAM 239 method (i.e. 16 mm sampling cowl) and 1.5 for the NIOSH 7400 method. There was no statistical dif ference between these ratios. Table I Total Airborne Fiber Concentrations Obtained by Using the NIOSH P&CAM 239 and 7400 "A" Methods (Combined) Fibers per Cubic Centimeter ALL FIBERS ITEM FOREHEARTH UHE PACKER BAGGER ROLLUP RERACK-CUBED RERACK-U1LLED ------- Filler*-------- Filters and Cowls # Exp. Sampled Value 96% LL 98% # Exp. UL Samples Value 96% LL 96% UL 20 0.017 0.003 0.028 2 0.003 0.000 0.032 30 0.028 0.017 0.040 8 0.023 0.012 0.033 2 0.021 0.001 0.041 9 0.024 0.008 0.040 4 0.040 0.013 0.088 19 0.026 0.003 0.04B 2 0.006 0.000 0.076 29 0.038 0.017 0.066 7 0.030 0.002 0.067 2 0.028 0.000 0.082 9 0.033 0.002 0.006 3 0.046 0.000 0.110 1355 Poster Session U .0-FILTER+COUL .+-FILTER LINEAR REGRESSION LINES Figure 1. Total fibers per cc--random field counts. Table H Ratio of (Fibers Deposited on Cowls + Fibers Deposited on Filters) Fibers Deposited on Filters for NIOSH P&CAM 239 and NIOSH 7400 Methods* METHOD 239 7400A # OF SAMPLES 162 160 AVERAGE 1.7 1.6 MEDIAN 1.6 1.4 STANDARD DEVIATION 0.90 0.62 NOTE: STATISTICALLY THE RATIOS FOR METHODS 239 AND 7400A ARE NOT DIFFERENT. 1356 Significant fiber deposition on samgling cowls has been reported previously by Seixas et. al.6 Also, in commenting on this phenomenon, some investigators have suggested that a high ratio of fibers detected on sampling cowls versus fibers found on filters is merely an artifact produced by undercount ing of fibers deposited on filters.7 This was not found to be the case, however, since the ratio, R, was consistently high for low as well as medium and high fiber counts. CONCLUSIONS The results obtained from our studies indicated that mean employee exposures to total fibers in Owens-Coming Fiberglas manufacturing facilities was 0.024 fibers/cc for filters only and 0.03 f/cc for filters and cowls combined. There was no statistically significant difference in the results obtained when the NIOSH P&CAM 239 and 7400 "A" methods were utilized. However, the study also demonstrated that there was a significant concentration of fibers deposited on the sampling cowls used for both methods which should conceivably be considered in determining the total level of exposures to fibrous glass. REFERENCES 1. Konzen, J.L. "Results of Environmental Air-Sampling Studies Con Poster Session U ducted in Owens-Coming Fiberglas Manufacturing Hants", pp. 115-120 in Occupational Exposure to Fibrous Glass: A Symposium, HEW Publication No. (NIOSH) 76-151, U.S. Department ofHealth, Educa tion, and Welfare, Public Health Service, Center for Disease Control, National Institute for Occupational Safety and Health. 2. Esman, N., Com, M. et. al. "Summary of Measurements of Employee Exposure to Airborne Dust and Fiber in Sixteen Facilities Producing Man-Made Mineral Fibers," American Industrial Hygiene Association Journal, 40:108-117. 3. Occupational Safety and Health Administration Standard--''Asbestos, Tremolite, Anthophyllite, and Acdonolite," 29CFR 1910.1001, 51 Federal Register, June 20, 1986. 4. Analytical Method P&CAM 239 for "Asbestos Fibers in Air" in NIOSH Manual of Analytical Methods-- Second Edition, DHEW (NIOSH) Publication No. 77-IS7, U.S- Department of Health Education and Welfare, Public Health Service, Center for Disease Control, National Institute for Occupational Safety and Health. 5. Analytical Method 7400 for "Fibers" in NIOSHManual ofAnalytical Methods--Third Edition, DHHS (NIOSH) Publication No. 84-100, U.S. Department of Health and Human Services, Public Health Service, Center for Disease Control, National Institute for Occupational Safety and Health. 6. Sexias, N. ehal. "Bias Introduced by NIOSH Method 7400 Quantitative Fiber Exposure Assessment," American Industrial Hygiene Associa tion Journal, 48:A242-243. 7. Keller, Richard, G., and Kestner, Chris, J. "Fiber Loss on Polycar bonate and Cellulose Ester Filters," Presentation at the American In dustrial Hygiene Association Conference on May 23, 1985. 1357