Document B5E4O3gBp64M2pepkvRpLbjXJ

Toxicity/Surface Characterization III RELEASE OF A "FIBROBLAST PROLIFERATION FACTOR" FROM HUMAN MACROPHAGES IN VITRO TREATED WITH QUARTZ DUST DQ 12 OR COAL MINE DUSTS N.H.SEEMAYER E. Maly Medical Institute of Environmental Hygiene at the University Gurlittstr. 53, D-4000 Dusseldorf, F.R.G. INTRODUCTION Alveolar macrophages are the primary target for die noxious effect of quartz--and coal mine dusts in human and animal lung. Macrophages produce a very large number ofmore than 50 "biofactors" or "mediators" which participate in various inflammation and immunological regulation processes.l6*26 Heppleston and Styles9 reported in 1967 that after incubation with quartz dust, peritoneal macrophages of die rat produce a factor which stimulates chicken fibroblasts to collagen syn thesis. After initially negative reports8 Heppleston's findings have been confirmed by various investigators using diverse in vivo and in vitro cell systems, iA,U2.27 Recendy, evidence is accumulating about die formation of mediators in human monocyte and macrophage cultures following stimulation with soluble and particulate agents which stimulate fibroblasts to cell replication.3*6*7*13*19 Various reports suggest, that human monocytes and macrophages have the ability to generate multiple "fibroblast activating factors," depending cm culture conditions, agents used for stimulation, cell type and assay employed for activity evaluation.3*6*7*13*19*26 As an extension ofour report of 1986,23 this paper presents results on die for mation of a "Fibroblast proliferation factor" in human monocyte/macrophage cultures following incubation with quartz dust DQ 12 and coal mine dusts TF-1 from die Ruhrarea (FRG). MATERIALS AND METHODS Cell Cultures Isolation ofhuman monocytes from peripheral blood in FicollHypaque gradient and cultivation ofmonocytes to maturation ofcells with characteristics ofmacrophages has already been described in detail elsewhere.22 The cell line FH-3 (human embryonal skin fibroblasts) and the cell line (FH-27) (human embryonal lung fibroblasts) were obtained from Biochrom, Berlin (FRG). The cell line WI-38 (human embryonal lung fibroblasts) were purchased from Flow Laboratories, Meckenheim, FRG and the cell line MRHF (human foreskin dermal fibroblasts) from Api-BioMerieux Niirtingen, FRG. The cell lines of human lung and dermal fibroblasts were cultured in Dulbecco's modified Minimum essential medium with 10% foetal calf serum and antibiotics (Pencillin 100 I.U/ml, Streptomycin 100 pg/ml). Mine Dust and Control Dust The TF-1 dust, fraction BAT-II from die Ruhr region was kindly supplied by the Hauptstelle for Staubbekampfung und 926 Pneumokonioseverhutung des Steinkohlenbergbauvereins in Essen. This is a mine dust with a high mineral content of 95 wt % and a quartz content of 10.6 wt %, with a particle size distribution of 0.5-2.5 pm.20 Quartz dust DQ 12 was used as the toxic dust (positive con trol). This is Dorentruper crystall quartz flour (grinding no. 12) with a particle size 5 pm. Preparation ofSupernatants from Cultures of Human Monocytes/Macrophages Dust samples were suspended in Iscove medium or RPMI-1640 medium without an addition or with 1 % newborn calf serum. The samples were subjected to ultrasonic treat ment (Sonifier B-12 from Branson Sonic Power Company, USA) in order to achieve a uniform distribution of the par ticles and to destroy germs. Suspended dust samples in con centrations as described under "Results" were added to cultures ofhuman monocytes/macrophages. After an incuba tion period of24 hours at 37C, the culture supernatants were centrifuged for 15 min at 3,000 rpm and then filtered through Millipore filters (pore size 0.45 pm). The supernatants were then deep frozen at -20C until used. Chemicals and Equipment Fibroblast growth factor (FGF) was purchased from Sigma, Munich (FRG) and Boehringer, Mannheim (FRG), Platelet derived growth factor (PDGF), porcine, Speywood Laboratories was obtained from Sebak Company, Aidenbach, FRG. Ultrafree PF Filter Units of 10.000 and 30.000 NMWL and Centrifugal Ultrafree Filter Units of 10.000 and 30.000 NMWL were commercially available from Millipore, Eschbom, FRG. Determination of Cell Growth of Human Dermal and Lung Fibroblasts Human fibroblast cell lines (FH-3, MRHF, FH-27, WI-38) were detached with a trypsin-Versene mixture and adjusted to a cell count of 4-6 x 104 cells/ml in Dulbecco's MEM with 1 % foetal calfserum or with 10% serum (2% foetal and 8% newborn calf serum), corresponding to dermal and lung fibroblasts, respectively. 1 ml ofthis cell suspension per well was transferred to a tissue culture plate with 24 wells (Falcon 3047 MultiWell tissue culture plate) or to LAB-Tissue Culture Chamber (4 chamber, LT-4804). 24 hours later cell cultures were re-fed with Dulbecco's MEM with 0.5% foetal calf serum or with 0.15% bovine serum albumin (Boehringer Mannheim, FRG) and kept for 2-4 days to obtain "quiescent" cultures. The macrophage supernatants were added in an amount of0.2 ml per well or chamber. In each case 4 cultures were used per measurement point. The protein determination according to Lowry in the modification of Oyama and Eagle18 was carried out as already described earlier.21 A pro tein calibration curve was plotted with "pure" bovine serum albumin from Serva, Heidelberg (FRG). For morphological evaluation ofcell cultures by light microscopy we used a stan dard procedure of fixation and staining with Bouin's solution and hematoxylin-eosin or with methanol and Giemsa. Statistical Analysis For statistical analysis data were computerized and mean values and limits ofconfidence were determined. Futhennore, Bartlett test for equal variances, one-way analysis ofvariance and Students t-test were performed. RESULTS Human fibroblast cultures, "quiescent" or showing only slight cell replication were used to quantify the "fibroblast proliferation activity" of supernatants from treated and un treated human macrophage cultures. The results of such an experimental set-up are shown on Figure 1. Human macrophages were cultivated for 7 days. Thereafter cells were treated with quartz dust DQ12 for 24 hours in Iscove medium without serum at a concentration of30 pg/ml per approximate ly 1 x 106 cells. Supernatants were collected as outlined in Materials and Methods. To cell cultures of human dermal fibroblasts (FH-3) supernatants ofuntreated and quartz dust DQ 12 treated macrophages were added. After a culture period of 6-8 days on an average, the protein content of the cultures was determined by the method of Lowry in the modification of Oyama and Eagle.18 Column 1 (Figure 1) shows an untreated FH-3 fibroblast culture. The amount of Taxiciry/Surface Characterization III protein in /tg/ml is given as a criterion of cell growth. Col umn 2 illustrates the cell growth of the fibroblast culture to which die supernatants of the untreated macrophage culture had been added. No increase in cell growth is seen in com parison with the control. Column 3-6 (Figure 1) represent the growth ofhuman dermal fibroblast culture FH-3 to which die supernatant of a macrophage culture treated with 30 jig/ml DQ 12 had been added in various dilutions. The potent "proliferation-stimulating activity" of this supernatant led nearly to doubling of the protein content of these cultures at a dilution of 1:5 in the course of 7 days in comparison with the control (Column 3). But also at higher dilutions of 1:10 up to 1:40 die "proliferation-stimulating activity" of this supernatant can be seen (Column 4-6). The "proliferation-stimulating activity" of supernatants of human macrophages (age 7 days) to which die coal mine dust TF-1, fraction BAT-IIhad been added for 24 hours was tested in further experiments. For this purpose, supernatants ofun treated human macrophage cultures and of those treated with coal mine dust TF-1 (30 pg/ml, 24 hours) were added to human fibroblast cultures (line FH-3) and cultured at 37C for 7 days. The results are shown on Figure 2. Column 1 represents the cell control, column 2 shows the cell growth ofthe fibroblasts after addition ofthe supernatant ofuntreated macrophages. Column 3-6 (Figure 2) represent the cell growth following addition of supernatant of macrophages treated with coal mine dust TF-1. In comparison with die cell and macrophage control, there is a significant increase in cell growth ofthe human dermal fibroblasts FH-3 which had been incubated with die supernatant of macrophages treated with coal mine dust TF-1. A similar "proliferation-stimulating ef fect" was observed with human diploid lung fibroblasts WI-38 treated with supernatants from another batch of human macrophages incubated with coal mine dust TF-1 (30 /ig/ml, 24 hours). We made further attempts to characterize die fac tor produced by quartz and coal mine dust exposed human macrophages. We found that the factor is still active after Figure 1. Cell growth ofhuman fibroplasts ofcell line FH-3 following incubation with the supernatant ofan un treated (column 2) human macrophage culture and one incubated with quartz dust DQ 12 (column 3-6) in various dilutions. Mean values and con fidence limits are shown. Figure 2. Cell growth ofhuman fibroplasts ofcell line FH-3 following incubation with the supernatant ofan un treated (column 2) and one treated with coal mine dust TF-1, BAT-II (column 3-6) in various dilu tions. Mean values and limits of confidence are presented. 927 ToMdty/Surface Characterization III incubating at S6C for 60 min. For estimation ofthe approx imate molecular weight ofthe factor we utilized Ultrafree PF Filter Units (Millipore) of 10.000 and 30.000 NMWL (nominal molecular weight limits) and corresponding Cen trifugal Ultrafree Filter Units (Millipore) of 10.000 and 30.000 NMWL. Results based on induced cell multiplication of non-replicating MRHF and FH-3 fibroblasts and on stimulation ofDNA synthesis ofWI-38 cells by the superna tant ofquartz dust DQ12 exposed human macrophages larger and smaller than 10.000 and 30.000 NMWL indicate a molecular weight ofdie factor of more than 30 KDa. In fur ther experiments we incubated non-replicating WI-38 cells for various lengths oftime (2,4,16 and 24 h, 7 days) with super natants ofquartz dust DQ 12 exposed macrophages. Results revealed that a continuous presence ofdie factor is necessary for cell replication. This assumption is also supported by measuring the number of DNA synthesizing cells of WI-38 cells exposed for various lengths oftime to supernatant. On ly continuous presence of the factor led to a high number of DNA synthesizing cells.10 After removal of the factor die DNA synthesis ceased very rapidly. To elucidate the nature ofthe "fibroblast proliferation factor" we performed "com plementation tests" according to Stiles et al.25 and Bitterman et al.3 Addition of fibroblast growth factor or of platelet derived growth factor (PDGF) to no replicating WI-38 or MRHF-cells enhanced the cell growth significantly in presence of supernatants ofquartz dust DQ 12-treated human macrophages. Similar results were obtained with WI-38 cells in presence of supernatant of coal mine dust TF-1 treated human macrophages. Addition of FGF or PDGF led to a remarkable enhancement of cell multiplication. DISCUSSION AND CONCLUSION Results presented demonstrate that human macrophages in culture obtained by cultivation and differentiation of blood monocytes, form a soluble factors) following incubation with quartz dust DQ 12 or coal mine dust TF-1. This factor stimulates "quiescent" or only moderately replicating human lung and dermal fibroblasts to a considerable cell multiplica tion. Therefore, we designated the "factor" as "fibroblast proliferation factor" (FPF). As we earlier reported,23'24 foe process of fibroblast multi plication stimulated by FPF could also be visualized by mor phological criteria, such as increased rate of DNA synthesis10 and of mitosis and by manifestation of high cell density of cultures. The "dual control model of growth regulation" suggests25 that growth factors can be classified either as "competence factors" or as "progression factors." While cells require only transient exposure to "competence factors," i.e. PDGF, FGF,"progression factors" are required continuously for DNA synthesis and cell replication. Results suggest a classification ofFPF as a "progression factor" because a con tinuous exposure was required for cell multiplication of fibroblasts and in view ofan enhanced growth after addition ofFGF or PDGF in a serum-free medium "complementation test." Bitterman et al.3 reported that human alveolar macrophages obtained by lung lavage and incubated in vitro with soluble 928 and particulate agents, release an alveolar macrophage derived growth factor (AMDGF), exhibiting activity as a "progres sion factor" for human lung fibroblasts. The molecular weight of AMDGF of 18 kDa differs from foe MW of FPF, preliminary estimated as more than 30 kDa. Thermal stabili ty ofFPF (56C, 60 min) is resembling stability ofan alveolar macrophage factor from silica instilled rats, isolated by Ben son et al.,2 inducing elevated DNA synthesis of rat lung fibroblasts. Beside FPF in supernatants of quartz DQ 12 or coal mine dust TF-1 treated human monocytes/macrophages a "Granulocyte activating Mediator" (GRAM) was detected14*15 causing a long lasting Lucigenin-dependent chemiluminescence of human granulocytes. In several studies "fibroblast growth factors" have been described, which were induced in cultures of human monocytes and macrophages by various soluble and par ticulate agents, i.e. by zymosan, phytohemagglutinine, concanavalin A, endotoxin, immune complexes, staphylococci and quartz dust (Bitterman et al.,3 Dohlman et al.,6 Glenn and Ross,7 Leslie et al.,13 Schmidt et al.,19 Seemayer et al.23*24 The growth stimulation of fibroblasts by activated or damaged macrophages is ofgreat importance for fibrotic lung processes especially silicosis and appears to be a generally applicable, pafoobiological principle.4 REFERENCES 1. Aalto, M., Kukmen, E., Ronneraaa, T., Sundstrom, C., Vilpo, J.: Liberation ofa fibrogenic factor from human blood monocytes, ascites cells, cultures histocytes and transformed mouse macrophages by treat ment with S02- Scand. J. Clin. Lab. Invest. 40:311-318 (1980). 2. Benson. S.C., Belton. J.S.. Scheve, L.G.: Regulation nftnng fibroblast proliferation and protein synthesis by broncboalveolar lavage in ex perimental silicosis. Environ. Res. 41:61-78 (1986). 3. Bitterman, P.B., Rennard, S.I., Hunningbake, G.W.: Human alveolar macrophage growth factor for fibroblasts. J. din. Invest. 70:806-822 (1982). 4. Bowden, D.H.: The alveolar macrophage. Environ. Health Persp. 55:327-341 (1984). 5. Burrell, R., Anderson, M.: The induction offibrogeoesis by silica-treated alveolar macrophages. Environ. Res. 6:389-391 (1973). 6. Dohlman, J.G., Payan, D.G., Goetzl, E.J.: Generation of a unique fibroblast-activating factor by human monocytes. Immunology 52:577-584 (1984). 7. Glenn, K.C., Ross, R.: Human monocyte-derived growth factorfs) for mesenchymal cells: Activation of secretion by endotoxin and coocanavalin A. Cell 25:603-615 (1981). 8. Harrington, J.S., Ritchie, M., King, P.C., Miller, K.: The in vitro ef fects of silica treated hamster macrophages on collagen production by hamster fibroblasts. J. Pathol. 109:21-37 (1973). 9. Heppleston, A-G., Styles, J.A.: Activity ofmacrophage factor in col lagen formation by silica. Nature 214:521-522 (1967). 10. Hiibner, K., Seemayer, N.H.: Sdnwlation ofDNA synthesis ofhuman diploid lung fibroblasts by mediators from quartz and coal mine dust treated human macrophages fin preparation). 11. Kilroe-Smith, T.A., Webster, I., van Drimmelen, M.,Marases, L.:An insoluble fibrogenic factor in macrophages from guinea pigs exposed to silica. Environ. Res. 6:290-305 (1973). 12. Kulonen, E., Aalto, M., Abo, S., Lehtinen, P., Podia, M.: The SK)2-liberated fibrogenic macrophage factors with reference to RNA. In: The In Vitro Effects ofMineral Dusts. Ed. by R.C. Brown, M. Chamberlain, R.Davies and I.P.Gormley. London: Acadmic Press 1980, pp. 282-287. 13. Leslie,C.C.,Musson,R^.,Henson,P.M.:Productionofgrowthfactor activity for fibroblasts by human monocyte-derived macrophages. J. Leukocyte Biol. 36:143-159(1984). 14. Maly, E., Braumann, A., Seemayer, N.H., Maly, F.E.: Induction of chemiluminescence of human granulocytes by supernatants of human mononuclear cultures after treatment with quartz dust DQ 12. in: Biochemiluminescence and Chemiluminescence, New Perspectives ed. by Scholmerich R. et al. J.Wiley & Sons, Chichester-New York 1987, pp 121-124. 15. Maly, E., Seemayer, N.H., Braumann, A., Behrendt, H., Maoojlovic, N., Maly, F.E.: The significance of immunomodulative cytokines for the development of silicosis. I. Biological characterisation of a granulocyte activating mediator from human monocytes/macrophages treated with quartz dust DQ 12. Nordrhein-Westfalen Silicosis Report Vol. 16:157-170(1987). 16. Nathan, C.F. Mai.: The macrophage as an effector cell. New England J. Med. 303:622-626 (1980). 17. Nourse, L.D., Nourse, P.N., Botes, H., Schwartz, H.M.: The effects ofmacrophages isolated from lungs ofguinea pigs dusted with silica on collagen biosynthesis by guinea pig fibroblasts in cell culture. Environ. Res. 9:115-127(1975). 18. Oyama, V.J., Eagle, H: Measurement of ceil growth in tissue culture with aphenol reagent (folin-ciocalteau). Proc. Soc. Exp. Biol 91:305-307 (1956). 19. Schmidt, J.A., Oliver, C.N., Lepe-Zuniga, J.L., Green, I., Gery, I.: Silica-stimulated monocytes release fibroblast proliferation factors iden tical to interleukin 1. A potential role for interleukin 1 inthe pathogenesis of silicosis. /. din. Invest. 73:1462-1472(1984). 20. Seemayer, N.H.: Importance ofgrain size and mineral content ofcoal mine dusts for cytotoxicity on macrophages in vitro. In: In Vitro Effects Toxicity/Surface Characterization 111 ofMineral Dusts Ed. by E.G. Beck and J. Bignon Springer-Verlag Berlin Heidelberg 1985, p. 497-504. 21. Seemayer, N.H., Manojlovic, N.: Cytotoxic effects ofair pollutants on mammalian cells in vitro. Toxicology 17:177-182 (1980). 22. Seemayer, N.H., Braumann, A.: Untersuchung uber die zytotoxische Wirkung von Quarz- und Grubenstauben aufmenschliche Makrophagen in vitro. In: Erqeb. Unters. Geb. Staub- u. Silikosebekampfimg im Steinkohlenbergbau. Nordrhein-Westfalen Silicosis Report, Vol 15, Essen: Steinkohlenbergbauverein 1985, p. 301-320. 23. Seemayer, N.H., Braumann, A., Maly, E., Behrendt, H., Maly, F.: Induction of fibroblast proliferation by supernatants from human monocytes/macrophages in vitrotreated with quartz dust DQ 12 and coal mine dust TF-1. Zbl. Bakt. Hyg. B 184:431 (1987). 24. Seemayer, N.H., Braumann, A., Maly, E.: Development of an "in vitro" test system with human macrophages and fibroblasts for analysis of the effect of quartz dusts and coal mine dusts. I. Formation of a fibroblast proliferation factor. Nordrhein-Westfalen SilicosisReportVol. 16:143-156 (1987). 25. Stiles, CD. Mai.: Dual growth control ofcell growth by somatomedins and platelet-derived growth factor Proc. Nat. Acad. Sci. 76:1279-1283 (1979). 26. Wahl, S.M.: The role oflymphokines and monokines in fibrosis. Ann. N.Y. Acad. Sci. 460:224-231 (1985). JNew address: Department ofAnatomy, The University ofArizona, Tucson, AZ 85724. 929 Toxicity/Surface Characterization III CISE OF A SENSITIVE ELECTRO-OPTICAL METHOD TO QUANTIFY SUPEROXIDE PRODUCTION FROM SINGLE PULMONARY ALVEOLAR MACROPHAGES EXPOSED TO DUSTS IN VITRO OR IN VIVO: SOME CURRENT EXPERIMENTAL AND MODEL RESULTS EUGENE V. CILENTO, Ph.D. * K. A. DiGregorio, Ph.D. R. Clark Lantz, Ph.D.1 Departments of Chemical Engineering and Anatomy, 413 Engineering Sciences, West Virginia University, Morgantown, WV 26506-6101, USA ABSTRACT This laboratory has developed a sensitive electro-optical method to quantify the initial rate (R) and total amount (MAX) of superoxide (O2) produced by single pulmonary alveolar macrophages (PAM). The method uses a microscope-based TV system to visualize PAM in culture, and to video record die images during the time die cells produce Of. MAX and R are calculated from measurement of temporal changes in optical density in the images due to precipitated diformazan formed by die reaction of Of, produced by each PAM, with nitroblue tetrazolium present in the culture medium. To date, values ofR and MAX, measured due to adherence of PAM to the dish, have been compared to values obtained when quartz, coal mine dust (CMD), and kaolin were added to the medium (in vitro). R and MAX have also been calculated for PAM lavaged from animals exposed to quartz and CMD in die WVU Inhalation Facility. Presently, experiments are being done using serum which will permit PAM to be restimulated by different dusts to help establish a dose response relation ship and a means to study the role ofhing surfactant cm modifying the acute toxicity ofinhaled duks. Ultimately, this methodology should provide useful insight into establishing die role of Of in PAM dysfunction due to inhalation of dusts, and in lung diseases such as pneumoconioses. INTRODUCTION PAM are free cells found in the lungs which protect die lungs by removing foreign debris and bacteria. This is accom plished, in part, by the process of phagocytosis, whereby foreign matter is internalized into vesicles known as primary phagosomes. Phagocytosis also involves the chemical breakdown of inhaled dusts and die killing of microbes. Detoxification is aided by the respiratory burst, which is a metabolic response of the cell to foreign substances, that results in die production of highly reactive oxidizing agents from the partial reduction ofextracellular oxygen to superox ide (Of) at the surface ofthe plasma membrane. Superoxide undergoes either spontaneous or enzyme catalyzed dismutation reactions to form hydrogen peroxide (H2O2) and subse quently hydroxyl radical (OH) and singlet oxygen (O21). However, while these oxygen metabolites aid in the killing ofmicrobes they also may destroy endogenous tissue. For ex ample, Of has been linked to die aging process and to many diseases including emphysema, diabetes, and cancer.4 Therefore, a better understanding ofdie production ofOf is extremely important since an abnormally low production could result in damage by inhaled dusts and bacteria while an abnormally high production could result in direct damage to the lung tissue by the phagocytes. Inhalation of respirable sized mineral dusts, such as quartz (silica), coal mine dusts (CMD), and asbestos, results in variouspulmonary disorders. PAM are thought to play an im portant role since evidence suggests that the first step in 930 fibrogenesis is an interaction of dust particles with PAM. Generally, in vivo exposure ofanimals to mineral dusts results in increased respiratory burst activity, migratory patterns, phagocytic behavior, and secretory potential ofPAM, and for this reason these have been implicated to be pivotal events in the pathogenesis of pulmonary diseases.1 In addition to stimulation ofresident PAM, an influx ofmonocytes into die lung and/or production ofnew macrophages generally occurs in response to fibrogenic dust inhalation. Further, PAM may attract and stimulate fibroblasts, which normally synthesize proteins and collagen during repair of tissue, by secreting chemoattractants and enzymes. Thus, PAM may be involved in pulmonary disorders through failure or partial loss oftheir defensive capability, or indirectly, through release of other mediators. The major objective of this project is to develop a better understanding for die role Of production by PAM plays in the development ofpulmonary disease following exposure to inhaled dusts. Specifically, it is not understood whether cer tain dusts (or constituents of dusts), inhaled over a period of time, can alter die ability of this cell-type to remove foreign material from the lung. Also, it is not known whether dysfunc tion of PAM occurs which may result in an underproduction or an overproduction of Of; either of which may be harm ful to normal lung tissue. A novel technique has been developed to quantify Of pro duction by single PAM which has permitted study of the effects of different dusts, concentrations ofdust, and time of exposure on Of release by PAM. A multifaceted approach has been used which encompasses the following: (1) study of Of production by single PAM isolated in culture, when con tacted directly with different concentrations ofdusts suspend ed in the medium (in vitro); (2) study of Of production by PAM after in vivo exposure to dusts in inhalation chambers for known periods of time; and (3) development of a mathematical model to describe the kinetics ofproduction by single cells. These studies ultimately will provide a rapid, quantitative assay to determine die effect oftoxic dusts on Of production by PAM. METHODOLOGY The methodology for measuring Of production from single PAM using NBT reduction was developed previously.2,3 Briefly, 3 ml of NBT solution (37 C) was placed in culture dishes and then 0.4 ml of a cell suspension (2-5 x 105 PAM) added. A layer ofparaffin oil was placed on top ofthe aqueous layer. The dish was placed on a temperature controlled stage of an inverted microscope and trans-illuminated at 550 nm. In die presence of a strong reducing agent, such as Of pro duced due to PAM adherence to die culture dish, soluble NBT is reduced to a diformazan precipitate which can be measured spectrophotometrically. PAM were visualized (20x) and im ages, containing at least 6 well-separated PAM, were televised and video recorded for 40 min. Recorded images were played back through electronic instrumentation which permitted determination ofoptical density (OD) changes for individual cells due to precipitated diformazan. Hie OD was converted to the mass ofdiformazan produced versus time, from which MAX was determined directly, and then the data fit to a phenomenological equation from which R was calculated. RESULTS In Vitro Experiments The effects of acute in vitro exposure to respirable quartz and kaolin on Of production during adherence of PAM to culture dishes were tested using low (0.025 mg/ml) and high (0.05 mg/ml) dust concentrations. The low dose ofquartz decreas ed the maximum amount of Of produced (MAX) 38 % com pared to control while die high dose did not. However, the maximum rate ofdiformazan production, R, decreased 31 % and 24% for the low and high dose, respectively. In vitro ex posure of PAM to the high dose of sonicated CMD suspen sions resulted in increased Of production. In contrast, kaolin, a non-fibrogenic dust did not significantly change either MAX or R. These results suggested that Of production may be a better indicator of pathogenicity and PAM dysfunction than cell death, which gives comparable results for quartz and kaolin. Interestingly, no changes in Of production were observed following in vitro exposure of PAM to quartz, kaolin, or CMD in die presence of a surfactant (tween 80), suggesting that lung surfactant may alter the acute toxicity of dusts. Previously, PAM could not be restimulated after adherence. However, it has been recently shown that Of production from Toxicity/Surface Characterization III adherent PAM is possible when cells are incubated with serum.3 Serum alone did not stimulate adherent PAM in dicating serum is necessary but not sufficient for stimulation. This finding supports the idea that in vivo serum may condi tion PAM to produce Of. In Vivo Experiments The effects ofin vivo exposure ofanimals to respirable quartz (20 mg/m3, 16 hr/d, 5 d/wk of MIN-U-SIL 10, 95% <5 ftm) was tested by housing animals (2-4 weeks) in die WVU inhalation facilities. Control (no quartz) animals also were kept in identical inhalation chambers for 2-4 weeks. Follow ing exposure, animals were removed from the inhalation' chambers and housed in animal-care facilities for 3, 10, or 31 days post-exposure. This approach permitted analysis of the effects oflength ofin vivo exposure and post-exposure time on PAM analyzed for Of production. Overall, respirable quartz increased MAX 36% and R 29% compared to control animals. Importantly, PAM from exposed animals showed an increased Of production for up to 10 days after 2-4 weeks ofexposure followed by a return to control levels by 31 days. Interestingly, the 3 day group suggested that there was activa tion and/or recruitment ofPAM. In vivo exposures performed using CMD (20 mg/m3,16 hr/d, 7 d/wk of Pittsburgh BOM Dust 2020,100 % 4-6 urn) also showed increased production at 10 days post-exposure, with large amounts of CMD phagocytosed by 31 days. Similarly, the data showed an ac tivation and/or recruitment of PAM. Theoretical Model Development A kinetic model was developed to describe the production of Of by single PAM. The kinetic model considered three reac tions: (1) the production of extracellular Of from the reduc tion of oxygen by NADPH oxidase using intracellular NADPH as the substrate, (2) the subsequent dismutation of Of to form H2O2, and (3) die reaction ofOf and NBT. NBT specificity of Of was analyzed by comparing experimental results, in the presence and absence of superoxide dismutase (SOD) which catalyzes the dismutation of Of to H2O2. Measured PAM heterogeneity (without SOD) was accounted for in the model by varying the concentration ofintracellular NADPH, its rate of depletion, and the concentration of NADPH oxidase. Model predictions compared well with ex perimental results except when SOD was present. Ex periments showed only a 50-60% decrease in diformazan pro duction using SOD. This discrepancy may be due to diffu sions! limitations which occur since SOD is a much larger molecule (34 kD) compared to NBT (818 D). In addition, die cell surface is both ruffled and negatively charged, which may introduce steric hindrances and/or electrostatic effects since SOD is also negatively charged. CONCLUSIONS In vitro assays on large numbers of cells in culture using hemolysis ofred blood cells or release ofenzymes from PAM following dust exposure have been used to analyze cytotox icity. In such systems, kaolin has been found to have an ac tivity comparable to quartz on a mass basis. However, in vivo. 931 Toxicity/Surface Characterization Ul quartz is highly fibrogenic resulting in silicosis while kaolin is not. Therefore, the assay results do not correlate with the in vivo effects of quartz and kaolin. However, in this study, in vitro exposure to kaolin did not significantly alter Of pro duction compared to control PAM. The results obtained cor relate more closely with in vivo exposure effects and help sup port the usefulness of this quantitative superoxide assay in evaluating the effects of respirable dusts on PAM. This sug gests Of production may play an important role in silicosis and other respiratory diseases. In addition, results from ex periments with surfactant support the concept that lung sur factant may alter the toxicity of dusts inhaled into die lung. Interestingly, in vivo exposure to quartz resulted in increased Of production rather than the decrease observed in vitro. While this difference may be due to a change in cellular func tion from the in vivo to the in vitro environment, it is also possible the in vitro response may be due to an initial (acute) response produced immediately after contacting the cells with dusts. In contrast, the in vivo responses may have resulted from die longer contact time since animals were exposed 2-4 weeks followed by a 3-31 day post-exposure period before Of analysis. Thus, it may be that quartz causes an initial injury to PAM resulting in decreased Of production followed by recruitment or activation of PAM having increased produc tion capabilities. This is supported by the foct that PAM analyzed 3 days post-exposure exhibited a wider range in Of production than the control or 10 and 31 day groups. Specifically, perhaps two populations of PAM are present: (1) cells injured by initial or long-term dust contact, resulting in decreased Of production release; and (2) recruited or ac tivated PAM with an increased Of production. In summary, a sensitive, quantitative assay to study individual PAM function related to Of production has been developed which shows that respirable dusts do affect Of release by PAM. The invitro results provide a basis for quantifying die acute effects ofdust-cell contact (or constituents ofdusts) on Of release by PAM. Importantly, the addition of serum to the culture medium permits restimulation ofthe same cell by different dusts after adherence ofthe cell to the culture dish. The in vivo results provide a basis for critically examining the effects of long-term exposure to airborne dusts on Of pro duction by PAM, and continued refinement of the methodology will provide a means to assess and improve pre sent understanding ofthe phagocytosis process in health and disease. Based on the results and conclusions obtained to date, con tinuing work is focused on several specific objectives. Ex perimental work is designed to critically examine the effects of repeated exposure to different dusts (and concentrations) on the ability ofthe same cell to produce and release Of. Con comitantly, the ability to maintain animals for long periods of time in die inhalation chambers will permit evaluation of the effects of chronic exposure to dusts. This approach has the potential to provide information not possible using popula tion measurements and to critically assess and ultimately lead to improved clinical therapies for treatment of pulmonary disorders. REFERENCES 1. Brain, J. D. Macrophage Damage in Relationto the Pathogenesis ofLung Diseases. Environ. Health Perspect. 35:21-28, (1980). 2. DiGregorio, K. A., Cilento, E.V., and Lantz, R. C.: Measurement of Superoxide Rdease from Single Pulmonary Alveolar Macrophages. Am. J. Physiol 252:0677-83 (1987). 3. DiGregorio, K.A.: Studies of Superoxide Production firm Single Pulmonary Alveolar Macrophages. West Virginia University, Ph.D. dissertation. Morgantown, WV (1988). 4. HalliweU, B. Oxygen is Poisonous: The Nature and Medical Importance ofOxygen radicals. Med. Lab. Sci. 41:157-171 (1984). ACKNOWLEDGEMENTS: The authors wish to acknowledge the sup port ofthe Department ofthe Interior's Mineral Institute Program ad ministered by the Bureau of Mines through the Generic Mineral Technology Cotter for Respirable Dust (G1135142). 932 Toxicity/Surface Characterization III AN ATTEMPT FOR EVALUATION OF THE ALVEOLAR DUST DEPOSITION ON THE BASE OF THE PARTICLE SIZE DISTRIBUTIONS OF LUNG DUSTS STOYNAIVANOVA-DJOtIBRILOVA, M.Sc. Petrana Tchemeva, M.Sc. Institute of Hygiene and Occupational Health, Sofia, Bulgaria INTRODUCTION The experimental and theoretical studies of die way human lungs behave at different size distributions ofthe offered dusts and form the alveolar depot--the major determinant of die development of lung diseases, did not offer completely de fined results. Regardless of the recent advances in this field, such as die derived curves ofextrathoracic, tracheobronchial and alveolar deposition of inhaled particles and the lot of unified parameters, the criteria for respirable dust sampling and assessment of dust hazards etc.9 many problems did not find satisfactory answers. The progress oftechnics and electronics eliminates the mass of difficulties in die particle size analysis and in the com plicated calculations ofthe selective deposition of particles in the respiratory tract. Presently, the most important problems derive from the im possibility to characterize precisely the entire dust inhaled by workers during their lifetime. The experimental studies in vivo on humans and animals, on models of the respiratory tract, as well as mathematical models providing a precise but valid information for the moment,6 are unable to assess yet die long-term retention of dust in the lungs as a response of the inhaled particles. A very specious solution of these problems seems to be die study ofdusts recovered post mortem from the lung tissue of dust-exposed workers and to compare them with die airborne dusts offered to die respiratory tract.Of course, a method of approach like this also has its disadvantages mainly in the assessment ofthe inhaled, resp. airborne dust. But it is possi ble after analyzing a significant number of airborne dust samples, collected in the work environment during a long period oftime and including all technological processes used on the areas where employees had worked, to obtain a more or less rough approximation to reality. So, the respiratory tract with its special features and intersubject variability depending on individuals and experimental design, can be considered as a "black box*' with known "inlet" and "oudet." Many authors have used lung dusts in their investigations and a lot of them support the reliability of the information received about the alveolar deposition of airborne particles.2,5*8 Another important limitation ofthis experimental design can be the impossibility for assessment of particle aggregations and the kinetic behaviour of aggregates. Possible errors can be eliminated to some extent by analyzing fully dispersed samples ofairborne and lung dusts using die same method for particle analysis.2 When the results from different studies of dust deposition in the respiratory tract are to be compared, it is particularly im portant to bear in mind that the main curves used nowadays as criteria for dust hazard evaluation9 are derived with monodispersed aerosols, while the determination ofalveolar dust deposition with lung dust studies (representing a long term retention of dust in lungs) is based on the inhalation of polydisperse aerosols. Diu and Yu3 proposed a new mathematical model of polydisperse aerosol deposition in human respiratory tract, proving more or less considerable differences varying with the polydispersity. The model needs a validation on ex perimental studies. Investigation oflung dusts should be con venient for this purpose. The mass particle size distributions of47 samples oftotal air borne dust in the work environment ofthree mines--24 from a polymetal ore mine (PMM), 18 from copper mine A (CMA) and 5 from copper mine B (CMB) were determined. Full-shift sampling was performed at normal passing technological pro cesses according to the actual standard in the country.1 Samples were collected in different intervals of time during 10 years. Fifty-nine dusts, recovered post mortem by the formamid method of Thomas and Stegemann12 from the lungs of miners with entire length of service in the same mine, were analyzed for determination of the particle size distributions. Forty three of them were from the PMM group, 11--from CMA and 5--from CMB groups. The particle size analyses were performed by two methods based on different principles: centrifugal sedimentation in Joyce-Loebl disc centrifuge4 analyzing particles in class in tervals of Stokes diameters 0.01-20 ptm and automatic count ing ofparticles in liquid media with Coulter Counter (CC) us ing tube aperture 50 pm, analyzing particles in class intervals of UDS diameter 0.7-25 pin. Dust samples were suspended in filtrated 0.1% solution of sodium hexametaphosphate in distillate water. A lot ofthe analyses were performed with die same suspension. The Stokes particle size distributions were calculated in aerodynamic diameters using the density data for every dust. Particle size distributions were plotted on logprobability graph paper. Mean values of the groups of airborne and lung dusts from die three mines were calculated, as well as the standard devia tions and die confidence limits intervals. The standard devia tions ofthe groups ofairborne and lung dust from every mine 933 Toxicity/Surface Characteriz/rrion U! were compared statistically by means ofthe Fischer criterion. The alveolar deposition was determined by die method described by Leiteritz, Einbrodt and Klcsterkotter.5 The enrichment offine sizes ofeach fraction ofthe lung dust par ticle size distribution was calculated as a ratio of die corre sponding airborne dust fraction. This is die so-called enrich ment factor. The relative alveolar deposition ofeach fraction is die quotient for the enrichment factors and the maximum enrichment factor which is taken to be = 1. RESULTS AND DISCUSSION Particle Size Distributions of Airborne and Lung Dusts It was found that the mean values of die mass median aerodynamic diameters of die particle size distributions (MMAD) of die three groups of mine airborne dusts are in die class intervals 3-5 pm, analyzed by both methods. The mean geometric standard deviations (og) ofdie groups were varying from 4.4 to 5.3 for Joyce analysis and from 2.2 to 2.3 for Coulter Counter analysis. The airborne dust samples analyzed with Joyce were reduced to 5 combined samples for each mine, because the analysis needs about 50 mg of dust. The mean value of MMAD of the particle size distributions of lung dusts ofdie three groups ofminers was varying from 1.9 to 2.6 pm by both analyses and og--from 3.3 to 3.6 and from 1.6 to 1.8 by Joyce and CC analyses, respectively. The maximum frequency percentage of die particle size distributions, as well as die other data about diem are presented in Table I. Relative Alveolar Deposition It was found that the maximum enrichment factors for the 3 groups of airborne and lung dusts are in die class interval 1.2-2.4pm. The mean values for each size range of die enrichment factor were calculated and a curve ofthe relative alveolar deposition was derived (Figure 1). The theoretical Figure 1. Curves of relative regional alveolar retention of airborne dusts from three ore mines derived on die basis of particle size distribution of lung dusts. LEGEND Mean retention curve derived by Joyce analysis Mean retention curve derived by CC analysis Deposition curve of ICRP9 Predictive deposition curve of Yu13 934 Toxicity/Surface Characterization III Table I Characteristics of the Mass Particle Size Distributions of Airborne Dusts and Lung Dusts of Miners from Three Ore Mines Analyzed with Joyce-Loebl Disc Centrifuge and Coulter Counter Mine Para meters Airborne dusts Joyce Coulter Lung dusts Joyce Coulter PMM Mean MMAD, Jim 3.30 3,95 Confid. 1 im. jim <rg - 3.01 5.30 2.45 2.08 Confid 1 im. - 2.68 0.66 Anal.No 5 24 Max.fr.% 32 37 Int. ,jim 1.6-6. 5 3.2-6.5 CMA Mean MMAD, Jim 2.90 3.74 Confid. 1 im. jim - 2.49 + 2.48 <T9 Confid. 1 im 5.00 - 2.66 2.00 + 0.71 Analirio 5 18 Max.fr % 34 36 Int;jim 1,6-6. 5 3.2-6.5 CMB Mean MMAD 3.92 4.82 Confid. 1 im. jim erg ~ 3.61 4.40 3.45 2.10 Conf.1im. 2.61 - 1.01 Anal.No 5 5 Max.fr.% 36 37 Int; jum 1.6-6. 5 3.2-6.5 2.13 2.49 - 1.60 3.60 - 1.50 1.66 - 2.26 43 23 1.6-3.2 0.41 43 53 1.6-3.2 1.86 2.31 - 1.70 1.08 2.35 1.83 1.52 - 0.51 11 42 0.8-3.2 11 44 1.6-3.2 2.53 2.57 - 3.48 3.60 2.57 5 21 1.6-3.2 - 2.34 1.63 - 0.66 5 50 1.6-3*2 935 Toxicity/Surface Characterization III curve of Yu13 and the ICRP curve ofTask Group9 were also plotted on Figure 1 for comparison, recalculated in the same way. The relative alveolar depositions with the minimum and max imum values of airborne and lung dusts of die three mines were calculated separately. The values' dispersion is shown on Figure 1 and the data are presented in Table n. DISCUSSION The data obtained showed some differences in the particle size distributions ofthe same dusts, analyzed by both methods. The MMAD determined by Joyce are finer than those determin ed by CC, while for og the contrary was proved. In principle this was expected because the instrumental limitations ofCC in sizing offine particles (smaller than 0.7 pm) as well as Joyce unreliability in sizing of coarse particles (larger than 15-20 pm) are known. But it is necessary to emphasize the fact for two reasons: on one hand to demonstrate the considerable in fluence on die calculated relative alveolar deposition of die different particle size distributions used, and, on the other hand--to draw die attention ofspecialists on the need ofgood knowledge about the limitations of every method or instru ment used for particle size analysis, when interpretation of data has to be performed. Thereby useless contradictions due to underestimation of the last reason could be avoided. Another fact of interest is die fine dispersity ofairborne dusts (mean MMAD vary from 3 to 5.0 pm, with real maximal value of die groups examined--5.2 pm by CC analysis). In most of the studies known these values are much higher but they concern coal mine dusts.2'5-8 Ore mine dusts are ex pected to be finer and the authors of this paper have many studies in this field.4-10 They have found a considerable presence of submicron fraction ofmineral origin in ore mine airborne dusts, as proved with X-ray analysis,11 the same be ing evidenced for lung dusts, also. The determination of the alveolar dust deposition by using lung dusts and work environment airborne dusts is connected with some preliminary assumptions: the particle sizes are the leading factor for the dust deposition in die respiratory tract; the "inlet" and the "oudet" dusts, e.g. die airborne and lung dusts are representative and reliable; alveolar deposition, the retention, respectively, ofdust is more real when lung dusts are used because die long-term clearance as a total and the changes ofdust parameters after continued staying in lung are included. The data obtained for alveolar deposition (more correctly it should be called alveolar long-term retention) differ to some extent from die referred in die main studies performed-- experimental, calculated and predicted. The maxima of die relative alveolar retentions calculated as mentioned above, are Mine PM Table n Size Range Intervals of die Calculated Maximal Enrichment Factors with the Mean and Real Values of MMAD and og of the Particle Size Distributions of Airborne and Lung Dusts from Three Ore Mines by Joyce and CC Analyses Values of Calculated maximal enrichment factors parameters used Joyce analysis size intervals CC analysis size intervals Bean Min Max 1.6-3.2 pm 0.4-0.8 jum 1.6-3.2 pm 1.6-3.2 jjm 0.4-0.8 jum 3.2-6.5 Jim CMA Mean Min Max 0.8-1.6 Jim 0.1-0.2 Jim 1.6-3.2 _jim 0.8-1.6 Jim 0.2-0.4 jim 0.4-0.8 pm CMB Mean Min Max 0.8-1.6 pm 0.1-0.2 Jim 0.8-1.6 jum 1.6-3.2 Jim 0.8-1.6 pm 1.6-3.3 pm 936 found to be in the same size range intervals as the maxima of the particle size distributions of lung dusts or are removed toward the fine size ranges--Figure 1 and Table II. These results are in correspondence with the mathematical model of Diu and Yu.3 To airborne dust with og significant ly higher than 2 results a different alveolar dust retention in comparison with the deposition curve of Yu,13 predicted for monodispersed aerosols--retention curve calculated with Joyce data on Figure 1. This curve is quite similar in the size range interval 0.5-1.6 /on to the ICRP curve9 derived from a lot of different studies. On the contrary, the CC retention curve does not differ in some ofthe size range intervals con siderably from the curve ofYu. The values of og of airborne dusts analyzed by CC are about 2. Morrow7 affirms that in haled dust with og < 2 have similar deposition in respiratory tract to this of monodispersed aerosols. CONCLUSIONS The experimental curve oflong-term alveolar retention ofin haled dust from the working environment derived on die basis of the particle size distributions of miners' lung dusts from three ore mines proved that the real alveolar deposition and retention is different from the predicted deposition of monodispersed aerosols. The study was performed with a comparatively great number of airborne and lung dust samples and can be regarded as reliable. It is obvious that the polydispersity ofdust samples has more considerable influence on the alveolar deposition and reten tion than it was estimated till now--a problem disregarded to some extent in the experimental studies. Modem technologies and the mechanizing and automation of working processes lead to increasing ofthe polydispersity of airborne dusts and namely ofthe fine particles share. This feet Toxicity/Surface Characterization III inevitably will lead to some changes in the understandings about the evolution of the hazard and imposes more attention to be paid to the problems ofparticle size analyses and the in terpretation data. REFERENCES 1. BDS 2200-85: Vredni veshtestva v vasduha na rabotnata sreda. Metodi za opredeliane concentraciata na praha. (In Bulg.) 2. Cartwright, J.: Airborne dust in coal mines: the particle size selection characteristics of the lung and the desirable characteristics of dust sampling instruments. Inhaled Particles and Vapours II. Rd. C.N. Davies, pp.393-406. Pergamon Press, (1967). 3. Diu, C.K., Yu, C.P.: Respiratory tract deposition of polydisperse aerosols in humans. Am. Ink. Hyg. Assoc. J. 44:62-66 (1983). 4. Ivanova-Djoubrilova, St.: Izsledvane na massovite dispersni razpredelenia na chastitsite nafini dispersniprahove, vkljuchvashti i submikronnata oblast. M.S.Thesis, Medical Academy. Sofia (1985) (in Bulg.). 5. Leisteritz, H., Einbrodt, H.J., Klosterkotter, W.: Gransize and mineral content of lung dust of coal miners compared with mine dust. Inhaled particles and vapours II. Ed. by C.N. Davies, pp. 381-390. Pergamon Press (1967). 6. Lippmann, M., Alschuler, B.: Regional deposition ofaerosols. Airpollution and the lung, Ed. by E.F. Aharoasonetal.,pp. 25-48. Halsted PressWilley, New York (1976). 7. Morrow, P.E.: Aerosol factor affecting respiratory deposition. Inter national Symposium in Deposition and Clearance ofAerosols in the Human Respiratory Tract. Bad Gleichenburg, Austria (May 1981). 8. Stober, W., Einbrodt, H.J., Klosterkotter, W.: Quantitative studies of dust retention in animal and human lungs after chronic inhalation. In haled Particles and Vapours II, Ed. by C.N. Davies, pp. 409-417. Pergamon Press (1967). 9. Task Group on Lung Dynamics: Deposition and retention models for interna dosimetry of the human respiratory tract. Health Phys. 12: 173-207 (1966). 10. Tcherneva, P.: Izsledvane na dispersnoto razpredelenie na belodrobnoto prahova depo. M.S. Thesis, Acadical Academy, Sofia (1983). 11. Tcherneva, P., Ivanova-Djoubrilova, St.: Sravnitelno izsledvane na dispersnite razpredelenia na prah ot rudnicnija vasduh i belite drobove na pochinali minjori ot silikosoopasni mini. International Symposium far Dust Prevention and Dust Diseases, Vidin, Bulgaria (Sept. 1981). 12. Thomas. K., Stegemann, H.: Beitr. Silikose-Forsch., No28,1,1954). 13. Yu, C.P.: A two-component theory of aerosol deposition in lung air ways. Bull. Math. Biol., 40: 693-706 (1978). 937 Toxicity/Surface Characterization III THE BIOLOGICAL EFFECT OF PARTICLE SIZE DISTRIBUTION OF QUARTZ COMPONENT IN POLYMINERAL DUSTS M. DOBREVA, Ph.D. N. Dancheva, Ph.D. S. Ivanova, Ph.D. R. Lukanova, Ph.D. Institute of Hygiene and Occupational Health, Sofia, Bulgaria Experimental and epidemiological studies have shown that not in all ofthe industrial dusts a correlation between the content of respirable quartz and fibrogenicity existed.1'9,14'21 This means that die mass of respirable quartz, which is the basis of contemporary norms for quartz containing dusts in a majority of countries, is not die solely sufficient criterion for their fibrogenicity. During the last 10-15 years the efforts ofa number ofauthors were directed toward the study ofdie properties of the surface of quartz dusts and finding quan tifiable indicators for their fibrogenic activity. A relationship was defined between pathogenic activity ofquartz dusts and: concentration ofhydroxyle groups,17 activating energy ofthe surface,12*13 free quartz surface,2*7,8 panicles size distribu tion.22*23 etc. 6 ,6 Most of the experiments studying die influence of the parti cle size ofquartz dust have been performed with pure quartz dusts. Results ofexperiments with coal quartz containing dust ofdifferent particle size are reported, but they do not present data on particles size distribution of the quartz content.2,15 The results ofinvestigations performed by different authors are consistent and could be summarized as follows: A max imum cytotoxic and fibrogenic effect is manifested by quartz particles of a size from 2-3 to 0.5-1 pm; at larger or smaller size a decrease in die activity is observed. Investigations on die size of respirable industrial dusts, as well as of lung dusts from deceased exposed workers show the presence in diem of mineral particles of a size within com paratively wide range (from more than 10 pm to 0.01 pm diameter).5,10'19 On die other hand, it is well known that the sizes of quartz particles usually differ more or less from those of the other components of the mixed dust. In the literature available to us we did not find research data on particle size distribution ofthe quartz component in mixed dusts. Gade and Luft,4 working out a IR-spectrophotometric method for quantitative determination ofquartz in industrial dusts by use of double absorption peak, found a relationship between particle size and die ratios ofdie absorbances at die double absorptions at 798"1 and 775"1 cm to the minimum absorbance between the two absorbance peaks. However, they proposed this method with a view at correction ofthe influence ofthe particle size upon die results at quantitative determina tion of die quartz. The above relationship was later proved by Dodgson and Whittaker3 for pure quartz. At mixed dusts the absorbance could be influenced also by interfering minerals,13*11 which would require die use of correction coefficients, making die method complicated and unreliable. The aim ofthe present work was to characterize the particle size distribution ofthe quartz component ofmixed dusts and to assess its biological role by an analysis of: lung dust from decreased exposed miners and some respirable industrial dusts. MATERIALS AND METHODS Dusts extracted from die lungs of26 workers from lead-zinc, copper and uranium mines and tunnels were investigated, as well as samples ofrespirable dust from the work environment in lead-zinc mines, dina plant and foundry. X-ray and morphological findings ofsilicotic changes ofdif ferent degrees were proved in almost all of die deceased miners with exception of 8 of diem, whose lungs contained less than 235 mg quartz. The lung dust was extracted by formamide digestion after Thomas and Stegemann. Then they were ashed at 600C and the residue subjected to an analysis ofits particle size distribu tion and mineral composition. For determination of the par ticle size distribution of the quartz in die mixed dusts the following procedure was applied: the dust sample, suspend ed in 0.1 % water solution ofsodium hexametaphosphate was fractionated within the range of 8.9-0.24 pm stockes diameters, calculated on the base ofthe specific weight ofdie quartz by sedimentation in a disc centrifuge. The suspensions ofthe fractions were run through Sartorius membrane filters of0.05 pm pores size. After that, the membrane filters with the fractions were ashed at 600C, die residues weighed and the quartz quantity for each fraction determined by IRspectroscopy. The absorption peak at 695 cm-1 was used, because ofits being less particle size dependent in the range of9 to less than 1 pm. To enhance the peak height the ordinate expansion of5 x was used. So a sensibility of0.03 mg quartz in die specially prepared pellet was attained. The quartz quan tities in it varied from 0.03 to 0.4 mg, die confidence limits at p = 95% being less than 0.011 mg. On die basis of the quartz quantities determined in die frac tions, the mass particle size distribution of die quartz com ponent was plotted on log-probability paper. 938 RESULTS The data of the total lung dust, as well as die parameters of its particle size distribution and that ofdie quartz components, in particular, are shown in Table I. It may be seen in die Table that die quantity of die extracted and ashed dust was varying from about 1 to 24 g, in 20 ofdie cases being between 1 and lOg. The mass median unit densi ty sphere (UDS) diameter ofthe majority ofthe dusts analyzed (17 from 26) is between 3 and 5 pm, in 5 of diem it is more than 5 up to a maximum of 6.8 pm and in 4--less than 3 pm with a minimum down to 2.4 pm. These data are consistent with the results obtained by Tcherneva18 in measuring equivalent volume diameter of lung dusts from deceased miners of similar exposure by using Coulter-counter. The quartz percentage ofthe dusts mentioned in Table I varies from about 6 to 40% and most frequendy between 10 and 30%, its quantity in 22 of the lungs being less than 3 g, in 12--less than 1 g and in 4--between 3 and 5 g. The mass particle size distribution of the quartz component plotted on die log-probability paper shows a log normal distribution. On Figure 1 the quartz component distribution in three of the dusts examined is illustrated. Taxicity/Surjuce Characterization III The particle size distribution in 19 ofdie samples investigated is characterized by a larger mass median UDS diameter as compared with that ofcorresponding total lung dust (See Table I). In 7 of the samples die mass median UDS diameter ofthe quartz component and that ofthe total dust, respectively, could be accepted as almost equal, since they differ only by 0.1 to 0.4 pm. The mass median UDS diameter of the quartz component in all 26 samples examined was over 3 pm; in 10 ofdie samples it was between 3 and S pm and in 16-larger than 5 pm. With very few exceptions, die median geometric standard deviations of the distribution of the quartz component were smaller than those of die corresponding total lung dust. Although that the quartz particles in general are larger than those of the other mineral components in die dust and are characterized with a more limited polydispersity, significant quantities of quartz particles with a UDS under 1 pm were found. The mass ofthe submicron fraction was varying from 3.2 to 15% from the whole mass of the quartz. In 16 of the samples it was between 5 and 10%; in six--more than 10% and only in 4 it was less than 5 %. These data are evidencing significant individual differences in the particle size distribu tion of quartz component in lung dusts. Table I Characteristics of Lung Dusts and Their Quartz Component Case No Whole extracted lung dust Hass *4 Hass median DOS diameter Jjm dg Quartz content t 43 1632 74 1148 75 1635 76 355 77 14897 107 24024 109 2950 no 8165 m 8450 1 12 2294 113 4482 114 5782 115 5796 117 1222 119 17882 120 15029 125 1083 126 1396 129 1240 140 11470 141 7209 142 3522 143 7068 144 2062 147 8626 148 7736 5.b 3.6 4.4 3.7 3.6 3.6 2.6 5.5 2.8 3.9 4.4 4.2 3.6 3.6 3.9 3.6 5.2 8.1 3.7 2.4 4.7 4.2 3.4 6.8 4.1 2.9 5.9 7.2 3.4 12.3 4.8 5.7 3.9 14.5 3.8 (3.5 3.8 17.8 5.6 17.8 4.7 29.9 4.1 28.1 4.6 16.0 4.8 35.8 3.9 27.6 4.3 43.8 4.3 17.4 4.2 28.0 4.3 27.5 6.3 13.2 6.8 16.8 5.3 13.6 3.8 26.5 5.9 15.3 7.8 17.4 4.6 21.9 6.0 13.3 4.4 *5s* 4.8 30.7 Quartz component Hass ,mg Hass median *9 UDS diameter jjm Submicron fraction % 118 141 92 51 2011 4276 525 2441 2374 367 1605 1596 2539 213 5007 4133 143 235 169 3Q40 1107 613 1548 274 223$ 2376 10.5 4.8 4.8 5.8 4.8 3.2 3.3 7.0 4.0 5.6 5.3 7.4 7.5 7.5 3.5 5.2 5.5 8.2 6.0 4.0 4.6 5.5 5.2 9.0 5.4 3.7 3.8 3.0 3.0 3.1 3.0 3.2 3.5 4.7 3.5 3.5 3.3 3.7 3.3 3.1 3.2 3.3 3.4 3.9 3.5 3.1 3.3 3.4 3.3 3.8 3.4 13.0 3.2 7.2 7.2 5.7 7.2 15.0 16.0 10.0 13.0 8.0 8.0 6.4 4.5 4.0 13.0 6.0 8.0 6.0 7.5 10.5 9.0 8.0 7.5 4.6 8.0 13.5 939 Toxicity/Surface Characterization HI Figure 1. 1. Mass particle size distribution curves of the quartz component of three lung dusts. Case No. 107---------------------74 --------------------- 120 .................... d -- Mass median UDS diameter % -- Mass percentage of quartz fraction 940 Research data for respirable industrial dusts are shown in Table II. In these samples significant differences were not ob tained between the mass median UDS diameters ofthe whole dust and ofthe quartz component. Comparatively near were proved to be the values for these parameters measured for die three sorts of dust (between 5,3 and 6.8 pm), in spite of the differences in their composition. The percentage ofquartz par ticles with UDS diameter less than 1 pm was between 6.8 and 13%. For assessment of the influence of the particle size of die quartz component on fibrogenicity of mixed dusts, their characteristic data and resulting fibrosis changes in die lung tissue were processed by the program product "Statgraf." The following dust parameters were included in die analysis: total quantity of the extracted dust, its residence time in the lungs, % of the free crystalline silica content, total free crystalline silica content and quantity ofthe fraction with sizes between 3 and 0.5 pm. Slightly higher correlation coefficient--0.84, was obtained for the quartz fraction within 3--0.5 pm, as compared with 0.83 for the total quartz quantity. Toxicity/Surface Characterization III DISCUSSION The results obtained illustrate the possibility ofdetermining the particle size ofquartz component in mixed dusts as a help for the better characterization of both lung and industrial or experimental dusts. Evidendy, this method has the all well-know shortcomings, related with the procedure of lung tissue digestion and dust extraction, ashing at 600C and preparing liquid suspension for centrifuging etc. In spite of these limitations, we are of the opinion that this method could be a help in elucidating the relationship between die properties ofquartz-containing dusts and their pathogenicity. In our attempt to assess the biological significance of this parameter on the basis of the 26 lung dusts investigated we did not obtain a significant increase ofthe coefficient of cor relation. This fact is perhaps due to the high coefficient of multiple correlation for a comparatively small number of sub jects on the background of the number of the variables in vestigated. The studies continue with a larger group of lung dust cases. Sort of dust Ore mine Dinas Foundry Table H Characteristics of Respirable Industrial Dusts and Their Quartz Component Silica content % 9.6 65.4 5.3 Parameters of particle size distribution Respirable dust Respirable quartz UDS mass me tfg. dian diameter urn 5.9 5.3 6.2 4.5 4.1 12.4 UDS mass median diameter sub micron fraction % 6.0 5.5 6.6 3.3 3.7 5.4 6.8 9.0 13.0 941 Toxicity/Surface Characterization UJ REFERENCES 1. Bruch, J., loos, M.-R., Reisner .M.T.R., Rosmanith. 1.: Unter suchungen zur spezifischen Schadlicbkeit vou Feinstauben der VF V*ayleighdyPjy^iw.wnrt'IWiiwil7rfhwysuchen. Ergebnisse wn Untersuchungen aufdem GebietderStaub and SlUdsebekangfung m Stemhohlenbergbau, 14:241-255 Gluckauf, Essen (1983) 2. Bruch, 1., Gade, M., Henke, A., Resner, M.T.R., Stopschinski, W.: Der der Korngrosse und minerologischen Zosammenzetzung von Grubensauben aufdie Zytotoxizitat Ergebnisse von Untersuchungen auf dem Gebiet der Staub- und Silikosebekampfung im Sleinkohlenbergbau. 14:301-312, Gluckauf, Essen (1983). 3. Dodgson, J., Whittaker, W.: TbeDetenmandoaofQuartz in Respirable Dust Samples by Infrared Spectrometry--I. Ann. Occup. Hyg. 16:373-387 (1973). 4. Gade, M,, Left, K. Fr.: DiealtrorotspectroscopischeQuaiTbestiirimnng insbesooders von Grubenstauben. Staub 23:353-357 (1963). 5. lvanova-Djubrikova, S.E.: bsiedvanenamascvitedupersnuuytredeiemja na casticite na finodispersni prahove vUyucvasti i submicromata oblast. Disertacia, Izd. Med. Akademia. Sofia (1985). 6. Yabuev, A. V., Velichkovskij, B.T., Deeva, N.V., Korkina, L.G.: Biologiceskoe znacenie poverfanostnyb svoistv fibrogamyh pylej. Gig. L Sand. (:34-37 (1986). 7. Kriegseis, W., Scharmann, A., Serafin, J., Beck, E.G., Hikes, F., Bruch, ].: DieAbhangigkehderZytotDxizitatSiOj-haltigenStaubevon fluen Oberflacbenreinbeit. Ergebnisse von Untersuchungen aufdem Gebiet der Staub- und Silikosebekampfung im Steinkohlenbergbau. 14:281-289. duckauf GMBH. Essen (1983). 8. Le Bouffant, L., Daniel, H., Martin, J.C., Bruyere, S.: Effect ofIm purities and Associated Minerals on Quartz Toxicity. Ann. Occup. Hyg. 26:625-634 (1982). 9. Leiteritz, H., Bauer, E.D.,Bn*danann,E.:Konzeutrazjooveriialtnisse imH mineralische Bcschaffenheh den Gmbenstaube imwestdeutscfaen Stemkohlbergbsu und Sue Bedeuhmg furdie Ettwickhmg derSteubhmge bei Kohlenhauem. Staub. Reinh. Lufi. 31:185-189 (1981). 10. Lippmann, M.: "Respirable" Dust Sampling. Am. buL Hyg. Assoc. J. 31:138-159 (1970). 11. 1 nlrnimva P T Pritnrhsni* wtn Mt-tp*. n*fitiwn*Uurtn knlimrvmn opredeljane na quartz iamorfen svoboden silicievdvuokis vpromishleni prahove. Disertacija, Med. Akademija. Sofia (1978). 12. Robock, K.: Die Wirkung mechanischer, tbermiscberund chemiscber Bebandlungen von SiUciumdioxid und Asbest-Stauben aufZytotoxizitatund Elektronenstruktur. Beitr. Siiikose-Forschung. 26:111-262 (1974). 13. Robock, K., IQosteikotter. W.: Untersurhungen zur Zhotoxizitat von SiOj Stauben. Ergebnisse von Untersuchungen auf dem Gebiet der Staub- und Silikosebekampfimgim Steinkohlenbergbau. Gluckhauf GMBH. Essen. 10:159-162 (1975). 14. Seemayer, N.H., Manojlovic, N.: Untersuchugen zur spezifischen Schadlichkdt von 20 Gmbenstaube an alvedaren Makrophagen in vitro. In: Ergebnisse von Untersuchungen aufdem Gebiet der Staub- und Silikasebekampfimgen im Sleinkohlenbergbau. 13:225-232. Verlag duckauf. Essen. (1981). 15. Seemayer, N.H.: Biological Effects ofCoal Mine Dusts on Macrophages in Vitro: Importance of Grain size and Mineral Content. Vlth bu. Pneumoconiosis Conference. 1985. pp. 513-527. Bergbau-- Berufsgeoossenschaft--ILO, Bochum. FRG (1983). 16. Stkova,W.A.,Aronova,G.V.,Wdkhkovsldj,B.T.:Ovlijanijkationov metallov adsorbirovannyh iz rastvorov solej na nekotoryje svojstva poverhnosd kremnesemsorderzbastih pylej. Prof. Bolezni Pylevoj Edoiogu. 3:138-144. Moskva (1975). 17. Sflrova, W.A.: Ometodahopredelenijaibiologiceskomznacenii gidroxibtogopokrova kremnezems oderzhasahpromyshlennyhpylej. Disertaci ja, Leningrad. (1976). 18. Tchemeva, P.: Izsledvane na dispersnata harakteristika na prabovoto depov bdi drobovena minyori. Disertacia. Med. Academia. Sofia (1983). 19. Walton, W.H., Dodgson, I. Hadden, G.G., Jacobsen, M.: The Effect ofQuartz and Other Noncoal Dusts in Coalworker's Pneumoconiosis. bthaled Particles IV:669-690. Pergamon Press. Oxford (1977). 20. Wdichkovskij,B.T., Arooova,G.V.,Starkov, P.P., Belobragina,G.V.: SravmteTno experomental'nyje issledovanija zavisimosti fibrinogetmosti kremnezema ot mechanizzna obrazovanija i dispersnosti pylevybcesdc. Borba s silikozom. 18 pp. 191-198. Nauka. Moskva (1970). 21. Wdichkovskij, B.T., Argunov, V.D., Kruglikov, G.G, Batsura, Ju.D., Fedorova, V.M.: O mechamzme samoodscenija lyoghih ot pyii. Gig. Tr. Prqf.ZaboL 4:31-36(1979). 942 Toxicity/Surface Characterization III CHEMILUMINESCENCE AND BIOLOGIC REACTIVITY OF FRESHLY FRACTURED SILICA N.S. DALAL V. Vallyathan N. Leelarasamee V. Castranova * K. Van Dyke Chemistry and Pharmacology Departments, West Virginia University, and Division of Respiratory Disease Studies, National Institute for Occupational Safety and Health, Morgantown, WV 26505, USA INTRODUCTION Silicosis is the chronic fibrosing disease of the lungs caused by the inhalation ofcrystalline silica. However, inhalation of crystalline silica, may induce three distinctly different disease patterns; i.e., chronic silicosis, accelerated silicosis, and acute silicosis. These three disease patterns differ in their pathologic characteristics, clinical symptoms, onset ofdisease, and mor tality. Many studies in the past have focused on the elucida tion of mechanisms involved in the development of chronic silicosis. We recently reported that freshly fractured silica has surface properties that could make it more reactive with lung tissue than aged silica, and that this unique reactivity offreshly fractured silica may be involved in the pathogenesis ofacute silicosis (Dalai et al., 1986; Vallyathan et al., 1988; Shi et al., 1988). Because freshly fractured silica could contain some moieties in an ` `excited state, ' ' due to the breakage ofsiliconoxygen bonds, we postulated that the de-excitation of these moieties could produce light which could be monitored by a luminescence measurement technique. In addition, ifthis "ex cited state' ' of silica could trigger a greater biologic response by phagocytic cells, this activation could be monitored by a chemiluminescence technique in die presence ofappropriate enhancers. This investigation describes for the first time the use of luminescence in the study of freshly fractured silica and its properties. Enhanced biologic reactivity of the freshly frac tured silica with alveolar macrophages was monitored by the chemiluminescence technique. MATERIALS AND METHODS Native silica was obtained from die dust bank ofthe Generic Respirable Dust Technology Center, Pennsylvania State University, State College, PA, and ground for 30 min in an agate ball mill. It was then sieved through a 20 micron mesh filter and used within 10 min as fresh silica or stored in glass botdes for aging. X-ray energy spectrometry and X-ray powder diffraction studies on representative samples were made to confirm the mineralogic purity. All die silica samples were found to be 99% pure with minimal detectable con tamination by metal ions. Luminescence offreshly ground silica was measured from 5 g samples in plastic scintillation vials using a Packard Liquid Scintillation Counter operated in the out-of-coincidence mode. After grinding, die samples were stored in dark for 10 min. and luminescence was monitored over a period of several hours and days. The effect ofaqueous solutions on the genera tion ofluminescence was studied in HEPES-buffered medium (145 mM NaCl, 5 mM KC1, and 10 mM HEPES, pH 7.4). Freshly ground silica (5 g) was added to 5 ml of HEPESbuffered medium, the vials stored in dark for 10 min, and samples counted at various time intervals. The effect of scavengers on luminescence was investigated by the addition of5 g freshly ground silica to HEPES-buffered medium con taining 125 /xg/ml superoxide dismutase (SOD), 125 pg/ml catalase, or 100 mM 5,5-dimethyl-l-pyrroline-l-oxide (DMPO). Biologic reactivity of freshly ground silica was monitored as lucigenin-enhanced chemiluminescence at 37C using a Berthold Luminometer, Model 9500. Alveolar macrophages were obtained by the broncho-pulmonary lavage of SpragueDawley male rats with calcium and magnesium-free Hank's balanced salt solution. Repetitive lavages were pooled (total volume of 80 ml) and was sedimented by centrifugation at 500 g for 5 min. Cells were washed and resuspended in HEPES-buffered medium containing 1 mM calcium and 5 mM glucose. With the aid of trypan-blue and hemocytometry, cell viability and counts were determined miscroscopically. Results of these studies indicate that approximately 90% of the lavaged cells were viable alveolar macrophages. Samples of alveolar macrophages (1 x 106) were then incubated with 20 /xg/ml silica in 0.5 ml HEPES-buffered medium at 37C. The buffer contained 2.5 x 10~8 M lucigenin as an enhancer of chemiluminescence. Silica-induced reactivity of alveolar macrophages was then monitored over time for 40 min. RESULTS Figure 1 shows the results ofluminescence studies on freshly ground silica in comparison with that ofaged dust. Aged dust, on the other hand, showed a steady minimal baseline intensi ty oflight emission, most likely due to "fluorescence." This intensity oflight emission was greater when samples were not dark equilibrated for 10 min. Therefore, we consider that, this basal emission to be due to fluorescence excited by the am bient light. The data clearly indicatethat freshly ground silica emitted substantially more light than aged silica. The inten sity of this light emission declined with time after grinding and exhibited half-life of approximately 40 min. In order to find whether contact with a biologic medium would quench die luminescence instantaneously, luminescence 943 Toxicity/Surface Characterization III TK AFTER GRIMING (MINJTES) Figure 1. Luminescence of freshly ground and aged silica compared to show the greater intensity of time dependent luminescence associated with freshness. Figure 2. Luminescence of freshly ground silica in air and in HEPES-buffered medium. 944 measurements were made on freshly ground silica dispersed in HEPES-buffered medium. It is evident from the data presented in Figure 2 that fresh silica suspended in the biologic medium emits substantial light suggesting that die reactive sur face sites on freshly ground silica are not immediately quenched after contact with biologic media. Effect of free radical scavengers on the light emission inten sity is presented in Figure 3. It is seen that luminescence of freshly ground silica was inhibited by approximately 71 % with SOD, while catalase and DMPO inhibited the light emission by 88% and 97%, respectively. Figure 4 shows the effect of freshly ground silica on alveolar macrophages stimulation and resulting release of reactive species of oxygen, monitored as chemiluminescence in the presence ofan enhancer, lucigenin. Alveolar macrophages in cubated with freshly ground silica (20 /ig/ml) generated chemiluminescence which peaked approximately 8 min after exposure to silica. This silica-induced activation of alveolar macrophages was substantially greater with freshly ground silica as compared to silica aged for 24 and 48 hours (Figure 4). DISCUSSION Data from the present study indicate that freshly fractured silica emits light which can be monitored by luminometry or a liquid scintillation technique. These studies also indicate dial the luminescence generated by silica is not quenched instant ly in biologic medium but can be inhibited to a substantial degree by SOD, catalase, and DMPO. These results suggest that excited surface sites result from die cleavage ofsilica and that these surface sites can react with aqueous media to form reactive oxygen species as a source of emission of light detected by the luminescence technique. We have previous ly shown that silicon-oxygen radicals and possibly O2 radicals are formed during grinding of silica (Dalai et al., 1986; Shi et al., 1988). We have also shown that these radicals undergo a time dependent decay in ambient air (Dalai et al., 1986; Shi et al., 1988). We tentatively assign the light emission from the freshly fractured silica to the de-excitation ofthese radicals Toxicity/Sutface Characterization III and/or the silicon-oxygen radicals. Our studies also indicate a correlation between light emission by freshly fractured silica and potential for biologic reactivi ty. The increased activation of alveolar macrophages induced by freshly ground silica as monitored by chemiluminescence indicates excessive secretion of reactive oxygen species dur ing phagocytosis. We have shown previously that generation of reactive species of oxygen on silica during grinding can cause the generation of OH radicals. The presence of these increased cytotoxicity reactive species has been related to lipid peroxidation (Vallyathanetal., 1988). Results ofthe present study support our hypothesis that the reactive species associated with fresh silica together with those generated by alveolar macrophages in response to fresh silica may induce an oxidant stress and overwhelm the protective anti-oxidant systems of lung in occupational exposures, such as sand blasting, tunnelling, drilling, or silica flour mills where freshly fractured silica dust is generated. We, therefore, conclude from these studies that oxidant stress may play a role in the etiology of acute silicosis. REFERENCES 1. Dalai, N.S., Suryan, M.M., Jafari, B., Shi, X., Vallyathan, V., and Green, F.H.Y.: Electron spin resonance detectionofreactive free radicals in fresh coal dust and quartz dust and its implication to pneumoconiosis and silicosis. In: Respirable Dust in the Mineral Industries: Health Ef fects Characterization and Control, Editors R.L. Frantz and R.V. Ramani, 1986. The Pennsylvania State University, University Park, PA. pp 24-32. 2. Vallyathan, V., Shi, X., Dalai, N.S., In, W., Castranova, V.: Siliconoxygen radicals and their role in acute silicosis. Am. Rev. Respir. Dis. 137:404, 1988. 3. Vallyathan, V., Shi. X., Dalai, N.S., In, W., Castranova, V.: Genera tion of free radicals from freshly fractured silica dust: potential role in acute silica-induced lung injury. Am. Rev. Respir. Dis. 138:1213-1219, 1988. 4. Shi, X., Dalai, N.S..Vallyathan, V.: ESR evidence for the hydroxyl radical formation in aqueous suspensions ofquartz particles and its possi ble significance to lipid peroxidation in silicosis. J. Environ. Toxicol. 25:237, 1988. 5. Dalai, N.S., Shi, X., Vallyathan, V.: Oxygenated radical formation by fresh quartz dust in a cell-free aqueous medium and its inhibition by scavengers. In: The Proceedings of VHth International Conference on Pneumoconioses, Pittsburgh, PA. Editor Molly Pickett-Harner. 945 Toxtcity/Swface Characterization HI CDffiO. SOD CRTFU6E OfO Figure 3. Effect of SOD, catalase, and DMPO on luminescence by freshly ground silica. Figure 4. Effect of freshly ground and aged silica on chemiluminescence by alveolar macrophages. 946 Toxicity/Surface Characterization III THE INJURIOUS EFFECT OF QUARTZ ON CELL MEMBRANES AND THE PREVENTIVE EFFECT OF ALUMINIUM CITRATE AGAINST QUARTZ CHENG J.CAO,* M.D. Shi J. Liu,* M.D. Ke C. Lin.t Ph.D. Dept, of Occupational Health, School of Public Health tDept. of Biophysics, School of Basic Medicine Beijing Medical University 100083, Beijing, China ABSTRACT The injurious effect ofquartz on die membranes ofmacrophages as well as erythrocytes and die anti-injurious effect ofaluminium citrate (A1 citrate) were examined. The comparative study with titanium dioxide was car ried out simutaneously. The results from the present study show that quartz can cause the increases of fluidity and permeability of macrophage membranes and reduce membrane-bound water of erythrocytes, resulting in the membrane dehydration. Furthermore, quartz can change electrophoretic behavour of macrophages by increasing the negative charge density and electrokinetic potential on these cells surface. The effect oftitanium dioxide on cell membranes however is very different from quartz in intensity and kinetics, and is not affected by A1 citrate. The relationship between these effects was discussed and a possible mechanism was proposed for the interaction of quartz with membrane lipids resulting in membrane damage. The preventive effect of A1 citrate against membrane damage by quartz was also demonstrated. In general, the addition ofA1 citrate can recover all alternations caused by quartz, so that the stability and order structure of cell membranes were maintained. A hypothesis about the action of A1 citrate on the surface of quartz par ticles to exert its anti-injurious effect was postulated in this paper. INTRODUCTION It is generally accepted that the cytotoxic effect of quartz on alveolar macrophages is a key step in the pathogenesis of silicosis. 1-2 The cytotoxic mechanism postulated by Allison depends on mainly the release of hydrolytic enzymes from lysosomes after the phagocytosis of quartz by macrophages followed by cell damage.3 However, a question that remains unanswered is whether die toxic particles directly damage the plasmic membranes ofmacrophages. It is well known that die contact of quartz with the cell membranes is die first event during die process of phagocytosis, no matter bow the par ticles are uptaken into the interior of these cells. For this reason, it is desirable to elucidate die molecular interactions between quartz and cell membranes from the viewpoint of membrane toxicology. The therapeutic effects of A] citrate on the experimental animal and patients with silicosis have been demonstrated in our previous experimental studies and clinical observations. It was also found that A1 citrate is able to prevent effectively macrophages from die cytotoxicity ofquartz instead ofdie in hibition of fibrosis.4-6 It is, therefore, necessary to clarify its pharmacology with the goal being to provide die experimen tal and theoretical evidence for screening die preventive measurments and therapeutic drugs for silicosis. On the other hand, titanium dioxide, a less toxic and usually classified as "inert dust,''7 was also studied in this work for comparsion. MATERIALS AND METHODS Macrophages were harvested from lung ofguinea pig through lavage. The erythrocyte membranes ofrabbit were prepared as described elsewhere.8 Quartz (99% pure) was supplied by Hygiene Institute of Chinese Prophylatic Medical Crater. Particles diameter is less than S (tm, among which 89.3 % is less than 2 pm. Titanium dioxide with the same purity and size was selected as a con trol. A1 citrate with A1 of 9.26% was supplied by Phar maceutical Factory of Beijing Medical University. Fluorescence probe, 1,6-diphenyl-1,3,5-hextriene (DPH) was purchased from Sigma. Adenosine 5 '-triphosphate disodium salt (ATP) was produced by Shanghai Biochemical Institute of Academia Sinica. Fluorescence polarization was determined by spectrophotofluometer Model MPF-4. Potassium (K+) content of cells was detected by Fire Atomic Absorption Spec trophotometer Model Y-3.9 Na+-K+-ATPase activity was determined using the method described by Pan H.Z.10 Viscosity of medium and surface charge of cells were measured by viscosimeter Model E and Cell Electrophoresis Autotimer Model SX-2, respectively." Membrane-bound water was determined employing the method of sorption isotherms and Nicolet Fourier Transform Infrared Spec trometer Model 5DX.812 There were on the average five samples in each group. Data 947 Toxicity/Surface Characterization HI were presented as mean + standard error and significance was estimated by analysis ofvariance. Pairing data about fluidity and permeability were treated by linear correlation and regression. RESULTS Cell Membrane Upid Fluidity We began with the examination ofmembrane fluidity ofmac rophages by measuring fluorescence polarization P and micro viscosity of membrane-bound DPH. As shown in Figure 2, P values ofquartz I and II groups dropped down continuous ly with the cultural time, resulting in more fluid membranes. It is important that the effect of quartz on membrane fluidity is not only time-dependent, but also dose-dependent (Figure 1). However, die change of membrane fluidity by titanium dioxide is much lower than that ofquartz group and tends to be recovered rapidly (Figure 1). Compared with quartz control, fluidity was decreased (e.g. P value raised) when quartz plus A1 citrate was added into die cells simutaneously, although A1 citrate did not affect mem brane fluidity alone. Similarly, die effect ofA1 citrate against quartz is dose-dependent (Figure 1). Permeability of Cell Membrane to K+ Table I presents die differences between die groups treated by several ways in membrane fluidity and permeability to K*. It is interesting that die increased pemeability ofmacrophage membranes to K+, that is, K+ content of the cells was reduced, by quartz was accompanied with increasing mem brane fluidity. Statistic analysis indicates the effects ofquartz on both these properties of macrophage membranes exhibit very siginificant correlation (for instance, using rj and K+ as X and Y, respectively, r=0.917, P< 0.001, Y=9.059X-0.011) (Figure 3). Like that on fluidity, Al citrate did not influence permeability of macrophage membranes to K+ by itself, but it prevented acting efflciendy against the effect of quartz, except that macrophages were pretreated with Al citrate (Table I). It is seen from Table I that membrane permeability oftitanium dioxide group was lowered only slightly and it seems that no exact relationship exists between the changes of fluidity and permeability. Another important difference from quartz is that die effects oftitanium dioxide are unable to be affected by Al citrate. Cmltr tla Figure 2. Kinetic curve of DPH fluorescence polarization labelled in macrophage membrane SiC>2l: the simultaneous addition of DPH and S1O2 to cell medium SiC>2lI: the addition of SiO^ to cell medium followed by the addition of DPH The dose of SiC>2 or Ti02 was 1 mg; the dose of Al citrate was 0.S mg Al. S10 or Al citrate dose * (fig or h Al) Figure 1. Dose-effect relationships of the effect of Si02 on fluorescence polarization (P) of macrophage membrane-bound DPH and lipid viscosity (q) and the antagonistic effect of Al citrate against SiC>2 ^ SiC>2: ^ g ^*^2 (1 mS) citrate 948 Figure 3. Scatter diagram ofmembrane lipid viscosity with K+ concentration of macrophages administrated with SiO?. It may be involved in K + permeability, however, no change in the activity of Na+-K+-ATPase could be found after the treatment ofthree cell preparations with quartz (Table II), in dicating that die increased permeability is related closely to the change in lipid fluidity. Membrane-bound Water Membrane hydration ofquartz group at die defferent relative humidities (RH), particularly at higher RH, was reduced markedly from sorption isotherms curve (Figure 4) and data listed in Table m. In IR spectra, VOH shifts largely to lower frequency (Figure 5) and the results represented in Figures 5 and 6 are identical, for instance, at 76% RH, the hydration and VOH peak position in both ofcontrol and quartz groups are 18.8% and 3535 cm-1, and 10.1% and 3447 cm-1, respectively. It is clear that the dehydration ofcell membranes was caused by quartz and has a significant does effect rela tionship (Table IV). Whereas membrane hydration in either quartz plus A1 citrate group or die pretreated quartz group with A1 citrate is higher than quartz control (Table HI) and their VOH peak position shifts towards the higher frequency (Figures 5,6). The effect of A1 citrate against quartz exists also a dose-effect relation ship (Table IV). Membrane-bound water under die treatment by the different ways is presented in Figure 7. The similar results are found from two quartz groups pretreated with A1 citrate and AICI3. However, die effect of titanium dioxide on membrane-bound water is not only lower than quartz, but also was not recovered by the pretreatment of A1 citrate (Figure 7). Toxicity/Surface Characterization III Figure 4. Sorption isotherm ofwater on red blood cell mem branes treated by different ways at 20C. 20mg S1O2: 8.333 mg A1(A1 citrate) Polarization (P) of Membrane-bound DPH and Its Lipid Microviscosity (rj) with K+ Content of Macrophages Groups Use tain) p TjSE rf itss r* 1*SE Control S10, 20 0.185*0.00? 1.352*0.042 11.557*0.149 60 0.184*0.00? 1.329*0.018 11.533*0.099 ?o 0.1*2*0.PO? 0.984*0.019 60 0.141*0.003 0.885*0.031 9.239*0.192 7.431*0.205 S10?*Al citrate 20 0.171*0.001 S109 pretreated with Al citrate 20 0.172*0.001 Cell pretreated with Al citrate 20 0.185*0.004 Cell pretreated with 20 0.154*0.003 Al cltrate*S102 1.183*0.007 1.195*0.010 1.350*0.043 1.000*0.029 10.612*0.227 10.694*0.254 11.608*0.181 9.498*0.205 fi09 TIOj+Al citrate T109 pretreated with Al citrate 20 0.164*0.002 1.104*0.019 10.616*0.264 60 0.163*0.00? 1.096*0.025 9.384*0.231 20 0.164*0.002 1.111*0.022 10.549*0.258 20 0.163*0.001 1.100*0.015 10.647*0.279 K+()ig/2*i06 cell)p lag S1C? or TiOjj 0.5ag Al 949 Toxicity/Surface Characterization U1 Table n Na+-K+-ATPase Activities (/tM Pi/mg protein) of Three Cell Preparations Groups Control sio2 Al citrate S102-Al citrate T10? adhesion cell TtSX 0.6720.099 0.6460.113 0.616+0.099 0.666*0.119 0.627*0.095 suspension cell XtSE 0.806*0.144 0.794*0.142 0.799*0.157 0.794*0.128 0.862*0.192 cell hoaogenate XiSE 1.156*0.190 1.099*0.25? 1.156*0.057 1.162*0.168 1.098*0.187 TIm doets of SlOj and Al citrate were 30Qug and I25;ig Al respectively; The ensyaatlc activities were determined at 1 hr. of culture. ' Table III Hydration of Erythrocyte Membranes of Several Groups at the Different Relative Humidity (RH) Croupe Control SiO? Al citrate S102+Al citrate 810* pretreated with Al citrate 95*RH 50.2 39.1 54.1 52.0 47.5 Rydration(jt) 61*RB ?6*RB 26.1 13.3 30.9 28.1 21.6 16.8 10.1 23.8 19.4 14.3 52J0M 9.2 5.8 14.7 10.6 9-1 20XRH 4.0 2.3 5.7 5.8 3.2 MftMWIM tvi` R-xa St $ns Hi l WWW tCt> R.n at 7 r-i* at stin r-ii at man Figure 5. 1. Membrane control 2. SiC>2 3. Al citrate 4. Al citrate + SiC>2 5. SiC>2 pretreated with Al citrate 2. Omg Si02: 0.833 mg Al 950 Toxicity/Surface Characterization HI JS4J iWj mwiM tn U MM, ooo. 3900.0 kqo.o zoso.o I CM-1 1 SOS for 5W - 10* M 000.0 9300.0 <500.0 1050.0 W*imw (Ot*l> SOB for 95% - *0* II 4000.0 9900.0 9*00.0 <050.0 WWCMJMCM IOf<l SO* for 7M - 20% 18 Figure 6. Table IV Hie Effects of Different Doses of SiC>2 and A1 Citrate on Membrane-bound Water SlO^Hg) V'oh<c-`,> 0.5 3395 1.0 3364 2.0 3314 3.0 3268 0.417 0.833 * 1.668 3419 3558 3460 3607 3493 * S102(2.0a)-frAl citrate tor Cell Membrane Charge As shown in Figure 8, electrophoretic mobility of macrophages sped up rapidly following the addition ofquartz. The result indicates that the interaction of quartz with macrophage surface causes increasing negative electropholdnetic potential (t-potential) and charge density on the membrane surface. Similar to that on membrane fluidity and permeability, the effect of quartz on membrane charge of macrophages has also significant time-dependent and dosedependent relationships (Figures 8, 9). A1 citrate can decrease electrophoretic mobility of macrophages by itself like its effect on membrane-bound water. The effect of quartz is almost abolished by the addi tion of a high dose of A1 citrate (Figure 9). Ofparticular in terest, the effect of quartz on membrane charge can be decreased by the pretreatment with A1 citrate (Figure 8). As illustrated in Figure 8, the increment by titanium dioxide is lower and its kinetics are veiy different from thatofquartz, although it increased also electrophoretic mobility of macrophages. Figure 7. PT-IR or erythrocyte membranes treated by die different ways at 76% RH. 2.0 mg Si02 or Ti02 951 Toxicity/Surface Characterization III DISCUSSION In previous studies, die cytotoxicity ofquartz on macrophages was evaluated usually by measuring the activities ofLDH and ACP and cell death rate.3-6*13"15 The enzymatic activities may reflect indirectly die palsmic and lysosomal membranes damage caused by quartz, but their changes did not occur until after one hour ofdie incubation ofcells with quartz. It is ob vious that the indirect interaction between quartz and macrophage membranes, particularly its early effect need to be observed in order to establish the injurious effect ofquartz on the membranes. It is for this purpose that the present studies was carried out. Membrane fluidity plays an important role in membrane func tion.16,17 Fluorescence Probe DPH used in this experiment can be inserted into the hydrocarbon region of lipid bilayers and fluorescence polarization depends on microviscosity of that region. The decrease of P under the action of quartz elucidates that die motion oflipidic molecules was increased due to the lowered membrane lipid microviscosity, resulting in disruption ofmembrane structure. Moreover, the feet that quartz can increase fluidity of liposomes prepared from lecithin and cholesterol also suggests that quartz interacts mainly with membrane lipids.18 In regard to the study ofpermeability,we have demonstrated that the reduction of K+ content in macrophages can an ticipate die enhanced activities of LDH and ACP in culture medium following the addition ofquartz to these cells and is responsible for the cytotoxicity.6 The present paper establishes further the correlation between both changes of permeability of macrophage membranes to K+ and their mambrane fluidity by quartz. Likewise, die mechanism ofthe increased permeability is considered to be associated with die effect of quartz on membrane lipids, but not on Na+-K+-ATPase. Bound water is a major component ofbiological membranes and is required for die structural stability oflipid bilayers and die normal function. A novel information about the effect of quartz on membrane "water structure" was obtained from die experiment of membrane-bound water of erythrocytes. The membrane IR spectra show hydration-dependent changes in the stretching vibration band ofbound water, namely VOH shifted to the lower frequency with decreasing hydration. The result from subtract spectra (SUB), which can exclude absor bance of several groups besides water at 3000-3800 cm*1 region, is consistent with die effect. A turning point ofmem branes hydration from sorption isotherms curve is at about 76% RH, at which hydration of normal erythrocytes mem branes is 18.8% and its VOH peak position is 3535 cm*1, whereas hydration of quartz group is only 10.1 %, and its VOH peak position exhibits red shift to 3447 cm-1. The decrease ofmembrane-bound water does not provide lipidic molecules with a necessary condition required for hydrophilic and hydrophobic interactions, so that die order degree of biomolecular layers was not maintained. Indeed, Clifford et al have found die changes ofstructure, such as phase separa tion of cholesterol from lipid, in membrane dehydration.19 Thus dehydration by quartz is associated with increasing fluidity or permeability. On die other hand, charges on the membrane surface will alter relatively because die dehydra tion has made water molecules separate from some groups on membranes which are bound to diem. This is further sup ported by die results from cell electrophoretic experiments. V O ia/aie/v/ei) Figure 8. Kinetic curve of electrophoretic mobility (V) of macrophages treated by different ways. 500/xg Si(>2 or Ti02i 250 fig Al 952 Figure 9. Dose-effect relationships of Si02, Al citrate and Si02 (500 #tg) + Al citrate on electrophoretic mobility (V) of macrophages. O--O Si02i A--A Al citrate; -- Si02+Al citrate It seems more possible that quartz interacts with the positively charged groups, such as -N+(CH3)3 riched in membrane phospholipids, so positive charge on the cell surface is neutralized partly and negative charge density increases relatively. Nash et a! and Depasse et al presented the indirect evidence that quartz is easy to attract amide phosphate and quatenary ammonium groups and suggested that die attrac tion is responsible for haemolysis of quartz toward erythrocytes.20*21 Compared to quartz, the effects of titanium dioxide on cell membranes are not only much lower in intensity, but also very different in kinetics, for instance, die changes of fluidity, permeability and cell electrophoresis can not enhance permanendy with the culture time, whereas tended to recover rapidly and remained constant. Of interest, the results from morphology is quite in accordance with biophysical and biochemical determinations.22 Whether the membrane damage is caused will depend on physical and chemical pro perties of different particles. The fact that the effects of titanium dioxide on cell membranes were not affected by Al citrate may give some insight to die difference between quartz and titanium dioxide in their surface structure and affinity for ions, such as -N+(CH3)3 and A13+. Another part ofthis paper focuses on the anti-injurious effect of Al citrate and its mechanism. In general, the increased membrane fluidity, permeability and negative charge densi ty were declined, but the decreased membrane hydration were enhanced following the addition ofAl citrate, so that the func tion, stability and order structure of cell membranes can be recovered and maintained. The observation by scanning elec tron microscope convinced us of the antagonistic effect of Al citrate once more.22 The mechanism is discussed through the compared an tagonistic effects of several ways of the administration. It seems that Al citrate will affect membrane-bound water and charge by it self if the addition of it into cell medium without washing, but the its effect of disappeared after the cells were washed.11 These findings suggest that Al citrate combines with certain membranes, even though the combination is not firm and matters little to its effect against quartz. No preven tive effect was found in fluidity and permeability experiments of macrophages pretreated with Al citrate. Moreover, Al citrate alone did not influence these properties of macrophage membranes. From these it is considered at least that the preventive effect ofAl citrate is not produced by its direct ac tion on cell membranes. The preventive effects of Al citrate and AICI3 were examined through the pretreatment of particles. The results show that this pretreatment way can effectively resist membrane damage by quartz. On the other hand, the fact that A1C13 exhibits Toxicity/Surface Characterization III a similar action indicates that the pharmalogical effective com ponent of Al citrate is mainly Al itself, which explains why many kinds of soluble Al agents processes a similar effect of treatment for silicosis. Attention should be paid to the poten tial significance ofthe special action ofAl on quartz in preven tive and therapeutic silicosis. REFERENCES 1. Marks, J. et al: A Study of Dust Toxicity Using a Quantitative Tissue Culture Technique. Brit. J. Ind. Med. 13:187 (1956). 2. Viglian, E.C., Penis, B.: An Immunological Approach to Silicosis. J. Occup. Med. 1:39(1959). 3. Allison, A.C. et al: An Examination of the Cytotoxic Effects of Silica on Macrophages. J. Exp. Med. 124:141 (1966). 4. 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