Journal of Toxicology and Environmental Health
ISSN: 0098-4108 (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/uteh19
Cytotoxic effects of quartz and chrysotile asbestos: In vitro interspecies comparison with alveolar macrophages J. Schimmelpfeng & A. Seidel To cite this article: J. Schimmelpfeng & A. Seidel (1991) Cytotoxic effects of quartz and chrysotile asbestos: In vitro interspecies comparison with alveolar macrophages, Journal of Toxicology and Environmental Health, 33:2, 131-140, DOI: 10.1080/15287399109531513 To link to this article: http://dx.doi.org/10.1080/15287399109531513
Published online: 19 Oct 2009.
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Date: 05 November 2015, At: 21:03
CYTOTOXIC EFFECTS OF QUARTZ AND CHRYSOTILE ASBESTOS: IN VITRO INTERSPECIES COMPARISON WITH ALVEOLAR MACROPHAGES J. Schimmelpfeng
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Hauptabteilung Sicherheit/Biophysik, Kernforschungszentrum Karlsruhe, Germany A. Seidel Institut für Genetik und Toxikologie, Kernforschungszentrum Karlsruhe, Germany
Cytotoxic effects of DQ12 quartz and chrysotile asbestos on alveolar macrophages of different animal species were compared in vitro. The type of cell reaction toward the cytotoxic dusts was always the same: a loss of cell viability (trypan blue dye exclusion test) was accompanied by the release of cytoplasmic and lysosomal enzymes. The extent of cellular destruction depended upon the amount of dust applied. In the range of 50-100 μg/ml quartz or chrysotile asbestos, species-specific variations were observed in the sensitivity of the cells. At this concentration alveolar macrophages of dogs, monkeys, and human patients were damaged to a greater extent than the cells from rats and cattle. Simultaneous incubation of the cells with quartz and L-αdipalmitoyl lecithin resulted in a reduction of the cytotoxic quartz effect. The extent of the protective effect varied according to the species. In the case of chrysotile asbestos no reduction of the fibers cytotoxicity was observed in the presence of L-αdipalmitoyl lecithin.
INTRODUCTION The inhalation of quartz and asbestos leads to pathological changes in the lung. Alveolar macrophages play a central role in the host defense mechanisms and in the development of lung fibrosis. In vitro investigations concerning the cytotoxic effects of quartz, asbestos, and other materials have been performed with macrophages of different species (Allison et al., 1966; Davies et al., 1974; Hill and Hobbs, 1982; White et al., 1983; Wallace et al., 1985; Lock et al., 1987; Hahon et al., 1986; Morrison et The authors are grateful to Prof. Dr. N. H. Seemayer (Düsseldorf) and Prof. Dr. K. Spurny (Schmallenberg-Grafschaft) for providing us with quartz and asbestos particles and also to Dr. W. G. Kreyling (München), Dr. F. Krombach (München) and Dr. G. Würtemberger (Freiburg) for their kind support. Requests for reprints should be sent to Prof. Dr. A. Seidel, Institut für Genetik und Toxikologie, Postfach 36 40, D-7500 Karlsruhe 1, Germany. 131 Journal of Toxicology and Environmental Health, 33:131-140, 1991 Copyright © 1991 by Hemisphere Publishing Corporation
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al., 1986). Up to now a systematic interspecies comparison has been missing. The aim of our studies was to compare the cytotoxic effects of native and surface-modified DQ12 quartz and chrysotile asbestos on alveolar macrophages of rat, cattle, dog, monkey, and human patients as well as on a human monocytic cell line (U937). In vitro studies with native dusts might lead to false, strong positive predictions concerning the cytotoxicity of the tested dust, due to the fact that in vivo inhaled dust particles become coated with protective surfactant. Lung surfactant is composed of proteins and lipids, mainly L-adipalmitoyl lecithin (DPL). Wallace et al. (1985) have shown that DPLcoated quartz particles were nontoxic in short-term cultures with rat alveolar macrophages. Simultaneous incubation of cells with dust and DPL represents a more realistic test system. In order to extend the studies of Wallace et al. we have examined the influence of DPL on the cytotoxicity of DQ12 quartz and chrysotile asbestos for longer periods of time with alveolar macrophages of different species. MATERIALS AND METHODS Alveolar Macrophages Alveolar macrophages of rat and cattle were obtained by post mortem lavage. Macrophages from dog (beagle), monkey (Macaca fascicularis), and human patients (one smoker and two sarcoidosis patients, one of them was under steroid treatment) were isolated by segmental bronchoalveolar lavage using routine procedures (for further details see Schimmelpfeng, 1989). Dog alveolar macrophages were received from Dr. W. G. Kreyling (GSF, München), monkey alveolar macrophages from Dr. F. Krombach (Klinikum Großhadern, München), and patients' macrophages from Dr. G. Würtemberger (Robert-Koch-Klinik, Freiburg). Cells (1-3 x 106) were cultured in 35-mm plastic dishes (Multiwell plates, Falcon) in 1 ml RPMI 1640 medium (Gibco, Paisley) with 100 U/ml penicillin and 100 ^g/ml streptomycin. Following a 2-h incubation period (37°C, 5% CO2, 95% humified air) the nonadherent cells were removed by a change of medium. Human Cell Line
Human, monocytic U937 cells were obtained from Prof. Dr. H. Rahmsdorf (KfK, Karlsruhe). The cells were grown in suspension in RPMI 1640 medium with 15% fetal calf serum (Gibco, Paisley), 100 ¿ig/ml ampicillin, and 10 /¿g/ml tetracyclin. During the 20-h interval of dust exposure, in RPMI medium without FCS, the U937 cells became adherent.
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Mineral Dusts
The international standard quartz DQ12 (Robock, 1973) was received from Prof. Dr. N. H. Seemayer (Medizinisches Institut für Umwelthygiene, Düsseldorf). The geometric diameter of the particles was 0.8 ± 0.2 ¡xm (evaluated from scanning electron microscopic pictures). Canadian UICC chrysotil A asbestos was supplied by Prof. Dr. K. Spurny (Fraunhofer Institut, Schmallenberg-Graïschaft; for details of preparation see Spurny et al., 1979). Approximately 95% of the fibers had a geometric diameter less than 0.5 ^m and 97% were shorter than 5 /¿m. The particles were suspended in medium, the quartz suspensions being sonicated on ice before use. Exposure of the Cultures
To determine the dose-response relationships, the cells were incubated for 20 h with different concentrations of the dusts (0-500 /¿g/ml). In separate experiments incubations with DQ12 quartz or chrysotile asbestos (250 /ig/ml) were performed in the presence of DPL (333 ^g/ml). The DPL suspension was sonicated on ice prior to use and was applied first to the cells, immediately followed by the dust suspension. DPL alone had no influence on the viability of the cells. Determination of Cell Viability and Enzyme Release
After 20 h of incubation the cell viability was tested by trypan blue dye exclusion test (0.25% in physiological saline, 3 min). For biochemical analysis (measurement of LDH and NAG) the cell cultures were treated as follows: medium and cells were collected separately. The medium was centrifuged to remove nonadherent cells and cellular debris from the supernatant to form a pellet. Cells and pellets were resuspended in 1 ml of medium. The three fractions (cells, debris, and supernatant) were sonicated prior to measurement of lactate dehydrogenase (LDH; LDH-Monotest, Boehringer, Mannheim) and N-acetyl-ßglucosaminidase (NAG; Andersen et al., 1982). The enzyme release into the supernatant (Figs. 2 and 3) was related to the sum of enzyme measured in the three fractions. Due to limited amounts of DQ12 quartz, chrysotile asbestos and cells from dogs, monkeys, and humans available for this study, the dose-response relationships were tested only a few times. Several repetitions were made with 250 fig dust/ml. RESULTS Incubation of alveolar macrophages from different species with DQ12 quartz or chrysotile asbestos led to qualitatively the same results.
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The viability of the cells decreased on increasing the dust concentration (Fig. 1a and 6). The number of trypan-blue-colored cells correlated with the portion of LDH released into the supernatant (Fig. 2). Correspondingly the intracellular amount of LDH decreased. Quantitatively species-specific differences appeared in the sensitivity of the cells toward DQ12 quartz up to a concentration of 100 ^g/ml (Fig. 1a). In the case of chrysotile asbestos, species-specific variations in sensitivity continued up to 500 /ig/ml (Fig. 16). After the incubation with 250 ¡xg quartz/ml (Fig. 26) alveolar macrophages of all species were damaged almost completely. Approximately 70-90% of the total LDH and 30-50% of the NAG were Downloaded by [Universite Laval] at 21:03 05 November 2015
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FIGURE 1. Decrease of cell viability after 20 h of exposure of alveolar macrophages of different species and U937 cells with different concentrations of (a) DQ12 quartz and (b) chrysotile asbestos; mean values and standard errors are shown, and the figures represent the number of tested individuals.
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FIGURE 2. Increase of nonviable cells and release of lactate dehydrogenase (LDH) and N-acetyl-/3glucosaminidase (NAG) after 20 h of incubation of alveolar macrophages of different species and U937 cells with 250 ¿tg dust/ml: (a) untreated control, (6) DQ12 quartz-treated, and (c) chrysotileasbestos-treated cells. Mean values and standard errors are shown; the figures in the base of the columns represent the number of tested individuals; the sum of enzyme content in supernatants, adherent, and nonadherent cells is 100%.
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found in the culture medium. The application of chrysotile asbestos (250 /ig/ml, Fig. 2c) led to a smaller release of LDH and NAG and a smaller decrease in cell viability than the same concentration of DQ12 quartz (Fig. 2b). Only monkey alveolar macrophages were damaged completely in both cases. The monocytic U937 cells were less sensitive toward quartz than the other cells (Figs. 1a and 2b) and resistant against chrysotile asbestos (Figs. 1b and 2c). In comparison to the release of LDH, the relative release of NAG was lower in all cases except for the smokers and bovine cells. Incubation of bovine alveolar macrophages (BAM) with quartz in the presence of DPL led to the complete abrogation of the cytotoxic quartz effect (Fig. 3a and c). The degree of the protective effect varied according to the species. It was highest in case of BAM and lowest for the human alveolar macrophages. With respect to enzyme release the amount of protection was quantitatively the same for LDH and NAG. Simultaneous incubation of cells with DPL and chrysotile asbestos led to no reduction of fiber cytotoxicity (Fig. 3b and d), not even in the presence of higher DPL concentrations (data not shown). DISCUSSION The main aim of our studies was to compare the sensitivity of alveolar macrophages from humans and different animal species toward quartz and chrysotile asbestos. According to our results there are no principal or systematic differences among the species tested. The response of the cells toward the toxic dusts was qualitatively the same with the exception of the U937 cells. In the range of 50-100 ¿ig quartz/ml bovine and rat alveolar macrophages were more resistant than the cells from the other animal species. Toward chrysotile asbestos the cells from cattle, rat, and one human smoker were less sensitive, whereas the cells from monkey, dog, and the human sarcoidosis patients were more sensitive. This study was primarily descriptive; it cannot give any explanations for the observed quantitative differences between the species. On the other hand, the fact that the reaction of cells from various species is qualitatively similar is important for the extrapolation of results from various laboratories working with different species. Our special interest was also to compare the reaction of bovine alveolar macrophages with that of other species since we are working mainly with bovine cells (alveolar macrophages can be obtained from bovine lungs in very large numbers). The human monocytic cell line U937 was used in the nondifferentiated form and was least sensitive to quartz and not at all sensitive to chrysotile asbestos. This is in agreement with our observation (Seidel et al., 1990) that another human cell line (HL-60, cells differentiated to macrophage-like cells) was also completely resistant toward quartz parti-
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Asbestos
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FIGURE 3. Effect of L-a-dipalmityl lecithin (DPL) o n t h e (a, c) DQ12 quartz-induced or (b, d) chrysotile asbestos-induced release of cytoplasmic lactate dehydrogenase (LDH, a, b) or lysosomal Nacetyl-|8-glucosaminidase (NAG, c, d). Alveolar macrophages of different species and U937 cells were incubated w i t h 250 ng dust/ml in t h e presence of 333 fig DPL/ml f o r 20 h; mean values and standard errors are s h o w n ; asterisk indicates statistically significantly different w i t h 2p < 0.05 f r o m quartz-treated cells and untreated control cells; d o u b l e asterisk indicates statistically not different f r o m untreated control cells. The figures in the base of t h e columns represent t h e n u m b e r of tested individuals; t h e sum o f enzyme content in supernatants, adherent, and nonadherent cells is 100%.
cles. We suggest that the differences in sensitivity toward quartz between the monocytic cell line (U937) and the macrophages are not due to former presence of fetal calf serum (in the case of U937 cells). Preincubation of bovine alveolar macrophages with protective DPL, followed by the exposure to native DQ12 quartz, led to the complete cytotoxic quartz effect—in contrast to a simultaneous exposure of cells to quartz in the presence of DPL (Schimmelpfeng, 1989). Again, we can only speculate as to the underlying mechanisms, but the results suggest that human myeloic cell lines are not suitable as models for working with quartz or asbestos. Behrendt et al. (1987) have also reported that human monocytederived macrophages are less sensitive toward quartz than alveolar macrophages. With only two exceptions, cattle and smoker, the relative release of LDH was higher than that of NAG. This suggests that the damage of the pericellular membrane is more accentuated and/or precedes that of the lysosomal membranes. As compared to the findings of other authors
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[Morgan and Allison (1980), human alveolar, mouse peritoneal, or guinea pig alveolar macrophages; Davies et al. (1974), mouse peritoneal macrophages; Jaurand et al. (1980), rabbit alveolar macrophages], our results with low concentrations of chrysotile asbestos are different. We did not observe a selective release of lysosomal enzymes without remarkable decrease in cell viability or release of LDH. However, other authors [White et al. (1983), bovine alveolar macrophages; Hahon et al. (1986) and Nadeau et al. (1987), rat alveolar macrophages] also found a combined release of LDH and lysosomal enzymes. We do not know the reason for these discrepancies, but they may be due to different properties (length, diameter, grade of contamination, etc.) of the chrysotile asbestos fibers used. Incubation of bovine alveolar macrophages with DQ12 quartz in the presence of DPL led to a complete abrogation of the cytotoxic quartz effect. No protection was found when bovine alveolar macrophages were first cultured with DPL alone, followed by the incubation with native quartz (Schimmelpfeng, 1989). Therefore the protective effect of DPL must be due to the interaction between DPL and the surface of the quartz particles (Wallace et al., 1985). Species-specific differences with respect to the amount of DPL protection might be due to variations in the speed of DPL digestion by lysosomal enzymes. According to Keane and co-workers (1988), DPL-coated particles became retoxified by the phagolysosomal enzyme phospholipase A2 in a cell-free system. In bovine alveolar macrophages, DPL-coated quartz remained nontoxic for at least 4 d (Pätzold, 1989), whereas the cellular damage of rat and human macrophages after 20 h of incubation with DQ12 quartz, in spite of the presence of DPL, indicates a retoxification process inside the cell. Incubation of alveolar macrophages with DPL and chrysotile asbestos led to no protective effect in any species. According to Bignon and Jaurand (1983), DPL adsorbs onto the surface of chrysotile asbestos. Thus the toxicity of chrysotile asbestos might depend on another parameter than its surface. Hahon and co-workers (1986) showed that longer fibers were more cytotoxic than shorter ones. As our fibers were very short in comparison to those used in the studies of Hahon and co-workers we suggest a further explanation: Jaurand et al. (1984) reported that about 40% of the fiber's magnesium was leached out and is removed in the acid environment of the cells phagolysosomes. The so-called leaching effect has been described by several other authors (Morgan and Holmes, 1986; Trosic and Stilinovic, 1987). This process even took place in the case of chrysotile asbestos fibers coated with membranes from erythrocytes. It is therefore possible that DPL-coated fibers become retoxified in phagolysosomes of the cells by removal of the outer DPL-brucite layer from the fiber with exposure of the underlying toxic silicate skeleton (Sample and Horn, 1979).
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REFERENCES Allison, A. C., Harington, J. S., and Birbeck, M. 1966. An examination of the cytotoxic effects of silica on macrophages. J. Exp. Med. 124:141-153. Andersen, O. K., Stenvold, S. E., and Volden, G. 1982. Optimalized assay conditions of 3T3 cell lysosomal hydrolases. Eur. Rev. Med. Pharmacol. Sci. IV:265-272. Behrendt, H., Seemayer, N. H., Braumann, A., and Nissen, H. 1987. Elektronen-mikroskopische Untersuchungen zur Wirkung von Quarzstaub DQ12 auf menschliche Monozyten/Makrophagen in vitro. Silikosebericht Nordrhein-Westfalen 16:213-222. Bignon, J., and Jaurand, M. C. 1983. Biological in vitro and in vivo responses of chrysotile versus amphiboles. Environ. Health Perspect. 51:73-80. Davies, P., Allison, A. C., Ackerman, J., Butterfield, A., and Williams, S. 1974. Asbestos induces selective release of lysosomal enzymes from mononuclear phagocytes. Nature (Lond.) 251:423-425. Hahon, N., Vallyathan, V., Booth, J. A., and Sepulveda, M. J. 1986. In vitro biologic responses to native and surface-modified asbestos. Environ. Res. 39:45-355. Hill, J. O., and Hobbs, C. H. 1982. Comparative cytotoxicity of DQ12-quartz and fly ash particles from coal combustion. Toxicol. Lett. 10:399-403. Jaurand, M. C., Magne, L., and Bignon, J. 1980. The significance of lysosomal enzyme release induced by asbestos. In The In Vitro Effects of Mineral Dusts, eds. R. C. Brown, M. Chamberlain, R. Davies, and I. P. Gormley, pp. 83-87. New York: Academic Press. Jaurand, M. C., Caudichet, A., Halpern, S., and Bignon, J. 1984. In vitro biodégradation of chrysotile fibres by alveolar macrophages and mesothelial cells in culture: Comparison with a pH effect. Br. J. Ind. Med. 41:389-395. Keane, M., Wallace, W., Seerha, M., Hill, C., Vallyathan, V, Raghootama, P., and Mike, P. 1988. Respirable particulate surface interactions with the lecithin component of pulmonary surfactant. In ILO Conference Proc., VIIth International Pneumoconiosis Conference, August 23-26, 1988, Pittsburgh. Lock, S. O., Jones, P. A., Friend, J. V., and Parish, W. E. 1987. Extracellular release of enzymes from macrophages in vitro for measuring cellular interaction with particulate and non-particulate materials. Toxic. in Vitro 1:77-83. Morgan, D. M. L., and Allison, A. C. 1980. Effects of silica and asbestos on alveolar and peritoneal macrophages: A comparative study. In The In Vitro Effects of Mineral Dusts, eds. R. C. Brown, M. Chamberlain, R. Davies, and I. P. Gormley, pp. 75-81. New York: Academic Press. Morgan, A., and Holmes, A. 1986. Solubility of asbestos and man-made mineral fibers in vitro and in vivo: Its significance in lung disease. Environ. Res. 39:475-484. Morrison, D. G., McLemore, T. L., Lawrence, E. C., Feuerbacher, D. G., Mace, M. L., Jr., Busbee, D. L., Griffin, A. C., and Marshall, M. V. 1986. In vitro cytotoxicity of chrysotile asbestos to human pulmonary alveolar macrophages is decreased by organosilane coating and surfactant. Cell Biol. Toxicol. 2:293-309. Nadeau, D., Fouquette-Couture, L., Paradis, D., Khorami, J., Lane, D., and Dunnigan, J. 1987. Cytotoxicity of respirable dusts from industrial materials: Comparison of two naturally occurring and two man-made silicates. Drug Chem. Toxicol. 10:49-86. Pätzold, S. 1989. Morphologische und biochemische Untersuchungen zur Wirkung von Quarz auf Rinderalveolarmakrophagen und deren Organellen. Report of Kernforschungszentrum Karlsruhe, KfK 4669. Sample, T. E., Jr., and Horn, J. M. 1979. Silane coated silicate minerals and method for preparing same. U.S. Patent 4137367, Jan. 30, 1979. Schimmelpfeng, J. 1989. Zytotoxische Wirkungen von Quarz und Asbest: Ein in vitro Interspeciesvergleich mit Alveolarmakrophagen. Report of Kernforschungszentrum Karlsruhe, KfK 4624. Seidel, A., Nieder, U., Pätzold, S., Schimmelpfeng, J., Schmidt, A., and Wilzcek, W. 1990. Effects of quartz and asbestos on alveolar macrophages: Interspecies comparison and cell biological
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studies. In Environmental Hygiene II, eds. N. H. Seemayer and W. Hadnagy, pp. 199-202. Berlin: Springer. Spumy, K. R., Stöber, W., Opiela, H., and Weiss, C. 1979. Size selective preparation of inorganic fibers for biological experiments. Am. Ind. Hyg. Assoc. J. 40:20-38. Trosic, I., and Stilinovic, L. 1987. Direct haemolysis of erythrocytes produced by chrysotile asbestos fibres and soluble constituents of chrysotile asbestos in vitro. Jugo. Physiol. Pharmacol. Acta 23:189-197. Wallace, W. E., Jr., Vallyathan, V., Keane, M. J., and Robinson, V. 1985. In vitro biologic toxicity of native and surface-modified silica and kaolin. J. Toxicol. Environ. Health 16:415-424. White, L. R., Marthinsen, A. B. L., Jakobsen, K., and Eik-Nees, B. 1983. Response of bovine alveolar macrophages in vitro to welding fume particles. Environ. Health Perspect. 51:211-215. Received July 6, 1990 Accepted December 14, 1990
ISSN: 0098-4108 (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/uteh19
Cytotoxic effects of quartz and chrysotile asbestos: In vitro interspecies comparison with alveolar macrophages J. Schimmelpfeng & A. Seidel To cite this article: J. Schimmelpfeng & A. Seidel (1991) Cytotoxic effects of quartz and chrysotile asbestos: In vitro interspecies comparison with alveolar macrophages, Journal of Toxicology and Environmental Health, 33:2, 131-140, DOI: 10.1080/15287399109531513 To link to this article: http://dx.doi.org/10.1080/15287399109531513
Published online: 19 Oct 2009.
Submit your article to this journal
View related articles
Citing articles: 9 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=uteh20 Download by: [Universite Laval]
Date: 05 November 2015, At: 21:03
CYTOTOXIC EFFECTS OF QUARTZ AND CHRYSOTILE ASBESTOS: IN VITRO INTERSPECIES COMPARISON WITH ALVEOLAR MACROPHAGES J. Schimmelpfeng
Downloaded by [Universite Laval] at 21:03 05 November 2015
Hauptabteilung Sicherheit/Biophysik, Kernforschungszentrum Karlsruhe, Germany A. Seidel Institut für Genetik und Toxikologie, Kernforschungszentrum Karlsruhe, Germany
Cytotoxic effects of DQ12 quartz and chrysotile asbestos on alveolar macrophages of different animal species were compared in vitro. The type of cell reaction toward the cytotoxic dusts was always the same: a loss of cell viability (trypan blue dye exclusion test) was accompanied by the release of cytoplasmic and lysosomal enzymes. The extent of cellular destruction depended upon the amount of dust applied. In the range of 50-100 μg/ml quartz or chrysotile asbestos, species-specific variations were observed in the sensitivity of the cells. At this concentration alveolar macrophages of dogs, monkeys, and human patients were damaged to a greater extent than the cells from rats and cattle. Simultaneous incubation of the cells with quartz and L-αdipalmitoyl lecithin resulted in a reduction of the cytotoxic quartz effect. The extent of the protective effect varied according to the species. In the case of chrysotile asbestos no reduction of the fibers cytotoxicity was observed in the presence of L-αdipalmitoyl lecithin.
INTRODUCTION The inhalation of quartz and asbestos leads to pathological changes in the lung. Alveolar macrophages play a central role in the host defense mechanisms and in the development of lung fibrosis. In vitro investigations concerning the cytotoxic effects of quartz, asbestos, and other materials have been performed with macrophages of different species (Allison et al., 1966; Davies et al., 1974; Hill and Hobbs, 1982; White et al., 1983; Wallace et al., 1985; Lock et al., 1987; Hahon et al., 1986; Morrison et The authors are grateful to Prof. Dr. N. H. Seemayer (Düsseldorf) and Prof. Dr. K. Spurny (Schmallenberg-Grafschaft) for providing us with quartz and asbestos particles and also to Dr. W. G. Kreyling (München), Dr. F. Krombach (München) and Dr. G. Würtemberger (Freiburg) for their kind support. Requests for reprints should be sent to Prof. Dr. A. Seidel, Institut für Genetik und Toxikologie, Postfach 36 40, D-7500 Karlsruhe 1, Germany. 131 Journal of Toxicology and Environmental Health, 33:131-140, 1991 Copyright © 1991 by Hemisphere Publishing Corporation
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al., 1986). Up to now a systematic interspecies comparison has been missing. The aim of our studies was to compare the cytotoxic effects of native and surface-modified DQ12 quartz and chrysotile asbestos on alveolar macrophages of rat, cattle, dog, monkey, and human patients as well as on a human monocytic cell line (U937). In vitro studies with native dusts might lead to false, strong positive predictions concerning the cytotoxicity of the tested dust, due to the fact that in vivo inhaled dust particles become coated with protective surfactant. Lung surfactant is composed of proteins and lipids, mainly L-adipalmitoyl lecithin (DPL). Wallace et al. (1985) have shown that DPLcoated quartz particles were nontoxic in short-term cultures with rat alveolar macrophages. Simultaneous incubation of cells with dust and DPL represents a more realistic test system. In order to extend the studies of Wallace et al. we have examined the influence of DPL on the cytotoxicity of DQ12 quartz and chrysotile asbestos for longer periods of time with alveolar macrophages of different species. MATERIALS AND METHODS Alveolar Macrophages Alveolar macrophages of rat and cattle were obtained by post mortem lavage. Macrophages from dog (beagle), monkey (Macaca fascicularis), and human patients (one smoker and two sarcoidosis patients, one of them was under steroid treatment) were isolated by segmental bronchoalveolar lavage using routine procedures (for further details see Schimmelpfeng, 1989). Dog alveolar macrophages were received from Dr. W. G. Kreyling (GSF, München), monkey alveolar macrophages from Dr. F. Krombach (Klinikum Großhadern, München), and patients' macrophages from Dr. G. Würtemberger (Robert-Koch-Klinik, Freiburg). Cells (1-3 x 106) were cultured in 35-mm plastic dishes (Multiwell plates, Falcon) in 1 ml RPMI 1640 medium (Gibco, Paisley) with 100 U/ml penicillin and 100 ^g/ml streptomycin. Following a 2-h incubation period (37°C, 5% CO2, 95% humified air) the nonadherent cells were removed by a change of medium. Human Cell Line
Human, monocytic U937 cells were obtained from Prof. Dr. H. Rahmsdorf (KfK, Karlsruhe). The cells were grown in suspension in RPMI 1640 medium with 15% fetal calf serum (Gibco, Paisley), 100 ¿ig/ml ampicillin, and 10 /¿g/ml tetracyclin. During the 20-h interval of dust exposure, in RPMI medium without FCS, the U937 cells became adherent.
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Mineral Dusts
The international standard quartz DQ12 (Robock, 1973) was received from Prof. Dr. N. H. Seemayer (Medizinisches Institut für Umwelthygiene, Düsseldorf). The geometric diameter of the particles was 0.8 ± 0.2 ¡xm (evaluated from scanning electron microscopic pictures). Canadian UICC chrysotil A asbestos was supplied by Prof. Dr. K. Spurny (Fraunhofer Institut, Schmallenberg-Graïschaft; for details of preparation see Spurny et al., 1979). Approximately 95% of the fibers had a geometric diameter less than 0.5 ^m and 97% were shorter than 5 /¿m. The particles were suspended in medium, the quartz suspensions being sonicated on ice before use. Exposure of the Cultures
To determine the dose-response relationships, the cells were incubated for 20 h with different concentrations of the dusts (0-500 /¿g/ml). In separate experiments incubations with DQ12 quartz or chrysotile asbestos (250 /ig/ml) were performed in the presence of DPL (333 ^g/ml). The DPL suspension was sonicated on ice prior to use and was applied first to the cells, immediately followed by the dust suspension. DPL alone had no influence on the viability of the cells. Determination of Cell Viability and Enzyme Release
After 20 h of incubation the cell viability was tested by trypan blue dye exclusion test (0.25% in physiological saline, 3 min). For biochemical analysis (measurement of LDH and NAG) the cell cultures were treated as follows: medium and cells were collected separately. The medium was centrifuged to remove nonadherent cells and cellular debris from the supernatant to form a pellet. Cells and pellets were resuspended in 1 ml of medium. The three fractions (cells, debris, and supernatant) were sonicated prior to measurement of lactate dehydrogenase (LDH; LDH-Monotest, Boehringer, Mannheim) and N-acetyl-ßglucosaminidase (NAG; Andersen et al., 1982). The enzyme release into the supernatant (Figs. 2 and 3) was related to the sum of enzyme measured in the three fractions. Due to limited amounts of DQ12 quartz, chrysotile asbestos and cells from dogs, monkeys, and humans available for this study, the dose-response relationships were tested only a few times. Several repetitions were made with 250 fig dust/ml. RESULTS Incubation of alveolar macrophages from different species with DQ12 quartz or chrysotile asbestos led to qualitatively the same results.
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The viability of the cells decreased on increasing the dust concentration (Fig. 1a and 6). The number of trypan-blue-colored cells correlated with the portion of LDH released into the supernatant (Fig. 2). Correspondingly the intracellular amount of LDH decreased. Quantitatively species-specific differences appeared in the sensitivity of the cells toward DQ12 quartz up to a concentration of 100 ^g/ml (Fig. 1a). In the case of chrysotile asbestos, species-specific variations in sensitivity continued up to 500 /ig/ml (Fig. 16). After the incubation with 250 ¡xg quartz/ml (Fig. 26) alveolar macrophages of all species were damaged almost completely. Approximately 70-90% of the total LDH and 30-50% of the NAG were Downloaded by [Universite Laval] at 21:03 05 November 2015
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FIGURE 1. Decrease of cell viability after 20 h of exposure of alveolar macrophages of different species and U937 cells with different concentrations of (a) DQ12 quartz and (b) chrysotile asbestos; mean values and standard errors are shown, and the figures represent the number of tested individuals.
CYTOTOXICITY OF QUARTZ A N D ASBESTOS
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Control
• Nonviable cells ES Release of LDH • Release of NAG
H 100 Quartz
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Smoker Rat
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Monkey
Sarcoid. Patients
U 937 Cell-line
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FIGURE 2. Increase of nonviable cells and release of lactate dehydrogenase (LDH) and N-acetyl-/3glucosaminidase (NAG) after 20 h of incubation of alveolar macrophages of different species and U937 cells with 250 ¿tg dust/ml: (a) untreated control, (6) DQ12 quartz-treated, and (c) chrysotileasbestos-treated cells. Mean values and standard errors are shown; the figures in the base of the columns represent the number of tested individuals; the sum of enzyme content in supernatants, adherent, and nonadherent cells is 100%.
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). SCHIMMELPFENG A N D A. SEIDEL
found in the culture medium. The application of chrysotile asbestos (250 /ig/ml, Fig. 2c) led to a smaller release of LDH and NAG and a smaller decrease in cell viability than the same concentration of DQ12 quartz (Fig. 2b). Only monkey alveolar macrophages were damaged completely in both cases. The monocytic U937 cells were less sensitive toward quartz than the other cells (Figs. 1a and 2b) and resistant against chrysotile asbestos (Figs. 1b and 2c). In comparison to the release of LDH, the relative release of NAG was lower in all cases except for the smokers and bovine cells. Incubation of bovine alveolar macrophages (BAM) with quartz in the presence of DPL led to the complete abrogation of the cytotoxic quartz effect (Fig. 3a and c). The degree of the protective effect varied according to the species. It was highest in case of BAM and lowest for the human alveolar macrophages. With respect to enzyme release the amount of protection was quantitatively the same for LDH and NAG. Simultaneous incubation of cells with DPL and chrysotile asbestos led to no reduction of fiber cytotoxicity (Fig. 3b and d), not even in the presence of higher DPL concentrations (data not shown). DISCUSSION The main aim of our studies was to compare the sensitivity of alveolar macrophages from humans and different animal species toward quartz and chrysotile asbestos. According to our results there are no principal or systematic differences among the species tested. The response of the cells toward the toxic dusts was qualitatively the same with the exception of the U937 cells. In the range of 50-100 ¿ig quartz/ml bovine and rat alveolar macrophages were more resistant than the cells from the other animal species. Toward chrysotile asbestos the cells from cattle, rat, and one human smoker were less sensitive, whereas the cells from monkey, dog, and the human sarcoidosis patients were more sensitive. This study was primarily descriptive; it cannot give any explanations for the observed quantitative differences between the species. On the other hand, the fact that the reaction of cells from various species is qualitatively similar is important for the extrapolation of results from various laboratories working with different species. Our special interest was also to compare the reaction of bovine alveolar macrophages with that of other species since we are working mainly with bovine cells (alveolar macrophages can be obtained from bovine lungs in very large numbers). The human monocytic cell line U937 was used in the nondifferentiated form and was least sensitive to quartz and not at all sensitive to chrysotile asbestos. This is in agreement with our observation (Seidel et al., 1990) that another human cell line (HL-60, cells differentiated to macrophage-like cells) was also completely resistant toward quartz parti-
CYTOTOXICITY O F Q U A R T Z A N D ASBESTOS
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Quartz
Asbestos
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fr
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Smoker Sarcoid. U937 , Patients Cell-line, Man
Rat
4
5
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Native Oust Dust + DPL
Cattle
Sarcoid. Patients Man
FIGURE 3. Effect of L-a-dipalmityl lecithin (DPL) o n t h e (a, c) DQ12 quartz-induced or (b, d) chrysotile asbestos-induced release of cytoplasmic lactate dehydrogenase (LDH, a, b) or lysosomal Nacetyl-|8-glucosaminidase (NAG, c, d). Alveolar macrophages of different species and U937 cells were incubated w i t h 250 ng dust/ml in t h e presence of 333 fig DPL/ml f o r 20 h; mean values and standard errors are s h o w n ; asterisk indicates statistically significantly different w i t h 2p < 0.05 f r o m quartz-treated cells and untreated control cells; d o u b l e asterisk indicates statistically not different f r o m untreated control cells. The figures in the base of t h e columns represent t h e n u m b e r of tested individuals; t h e sum o f enzyme content in supernatants, adherent, and nonadherent cells is 100%.
cles. We suggest that the differences in sensitivity toward quartz between the monocytic cell line (U937) and the macrophages are not due to former presence of fetal calf serum (in the case of U937 cells). Preincubation of bovine alveolar macrophages with protective DPL, followed by the exposure to native DQ12 quartz, led to the complete cytotoxic quartz effect—in contrast to a simultaneous exposure of cells to quartz in the presence of DPL (Schimmelpfeng, 1989). Again, we can only speculate as to the underlying mechanisms, but the results suggest that human myeloic cell lines are not suitable as models for working with quartz or asbestos. Behrendt et al. (1987) have also reported that human monocytederived macrophages are less sensitive toward quartz than alveolar macrophages. With only two exceptions, cattle and smoker, the relative release of LDH was higher than that of NAG. This suggests that the damage of the pericellular membrane is more accentuated and/or precedes that of the lysosomal membranes. As compared to the findings of other authors
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[Morgan and Allison (1980), human alveolar, mouse peritoneal, or guinea pig alveolar macrophages; Davies et al. (1974), mouse peritoneal macrophages; Jaurand et al. (1980), rabbit alveolar macrophages], our results with low concentrations of chrysotile asbestos are different. We did not observe a selective release of lysosomal enzymes without remarkable decrease in cell viability or release of LDH. However, other authors [White et al. (1983), bovine alveolar macrophages; Hahon et al. (1986) and Nadeau et al. (1987), rat alveolar macrophages] also found a combined release of LDH and lysosomal enzymes. We do not know the reason for these discrepancies, but they may be due to different properties (length, diameter, grade of contamination, etc.) of the chrysotile asbestos fibers used. Incubation of bovine alveolar macrophages with DQ12 quartz in the presence of DPL led to a complete abrogation of the cytotoxic quartz effect. No protection was found when bovine alveolar macrophages were first cultured with DPL alone, followed by the incubation with native quartz (Schimmelpfeng, 1989). Therefore the protective effect of DPL must be due to the interaction between DPL and the surface of the quartz particles (Wallace et al., 1985). Species-specific differences with respect to the amount of DPL protection might be due to variations in the speed of DPL digestion by lysosomal enzymes. According to Keane and co-workers (1988), DPL-coated particles became retoxified by the phagolysosomal enzyme phospholipase A2 in a cell-free system. In bovine alveolar macrophages, DPL-coated quartz remained nontoxic for at least 4 d (Pätzold, 1989), whereas the cellular damage of rat and human macrophages after 20 h of incubation with DQ12 quartz, in spite of the presence of DPL, indicates a retoxification process inside the cell. Incubation of alveolar macrophages with DPL and chrysotile asbestos led to no protective effect in any species. According to Bignon and Jaurand (1983), DPL adsorbs onto the surface of chrysotile asbestos. Thus the toxicity of chrysotile asbestos might depend on another parameter than its surface. Hahon and co-workers (1986) showed that longer fibers were more cytotoxic than shorter ones. As our fibers were very short in comparison to those used in the studies of Hahon and co-workers we suggest a further explanation: Jaurand et al. (1984) reported that about 40% of the fiber's magnesium was leached out and is removed in the acid environment of the cells phagolysosomes. The so-called leaching effect has been described by several other authors (Morgan and Holmes, 1986; Trosic and Stilinovic, 1987). This process even took place in the case of chrysotile asbestos fibers coated with membranes from erythrocytes. It is therefore possible that DPL-coated fibers become retoxified in phagolysosomes of the cells by removal of the outer DPL-brucite layer from the fiber with exposure of the underlying toxic silicate skeleton (Sample and Horn, 1979).
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REFERENCES Allison, A. C., Harington, J. S., and Birbeck, M. 1966. An examination of the cytotoxic effects of silica on macrophages. J. Exp. Med. 124:141-153. Andersen, O. K., Stenvold, S. E., and Volden, G. 1982. Optimalized assay conditions of 3T3 cell lysosomal hydrolases. Eur. Rev. Med. Pharmacol. Sci. IV:265-272. Behrendt, H., Seemayer, N. H., Braumann, A., and Nissen, H. 1987. Elektronen-mikroskopische Untersuchungen zur Wirkung von Quarzstaub DQ12 auf menschliche Monozyten/Makrophagen in vitro. Silikosebericht Nordrhein-Westfalen 16:213-222. Bignon, J., and Jaurand, M. C. 1983. Biological in vitro and in vivo responses of chrysotile versus amphiboles. Environ. Health Perspect. 51:73-80. Davies, P., Allison, A. C., Ackerman, J., Butterfield, A., and Williams, S. 1974. Asbestos induces selective release of lysosomal enzymes from mononuclear phagocytes. Nature (Lond.) 251:423-425. Hahon, N., Vallyathan, V., Booth, J. A., and Sepulveda, M. J. 1986. In vitro biologic responses to native and surface-modified asbestos. Environ. Res. 39:45-355. Hill, J. O., and Hobbs, C. H. 1982. Comparative cytotoxicity of DQ12-quartz and fly ash particles from coal combustion. Toxicol. Lett. 10:399-403. Jaurand, M. C., Magne, L., and Bignon, J. 1980. The significance of lysosomal enzyme release induced by asbestos. In The In Vitro Effects of Mineral Dusts, eds. R. C. Brown, M. Chamberlain, R. Davies, and I. P. Gormley, pp. 83-87. New York: Academic Press. Jaurand, M. C., Caudichet, A., Halpern, S., and Bignon, J. 1984. In vitro biodégradation of chrysotile fibres by alveolar macrophages and mesothelial cells in culture: Comparison with a pH effect. Br. J. Ind. Med. 41:389-395. Keane, M., Wallace, W., Seerha, M., Hill, C., Vallyathan, V, Raghootama, P., and Mike, P. 1988. Respirable particulate surface interactions with the lecithin component of pulmonary surfactant. In ILO Conference Proc., VIIth International Pneumoconiosis Conference, August 23-26, 1988, Pittsburgh. Lock, S. O., Jones, P. A., Friend, J. V., and Parish, W. E. 1987. Extracellular release of enzymes from macrophages in vitro for measuring cellular interaction with particulate and non-particulate materials. Toxic. in Vitro 1:77-83. Morgan, D. M. L., and Allison, A. C. 1980. Effects of silica and asbestos on alveolar and peritoneal macrophages: A comparative study. In The In Vitro Effects of Mineral Dusts, eds. R. C. Brown, M. Chamberlain, R. Davies, and I. P. Gormley, pp. 75-81. New York: Academic Press. Morgan, A., and Holmes, A. 1986. Solubility of asbestos and man-made mineral fibers in vitro and in vivo: Its significance in lung disease. Environ. Res. 39:475-484. Morrison, D. G., McLemore, T. L., Lawrence, E. C., Feuerbacher, D. G., Mace, M. L., Jr., Busbee, D. L., Griffin, A. C., and Marshall, M. V. 1986. In vitro cytotoxicity of chrysotile asbestos to human pulmonary alveolar macrophages is decreased by organosilane coating and surfactant. Cell Biol. Toxicol. 2:293-309. Nadeau, D., Fouquette-Couture, L., Paradis, D., Khorami, J., Lane, D., and Dunnigan, J. 1987. Cytotoxicity of respirable dusts from industrial materials: Comparison of two naturally occurring and two man-made silicates. Drug Chem. Toxicol. 10:49-86. Pätzold, S. 1989. Morphologische und biochemische Untersuchungen zur Wirkung von Quarz auf Rinderalveolarmakrophagen und deren Organellen. Report of Kernforschungszentrum Karlsruhe, KfK 4669. Sample, T. E., Jr., and Horn, J. M. 1979. Silane coated silicate minerals and method for preparing same. U.S. Patent 4137367, Jan. 30, 1979. Schimmelpfeng, J. 1989. Zytotoxische Wirkungen von Quarz und Asbest: Ein in vitro Interspeciesvergleich mit Alveolarmakrophagen. Report of Kernforschungszentrum Karlsruhe, KfK 4624. Seidel, A., Nieder, U., Pätzold, S., Schimmelpfeng, J., Schmidt, A., and Wilzcek, W. 1990. Effects of quartz and asbestos on alveolar macrophages: Interspecies comparison and cell biological
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studies. In Environmental Hygiene II, eds. N. H. Seemayer and W. Hadnagy, pp. 199-202. Berlin: Springer. Spumy, K. R., Stöber, W., Opiela, H., and Weiss, C. 1979. Size selective preparation of inorganic fibers for biological experiments. Am. Ind. Hyg. Assoc. J. 40:20-38. Trosic, I., and Stilinovic, L. 1987. Direct haemolysis of erythrocytes produced by chrysotile asbestos fibres and soluble constituents of chrysotile asbestos in vitro. Jugo. Physiol. Pharmacol. Acta 23:189-197. Wallace, W. E., Jr., Vallyathan, V., Keane, M. J., and Robinson, V. 1985. In vitro biologic toxicity of native and surface-modified silica and kaolin. J. Toxicol. Environ. Health 16:415-424. White, L. R., Marthinsen, A. B. L., Jakobsen, K., and Eik-Nees, B. 1983. Response of bovine alveolar macrophages in vitro to welding fume particles. Environ. Health Perspect. 51:211-215. Received July 6, 1990 Accepted December 14, 1990
