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In vitro effects of incinerator fly ash on pulmonary macrophages and epithelial cells1
International Journal of Hygiene and Environmental Health
Int. J. Hyg. Environ. Health 204, 323 ± 326 (2002) ¹ Urban & Fischer Verlag http: // www.urbanfischer.de/journals/intjhyg
In vitro effects of incinerator fly ash on pulmonary macrophages and epithelial cells* Silvia Diabate¬a, Sonja M¸lhoptb, Hanns-R. Paurb, Ralf Wottricha, Harald F. Kruga a b
Institute of Toxicology and Genetics, Forschungszentrum Karlsruhe, Karlsruhe, Germany Institute of Technical Chemistry, Forschungszentrum Karlsruhe, Karlsruhe, Germany
Abstract Fly ash from a municipal waste incinerator was used as a model for atmospheric particles in order to identify parameters relevant for the induction of adverse health effects. The aim of this study was to compare the biological effects of the total incinerator fly ash (IFA), the soluble and the insoluble fraction with the effects of quartz by in vitro toxicity studies. The previously sized fly ash (< 20 mm) was characterized by elemental composition and particle size distribution. The particles were administered to rat alveolar macrophages (NR8383) and human bronchial epithelial cells (BEAS-2B) at different amounts via the medium. The total IFA and its insoluble fraction were shown to induce cytotoxicity and cytokine release in a dose-dependent manner. The soluble fraction was nearly unable to induce cytotoxicity and TNF-a release but showed potent induction of IL-8 release in BEAS-2B cells at increasing concentrations. Quartz caused similar effects compared to IFA in NR8383 but was less effective in BEAS-2B. Key words: fly ash ± in vitro ± alveolar macrophages ± lung epithelial cells
Introduction Particulate air pollution is a complex mixture of organic and inorganic compounds of different origin. Anthropogenic emissions including fly ash from combustion processes contribute in particular to the PM2.5 fraction which is under suspicion to threaten public health. Industrial combustion of coal or waste are well studied processes and the fly ash produced is frequently used as a model particle for PM10 and PM2.5 in toxicological studies (e.g. Smith et al., 1998). In vitro studies concentrate on pulmonary cells like alveolar macrophages which are in the first line of defence in the lung against inhaled
environmental pollutants. They are able to engulf particles and to produce biological mediators including reactive oxygen species, cytokines or growth factors to neutralize the pathological agents. Tumor necrosis factor alpha (TNF-a) plays a key role in this process (Driscoll et al., 1997). Primary effectors of cytokines are epithelial cells of the airways amplifying the inflammatory process by production of cytokines like IL-8 or IL-6. The purpose of this study was to examine the acute toxicity of IFA and its soluble and insoluble fractions as well as its potential to induce the release of inflammatory mediators in alveolar macrophage and bronchial epithelial cell lines. These results obtained by suspension of the fly
* Following a presentation given as part of the International Congress on Environmental Health and the 4th Annual Meeting of the International Society of Environmental Medicine (ISEM), 1-4 October, 2000, Hannover, Germany. Corresponding author: Silvia Diabate¬, Forschungszentrum Karlsruhe GmbH, Institute of Toxicology and Genetics, P.O. Box 3640, D-76021 Karlsruhe, Germany, Phone: 49 7247 822692, Fax: 49 7247 823557, E-mail: [email protected]
1438-4639/02/204-323 $ 15.00/0
324
S. Diabate¬ et al.
ash in culture medium will be compared to exposures to fly ash aerosol at the air-liquid interface in a later study.
Materials and methods Particles The fly ash obtained from a commercial municipal waste incinerator was collected in a bag filter. The sample was pre-sized and contained only particles < 20 mm. The crystalline quartz DQ12 (particle size < 5 mm, kindly provided by Prof. Dr. N. H. Seemayer, D¸sseldorf, Germany) was used as reference particle with well known cytotoxic properties. All particle suspensions were freshly prepared in the culture medium for the respective cells (10 mg/ml) and sonified. Insoluble and water soluble fractions were separated by centrifugation at 1000 g. Cell culture The NR8383 alveolar macrophage cell line from rat (ATCC, Rockville, MD) was maintained in Ham's F-12K medium containing 15% fetal bovine serum (both Life Sciences, Karlsruhe), 100 mg/ml penicillin and 100 U/ml streptomycin. BEAS-2B, immortalised cells of normal human bronchial epithelium, were maintained in serumfree growth medium (KGM, CellSystems, Remagen) (Mˆgel et al., 1998). For exposure to particles, the cells were seeded onto 96-well plates and allowed to adhere overnight. For nitrite release tests NR8383 macrophages have been pre-stimulated with 1 mg/ml LPS (Lipopolysaccharide from Pseudomonas aeruginosa, Sigma) 20 h before exposure. After 24 h incubation with particles, the supernatant medium was subsequently analyzed for LDH or nitrite. For cytokine determination, the supernates were stored at 208C until analysis. MTT assay The viability of cells was determined by the MTT test measuring the absorbance of the formazan product (Mossman, 1983).
LDH LDH activity was assayed in the supernate of cells using a kit from Roche (Mannheim) according the suppliers instructions. TNF-a TNF-a concentration was determined by detecting the TNF-induced cell permeability in actinomycin D-sensitized WEHI 164 cells using SYTOX¾ green nucleic acid stain (Molecular probes) in a fluorescence microplatebased bioassay according the manufactors instructions. Nitrite Nitrite in the supernatant medium was quantified as an indicator of NO production by the Griess reaction (Hˆckele et al., 1998). IL-8 IL-8 was detected by sandwich ELISA using the appropriate antibody pairs and recombinant human IL-8 as standard (PharMingen, Heidelberg).
Results Fly ash characteristics The size-fractionated IFA has been characterized by elemental composition and particle size distribution. Briefly, 70% by weight are water soluble representing chlorides and sulfates of Na, K or Ca. About 6% are metals, mainly (mg/g) Cd, 0.67; Cu, 2.55; Fe, 7.04; Pb, 23.35; Sb, 1.26; Sn, 2.7; Ti, 1.64; Zn, 21.39. 10 mg/g carbon and 1.212 mg/g PCDD/PCDF were detected. Figure 1 shows that the number concentration of the particles resuspended in air is dominated by fine particles with a modal value of 200 nm.
Fig. 1. Number concentration versus particle size of resuspended fly ash measured by SMPS (scanning mobility particle sizer).
In vitro effects of fly ash
Cytotoxicity tests The total IFA and the insoluble fraction were found to be highly toxic with EC50 of 150 mg/ml for NR8383 and 100 mg/ml for BEAS-2B in the MTT assay after 24 h exposure (Fig. 2). The soluble fraction was only slightly toxic at high concentrations. The toxicity of quartz was comparable to that of IFA in NR8383 but much lower in BEAS-2B at all concentrations tested. The LDH release test revealed similar EC50 values for both cell lines. The prestimulation of NR8383 with LPS and of BEAS-2B with TNF-a showed no significant effect in particle treated cells compared to corresponding controls. Cytokine release Treatment of NR8383 and BEAS-2B with IFA induces the release of cytokines in both cell types
325
(Fig. 3). The effect of quartz on TNF-a release from macrophages was higher compared to total IFA and its insoluble fraction. Both induced TNF-a release to the same extent whereas in the cells treated with the soluble fraction and in the controls TNF-a was below its detection limit. LPS (1 mg/ml) was highly effective in inducing the production of TNF-a (5200 420 pg/ml) and additional treatment with quartz, IFA or its fractions enhanced TNF-a formation (preliminary results). BEAS-2B cells were induced to release IL-8 after treatment with all fractions of IFA as well as with the soluble and insoluble fractions (Fig. 3). The highest effect was observed at 100 mg/ml of the total and the insoluble fraction. The effect of the soluble fraction at the same concentration was only 15% of that of the total and insoluble fractions. IL-8 production at higher particle concentrations was reduced besides by the soluble fraction which induced increasing IL-8
Fig. 2. The effect of total fly ash, the soluble and insoluble fraction on the absorbance of reduced MTT produced by NR8383 and BEAS-2B after 24 h exposure to different concentrations. The data related to control present means of three to four experiments SEM (each experiment was carried out in triplicate).
Fig. 3. The effect of total fly ash, the soluble and insoluble fraction on the production of TNF-a in NR8383 and of IL-8 in BEAS-2B after 24 h exposure to different concentrations. The data represent means of two experiments SEM (each experiment was carried out in triplicate).
326
S. Diabate¬ et al.
production. This can be explained by the higher toxicity of the total and insoluble fraction.
Acknowledgement. The authors thank Brigitte Eggmann for her excellent technical assistance.
Nitrite production IFA and its fractions were neither able to induce nitrite production in NR8383 cells nor did they affect the nitrite release in LPS pre-stimulated cells. 1 mg/ml LPS induced the release of 62 0.6 mM nitrite per 1 105 cells after 24 h indicating the ability of the cells to induce the iNOS.
Discussion The results presented here show that the IFA fraction which was insoluble at neutral pH was much more toxic and effective in inducing the release of cytokines than the soluble fraction which amounts to 70% of weight. Studies with other fly ashes like residual oil fly ash (ROFA, Dreher et al., 1997) or coal fly ash (CFA, van Maanen et al., 1999; Smith et al., 2000) showed that bioavailable transition metals such as Fe, Cu, Ni or V seem to play an important role in the induction of cytotoxic and inflammatory effects. These metals are known to induce Fenton-like reactions and catalyze the production of reactive oxygen species. In the studies with CFA, iron was mobilized by chelating with citrate for 24 h. Here the soluble fraction was obtained at neutral pH directly after having prepared the particle suspension. Concluding the preliminary results of this study, the fly ash used seems to act due to chemical or physical properties of the insoluble particle core. The proinflammatory responses of the cells could be induced by metals associated to the particle surface or by the large number of the fine and ultrafine particles which is associated with a large surface/volume ratio. The role of transition metals and particle size in proinflammatory responses are subject of further studies.
References Dreher, K. L., Richard, H. J., Lehmann, J. R., Richards, J. H., McGee, J. K.: Soluble transition metals mediate residual oil fly ash induced acute lung injury. J. Toxicol. Environ. Health 50, 285 ± 305 (1997). Driscoll, K. E., Carter, J. M., Hassenbein, D. G., Howard, B.: Cytokines and particle-induced inflammatory cell recruitment. Environ. Health Perspect. 105: 1159 ± 1164 (1997). Hˆckele, V., Kr¸ger, E., Krug, H. F. Seidel, A.: In vitro effects of nitrogen dioxide on the release of nitric oxide by bovine alveolar macrophages. Toxicol. Lett. 96/97: 53 ± 57 (1998). Mossman, T.: Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J. Immunol. Meth. 65, 55 ± 63 (1983). Mˆgel, M., Kr¸ger, E., Krug, H. F. Seidel, A.: A new coculture-system of bronchial epithelial and endothelial cells as a model for studying ozone effects on airway tissue. Toxicol. Lett. 96/97: 25 ± 32 (1998). Smith, K. R., Veranth, J. M., Hu, A. A., Lighty, J. S., Aust, A. E.: Interleukin-8 levels in human lung epithelial cells are increased in response to coal fly ash and vary with the bioavailability of iron, as a function of particle size and source of coal. Chem. Res. Toxicol. 13, 118 ± 125 (2000). Smith, K. R., Veranth, J. M., Lighty, J. S. Aust, A. E.: Mobilization of iron from coal fly ash was dependent upon the particle size and the source of coal. Chem. Res. Toxicol. 11, 1494 ± 1500 (1998). van Maanen, J. M. S., Borm, P. J. A., Knaapen, A., van Herwijnen, M., Schildermann, P. A. E. L., Smith, K. R., Aust, A. E., Tomatis, M.: In vitro effects of coal fly ashes: Hydroxyl radical generation, iron release, and DNA damage and toxicity in rat lung epithelial cells. Inhal. Toxicol. 11, 1123 ± 1141 (1999).
Int. J. Hyg. Environ. Health 204, 323 ± 326 (2002) ¹ Urban & Fischer Verlag http: // www.urbanfischer.de/journals/intjhyg
In vitro effects of incinerator fly ash on pulmonary macrophages and epithelial cells* Silvia Diabate¬a, Sonja M¸lhoptb, Hanns-R. Paurb, Ralf Wottricha, Harald F. Kruga a b
Institute of Toxicology and Genetics, Forschungszentrum Karlsruhe, Karlsruhe, Germany Institute of Technical Chemistry, Forschungszentrum Karlsruhe, Karlsruhe, Germany
Abstract Fly ash from a municipal waste incinerator was used as a model for atmospheric particles in order to identify parameters relevant for the induction of adverse health effects. The aim of this study was to compare the biological effects of the total incinerator fly ash (IFA), the soluble and the insoluble fraction with the effects of quartz by in vitro toxicity studies. The previously sized fly ash (< 20 mm) was characterized by elemental composition and particle size distribution. The particles were administered to rat alveolar macrophages (NR8383) and human bronchial epithelial cells (BEAS-2B) at different amounts via the medium. The total IFA and its insoluble fraction were shown to induce cytotoxicity and cytokine release in a dose-dependent manner. The soluble fraction was nearly unable to induce cytotoxicity and TNF-a release but showed potent induction of IL-8 release in BEAS-2B cells at increasing concentrations. Quartz caused similar effects compared to IFA in NR8383 but was less effective in BEAS-2B. Key words: fly ash ± in vitro ± alveolar macrophages ± lung epithelial cells
Introduction Particulate air pollution is a complex mixture of organic and inorganic compounds of different origin. Anthropogenic emissions including fly ash from combustion processes contribute in particular to the PM2.5 fraction which is under suspicion to threaten public health. Industrial combustion of coal or waste are well studied processes and the fly ash produced is frequently used as a model particle for PM10 and PM2.5 in toxicological studies (e.g. Smith et al., 1998). In vitro studies concentrate on pulmonary cells like alveolar macrophages which are in the first line of defence in the lung against inhaled
environmental pollutants. They are able to engulf particles and to produce biological mediators including reactive oxygen species, cytokines or growth factors to neutralize the pathological agents. Tumor necrosis factor alpha (TNF-a) plays a key role in this process (Driscoll et al., 1997). Primary effectors of cytokines are epithelial cells of the airways amplifying the inflammatory process by production of cytokines like IL-8 or IL-6. The purpose of this study was to examine the acute toxicity of IFA and its soluble and insoluble fractions as well as its potential to induce the release of inflammatory mediators in alveolar macrophage and bronchial epithelial cell lines. These results obtained by suspension of the fly
* Following a presentation given as part of the International Congress on Environmental Health and the 4th Annual Meeting of the International Society of Environmental Medicine (ISEM), 1-4 October, 2000, Hannover, Germany. Corresponding author: Silvia Diabate¬, Forschungszentrum Karlsruhe GmbH, Institute of Toxicology and Genetics, P.O. Box 3640, D-76021 Karlsruhe, Germany, Phone: 49 7247 822692, Fax: 49 7247 823557, E-mail: [email protected]
1438-4639/02/204-323 $ 15.00/0
324
S. Diabate¬ et al.
ash in culture medium will be compared to exposures to fly ash aerosol at the air-liquid interface in a later study.
Materials and methods Particles The fly ash obtained from a commercial municipal waste incinerator was collected in a bag filter. The sample was pre-sized and contained only particles < 20 mm. The crystalline quartz DQ12 (particle size < 5 mm, kindly provided by Prof. Dr. N. H. Seemayer, D¸sseldorf, Germany) was used as reference particle with well known cytotoxic properties. All particle suspensions were freshly prepared in the culture medium for the respective cells (10 mg/ml) and sonified. Insoluble and water soluble fractions were separated by centrifugation at 1000 g. Cell culture The NR8383 alveolar macrophage cell line from rat (ATCC, Rockville, MD) was maintained in Ham's F-12K medium containing 15% fetal bovine serum (both Life Sciences, Karlsruhe), 100 mg/ml penicillin and 100 U/ml streptomycin. BEAS-2B, immortalised cells of normal human bronchial epithelium, were maintained in serumfree growth medium (KGM, CellSystems, Remagen) (Mˆgel et al., 1998). For exposure to particles, the cells were seeded onto 96-well plates and allowed to adhere overnight. For nitrite release tests NR8383 macrophages have been pre-stimulated with 1 mg/ml LPS (Lipopolysaccharide from Pseudomonas aeruginosa, Sigma) 20 h before exposure. After 24 h incubation with particles, the supernatant medium was subsequently analyzed for LDH or nitrite. For cytokine determination, the supernates were stored at 208C until analysis. MTT assay The viability of cells was determined by the MTT test measuring the absorbance of the formazan product (Mossman, 1983).
LDH LDH activity was assayed in the supernate of cells using a kit from Roche (Mannheim) according the suppliers instructions. TNF-a TNF-a concentration was determined by detecting the TNF-induced cell permeability in actinomycin D-sensitized WEHI 164 cells using SYTOX¾ green nucleic acid stain (Molecular probes) in a fluorescence microplatebased bioassay according the manufactors instructions. Nitrite Nitrite in the supernatant medium was quantified as an indicator of NO production by the Griess reaction (Hˆckele et al., 1998). IL-8 IL-8 was detected by sandwich ELISA using the appropriate antibody pairs and recombinant human IL-8 as standard (PharMingen, Heidelberg).
Results Fly ash characteristics The size-fractionated IFA has been characterized by elemental composition and particle size distribution. Briefly, 70% by weight are water soluble representing chlorides and sulfates of Na, K or Ca. About 6% are metals, mainly (mg/g) Cd, 0.67; Cu, 2.55; Fe, 7.04; Pb, 23.35; Sb, 1.26; Sn, 2.7; Ti, 1.64; Zn, 21.39. 10 mg/g carbon and 1.212 mg/g PCDD/PCDF were detected. Figure 1 shows that the number concentration of the particles resuspended in air is dominated by fine particles with a modal value of 200 nm.
Fig. 1. Number concentration versus particle size of resuspended fly ash measured by SMPS (scanning mobility particle sizer).
In vitro effects of fly ash
Cytotoxicity tests The total IFA and the insoluble fraction were found to be highly toxic with EC50 of 150 mg/ml for NR8383 and 100 mg/ml for BEAS-2B in the MTT assay after 24 h exposure (Fig. 2). The soluble fraction was only slightly toxic at high concentrations. The toxicity of quartz was comparable to that of IFA in NR8383 but much lower in BEAS-2B at all concentrations tested. The LDH release test revealed similar EC50 values for both cell lines. The prestimulation of NR8383 with LPS and of BEAS-2B with TNF-a showed no significant effect in particle treated cells compared to corresponding controls. Cytokine release Treatment of NR8383 and BEAS-2B with IFA induces the release of cytokines in both cell types
325
(Fig. 3). The effect of quartz on TNF-a release from macrophages was higher compared to total IFA and its insoluble fraction. Both induced TNF-a release to the same extent whereas in the cells treated with the soluble fraction and in the controls TNF-a was below its detection limit. LPS (1 mg/ml) was highly effective in inducing the production of TNF-a (5200 420 pg/ml) and additional treatment with quartz, IFA or its fractions enhanced TNF-a formation (preliminary results). BEAS-2B cells were induced to release IL-8 after treatment with all fractions of IFA as well as with the soluble and insoluble fractions (Fig. 3). The highest effect was observed at 100 mg/ml of the total and the insoluble fraction. The effect of the soluble fraction at the same concentration was only 15% of that of the total and insoluble fractions. IL-8 production at higher particle concentrations was reduced besides by the soluble fraction which induced increasing IL-8
Fig. 2. The effect of total fly ash, the soluble and insoluble fraction on the absorbance of reduced MTT produced by NR8383 and BEAS-2B after 24 h exposure to different concentrations. The data related to control present means of three to four experiments SEM (each experiment was carried out in triplicate).
Fig. 3. The effect of total fly ash, the soluble and insoluble fraction on the production of TNF-a in NR8383 and of IL-8 in BEAS-2B after 24 h exposure to different concentrations. The data represent means of two experiments SEM (each experiment was carried out in triplicate).
326
S. Diabate¬ et al.
production. This can be explained by the higher toxicity of the total and insoluble fraction.
Acknowledgement. The authors thank Brigitte Eggmann for her excellent technical assistance.
Nitrite production IFA and its fractions were neither able to induce nitrite production in NR8383 cells nor did they affect the nitrite release in LPS pre-stimulated cells. 1 mg/ml LPS induced the release of 62 0.6 mM nitrite per 1 105 cells after 24 h indicating the ability of the cells to induce the iNOS.
Discussion The results presented here show that the IFA fraction which was insoluble at neutral pH was much more toxic and effective in inducing the release of cytokines than the soluble fraction which amounts to 70% of weight. Studies with other fly ashes like residual oil fly ash (ROFA, Dreher et al., 1997) or coal fly ash (CFA, van Maanen et al., 1999; Smith et al., 2000) showed that bioavailable transition metals such as Fe, Cu, Ni or V seem to play an important role in the induction of cytotoxic and inflammatory effects. These metals are known to induce Fenton-like reactions and catalyze the production of reactive oxygen species. In the studies with CFA, iron was mobilized by chelating with citrate for 24 h. Here the soluble fraction was obtained at neutral pH directly after having prepared the particle suspension. Concluding the preliminary results of this study, the fly ash used seems to act due to chemical or physical properties of the insoluble particle core. The proinflammatory responses of the cells could be induced by metals associated to the particle surface or by the large number of the fine and ultrafine particles which is associated with a large surface/volume ratio. The role of transition metals and particle size in proinflammatory responses are subject of further studies.
References Dreher, K. L., Richard, H. J., Lehmann, J. R., Richards, J. H., McGee, J. K.: Soluble transition metals mediate residual oil fly ash induced acute lung injury. J. Toxicol. Environ. Health 50, 285 ± 305 (1997). Driscoll, K. E., Carter, J. M., Hassenbein, D. G., Howard, B.: Cytokines and particle-induced inflammatory cell recruitment. Environ. Health Perspect. 105: 1159 ± 1164 (1997). Hˆckele, V., Kr¸ger, E., Krug, H. F. Seidel, A.: In vitro effects of nitrogen dioxide on the release of nitric oxide by bovine alveolar macrophages. Toxicol. Lett. 96/97: 53 ± 57 (1998). Mossman, T.: Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J. Immunol. Meth. 65, 55 ± 63 (1983). Mˆgel, M., Kr¸ger, E., Krug, H. F. Seidel, A.: A new coculture-system of bronchial epithelial and endothelial cells as a model for studying ozone effects on airway tissue. Toxicol. Lett. 96/97: 25 ± 32 (1998). Smith, K. R., Veranth, J. M., Hu, A. A., Lighty, J. S., Aust, A. E.: Interleukin-8 levels in human lung epithelial cells are increased in response to coal fly ash and vary with the bioavailability of iron, as a function of particle size and source of coal. Chem. Res. Toxicol. 13, 118 ± 125 (2000). Smith, K. R., Veranth, J. M., Lighty, J. S. Aust, A. E.: Mobilization of iron from coal fly ash was dependent upon the particle size and the source of coal. Chem. Res. Toxicol. 11, 1494 ± 1500 (1998). van Maanen, J. M. S., Borm, P. J. A., Knaapen, A., van Herwijnen, M., Schildermann, P. A. E. L., Smith, K. R., Aust, A. E., Tomatis, M.: In vitro effects of coal fly ashes: Hydroxyl radical generation, iron release, and DNA damage and toxicity in rat lung epithelial cells. Inhal. Toxicol. 11, 1123 ± 1141 (1999).