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Investigation of the Cytotoxic and Proinflammatory Effects of Cement Dusts in Rat Alveolar Macrophages Damien van Berlo, Petra Haberzettl, Kirsten Gerloff, Hui Li, Agnes M. Scherbart, Catrin Albrecht, and Roel P. F. Schins* IUF-Institut fu¨r Umweltmedizinische Forschung, Auf’m Hennekamp 50, D-40225 Du¨sseldorf, Germany ReceiVed February 4, 2009
Exposure to cement dust, a specifically alkaline and irritant dust, is one of the most common occupational dust exposures worldwide. Although several adverse respiratory health effects have been associated with cement dust exposure, the evidence is not conclusive. In the current study, cytotoxic and pro-inflammatory effects as well as oxidative stress elicited by a number of cement dusts, including a limestone and cement clinker sample, were tested using the NR8383 rat alveolar macrophage cell line and primary rat alveolar macrophages. DQ12 quartz and TiO2 were included as positive and negative controls, respectively. Cytotoxicity was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide assay and the lactate dehydrogenase assay, oxidative stress was determined by measurement of the depletion of total cellular glutathione, and electron spin resonance was applied to determine reactive oxygen species (ROS) generation. The release of the cytokines tumor necrosis factor-R (TNFR), interleukin-1β (IL-1β), and macrophage inflammatory protein-2 (MIP-2) was determined by enzyme-linked immunosorbent assay. None of the dust samples were found to cause toxicity to the macrophages or notable glutathione depletion when compared to DQ12. The cement samples also failed to activate macrophages for the generation of ROS and the production of inflammatory cytokines IL-1β and MIP-2. In contrast, however, most of the cement dusts were found to activate macrophage TNFR production, and this was significantly associated with their content of CaO. Further research is needed to determine the relevance of these in vitro observations for occupational cement dust exposure settings. Introduction Exposure to cement dust, a specifically alkaline and irritant dust, is one of the most common occupational dust exposures worldwide. Portland cement, the most abundant form, is produced by grinding calcareous materials such as limestone or chalk and argillaceous materials like clay or shale. The mixture is then calcined to produce clinker, which consists mainly of calcium silicates, aluminates, and alumino-ferrites. Then, it is either finely ground once more to obtain the final cement product or mixed with further constituents such as gravel to produce concrete. Inhalation of dust represents the main hazard in cement processing. Several adverse respiratory effects have been associated with cement dust exposure; the evidence for this association is, however, not conclusive. Chronic bronchitis has been linked to cement dust inhalation (1), and some studies suggest that there is a mildly increased risk for lung cancer among cement workers (2, 3). However, conflicting results have been observed in various studies. In a number of investigations, an increased prevalence of respiratory symptoms and airflow obstruction have been shown (4-8), whereas other studies could not show significant effects of cement dust exposure on respiratory parameters (9, 10). Therefore, further investigation of the adverse health effects of cement dust is required. Inflammation is nowadays considered to represent a fundamental mechanism mediating the pulmonary toxicity of particles and the development of pathogenic conditions such as chronic * To whom correspondence should be addressed. Tel.: +49-211-3389269. Fax: +49-211-3389-331. E-mail:
[email protected].
obstructive pulmonary disease (COPD)1 and lung fibrosis (11). Upon inhalation, respirable (fine and ultrafine) particles can circumvent the defense mechanisms of the upper respiratory tract (e.g., coughing, sneezing, and mucociliary clearance), thereby reaching the bronchi and alveolar sacs. In the alveoli, generally, the first cell to come into contact with inhaled particles is the alveolar macrophage (AM), considered as a key cell in the orchestration of inflammatory responses within the respiratory tract. AMs are involved in the clearance through phagocytosis of potentially damaging factors such as bacteria or particles. Besides their ability to clear particles from the airways into the lymph nodes or along the mucociliary escalator, activated AMs secrete a wide range of products including reactive oxygen species (ROS) and nitrogen species (RNS), bioactive lipids, cytokines, chemokines, and proteases, which can contribute to lung toxicity (12). Among these products, ROS, as well as cytokines such as tumor necrosis factor-R (TNFR), interleukin-1β (IL-1β), and macrophage inflammatory protein-2 (MIP-2), are considered to be important mediators of particleinduced pulmonary inflammation and toxicity (12, 13). The release of these inflammatory mediators and products is known to be driven by the induction of cellular oxidative stress and associated activation of redox-sensitive transcription factors, with nuclear factor-κB (NF-κB) being a key player. Whereas 1 Abbreviations: DQ12, Do¨rentrup quartz “ground product nr. 12”; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide; LDH, lactate dehydrogenase; ROS, reactive oxygen species; TNFR, tumor necrosis factor R; IL-1β, interleukin-1β; MIP-2, macrophage inflammatory protein-2; COPD, chronic obstructive pulmonary disease; AM, alveolar macrophage; RNS, reactive nitrogen species; NF-κB, nuclear factor κB; BAL, bronchoalveolar lavage; DMSO, dimethylsulfoxide; HBSS, Hank’s buffered salt solution; DMPO, 5,5-dimethyl-1-pyrroline N-oxide.
10.1021/tx900046x CCC: $40.75 2009 American Chemical Society Published on Web 08/21/2009
Effects of Cement Dusts in AlVeolar Macrophages
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Table 1. Chemical Characterization of Cement Dusts % code
sample
loss on ignition
SiO2
Al2O3
TiO2
P2O5
Fe2O3
Mn2O3
MgO
CaO
K2O
SO3
Na2O
cCli cA cB cC cD cG cH1 cJ cK cL LM
cement clinker cement dust A cement dust B cement dust C cement dust D cement dust G cement dust H1 cement dust J cement dust K cement dust L limestone powder
1.44 4.20 9.06 17.88 5.18 8.94 7.36 5.41 17.43 13.59 44.06
19.45 29.01 20.12 14.76 17.51 29.92 16.26 25.97 11.36 15.10 0.61
5.70 8.11 7.62 3.25 5.49 11.30 3.94 9.98 3.18 6.05 0.30
0.27 0.33 0.35 0.17 0.34 0.50 0.20 0.66 0.16 0.23 0.04
0.23 0.26 0.14 0.09 0.09 0.17 0.25 0.13 0.06 0.06 0.01
2.79 3.31 3.78 1.24 2.73 4.74 2.22 1.27 2.55 2.88 0.07
0.06 0.10 0.06 0.04 0.15 0.17 0.05 0.15 0.07 0.04 0.04
3.44 1.00 1.84 0.73 3.39 1.83 2.27 4.07 1.34 1.67 0.49
60.25 48.52 45.35 53.91 54.59 33.48 60.85 47.64 55.26 48.24 54.18
2.78 1.70 2.34 1.05 2.22 3.39 2.37 0.70 1.35 4.17 0.09
3.20 2.28 9.10 6.74 7.89 4.50 3.59 3.76 7.18 7.51 0.05
0.41 1.14 0.29 0.20 0.47 1.01 0.62 0.36 0.12 0.33 0.03
the release of cytokines and chemokines such as TNFR and MIP-2 triggers the inflammatory axis via recruitment and activation of inflammatory cells into the lung, the production of ROS is additionally implicated as a major factor in lung tissue damage and remodeling. On a cellular level, ROS are considered to trigger signaling pathways involved in inflammatory mediator release and proliferation and to induce oxidative damage, for example, to membrane constituents, intracellular proteins, and the genomic DNA (14). In the present study, a set of respirable cements, including a limestone and cement clinker sample, were investigated in vitro for their cytotoxic effects as well as their oxidative stressinducing and inflammogenic properties using the well-established rat alveolar macrophage cell line NR8383. Respirable crystalline silica particles and TiO2, known for their contrasting inflammatory and toxic effects in the lungs (15, 16), were used as positive and negative particulate controls, respectively. Two selected cement dust samples were further investigated using rat primary AMs. The present study was performed within the framework of Comprehensive Health Risk Study sponsored by CEMBUREAU (Brussels, Belgium).
Materials and Methods Dust Samples. A set of commonly used cements was selected. For each sample, a fine respirable fraction was prepared and provided by VDZ [Verein Deutscher Zementwerke e.V., Du¨sseldorf, Germany]. For all cement samples, the size was found to be
