- Email: [email protected]
Pulmonary functional and morphological damage after exposure to tripoli dust
Respiratory Physiology & Neurobiology 196 (2014) 17–24
Contents lists available at ScienceDirect
Respiratory Physiology & Neurobiology journal homepage: www.elsevier.com/locate/resphysiol
Pulmonary functional and morphological damage after exposure to tripoli dust Mariana Nascimento Machado a , Aline Cunha Schmidt a , Paulo Hilário Nascimento Saldiva b , Débora Souza Faffe a , Walter Araujo Zin a,∗ a b
Universidade Federal do Rio de Janeiro, Instituto de Biofísica Carlos Chagas Filho, Rio de Janeiro, Brazil Universidade de São Paulo, Faculdade de Medicina, São Paulo, Brazil
a r t i c l e
i n f o
Article history: Received 22 November 2013 Received in revised form 13 February 2014 Accepted 13 February 2014 Keywords: Silicosis Inflammation Granuloma Tripoli dust Lung mechanics
a b s t r a c t Tripoli is a microcrystalline siliceous rock used to polish metals and precious stones. Its inhalation has been associated with increased prevalence of breathing complaints and pneumoconiosis. However, its acute human exposure has not been so far studied. We aimed at evaluating the putative mechanical, morphological, biochemical and inflammatory lung damage in mice acutely exposed to Tripoli dust. BALB/c mice were randomly assigned to 2 groups: In control group (CTRL, n = 6) animals received intratracheally (i.t.) 0.9% NaCl (50 l), while Tripoli group (TRIP, n = 15) received 20 mg of Tripoli powder diluted in 50 L of saline i.t. The experiments were done 15 days later. TRIP mice showed higher pulmonary mechanical impedance, polymorphonuclear cells, TNF-␣, IL1- and IL-6 than CTRL. TRIP presented granulomatous nodules containing collagenous fibers that occupied 35% of the lung tissue area. In conclusion, acute exposure to Tripoli dust triggered important lung damage in mice lungs that if found in human workers could trigger severe illness. © 2014 Elsevier B.V. All rights reserved.
Introduction The chronic inhalation of crystalline silicon dioxide (SiO2 ) is associated with the occurrence of silicosis. Despite being one of the firstly recognized occupational lung diseases, silicosis remains an important cause of morbidity and mortality worldwide (Martínez et al., 2010). This pneumoconiosis displays persistent inflammation, fibroblast proliferation, and excessive collagen deposition (Thakur et al., 2009). Furthermore, the cytotoxic effects of silica in lung tissue yield macrophage death, subsequent release of inflammatory cytokines, such as TNF-␣, IL-1 and IL-6, and many other substances. As a net result fibrosis (Piguet et al., 1990; Davis et al., 1998; Mossman and Churg, 1998; Srivastava et al., 2002; Rimal et al., 2005; Hamilton et al., 2008; Sirajuddin and Kanne, 2009) and apoptosis (Borges et al., 2002; Srivastava et al., 2002; Langley et al., 2010) ensue. The continuous recruitment and activation of macrophages and granulocytes contributes to the chronic
∗ Corresponding author at: Laboratório de Fisiologia da Respirac¸ão, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Av Carlos Chagas Filho 373, CCS, Rm G2-042, Ilha do Fundão, 21941-902 - Rio de Janeiro, RJ, Brazil. Tel.: +55 21 2562 6557; fax: +55 21 2280 8193. E-mail addresses: walter [email protected], [email protected] (W.A. Zin). http://dx.doi.org/10.1016/j.resp.2014.02.007 1569-9048/© 2014 Elsevier B.V. All rights reserved.
inflammatory process and, thus, to tissue remodeling (Scabilloni et al., 2005; Delgado et al., 2006). As part of the fibrotic process silicotic nodules or granulomas (Scabilloni et al., 2005) are formed. Increased pulmonary mechanical impedance represents the functional counterpart of the morphological changes (Ebihara et al., 2000; Borges et al., 2001; Faffe et al., 2001; Hertzberg et al., 2002). Crystalline silica is found in sand and several rocks, like sandstone, granite and silex and presents polymorphisms, the principal naturally occurring crystalline silica being quartz (Moore, 1999). Cristobalite, tridymite and Tripoli constitute the three other forms of crystalline silica. Tripoli presents unique applications as an abrasive owing to its hardness and because its grain structure lacks distinct edges and corners. It is a mild abrasive, making it suitable for use in toothpaste and tooth polishing compounds, industrial soaps, metal/jewelry polishing mixtures, resins, ceramics, paints, rubber, and cement (Keller, 1978). Along the processing and use of Tripoli powder the dust generated can be inhaled by human beings, not only workers but the general population as well, and may cause an inflammatory lung disease. It should be noted that Tripoli dust contains more components than SiO2 , what could induce a different harmful outcome. However, no study on the detailed acute functional respiratory impairment secondary to exposure to Tripoli dust has been reported so far either in human beings or in experimental animals. Furthermore, no epidemiological work on the prevalence
18
M.N. Machado et al. / Respiratory Physiology & Neurobiology 196 (2014) 17–24
or incidence of Tripoli-triggered disease has been published. In other words, Tripoli powder may be generating more harm than so far recognized by physicians and public health authorities. The aim of the present study was to describe the physical and chemical characteristics of the Tripoli dust used, and to verify whether the exposure to Tripoli dust induces lung morphological, inflammatory and mechanical burdens that could resemble those of chronic silicosis, thus calling physicians and environmental health officers’ attention to the Tripoli issue. Methods Animal and experimental protocol Twenty-one female BALB/c mice (20–25 g) were randomly divided into 2 groups. In control group (CTRL, n = 6) mice were intratracheally (i.t.) instilled with 0.05 mL of sterile saline solution (0.9% NaCl), whereas Tripoli-administered animals (TRIP, n = 15) received intratracheally 20 mg of Tripoli dust vortexed in 0.05 mL of saline, respectively, as previously described in murine models of acute silicosis (Faffe et al., 2001; Borges et al., 2001, 2002). Before the administration of the latter suspension, a stock solution of 600 mg of Tripoli was placed in a 2-mL Eppendorf tube and saline solution was added to reach a final volume of 1.5 mL. The suspension was vortex-mixed for 10 min and 0.05 mL of it was then collected with a Gilson precision micropipette (Gilson, Inc., Middleton, WI, USA) and immediately given to the animal. The vortexing was repeated for every TRIP animal. Fifteen days after saline or Tripoli administration the animals were analyzed. Particle analysis The dust sample was kindly provided by a gemstone-polishing company in São Lourenc¸o, Brazil. It was taken from the same supply being at the polishers’ disposal. It was dried in an oven at 50 ◦ C until completely de-hydrated. Elements were determined by an energy dispersive X-ray fluorescence spectrometer (EDX 700HS, Shimadzu Corp, Analytical Instruments Division, Kyoto, Japan). Aluminum (Al), cobalt (Co), iron (Fe), potassium (K), magnesium (Mg), sodium (Na), sulfur (S), silicon (Si), aluminum oxide (Al2 O3 ), calcium oxide (CaO), red iron oxide (Fe2 O3 ), potassium oxide (K2 O), magnesium oxide (MgO), manganese oxide (MnO), sodium oxide (Na2 O), titanium dioxide (TiO2 ) and silicon dioxide (SiO2 ) were determined and the results expressed as percent composition (wt %) of particles. The trace elements bromine (Br), copper (Cu), germanium (Ge), lutecium (Lu), manganese (Mn), nickel (Ni), rubidium (Rb), selenium (Se), tin (Sn), strontium (Sr), titanium (Ti), zinc (Zn), and zirconium (Zr) were measured and the results expressed as particles per million (ppm) of particles. Three independent samples of the particulate matter were analyzed for this purpose. The distribution of particle sizes, as measured by their volume and surface, and the diameters encompassing 90%, 50% and 10% of the particulate matter were determined by laser diffraction (Long Bench Mastersizer S, Malvern Instruments Ltd, Malvern, Worcestershire, UK). The particulate matter was visualized by scanning electron microscopy (JEOL 5310, Tokyo, Japan).
tidal volume of 0.2 mL, flow equal to 1 mL s−1 , and positive endexpiratory pressure amounting to 2 cmH2 O. The anterior chest wall was surgically removed. A pneumotachograph (1.5-mm ID, length = 4.2 cm, distance between side ports = 2.1 cm) was connected to the tracheal cannula for the measurement of airflow (V ). Changes in lung volume were obtained by flow signal digital integration. The pressure gradient across the pneumotachograph was determined by means of a Validyne MP45-2 differential pressure transducer (Engineering Corp, Northridge, CA, USA). Equipment resistive pressure (=Req.V ) was subtracted from pulmonary resistive pressure so that the present results represent intrinsic values. Transpulmonary pressure was measured with a Validyne MP-45 differential pressure transducer (Engineering Corp, Northridge, CA, USA). Briefly, we determined lung resistive (P1 ) and viscoelastic/inhomogeneous (P2 ) pressures, static elastance (Est ), and viscoelastic component of elastance (E) by the end-inflation occlusion method (Bates et al., 1985). P1 selectively reflects airway resistance, and P2 represents stress relaxation or viscoelastic properties and mechanical heterogeneities of the lung (Bates et al., 1989; Saldiva et al., 1992). Lung mechanics were measured 10–15 times in each animal.
Histological study Heparin (1000 IU) was intravenously injected immediately after the determination of respiratory mechanics. The trachea was clamped at end expiration, and the abdominal aorta and vena cava were sectioned, yielding a massive hemorrhage that quickly euthanized the mice. The right lungs were removed en bloc and quick-frozen by immersion in liquid nitrogen and fixed with Carnoy’s solution (Nagase et al., 1992). After fixation, the tissue was embedded in paraffin. Four-m-thick slices were cut and stained with hematoxylin-eosin or picrosirius red. Morphometric analysis was performed with an integrating eyepiece with a coherent system made of a 100-point and 50 lines (known length) grid coupled to a conventional light microscope (Axioplan, Zeiss, Oberkochen, Germany) in granuloma free areas. The volume fraction of collapsed and normal alveoli was determined in each sample by the point-counting technique (Gundersen et al., 1988) across 10 random non-overlapping microscopic fields at ×400 magnification. The total amount of points also included those falling on tissue, airways and other non-alveolar structures. The number of mononuclear (MN) and polymorphonuclear (PMN) cells in the pulmonary tissue was counted in each animal across 10 random non-overlapping microscopic fields at ×1000 magnification in a 10,000 m2 granuloma free area; in the same field the amount of points that fell on lung tissue was also counted, so that cellularity was expressed as percentage of lung tissue area (Gundersen et al., 1988; Capelozzi et al., 1997). The fraction area of the granulomas was determined using the point-counting technique across 20 random non-coincident microscopic fields per animal at a magnification of ×200. Percentage of lung tissue occupied by granulomatous nodules was scored as following: phase 1, nodules present only in the lung parenchyma; phase 2, nodules around the airways; phase 3, nodules obstructing the airway; and, phase 4, lung nodules in various structures.
Pulmonary mechanics Fifteen days after saline or Tripoli dust administration, the animals were sedated with diazepam (1 mg i.p.) and anesthetized with pentobarbital sodium (20 mg kg body weight−1 i.p.), paralyzed with pancuronium bromide (0.1 mg kg body weight−1 i.v.), and mechanically ventilated (Samay VR15, Universidad de la Republica, Montevideo, Uruguay) with a frequency of 100 breaths min−1 ,
Analysis of cytokines Samples of lung cytosol were analyzed by ELISA for the detection of the inflammatory cytokines TNF-␣, IL-1, IL-6 (ELISA kits, R&D Systems Europe, Abingdon, UK) with detection limits of 5.1 pg/mL, 1.6 pg/mL and 3.0 pg/mL respectively.
M.N. Machado et al. / Respiratory Physiology & Neurobiology 196 (2014) 17–24
19
Fig. 1. (A) Electron scanning micrographs of Tripoli powder. White bars: 5, 10 and 50 m (left, middle and right panels, respectively). (B) Histogram of the frequency distribution of particle diameters (columns) and accumulated frequency (solid line).
Statistical analysis SigmaPlot 11 statistical package (Systat Software, Inc., Chicago, IL, USA) was used. The normality of the data (Kolmogorov–Smirnov test with Lilliefors’ correction) and the homogeneity of variances (Levene median test) were tested. When both conditions were satisfied Student’s t-test was used. Otherwise Mann-Whitney test was employed. The significance level was set at 5%.
Results Particle analysis Scanning electron micrographs of particles are shown in Fig. 1A. Tripoli dust particles were not spherical in shape, displaying an irregular form and aggregates. Fig. 1B depicts the frequency distribution of particle diameters. Ninety percent of the particles presented a diameter below 17.28 m, being 50% below 5.09 m and 10% below 1.54 m. The average sizes of the particles according to their volume and surface were 7.57 and 3.47 m, respectively. Metals and oxides in the Tripoli dust are presented in Fig. 2; a high concentration of the element Si was found as SiO2 .
Pulmonary mechanics TRIP group presented significantly higher lung mechanical parameters than CTRL, excluding P1 that was not different between the groups (Table 1).
Table 1 Mechanics, histology and inflammation markers in lung parenchyma. CTRL
TRIP
Mechanics P1 (cmH2 O) P2 (cmH2 O) Est (cmH2 O/mL) E (cmH2 O/mL)
0.40 (0.38–0.50) 1.22 (1.06–1.48) 24.04 ± 1.60 6.17 (5.18–7.46)
0.52 (0.44–0.80) 1.66 (1.29–2.22)* 33.07 ± 2.64* 8.26 (6.35–11.17)*
Histology Normal area (%) Alveolar collapse (%) PMN (cel × 10−3 /m2 ) MN (cel × 10−3 /m2 )
70.00 ± 0.80 14.00 ± 0.50 4.00 ± 0.01 11.00 ± 1.00
11.00 ± 1.10* 37.00 ± 0.70* 6.00 ± 0.01* 12.00 ± 1.00
Cytokines TNF-␣ (pg/mL) IL-1 (pg/mL) IL-6 (pg/mL)
79.98 ± 2.67 129.71 ± 11.27 55.59 ± 1.90
115.08 ± 2.41* 609.09 ± 22.98* 81.90 ± 4.87*
Values are mean ± SEM or median (25–75%). Control mice (CTRL, n = 6) and those instilled with 20 mg of Tripoli dust/50 L saline (TRIP, n = 15); P1 and P2 , resistive and viscoelastic/inhomogeneous pressures, respectively; Est , static elastance; E, viscoelastic component of elastance; PMN and MN, polymorpho- and mononuclear cells, respectively; percentage of normal and collapsed areas in pulmonary tissue. * Significantly different from CTRL group (p < 0.05).
Histology Representative photomicrographs of lung parenchyma (picrosirius red) in control mice (Panel A) and in animals intratracheally instilled with 20 mg of Tripoli (Panels B–F) are depicted in Fig. 3. In panel B phase 1 granuloma shows accumulation of macrophages containing dust particles, eliciting a slight fibrotic response (more intense red staining); in panel C (phase 2), the
20
M.N. Machado et al. / Respiratory Physiology & Neurobiology 196 (2014) 17–24
parenchyma (Table 1). Spearman correlation was used to evaluate the association between static elastance and percentage of collapsed air spaces. Significant correlation between alveolar collapse and Est was found: = 0.611, p < 0.0043. Cytokines TNF-␣, IL-1, and IL-6 were significantly higher (+43.9%, +369.6%, and +47.3%, respectively) in lung homogenates of Tripoliinstilled mice than in control group (Table 1). Discussion
Fig. 2. Metals (panels A and B) and oxides (panel C) in Tripoli dust. Aluminum (Al), cobalt (Co), iron (Fe), potassium (K), magnesium (Mg), sodium (Na), sulfur (S), silicon (Si), aluminum oxide (Al2 O3 ), calcium oxide (CaO), red iron oxide (Fe2 O3 ), potassium oxide (K2 O), magnesium oxide (MgO), manganese oxide (MnO), sodium oxide (Na2 O), titanium dioxide (TiO2 ) and silicon dioxide (SiO2 ) were measured and the results expressed as WT (%) of particles. Trace elements bromine (Br), copper (Cu), germanium (Ge), lutecium (Lu), manganese (Mn), nickel (Ni), rubidium (Rb), selenium (Se), tin (Sn), strontium (Sr), titanium (Ti), zinc (Zn), and zirconium (Zr) were measured and the results expressed as ppm of particles or WT (%). ppm, part per million; WT, percent composition by weight.
alveoli around the granuloma exhibit an acute inflammatory response and contain dust particles within macrophages; in panel D (phase 3), obliterative bronchiolitis shows a fibrotic inflammatory polip protruding into the bronchiolar lumen; and in panel E (phase 4), multiple dust nodules and inflammatory reaction composed by dust particles, large number of macrophages, parenchymal collapse and fibrosis are present in the gas exchanging territory. Panel F displays lung parenchyma under polarized light showing the birefringent particles of Tripoli dust as bright spots. The overall area fraction of granulomatous nodules was 35.10 ± 0.05% in TRIP group. The percentages of nodules in lung tissue found in phases 1, 2, 3, and 4 were 38.7 ± 16.7%, 20.0 ± 9.7%, 6.0 ± 6.1%, and 35.4 ± 16.7% (mean ± SD), respectively. TRIP animals presented increased alveolar collapse (Table 1) with polymorphonuclear (PMN) and total cells influx in lung
Our results demonstrated that acute exposure to Tripoli dust was done with particles presenting various shapes with a mean aerodynamic diameter of about 5 m. Due to their elevated content of silica and silicon dioxide these particles triggered lung morphological changes similar to classical acute silicosis. Intratracheal instillation of 20 mg of Tripoli induced pulmonary influx of inflammatory cells, increased release of pro-inflammatory cytokines such as TNF-␣, IL-1 and IL-6, collapsed alveoli, and granulomas with expression of collagen fibers in the lung parenchyma. As a result pulmonary mechanical impedance was impaired mainly at tissue level, turning the lung stiffer and offering a higher resistance to movement. Once inhaled, silica particles are deposited in different parts of the respiratory system, according to their size and initiate the pathological process. Toxicity varies according to particle diameter. In humans toxicity becomes very important when the aerodynamic diameter of the particles is less than 10 m, which enables them to reach the pulmonary alveoli. In rats and mice this value approximates 2 m for intratracheally instilled silica (Wiessner et al., 1989; Takayoshi et al., 2007). Our results showed that 90% of Tripoli dust particles were below 17.28 m diameter, being 50% below 5.09 m and 10% below 1.54 m. Thus, at least part of the administered Tripoli dust reached the lung periphery. It should be stressed that the fine and ultrafine particles are known as “breathable” and are able to penetrate the airways, reaching the alveoli (Dusseldorp et al., 1995; Peters et al., 1997; Brown et al., 2002; Tao et al., 2003). Otherwise, they can be eliminated by the mucociliary system or, when deposited in the alveolar regions, can be phagocytosed by macrophages. It is believed that these fine and ultrafine particles can still penetrate into the lung parenchyma and reach the bloodstream (Donaldson et al., 2001a, 2001b; Donaldson and Stone, 2003). As depicted in Fig. 2 our particulate matter showed a very high concentration of the element silicon and silicon dioxide, as well as other elements and oxides. Thus, Tripoli dust is not pure silicon dioxide, the silica dust most frequently used to produce experimental models of silicosis. Metal containing particles induced pulmonary changes, reinforcing their potential role in determining particle toxicity (Soukup et al., 2000; Dye et al., 2001; Molinelli et al., 2002). Mazzoli-Rocha et al. (2010) demonstrated that a single aerosolization of small quantities of particles containing mainly aluminum induced acute respiratory inflammation, as suggested by changes in lung tissue mechanics, reflected in increased Est , E and P2 , but no significant change in P1 and lung histology. These studies demonstrate the impact of such metals in pulmonary mechanics. Kim et al. (2010) assessed and compared the in vitro toxicity of four different oxide nanoparticles (Al2 O3 , CeO2 , TiO2 and ZnO) to human lung epithelial cells. Among four tested nanoparticles, ZnO exhibited the highest cytotoxicity in terms of cell proliferation, cell viability, membrane integrity, and colony formation. Al2 O3 , CeO2 and TiO2 showed little adverse effects on cell proliferation
M.N. Machado et al. / Respiratory Physiology & Neurobiology 196 (2014) 17–24
21
Fig. 3. Representative photomicrographs of lung parenchyma (picrosirius red) in control animals (panel A), and in mice intratracheally instilled with a suspension of 20 mg of Tripoli dust in 0.05 mL of saline solution: in panel B, nodule is seen only in the lung parenchyma featuring an early stage of disease; in panel C, nodules are found around the airways; in panel D, nodule obstructs the airway; in panel E, nodules are present in various lung structures featuring an advanced stage of the disease. Image obtained with polarized light (panel F) shows scattered interstitial Tripoli particles as bright spots. Tripoli particles can be seen in the nodules in panels B–E. Twenty random non-coincident microscopic fields were evaluated per animal at a magnification of ×200. Bar equals 100 m.
and cell viability. However, TiO2 induced oxidative stress in a concentration- and time-dependent manner. Al2 O3 seems to be less toxic than the other nanoparticles even after long time exposure. These findings suggest the relevance of chemical components on the toxicity of Tripoli powder. However, it is not possible to exclude the contribution of non-determined constituents of the particle composition. As observed in other murine models of fibrosis, the response is strain-dependent, being BALB/c mice more resistant to silica instillation (Moore and Hogaboam, 2008). In this context, 2.5 mg of silica in/60 L of saline were enough to trigger lung inflammation and fibrosis in C57BL/6 mice (Giordano et al., 2010), whereas our animals were exposed to 20 mg of Tripoli dust in 50 L of saline instilled directly into the trachea, a dose very close to those reported by others, who used silicon dioxide in mice and rats (Gross et al., 1984; Wright et al., 1988; Borges et al., 2001; Faffe et al., 2001; Borges et al., 2002; Lassance et al., 2009). However, considering that only circa 50% of our dust presented a diameter less than 5 m, our effective dose of PM5 was half theirs. Indeed Tripoli dust presents particle diameters larger than those found in commercially available silicon dioxide (1–5 m). A limit exposure to Tripoli dust has not been described yet, but the permissible exposure limit for dust containing respirable crystalline silica is 0.1 mg/m3 (NIOSH, 2011). The Occupational Safety and Health Administration (OSHA) has determined that the exposure limit of quartz from 0.08 to 0.1 mg/m3 can be used as a basis to limit exposure to the Tripoli particles (NIOSH, 2011). However, even these levels may not be low enough to prevent chronic disease. In this line, Greaves (2000) concluded that the 0.1 mg/m3 recommended exposure limit of the NIOSH might not be sufficiently protective for a substantial proportion of workers. Additionally, Steenland et al. (2001) described that the estimated excess lifetime risk (through age 75) of lung cancer for a worker exposed from age 20 to 65 at 0.1 mg/m3 respirable crystalline silica was 1.1–1.7%, above background risks of 3–6%, and Mannetje et al. (2002) described that the risk of death from silicosis is 13 per 1000
workers to a limit exposure of 0.1 mg/m3 of respirable silica in the environment. Therefore we used a dose of 20 mg of Tripoli to evaluate possible pulmonary complications owing to exposure to this dust. The time lag between exposure and measurement represents another factor involved in the response. Although silicosis develops over years in humans exposed to silica dust, experimental studies in BALB/c mice showed that even a single exposure leads to functional and histological impairment (Borges et al., 2001; Faffe et al., 2001; Borges et al., 2002). Some studies report the development of fibrosis within the first month after exposure (Lardot et al., 1998; Borges et al., 2001; Faffe et al., 2001; Lakatos et al., 2006; Borges et al., 2002), or even after longer periods (Barbarin et al., 2005; Giordano et al., 2010). To the best of our knowledge, we report for the first time the acute effects of the exposure to Tripoli dust. Exposure to Tripoli dust resulted in polymorphonuclear cell influx into the lung parenchyma as previously reported with silica (Faffe et al., 2001). These authors showed progressive histological changes in a murine model of silicosis. We observed that TNF-␣, IL-1 and IL-6 levels in the lung tissue increased significantly after acute exposure to Tripoli (Table 1). In a murine model of silicosis, IL-1 was associated with mononuclear cell-related inflammation and collagen deposition (Zhang et al., 1993). We might advance that the cytotoxic effects of Tripoli particles in the lung could be related to the following cascade, similar to that of silica: death of alveolar macrophages, subsequent release of inflammatory cytokines (TNF-␣, IL-1 and IL-6 among others), stimulation of fibroblasts activity, development of granulomas and subsequent pulmonary fibrosis (Piguet et al., 1990; Davis et al., 1998; Mossman and Churg, 1998; Srivastava et al., 2002; Rimal et al., 2005; Hamilton et al., 2008; Sirajuddin and Kanne, 2009). Davis et al. (1998) showed that the increased production of TNF␣ and IL-1 occurs within the silicotic lesions and bronchoalveolar lavage fluid. These mediators seem to be involved mainly in early disease, since they precede inflammation and fibrosis (Driscoll
22
M.N. Machado et al. / Respiratory Physiology & Neurobiology 196 (2014) 17–24
et al., 1995). However, the alveolar macrophages are related to the maintenance of silicotic inflammation in a vicious cycle: they phagocytize the silica particles, are activated and damaged, releasing the silica particle back into the lung environment. Then another macrophage engulfs the same particle and perpetuates the process (Greenberg et al., 2007; Gilberti et al., 2008). Despite the important role of inflammation in fibrogenesis, some evidence suggests that inflammation is not necessarily related to the fibrotic response and that additional pathogenic pathways may be responsible for development of the fibrotic response in the lung. Some studies show that lung inflammation is not always followed by fibrotic disease (Adamson et al., 1992; Huaux et al., 1998; Munger et al., 1999), whereas other works reveal that control of inflammation is not always associated with reduced fibrosis (Tanino et al., 2002; Sakamoto et al., 2002). Chronic exposure to silica particles is histologically characterized by the presence of granulomas, alveolar septal thickening and accumulation of inflammatory cells such as macrophages (Delgado et al., 2006). Ortiz et al. (2001) intratracheally instilled mice with silica (0.2 g/kg, average of 5 mg of silica per animal, particles presenting an average diameter of 1 m) and observed granulomas in the lung parenchyma 28 days after the challenge, but qualitatively these nodules were not as important as those presented in our study. Park et al. (2011) evaluated changes in lung tissue 1, 7, 14 and 28 days after a single intratracheal instillation of silica nanoparticles (1 mg/kg) and found inflammatory cell migration to the alveolar space and infiltration in alveolar wall. In the present Tripoli-exposed lungs there were granulomas containing large numbers of mononuclear cells, fibroblasts and collagen fibers arranged in a circular orientation, showing features of immature silicotic nodules (Fig. 3). More advanced stages of tissue remodelling display the classical formation of fibrotic nodules with collagen fibers concentrically aligned, a central hyaline core, and lumping and agglomeration of small silicotic nodules (Castranova and Vallyathan, 2000; Ding et al., 2002; Rimal et al., 2005). The fraction area of pulmonary tissue occupied by granulomas in the TRIP group was 35.1%. In our experimental model these granulomas were present in four phases. Since 80% of the macrophages die within 12 h after exposure to silica and silica internalization by macrophages occurs even faster (Gilberti et al., 2008), our results may suggest a still active inflammatory process as indicated by the high percentage of phase 1 granulomas together with well established ones. This hypothesis is supported by our findings of elevated levels of pro-inflammatory cytokines (Table 1). The aforementioned histological and immunological phenomena brought about significant changes in mechanical parameters (Table 1). Our results demonstrated that intratracheal instillation of Tripoli dust induced lung injury and increased lung impedance determined on the 15th day after exposure. In accordance with our results Borges et al. (2001) intratracheally instilled BALB/c mice with silica dust and reported an elastic component of pulmonary impedance similar to ours. Control groups in both studies presented similar results. We did not find changes in the pressure spent against central airway resistance (Table 1); in this context, Hnizdo et al. (1994) reported that large airway disease was not positively associated with silicosis in 242 miners that never smoked. Central airway resistance can increase in silicosis when lymph nodes and/or granulomas protrude into or compress the airways, which seems to be of minor importance in the present study, since we found only 6% phase 3 granulomatous nodules. Borges et al. (2001) reported an increased P1 in mice, but they did not evaluate the categories of granulomas and found intrabronchial cellular infiltration obstructing the lumen. We found that both elastic and viscoelastic components of lung mechanics were increased in TRIP mice (Table 1); supporting our results, Faffe
et al. (2001) measured lung tissue mechanics in silica-exposed animals and found a similar result. Our study presents some limitations: (1) it should be stressed that a large part of non-respirable particles was detected in the Tripoli dust (more than 50% with a diameter above 5 m). In mice most particles larger than 2 m would not reach the lung periphery and could lead to an overload of particles possibly overwhelming the physiological clearance mechanisms. To prove this point lung burden analyses are required; (2) we did not study a dose-response curve and used a single dosis according with our previous studies on models of silicosis; (3) ours is a study focused on the acute outcomes of the exposure to Tripoli dust and did not address chronic effects; (4) no animals exposed to pure silicon were included as a group. Conclusions We demonstrated for the first time that exposure to Tripoli dust, a powder different from pure silicon dioxide and commonly used in industry and households, acutely damaged mice lungs, as suggested by an increased presence of inflammatory cells and mediators in the lung, collapse of airspaces, and remodeling consisting of granuloma formation and collagen fibers deposition. Altogether these responses impaired pulmonary elastic and viscoelastic mechanical properties. The disease profile is similar to that of chronic silicosis, but it should be stressed that Tripoli powder can be more frequently found in indoor environments than silica. Conflict of interest The authors declare that they have no conflict of interest. Acknowledgements We would like to express our gratitude to Mr. Antônio Carlos de Souza Quaresma and Mr. João Luiz Coelho Rosas Alves for their skillful technical assistance and to Julyana Costa Vieira for her participation in gathering respiratory mechanics data. This study was supported by: The Centers of Excellence Program (PRONEX-MCT/FAPERJ), The Brazilian Council for Scientific and Technological Development (CNPq/MCT), The Carlos Chagas Filho Rio de Janeiro State Research Supporting Foundation (FAPERJ), and Financing for Studies and Projects (FINEP). This study was approved by the Ethics Committee on the Use of Animals, Health Sciences Center, Federal University of Rio de Janeiro (protocol no. IBCCF 046). All animals received humane care in compliance with the “Principles of Laboratory Animal Care” formulated by the National Society for Medical Research and with the “Guide for the Care and Use of Laboratory Animals” prepared by the National Academy of Sciences, USA. References Adamson, I.Y.R., Prieditis, H., Bowden, D.H., 1992. Instillation of chemotactic factor to silica-injected lungs lowers interstitial particle content and reduces pulmonary fibrosis. Am. J. Pathol. 141, 319–326. Barbarin, V., Nihoul, A., Misson, P., Arras, M., Delos, M., Leclereq, I., Lison, D., Huaux, F., 2005. The role of pro- and anti-inflammatory responses in sílica-induced lung fibrosis. Respir. Res. 6, 112. Bates, J.H.T., Rossi, A., Milic-Emili, J., 1985. Analysis of the behavior of the respiratory system with constant inspiratory flow. J. Appl. Physiol. 58, 1840–1848. Bates, J.H.T., Abe, T., Romero, P.V., Soto, J., 1989. Measurement of alveolar pressure in closed-chest dogs during flow interruption. J. Appl. Physiol. 67, 488–492. Borges, V.M., Falcão, H., Leite-Júnior, J.H., Alvim, L., Teixeira, G.P., Russo, M., Nóbrega, A.F., Lopes, M.F., Rocco, P.M., Davidson, W.F., Linden, R., Yagita, H., Zin, W.A., Dosreis, G.A., 2001. Fas ligand triggers pulmonary silicosis. J. Exp. Med. 194, 155–163.
M.N. Machado et al. / Respiratory Physiology & Neurobiology 196 (2014) 17–24 Borges, V.M., Lopes, M.F., Falcão, H., Leite-Júnior, J.H., Rocco, P.M., Davidson, W.F., Linden, R., Zin, W.A., Dosreis, G.A., 2002. Apoptosis underlies immunopathogenic mechanisms in acute silicosis. Am. J. Respir. Cell. Mol. Biol. 27, 78–84. Brown, J.S., Zeman, K.L., Bennett, W.D., 2002. Ultrafine particle deposition and clearance in the healthy and obstructed lung. Am. J. Respir. Crit. Care Med. 166, 1240–1247. Capelozzi, V.L., Saldiva, P.H.N., Antonângelo, L., Carvalho, T.S., Logulo, A., Carvalho, C.R.R., Deheinzelin, D., 1997. Quantitation in inflammatory pleural disease to distinguish tuberculous and paramalignant from chronic non-specific pleuritis. J. Clin. Pathol. 50, 935–940. Castranova, V., Vallyathan, V., 2000. Silicosis and coal workers’ pneumoconiosis. Evironl. Health. Perspect. 108, 675–684. Davis, G.S., Pfeiffer, L.M., Hemenway, D.R., 1998. Persistent overexpression of interleukin-1 beta and tumor necrosis factor-alpha in murine silicosis. J. Environ. Toxicol. Onco. 17, 99–114. Delgado, L., Parra, E.R., Capelozzi, V.L., 2006. Apoptosis and extracellular matrix remodelling in human silicosis. Histopathology 49, 283–289. Ding, M., Chen, F., Shi, X., Yucesoy, B., Mossman, B., Vallyathan, V., 2002. Diseases caused by silica: mechanisms of injury and disease development. Int. Immunopharmacol. 2, 173–182. Donaldson, K., Stone, V., 2003. Current hypotheses on the mechanisms of toxicity of ultrafine particles. Ann. Ist. Super Sanita. 9, 405–410. Donaldson, K., Stone, V., Clouter, A., Renwick, L., MacNee, W., 2001a. Ultrafine particles. Occup. Environ. Med. 58, 211–216. Donaldson, K., Stone, V., Seaton, A., MacNee, W., 2001b. Ambient particle inhalation and the cardiovascular system: potential mechanisms. Environ. Health Perspect. 109, 523–527. Driscoll, K.E., Hassenbein, D.G., Carter, J.M., Kunkel, S.L., Quinlan, T.R., Mossman, B.T., 1995. TNF-␣ and increased chemokine expression in rat lung after particle exposure. Toxicol. Lett. 82–83, 483–489. Dusseldorp, A., Kruize, H., Brunekreef, B., Hofschreuder, P., de Meer, G., van Oudvorst, A.B., 1995. Associations of PM10 and airborne iron with respiratory health of adults living near a steel factory. Am. J. Respir. Crit. Care Med. 152, 1932– 1939. Dye, J.A., Lehmann, J.R., McGee, J.K., Winsett, D.W., Ledbetter, A.D., Everitt, J.I., Ghio, A.J., Costa, D.L., 2001. Acute pulmonary toxicity of particulate matter filter extracts in rats: coherence with epidemiologic studies in Utah Valley residents. Environ. Health. Perspect. 109, 395–403. Ebihara, T., Venkatesan, N., Tanaka, R., Ludwig, M.S., 2000. Changes in extracellular matrix and tissue viscoelasticity in bleomycin-induced lung fibrosis. Am. J. Respir. Crit. Care Med. 162, 1569–1576. Faffe, D.S., Silva, G.H., Kurtz, P.M., Negri, E.M., Capelozzi, V.L., Rocco, P.R., Zin, W.A., 2001. Lung tissue mechanics and extracellular matrix composition in a murine model of silicosis. J. Appl. Physiol. 90, 1400–1406. Gilberti, R.M., Joshi, G.N., Knecht, D.A., 2008. The phagocytosis of crystalline silica particles by macrophages. Am. J. Respir. Cell. Mol. Biol. 39, 619–627. Giordano, G., van den Brûle, S., Lo Re, S., Triqueneaux, P., Uwambayinema, F., Yakoub, Y., Couillin, I., Ryffel, B., Michiels, T., Renauld, J.C., Lison, D., Huaux, F., 2010. Type I interferon signaling contributes to chronic inflammation in a murine model of silicosis. Toxicol. Sci. 116, 682–692. Greaves, I.A., 2000. No-so-simple silicosis: a case for public health action. Am. J. Ind. Med. 37, 245–251. Greenberg, M.I., Waksman, J., Curtis, J., 2007. Silicosis: a review. Dis. Mon. 53, 394–416. Gross, K.B., White, H.J., Smiler, K.L., 1984. Functional and morphologic changes in the lungs after a single intratracheal instillation of silica. Am. Rev. Respir. Dis. 129, 833–839. Gundersen, H.J.G., Bagger, P., Bendtsen, T.F., Evans, S.M., Korbo, L., Marcussen, N., Møller, A., Nielsen, K., Nyengaard, J.R., Pakkenberg, B., Sorensen, S.B., Vesterby, A., West, M.J., 1988. The new stereological tools: disector, fractionator, nucleator and point sampled intercepts and their use in pathological research and diagnosis. APMIS 96, 857–881. Hamilton Jr., R.F., Thakur, S.A., Holian, A., 2008. Silica binding and toxicity in macrophages. Free. Radic. Biol. Med. 44, 1246–1258. Hertzberg, V.S., Rosenman, K.D., Reilly, M.J., Rice, C.H., 2002. Effect of occupational silica exposure on pulmonary function. Chest 122, 721–728. Hnizdo, E., Sluis-Cremer, G.K., Baskind, E., Murray, J., 1994. Emphysema and airway obstruction in non-smoking south African gold miners with long exposure to silica dust. Occup. Environ. Med. 51, 557–563. Huaux, F., Louahed, J., Hudspith, B., Meredith, C., Delos, M., Renauld, J.C., Lison, D., 1998. Role of interleukin-10 in the lung response to silica in mice. Am. J. Respir. Cell Mol. Biol. 18, 51–59. Keller, W.D., 1978. Textures of Tripoli illustrated by scanning electron micrographs. Econ. Geol. 73, 442–446. Kim, I.S., Baek, M., Choi, S.J., 2010. Comparative cytotoxicity of Al2 O3 , CeO2 , TiO2 and ZnO nanoparticles to human lung cells. J. Nanosci. Nanotechnol. 10, 3453–3458. Lakatos, H.F., Burgess, H.A., Thatcher, T.H., Redonnet, M.R., Hernady, E., Williams, J.P., Sime, P.J., 2006. Oropharyngeal aspiration of a silica suspension produces a superior model of silicosis in the mouse when compared to intratracheal instillation. Exp. Lung Res. 32, 181–199. ˜ J.C., Hutt, J.A., Sopori, M.L., 2010. GranuLangley, R.J., Mishra, N.C., Pena-Philippides, loma formation induced by low-dose chronic silica inhalation is associated with an anti-apoptotic response in Lewis rats. J. Toxicol. Environ. Health 73, 669–683. Lardot, C.G., Huaux, F.A., Broeckaert, F.R., Declerck, P.J., Delos, M., Fubini, B., Lison, D.F., 1998. Role of urokinase in the fibrogenic response of the lung to mineral particles. Am. J. Respir. Crit. Care Med. 157, 617–628.
23
Lassance, R.M., Prota, L.F., Maron-Gutierrez, T., Garcia, C.S., Abreu, S.C., Pássaro, C.P., Xisto, D.G., Castiglione, R.C., Carreira Jr., H., Ornellas, D.S., Santana, M.C., Souza, S.A., Gutfilen, B., Fonseca, L.M., Rocco, P.R., Morales, M.M., 2009. Intratracheal instillation of boné marrow-derived cell in na experimental modelo f silicosis. Respir. Physiol. Neurobiol. 169, 227–233. Mannetje, A., Steenland, K., Attfield, M., Boffetta, P., Checkoway, H., DeKlerk, N., Koskela, R.S., 2002. Exposure-response analysis and risk assessment for silica and silicosis mortality in apooled analysis of six cohorts. Occup. Environ. Med. 59, 721–722. Martínez, C., Prieto, A., García, L., Quero, A., González, S., Casan, P., 2010. Silicosis, una enfermedad con presente activo. Arch. Bronconeumol. 46, 97–100. Mazzoli-Rocha, F., Dos Santos, A.N., Fernandes, S., Ferreira Normando, V.M., Malm, O., Nascimento Saldiva, P.H., Wanderley Picanc¸o-Diniz, D.L., Faffe, D.S., Zin, W.A., 2010. Pulmonary function and histological impairment in mice after acute exposure to aluminum dust. Inhal. Toxicol. 22, 861–867. Molinelli, A.R., Madden, M.C., McGee, J.K., Stonehuerner, J.G., Ghio, A.J., 2002. Effect of metal removal on the toxicity of airborne particulate matter from the Utah Valley. Inhal. Toxicol. 14, 1069–1086. Moore, M., 1999. Crystalline silica: occurrence and use. Indoor Built Environ. 8, 82–88. Moore, B.B., Hogaboam, C.M., 2008. Murine models of pulmonary fibrosis. Am. J. Physiol. Cell Mol. Physiol. 294, L152–L160. Mossman, B.T., Churg, A., 1998. Mechanisms in the pathogenesis of asbestosis and silicosis. Am. J. Respir. Crit. Care Med. 157, 1666–1680. Munger, J.S., Huang, X., Kawakatsu, H., Griffiths, M.J., Dalton, S.L., Wu, J., Pittet, J.F., Kaminski, N., Garat, C., Matthay, M.A., Rifkin, D.B., Sheppard, D., 1999. The integrin alpha v beta 6 binds and activates latent TGF beta 1: a mechanism for regulating pulmonary inflammation and fibrosis. Cell 96, 319–328. Nagase, T., Lei, M., Robatto, F.M., Eidelman, D.H., Ludwig, M.S., 1992. Tissue viscance during induced constriction in rabbit lung: morphological–physiological correlation. J. Appl. Physiol. 73, 1900–1907. NIOSH: National Institute for Occupational Safety and Health. Silica, Crystal, http://www.cdc.gov/niosh/pel88/14808-60.html (2011, accessed 01.4.13). Ortiz, L.A., Lasky, J., Gozal, E., Ruiz, V., Lungarella, G., Cavarra, E., Brody, A.R., Friedman, M., Pardo, A., Selman, M., 2001. Tumor necrosis factor receptor deficiency alters matrix metalloproteinase 13/tissue inhibitor of metalloproteinase 1 expression in murine silicosis. Am. J. Respir. Crit. Care Med. 163, 244–252. OSHA: Occupational Safety and Health Administration. Silica Crystalline-Tripoli, 1989, www.cdc.gov/niosh/pel88/1317-95.html (2011, accessed 01.4.13). Park, E.J., Roh, J., Kim, Y., Choi, K., 2011. A single instillation of amorphous silica nanoparticles induced inflammatory responses and tissue damage until day 28 after exposure. J. Health Sci. 57, 60–71. Peters, A., Wichmann, H.E., Tuch, T., Heinrich, J., Heyder, J., 1997. Respiratory effects are associated with the number of ultrafine particles. Am. J. Respir. Crit. Care Med. 155, 1376–1383. Piguet, P.F., Collart, M.A., Grau, G.E., Sappino, A.P., Vassalli, P., 1990. Requirement of tumour necrosis factor for development of silica-induced pulmonary fibrosis. Nature 344, 245–247. Rimal, B., Greenberg, A.K., Rom, W.N., 2005. Basic pathogenic mechanisms in silicosis: current understanding. Curr. Opin. Pulm. Med. 11, 169–173. Sakamoto, H., Zhao, L.H., Jain, F., Kradin, R., 2002. IL-12p40(−/−) mice treated with intratracheal bleomycin exhibit decreased pulmonary inflammation and increased fibrosis. Exp. Mol. Pathol. 72, 1–9. Saldiva, P.H.N., Zin, W.A., Santos, R.L.B., Eidelman, D.H., Milic-Emili, J., 1992. Alveolar pressure measurement in open-chest rats. J. Appl. Physiol. 72, 302– 306. Scabilloni, J.F., Wang, L., Antonini, J.M., Roberts, J.R., Castranova, V., Mercer, R.R., 2005. Matrix metalloproteinase induction in fibrosis and fibrotic nodule formation due to silica inhalation. Am. J. Physiol. Lung Cell. Mol. Physiol. 288, L709–L717. Sirajuddin, A., Kanne, J.P., 2009. Occupational lung disease. J. Thorac. Imaging 24, 310–320. Soukup, J.M., Ghio, A.J., Becker, S., 2000. Soluble components of Utah Valley particulate pollution alter alveolar macrophage function in vivo and in vitro. Inhal. Toxicol. 12, 401–414. Srivastava, K.D., Rom, W.N., Jagirdar, J., Yie, T.A., Gordon, T., Tchou-Wong, K.M., 2002. Crucial role of interleukin-1beta and nitric oxide synthase in silica-induced inflammation and apoptosis in mice. Am. J. Respir. Crit. Care Med. 165, 527– 533. Steenland, K., Mannetje, A., Boffetta, P., 2001. Pooled exposure-response analyses and risk assessment for lung cancer in 10 cohorts of silica-exposed workers: an IARC multicentre study. Can. Caus. Cont. 12, 773–784. Takayoshi, K., Ogami, A., Yamato, H., Oyabu, T., Mormoto, Y., Tanaka, I., 2007. Effect of particle size of intratracheally instilled crystallina silica on pulmonary inflammation. J. Occup. Health 49, 88–94. Tanino, Y., Makita, H., Miyamoto, K., Betsuyaku, T., Ohtsuka, Y., Nishihira, J., Nishimura, M., 2002. Role of macrophage migration inhibitory factor in bleomycin-induced lung injury and fibrosis in mice. Am. J. Physiol. Lung Cell Mol. Physiol. 283, L156–L162. Tao, F., Gonzalez-Flecha, B., Kobzik, L., 2003. Reactive oxygen species in pulmonary inflammation by ambient particulates. Free Radic. Biol. Med. 35, 327– 340. Thakur, S.A., Beamer, C.A., Migliaccio, C.T., Holian, A., 2009. Critical role of MARCO in crystalline silica–induced pulmonary inflammation. Toxicol. Sci. 108, 462– 471.
24
M.N. Machado et al. / Respiratory Physiology & Neurobiology 196 (2014) 17–24
Wiessner, J.H., Mandel, N.S., Sohnle, P.G., Mandel, G.S., 1989. Effect of particle size on quartz-induced hemolysis and on lung inflammation and fibrosis. Exp. Lung Res. 15, 801–812. Wright, J.L., Harrison, N., Wiggs, B., Churg, A., 1988. Quartz but not iron oxide causes air-flow obstruction, emphysema, and small airways lesions in the rat. Am. Rev. Respir. Dis. 138, 129–135.
Zhang, Y., Lee, T.C., Guillemin, B., Yu, M.C., Rom, W.N., 1993. Enhanced IL-1 beta and tumor necrosis factor-alpha release and messenger RNA expression in macrophages from idiopathic pulmonary fibrosis or after asbestos exposure. J. Immunol. 50, 4188–4196.
Contents lists available at ScienceDirect
Respiratory Physiology & Neurobiology journal homepage: www.elsevier.com/locate/resphysiol
Pulmonary functional and morphological damage after exposure to tripoli dust Mariana Nascimento Machado a , Aline Cunha Schmidt a , Paulo Hilário Nascimento Saldiva b , Débora Souza Faffe a , Walter Araujo Zin a,∗ a b
Universidade Federal do Rio de Janeiro, Instituto de Biofísica Carlos Chagas Filho, Rio de Janeiro, Brazil Universidade de São Paulo, Faculdade de Medicina, São Paulo, Brazil
a r t i c l e
i n f o
Article history: Received 22 November 2013 Received in revised form 13 February 2014 Accepted 13 February 2014 Keywords: Silicosis Inflammation Granuloma Tripoli dust Lung mechanics
a b s t r a c t Tripoli is a microcrystalline siliceous rock used to polish metals and precious stones. Its inhalation has been associated with increased prevalence of breathing complaints and pneumoconiosis. However, its acute human exposure has not been so far studied. We aimed at evaluating the putative mechanical, morphological, biochemical and inflammatory lung damage in mice acutely exposed to Tripoli dust. BALB/c mice were randomly assigned to 2 groups: In control group (CTRL, n = 6) animals received intratracheally (i.t.) 0.9% NaCl (50 l), while Tripoli group (TRIP, n = 15) received 20 mg of Tripoli powder diluted in 50 L of saline i.t. The experiments were done 15 days later. TRIP mice showed higher pulmonary mechanical impedance, polymorphonuclear cells, TNF-␣, IL1- and IL-6 than CTRL. TRIP presented granulomatous nodules containing collagenous fibers that occupied 35% of the lung tissue area. In conclusion, acute exposure to Tripoli dust triggered important lung damage in mice lungs that if found in human workers could trigger severe illness. © 2014 Elsevier B.V. All rights reserved.
Introduction The chronic inhalation of crystalline silicon dioxide (SiO2 ) is associated with the occurrence of silicosis. Despite being one of the firstly recognized occupational lung diseases, silicosis remains an important cause of morbidity and mortality worldwide (Martínez et al., 2010). This pneumoconiosis displays persistent inflammation, fibroblast proliferation, and excessive collagen deposition (Thakur et al., 2009). Furthermore, the cytotoxic effects of silica in lung tissue yield macrophage death, subsequent release of inflammatory cytokines, such as TNF-␣, IL-1 and IL-6, and many other substances. As a net result fibrosis (Piguet et al., 1990; Davis et al., 1998; Mossman and Churg, 1998; Srivastava et al., 2002; Rimal et al., 2005; Hamilton et al., 2008; Sirajuddin and Kanne, 2009) and apoptosis (Borges et al., 2002; Srivastava et al., 2002; Langley et al., 2010) ensue. The continuous recruitment and activation of macrophages and granulocytes contributes to the chronic
∗ Corresponding author at: Laboratório de Fisiologia da Respirac¸ão, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Av Carlos Chagas Filho 373, CCS, Rm G2-042, Ilha do Fundão, 21941-902 - Rio de Janeiro, RJ, Brazil. Tel.: +55 21 2562 6557; fax: +55 21 2280 8193. E-mail addresses: walter [email protected], [email protected] (W.A. Zin). http://dx.doi.org/10.1016/j.resp.2014.02.007 1569-9048/© 2014 Elsevier B.V. All rights reserved.
inflammatory process and, thus, to tissue remodeling (Scabilloni et al., 2005; Delgado et al., 2006). As part of the fibrotic process silicotic nodules or granulomas (Scabilloni et al., 2005) are formed. Increased pulmonary mechanical impedance represents the functional counterpart of the morphological changes (Ebihara et al., 2000; Borges et al., 2001; Faffe et al., 2001; Hertzberg et al., 2002). Crystalline silica is found in sand and several rocks, like sandstone, granite and silex and presents polymorphisms, the principal naturally occurring crystalline silica being quartz (Moore, 1999). Cristobalite, tridymite and Tripoli constitute the three other forms of crystalline silica. Tripoli presents unique applications as an abrasive owing to its hardness and because its grain structure lacks distinct edges and corners. It is a mild abrasive, making it suitable for use in toothpaste and tooth polishing compounds, industrial soaps, metal/jewelry polishing mixtures, resins, ceramics, paints, rubber, and cement (Keller, 1978). Along the processing and use of Tripoli powder the dust generated can be inhaled by human beings, not only workers but the general population as well, and may cause an inflammatory lung disease. It should be noted that Tripoli dust contains more components than SiO2 , what could induce a different harmful outcome. However, no study on the detailed acute functional respiratory impairment secondary to exposure to Tripoli dust has been reported so far either in human beings or in experimental animals. Furthermore, no epidemiological work on the prevalence
18
M.N. Machado et al. / Respiratory Physiology & Neurobiology 196 (2014) 17–24
or incidence of Tripoli-triggered disease has been published. In other words, Tripoli powder may be generating more harm than so far recognized by physicians and public health authorities. The aim of the present study was to describe the physical and chemical characteristics of the Tripoli dust used, and to verify whether the exposure to Tripoli dust induces lung morphological, inflammatory and mechanical burdens that could resemble those of chronic silicosis, thus calling physicians and environmental health officers’ attention to the Tripoli issue. Methods Animal and experimental protocol Twenty-one female BALB/c mice (20–25 g) were randomly divided into 2 groups. In control group (CTRL, n = 6) mice were intratracheally (i.t.) instilled with 0.05 mL of sterile saline solution (0.9% NaCl), whereas Tripoli-administered animals (TRIP, n = 15) received intratracheally 20 mg of Tripoli dust vortexed in 0.05 mL of saline, respectively, as previously described in murine models of acute silicosis (Faffe et al., 2001; Borges et al., 2001, 2002). Before the administration of the latter suspension, a stock solution of 600 mg of Tripoli was placed in a 2-mL Eppendorf tube and saline solution was added to reach a final volume of 1.5 mL. The suspension was vortex-mixed for 10 min and 0.05 mL of it was then collected with a Gilson precision micropipette (Gilson, Inc., Middleton, WI, USA) and immediately given to the animal. The vortexing was repeated for every TRIP animal. Fifteen days after saline or Tripoli administration the animals were analyzed. Particle analysis The dust sample was kindly provided by a gemstone-polishing company in São Lourenc¸o, Brazil. It was taken from the same supply being at the polishers’ disposal. It was dried in an oven at 50 ◦ C until completely de-hydrated. Elements were determined by an energy dispersive X-ray fluorescence spectrometer (EDX 700HS, Shimadzu Corp, Analytical Instruments Division, Kyoto, Japan). Aluminum (Al), cobalt (Co), iron (Fe), potassium (K), magnesium (Mg), sodium (Na), sulfur (S), silicon (Si), aluminum oxide (Al2 O3 ), calcium oxide (CaO), red iron oxide (Fe2 O3 ), potassium oxide (K2 O), magnesium oxide (MgO), manganese oxide (MnO), sodium oxide (Na2 O), titanium dioxide (TiO2 ) and silicon dioxide (SiO2 ) were determined and the results expressed as percent composition (wt %) of particles. The trace elements bromine (Br), copper (Cu), germanium (Ge), lutecium (Lu), manganese (Mn), nickel (Ni), rubidium (Rb), selenium (Se), tin (Sn), strontium (Sr), titanium (Ti), zinc (Zn), and zirconium (Zr) were measured and the results expressed as particles per million (ppm) of particles. Three independent samples of the particulate matter were analyzed for this purpose. The distribution of particle sizes, as measured by their volume and surface, and the diameters encompassing 90%, 50% and 10% of the particulate matter were determined by laser diffraction (Long Bench Mastersizer S, Malvern Instruments Ltd, Malvern, Worcestershire, UK). The particulate matter was visualized by scanning electron microscopy (JEOL 5310, Tokyo, Japan).
tidal volume of 0.2 mL, flow equal to 1 mL s−1 , and positive endexpiratory pressure amounting to 2 cmH2 O. The anterior chest wall was surgically removed. A pneumotachograph (1.5-mm ID, length = 4.2 cm, distance between side ports = 2.1 cm) was connected to the tracheal cannula for the measurement of airflow (V ). Changes in lung volume were obtained by flow signal digital integration. The pressure gradient across the pneumotachograph was determined by means of a Validyne MP45-2 differential pressure transducer (Engineering Corp, Northridge, CA, USA). Equipment resistive pressure (=Req.V ) was subtracted from pulmonary resistive pressure so that the present results represent intrinsic values. Transpulmonary pressure was measured with a Validyne MP-45 differential pressure transducer (Engineering Corp, Northridge, CA, USA). Briefly, we determined lung resistive (P1 ) and viscoelastic/inhomogeneous (P2 ) pressures, static elastance (Est ), and viscoelastic component of elastance (E) by the end-inflation occlusion method (Bates et al., 1985). P1 selectively reflects airway resistance, and P2 represents stress relaxation or viscoelastic properties and mechanical heterogeneities of the lung (Bates et al., 1989; Saldiva et al., 1992). Lung mechanics were measured 10–15 times in each animal.
Histological study Heparin (1000 IU) was intravenously injected immediately after the determination of respiratory mechanics. The trachea was clamped at end expiration, and the abdominal aorta and vena cava were sectioned, yielding a massive hemorrhage that quickly euthanized the mice. The right lungs were removed en bloc and quick-frozen by immersion in liquid nitrogen and fixed with Carnoy’s solution (Nagase et al., 1992). After fixation, the tissue was embedded in paraffin. Four-m-thick slices were cut and stained with hematoxylin-eosin or picrosirius red. Morphometric analysis was performed with an integrating eyepiece with a coherent system made of a 100-point and 50 lines (known length) grid coupled to a conventional light microscope (Axioplan, Zeiss, Oberkochen, Germany) in granuloma free areas. The volume fraction of collapsed and normal alveoli was determined in each sample by the point-counting technique (Gundersen et al., 1988) across 10 random non-overlapping microscopic fields at ×400 magnification. The total amount of points also included those falling on tissue, airways and other non-alveolar structures. The number of mononuclear (MN) and polymorphonuclear (PMN) cells in the pulmonary tissue was counted in each animal across 10 random non-overlapping microscopic fields at ×1000 magnification in a 10,000 m2 granuloma free area; in the same field the amount of points that fell on lung tissue was also counted, so that cellularity was expressed as percentage of lung tissue area (Gundersen et al., 1988; Capelozzi et al., 1997). The fraction area of the granulomas was determined using the point-counting technique across 20 random non-coincident microscopic fields per animal at a magnification of ×200. Percentage of lung tissue occupied by granulomatous nodules was scored as following: phase 1, nodules present only in the lung parenchyma; phase 2, nodules around the airways; phase 3, nodules obstructing the airway; and, phase 4, lung nodules in various structures.
Pulmonary mechanics Fifteen days after saline or Tripoli dust administration, the animals were sedated with diazepam (1 mg i.p.) and anesthetized with pentobarbital sodium (20 mg kg body weight−1 i.p.), paralyzed with pancuronium bromide (0.1 mg kg body weight−1 i.v.), and mechanically ventilated (Samay VR15, Universidad de la Republica, Montevideo, Uruguay) with a frequency of 100 breaths min−1 ,
Analysis of cytokines Samples of lung cytosol were analyzed by ELISA for the detection of the inflammatory cytokines TNF-␣, IL-1, IL-6 (ELISA kits, R&D Systems Europe, Abingdon, UK) with detection limits of 5.1 pg/mL, 1.6 pg/mL and 3.0 pg/mL respectively.
M.N. Machado et al. / Respiratory Physiology & Neurobiology 196 (2014) 17–24
19
Fig. 1. (A) Electron scanning micrographs of Tripoli powder. White bars: 5, 10 and 50 m (left, middle and right panels, respectively). (B) Histogram of the frequency distribution of particle diameters (columns) and accumulated frequency (solid line).
Statistical analysis SigmaPlot 11 statistical package (Systat Software, Inc., Chicago, IL, USA) was used. The normality of the data (Kolmogorov–Smirnov test with Lilliefors’ correction) and the homogeneity of variances (Levene median test) were tested. When both conditions were satisfied Student’s t-test was used. Otherwise Mann-Whitney test was employed. The significance level was set at 5%.
Results Particle analysis Scanning electron micrographs of particles are shown in Fig. 1A. Tripoli dust particles were not spherical in shape, displaying an irregular form and aggregates. Fig. 1B depicts the frequency distribution of particle diameters. Ninety percent of the particles presented a diameter below 17.28 m, being 50% below 5.09 m and 10% below 1.54 m. The average sizes of the particles according to their volume and surface were 7.57 and 3.47 m, respectively. Metals and oxides in the Tripoli dust are presented in Fig. 2; a high concentration of the element Si was found as SiO2 .
Pulmonary mechanics TRIP group presented significantly higher lung mechanical parameters than CTRL, excluding P1 that was not different between the groups (Table 1).
Table 1 Mechanics, histology and inflammation markers in lung parenchyma. CTRL
TRIP
Mechanics P1 (cmH2 O) P2 (cmH2 O) Est (cmH2 O/mL) E (cmH2 O/mL)
0.40 (0.38–0.50) 1.22 (1.06–1.48) 24.04 ± 1.60 6.17 (5.18–7.46)
0.52 (0.44–0.80) 1.66 (1.29–2.22)* 33.07 ± 2.64* 8.26 (6.35–11.17)*
Histology Normal area (%) Alveolar collapse (%) PMN (cel × 10−3 /m2 ) MN (cel × 10−3 /m2 )
70.00 ± 0.80 14.00 ± 0.50 4.00 ± 0.01 11.00 ± 1.00
11.00 ± 1.10* 37.00 ± 0.70* 6.00 ± 0.01* 12.00 ± 1.00
Cytokines TNF-␣ (pg/mL) IL-1 (pg/mL) IL-6 (pg/mL)
79.98 ± 2.67 129.71 ± 11.27 55.59 ± 1.90
115.08 ± 2.41* 609.09 ± 22.98* 81.90 ± 4.87*
Values are mean ± SEM or median (25–75%). Control mice (CTRL, n = 6) and those instilled with 20 mg of Tripoli dust/50 L saline (TRIP, n = 15); P1 and P2 , resistive and viscoelastic/inhomogeneous pressures, respectively; Est , static elastance; E, viscoelastic component of elastance; PMN and MN, polymorpho- and mononuclear cells, respectively; percentage of normal and collapsed areas in pulmonary tissue. * Significantly different from CTRL group (p < 0.05).
Histology Representative photomicrographs of lung parenchyma (picrosirius red) in control mice (Panel A) and in animals intratracheally instilled with 20 mg of Tripoli (Panels B–F) are depicted in Fig. 3. In panel B phase 1 granuloma shows accumulation of macrophages containing dust particles, eliciting a slight fibrotic response (more intense red staining); in panel C (phase 2), the
20
M.N. Machado et al. / Respiratory Physiology & Neurobiology 196 (2014) 17–24
parenchyma (Table 1). Spearman correlation was used to evaluate the association between static elastance and percentage of collapsed air spaces. Significant correlation between alveolar collapse and Est was found: = 0.611, p < 0.0043. Cytokines TNF-␣, IL-1, and IL-6 were significantly higher (+43.9%, +369.6%, and +47.3%, respectively) in lung homogenates of Tripoliinstilled mice than in control group (Table 1). Discussion
Fig. 2. Metals (panels A and B) and oxides (panel C) in Tripoli dust. Aluminum (Al), cobalt (Co), iron (Fe), potassium (K), magnesium (Mg), sodium (Na), sulfur (S), silicon (Si), aluminum oxide (Al2 O3 ), calcium oxide (CaO), red iron oxide (Fe2 O3 ), potassium oxide (K2 O), magnesium oxide (MgO), manganese oxide (MnO), sodium oxide (Na2 O), titanium dioxide (TiO2 ) and silicon dioxide (SiO2 ) were measured and the results expressed as WT (%) of particles. Trace elements bromine (Br), copper (Cu), germanium (Ge), lutecium (Lu), manganese (Mn), nickel (Ni), rubidium (Rb), selenium (Se), tin (Sn), strontium (Sr), titanium (Ti), zinc (Zn), and zirconium (Zr) were measured and the results expressed as ppm of particles or WT (%). ppm, part per million; WT, percent composition by weight.
alveoli around the granuloma exhibit an acute inflammatory response and contain dust particles within macrophages; in panel D (phase 3), obliterative bronchiolitis shows a fibrotic inflammatory polip protruding into the bronchiolar lumen; and in panel E (phase 4), multiple dust nodules and inflammatory reaction composed by dust particles, large number of macrophages, parenchymal collapse and fibrosis are present in the gas exchanging territory. Panel F displays lung parenchyma under polarized light showing the birefringent particles of Tripoli dust as bright spots. The overall area fraction of granulomatous nodules was 35.10 ± 0.05% in TRIP group. The percentages of nodules in lung tissue found in phases 1, 2, 3, and 4 were 38.7 ± 16.7%, 20.0 ± 9.7%, 6.0 ± 6.1%, and 35.4 ± 16.7% (mean ± SD), respectively. TRIP animals presented increased alveolar collapse (Table 1) with polymorphonuclear (PMN) and total cells influx in lung
Our results demonstrated that acute exposure to Tripoli dust was done with particles presenting various shapes with a mean aerodynamic diameter of about 5 m. Due to their elevated content of silica and silicon dioxide these particles triggered lung morphological changes similar to classical acute silicosis. Intratracheal instillation of 20 mg of Tripoli induced pulmonary influx of inflammatory cells, increased release of pro-inflammatory cytokines such as TNF-␣, IL-1 and IL-6, collapsed alveoli, and granulomas with expression of collagen fibers in the lung parenchyma. As a result pulmonary mechanical impedance was impaired mainly at tissue level, turning the lung stiffer and offering a higher resistance to movement. Once inhaled, silica particles are deposited in different parts of the respiratory system, according to their size and initiate the pathological process. Toxicity varies according to particle diameter. In humans toxicity becomes very important when the aerodynamic diameter of the particles is less than 10 m, which enables them to reach the pulmonary alveoli. In rats and mice this value approximates 2 m for intratracheally instilled silica (Wiessner et al., 1989; Takayoshi et al., 2007). Our results showed that 90% of Tripoli dust particles were below 17.28 m diameter, being 50% below 5.09 m and 10% below 1.54 m. Thus, at least part of the administered Tripoli dust reached the lung periphery. It should be stressed that the fine and ultrafine particles are known as “breathable” and are able to penetrate the airways, reaching the alveoli (Dusseldorp et al., 1995; Peters et al., 1997; Brown et al., 2002; Tao et al., 2003). Otherwise, they can be eliminated by the mucociliary system or, when deposited in the alveolar regions, can be phagocytosed by macrophages. It is believed that these fine and ultrafine particles can still penetrate into the lung parenchyma and reach the bloodstream (Donaldson et al., 2001a, 2001b; Donaldson and Stone, 2003). As depicted in Fig. 2 our particulate matter showed a very high concentration of the element silicon and silicon dioxide, as well as other elements and oxides. Thus, Tripoli dust is not pure silicon dioxide, the silica dust most frequently used to produce experimental models of silicosis. Metal containing particles induced pulmonary changes, reinforcing their potential role in determining particle toxicity (Soukup et al., 2000; Dye et al., 2001; Molinelli et al., 2002). Mazzoli-Rocha et al. (2010) demonstrated that a single aerosolization of small quantities of particles containing mainly aluminum induced acute respiratory inflammation, as suggested by changes in lung tissue mechanics, reflected in increased Est , E and P2 , but no significant change in P1 and lung histology. These studies demonstrate the impact of such metals in pulmonary mechanics. Kim et al. (2010) assessed and compared the in vitro toxicity of four different oxide nanoparticles (Al2 O3 , CeO2 , TiO2 and ZnO) to human lung epithelial cells. Among four tested nanoparticles, ZnO exhibited the highest cytotoxicity in terms of cell proliferation, cell viability, membrane integrity, and colony formation. Al2 O3 , CeO2 and TiO2 showed little adverse effects on cell proliferation
M.N. Machado et al. / Respiratory Physiology & Neurobiology 196 (2014) 17–24
21
Fig. 3. Representative photomicrographs of lung parenchyma (picrosirius red) in control animals (panel A), and in mice intratracheally instilled with a suspension of 20 mg of Tripoli dust in 0.05 mL of saline solution: in panel B, nodule is seen only in the lung parenchyma featuring an early stage of disease; in panel C, nodules are found around the airways; in panel D, nodule obstructs the airway; in panel E, nodules are present in various lung structures featuring an advanced stage of the disease. Image obtained with polarized light (panel F) shows scattered interstitial Tripoli particles as bright spots. Tripoli particles can be seen in the nodules in panels B–E. Twenty random non-coincident microscopic fields were evaluated per animal at a magnification of ×200. Bar equals 100 m.
and cell viability. However, TiO2 induced oxidative stress in a concentration- and time-dependent manner. Al2 O3 seems to be less toxic than the other nanoparticles even after long time exposure. These findings suggest the relevance of chemical components on the toxicity of Tripoli powder. However, it is not possible to exclude the contribution of non-determined constituents of the particle composition. As observed in other murine models of fibrosis, the response is strain-dependent, being BALB/c mice more resistant to silica instillation (Moore and Hogaboam, 2008). In this context, 2.5 mg of silica in/60 L of saline were enough to trigger lung inflammation and fibrosis in C57BL/6 mice (Giordano et al., 2010), whereas our animals were exposed to 20 mg of Tripoli dust in 50 L of saline instilled directly into the trachea, a dose very close to those reported by others, who used silicon dioxide in mice and rats (Gross et al., 1984; Wright et al., 1988; Borges et al., 2001; Faffe et al., 2001; Borges et al., 2002; Lassance et al., 2009). However, considering that only circa 50% of our dust presented a diameter less than 5 m, our effective dose of PM5 was half theirs. Indeed Tripoli dust presents particle diameters larger than those found in commercially available silicon dioxide (1–5 m). A limit exposure to Tripoli dust has not been described yet, but the permissible exposure limit for dust containing respirable crystalline silica is 0.1 mg/m3 (NIOSH, 2011). The Occupational Safety and Health Administration (OSHA) has determined that the exposure limit of quartz from 0.08 to 0.1 mg/m3 can be used as a basis to limit exposure to the Tripoli particles (NIOSH, 2011). However, even these levels may not be low enough to prevent chronic disease. In this line, Greaves (2000) concluded that the 0.1 mg/m3 recommended exposure limit of the NIOSH might not be sufficiently protective for a substantial proportion of workers. Additionally, Steenland et al. (2001) described that the estimated excess lifetime risk (through age 75) of lung cancer for a worker exposed from age 20 to 65 at 0.1 mg/m3 respirable crystalline silica was 1.1–1.7%, above background risks of 3–6%, and Mannetje et al. (2002) described that the risk of death from silicosis is 13 per 1000
workers to a limit exposure of 0.1 mg/m3 of respirable silica in the environment. Therefore we used a dose of 20 mg of Tripoli to evaluate possible pulmonary complications owing to exposure to this dust. The time lag between exposure and measurement represents another factor involved in the response. Although silicosis develops over years in humans exposed to silica dust, experimental studies in BALB/c mice showed that even a single exposure leads to functional and histological impairment (Borges et al., 2001; Faffe et al., 2001; Borges et al., 2002). Some studies report the development of fibrosis within the first month after exposure (Lardot et al., 1998; Borges et al., 2001; Faffe et al., 2001; Lakatos et al., 2006; Borges et al., 2002), or even after longer periods (Barbarin et al., 2005; Giordano et al., 2010). To the best of our knowledge, we report for the first time the acute effects of the exposure to Tripoli dust. Exposure to Tripoli dust resulted in polymorphonuclear cell influx into the lung parenchyma as previously reported with silica (Faffe et al., 2001). These authors showed progressive histological changes in a murine model of silicosis. We observed that TNF-␣, IL-1 and IL-6 levels in the lung tissue increased significantly after acute exposure to Tripoli (Table 1). In a murine model of silicosis, IL-1 was associated with mononuclear cell-related inflammation and collagen deposition (Zhang et al., 1993). We might advance that the cytotoxic effects of Tripoli particles in the lung could be related to the following cascade, similar to that of silica: death of alveolar macrophages, subsequent release of inflammatory cytokines (TNF-␣, IL-1 and IL-6 among others), stimulation of fibroblasts activity, development of granulomas and subsequent pulmonary fibrosis (Piguet et al., 1990; Davis et al., 1998; Mossman and Churg, 1998; Srivastava et al., 2002; Rimal et al., 2005; Hamilton et al., 2008; Sirajuddin and Kanne, 2009). Davis et al. (1998) showed that the increased production of TNF␣ and IL-1 occurs within the silicotic lesions and bronchoalveolar lavage fluid. These mediators seem to be involved mainly in early disease, since they precede inflammation and fibrosis (Driscoll
22
M.N. Machado et al. / Respiratory Physiology & Neurobiology 196 (2014) 17–24
et al., 1995). However, the alveolar macrophages are related to the maintenance of silicotic inflammation in a vicious cycle: they phagocytize the silica particles, are activated and damaged, releasing the silica particle back into the lung environment. Then another macrophage engulfs the same particle and perpetuates the process (Greenberg et al., 2007; Gilberti et al., 2008). Despite the important role of inflammation in fibrogenesis, some evidence suggests that inflammation is not necessarily related to the fibrotic response and that additional pathogenic pathways may be responsible for development of the fibrotic response in the lung. Some studies show that lung inflammation is not always followed by fibrotic disease (Adamson et al., 1992; Huaux et al., 1998; Munger et al., 1999), whereas other works reveal that control of inflammation is not always associated with reduced fibrosis (Tanino et al., 2002; Sakamoto et al., 2002). Chronic exposure to silica particles is histologically characterized by the presence of granulomas, alveolar septal thickening and accumulation of inflammatory cells such as macrophages (Delgado et al., 2006). Ortiz et al. (2001) intratracheally instilled mice with silica (0.2 g/kg, average of 5 mg of silica per animal, particles presenting an average diameter of 1 m) and observed granulomas in the lung parenchyma 28 days after the challenge, but qualitatively these nodules were not as important as those presented in our study. Park et al. (2011) evaluated changes in lung tissue 1, 7, 14 and 28 days after a single intratracheal instillation of silica nanoparticles (1 mg/kg) and found inflammatory cell migration to the alveolar space and infiltration in alveolar wall. In the present Tripoli-exposed lungs there were granulomas containing large numbers of mononuclear cells, fibroblasts and collagen fibers arranged in a circular orientation, showing features of immature silicotic nodules (Fig. 3). More advanced stages of tissue remodelling display the classical formation of fibrotic nodules with collagen fibers concentrically aligned, a central hyaline core, and lumping and agglomeration of small silicotic nodules (Castranova and Vallyathan, 2000; Ding et al., 2002; Rimal et al., 2005). The fraction area of pulmonary tissue occupied by granulomas in the TRIP group was 35.1%. In our experimental model these granulomas were present in four phases. Since 80% of the macrophages die within 12 h after exposure to silica and silica internalization by macrophages occurs even faster (Gilberti et al., 2008), our results may suggest a still active inflammatory process as indicated by the high percentage of phase 1 granulomas together with well established ones. This hypothesis is supported by our findings of elevated levels of pro-inflammatory cytokines (Table 1). The aforementioned histological and immunological phenomena brought about significant changes in mechanical parameters (Table 1). Our results demonstrated that intratracheal instillation of Tripoli dust induced lung injury and increased lung impedance determined on the 15th day after exposure. In accordance with our results Borges et al. (2001) intratracheally instilled BALB/c mice with silica dust and reported an elastic component of pulmonary impedance similar to ours. Control groups in both studies presented similar results. We did not find changes in the pressure spent against central airway resistance (Table 1); in this context, Hnizdo et al. (1994) reported that large airway disease was not positively associated with silicosis in 242 miners that never smoked. Central airway resistance can increase in silicosis when lymph nodes and/or granulomas protrude into or compress the airways, which seems to be of minor importance in the present study, since we found only 6% phase 3 granulomatous nodules. Borges et al. (2001) reported an increased P1 in mice, but they did not evaluate the categories of granulomas and found intrabronchial cellular infiltration obstructing the lumen. We found that both elastic and viscoelastic components of lung mechanics were increased in TRIP mice (Table 1); supporting our results, Faffe
et al. (2001) measured lung tissue mechanics in silica-exposed animals and found a similar result. Our study presents some limitations: (1) it should be stressed that a large part of non-respirable particles was detected in the Tripoli dust (more than 50% with a diameter above 5 m). In mice most particles larger than 2 m would not reach the lung periphery and could lead to an overload of particles possibly overwhelming the physiological clearance mechanisms. To prove this point lung burden analyses are required; (2) we did not study a dose-response curve and used a single dosis according with our previous studies on models of silicosis; (3) ours is a study focused on the acute outcomes of the exposure to Tripoli dust and did not address chronic effects; (4) no animals exposed to pure silicon were included as a group. Conclusions We demonstrated for the first time that exposure to Tripoli dust, a powder different from pure silicon dioxide and commonly used in industry and households, acutely damaged mice lungs, as suggested by an increased presence of inflammatory cells and mediators in the lung, collapse of airspaces, and remodeling consisting of granuloma formation and collagen fibers deposition. Altogether these responses impaired pulmonary elastic and viscoelastic mechanical properties. The disease profile is similar to that of chronic silicosis, but it should be stressed that Tripoli powder can be more frequently found in indoor environments than silica. Conflict of interest The authors declare that they have no conflict of interest. Acknowledgements We would like to express our gratitude to Mr. Antônio Carlos de Souza Quaresma and Mr. João Luiz Coelho Rosas Alves for their skillful technical assistance and to Julyana Costa Vieira for her participation in gathering respiratory mechanics data. This study was supported by: The Centers of Excellence Program (PRONEX-MCT/FAPERJ), The Brazilian Council for Scientific and Technological Development (CNPq/MCT), The Carlos Chagas Filho Rio de Janeiro State Research Supporting Foundation (FAPERJ), and Financing for Studies and Projects (FINEP). This study was approved by the Ethics Committee on the Use of Animals, Health Sciences Center, Federal University of Rio de Janeiro (protocol no. IBCCF 046). All animals received humane care in compliance with the “Principles of Laboratory Animal Care” formulated by the National Society for Medical Research and with the “Guide for the Care and Use of Laboratory Animals” prepared by the National Academy of Sciences, USA. References Adamson, I.Y.R., Prieditis, H., Bowden, D.H., 1992. Instillation of chemotactic factor to silica-injected lungs lowers interstitial particle content and reduces pulmonary fibrosis. Am. J. Pathol. 141, 319–326. Barbarin, V., Nihoul, A., Misson, P., Arras, M., Delos, M., Leclereq, I., Lison, D., Huaux, F., 2005. The role of pro- and anti-inflammatory responses in sílica-induced lung fibrosis. Respir. Res. 6, 112. Bates, J.H.T., Rossi, A., Milic-Emili, J., 1985. Analysis of the behavior of the respiratory system with constant inspiratory flow. J. Appl. Physiol. 58, 1840–1848. Bates, J.H.T., Abe, T., Romero, P.V., Soto, J., 1989. Measurement of alveolar pressure in closed-chest dogs during flow interruption. J. Appl. Physiol. 67, 488–492. Borges, V.M., Falcão, H., Leite-Júnior, J.H., Alvim, L., Teixeira, G.P., Russo, M., Nóbrega, A.F., Lopes, M.F., Rocco, P.M., Davidson, W.F., Linden, R., Yagita, H., Zin, W.A., Dosreis, G.A., 2001. Fas ligand triggers pulmonary silicosis. J. Exp. Med. 194, 155–163.
M.N. Machado et al. / Respiratory Physiology & Neurobiology 196 (2014) 17–24 Borges, V.M., Lopes, M.F., Falcão, H., Leite-Júnior, J.H., Rocco, P.M., Davidson, W.F., Linden, R., Zin, W.A., Dosreis, G.A., 2002. Apoptosis underlies immunopathogenic mechanisms in acute silicosis. Am. J. Respir. Cell. Mol. Biol. 27, 78–84. Brown, J.S., Zeman, K.L., Bennett, W.D., 2002. Ultrafine particle deposition and clearance in the healthy and obstructed lung. Am. J. Respir. Crit. Care Med. 166, 1240–1247. Capelozzi, V.L., Saldiva, P.H.N., Antonângelo, L., Carvalho, T.S., Logulo, A., Carvalho, C.R.R., Deheinzelin, D., 1997. Quantitation in inflammatory pleural disease to distinguish tuberculous and paramalignant from chronic non-specific pleuritis. J. Clin. Pathol. 50, 935–940. Castranova, V., Vallyathan, V., 2000. Silicosis and coal workers’ pneumoconiosis. Evironl. Health. Perspect. 108, 675–684. Davis, G.S., Pfeiffer, L.M., Hemenway, D.R., 1998. Persistent overexpression of interleukin-1 beta and tumor necrosis factor-alpha in murine silicosis. J. Environ. Toxicol. Onco. 17, 99–114. Delgado, L., Parra, E.R., Capelozzi, V.L., 2006. Apoptosis and extracellular matrix remodelling in human silicosis. Histopathology 49, 283–289. Ding, M., Chen, F., Shi, X., Yucesoy, B., Mossman, B., Vallyathan, V., 2002. Diseases caused by silica: mechanisms of injury and disease development. Int. Immunopharmacol. 2, 173–182. Donaldson, K., Stone, V., 2003. Current hypotheses on the mechanisms of toxicity of ultrafine particles. Ann. Ist. Super Sanita. 9, 405–410. Donaldson, K., Stone, V., Clouter, A., Renwick, L., MacNee, W., 2001a. Ultrafine particles. Occup. Environ. Med. 58, 211–216. Donaldson, K., Stone, V., Seaton, A., MacNee, W., 2001b. Ambient particle inhalation and the cardiovascular system: potential mechanisms. Environ. Health Perspect. 109, 523–527. Driscoll, K.E., Hassenbein, D.G., Carter, J.M., Kunkel, S.L., Quinlan, T.R., Mossman, B.T., 1995. TNF-␣ and increased chemokine expression in rat lung after particle exposure. Toxicol. Lett. 82–83, 483–489. Dusseldorp, A., Kruize, H., Brunekreef, B., Hofschreuder, P., de Meer, G., van Oudvorst, A.B., 1995. Associations of PM10 and airborne iron with respiratory health of adults living near a steel factory. Am. J. Respir. Crit. Care Med. 152, 1932– 1939. Dye, J.A., Lehmann, J.R., McGee, J.K., Winsett, D.W., Ledbetter, A.D., Everitt, J.I., Ghio, A.J., Costa, D.L., 2001. Acute pulmonary toxicity of particulate matter filter extracts in rats: coherence with epidemiologic studies in Utah Valley residents. Environ. Health. Perspect. 109, 395–403. Ebihara, T., Venkatesan, N., Tanaka, R., Ludwig, M.S., 2000. Changes in extracellular matrix and tissue viscoelasticity in bleomycin-induced lung fibrosis. Am. J. Respir. Crit. Care Med. 162, 1569–1576. Faffe, D.S., Silva, G.H., Kurtz, P.M., Negri, E.M., Capelozzi, V.L., Rocco, P.R., Zin, W.A., 2001. Lung tissue mechanics and extracellular matrix composition in a murine model of silicosis. J. Appl. Physiol. 90, 1400–1406. Gilberti, R.M., Joshi, G.N., Knecht, D.A., 2008. The phagocytosis of crystalline silica particles by macrophages. Am. J. Respir. Cell. Mol. Biol. 39, 619–627. Giordano, G., van den Brûle, S., Lo Re, S., Triqueneaux, P., Uwambayinema, F., Yakoub, Y., Couillin, I., Ryffel, B., Michiels, T., Renauld, J.C., Lison, D., Huaux, F., 2010. Type I interferon signaling contributes to chronic inflammation in a murine model of silicosis. Toxicol. Sci. 116, 682–692. Greaves, I.A., 2000. No-so-simple silicosis: a case for public health action. Am. J. Ind. Med. 37, 245–251. Greenberg, M.I., Waksman, J., Curtis, J., 2007. Silicosis: a review. Dis. Mon. 53, 394–416. Gross, K.B., White, H.J., Smiler, K.L., 1984. Functional and morphologic changes in the lungs after a single intratracheal instillation of silica. Am. Rev. Respir. Dis. 129, 833–839. Gundersen, H.J.G., Bagger, P., Bendtsen, T.F., Evans, S.M., Korbo, L., Marcussen, N., Møller, A., Nielsen, K., Nyengaard, J.R., Pakkenberg, B., Sorensen, S.B., Vesterby, A., West, M.J., 1988. The new stereological tools: disector, fractionator, nucleator and point sampled intercepts and their use in pathological research and diagnosis. APMIS 96, 857–881. Hamilton Jr., R.F., Thakur, S.A., Holian, A., 2008. Silica binding and toxicity in macrophages. Free. Radic. Biol. Med. 44, 1246–1258. Hertzberg, V.S., Rosenman, K.D., Reilly, M.J., Rice, C.H., 2002. Effect of occupational silica exposure on pulmonary function. Chest 122, 721–728. Hnizdo, E., Sluis-Cremer, G.K., Baskind, E., Murray, J., 1994. Emphysema and airway obstruction in non-smoking south African gold miners with long exposure to silica dust. Occup. Environ. Med. 51, 557–563. Huaux, F., Louahed, J., Hudspith, B., Meredith, C., Delos, M., Renauld, J.C., Lison, D., 1998. Role of interleukin-10 in the lung response to silica in mice. Am. J. Respir. Cell Mol. Biol. 18, 51–59. Keller, W.D., 1978. Textures of Tripoli illustrated by scanning electron micrographs. Econ. Geol. 73, 442–446. Kim, I.S., Baek, M., Choi, S.J., 2010. Comparative cytotoxicity of Al2 O3 , CeO2 , TiO2 and ZnO nanoparticles to human lung cells. J. Nanosci. Nanotechnol. 10, 3453–3458. Lakatos, H.F., Burgess, H.A., Thatcher, T.H., Redonnet, M.R., Hernady, E., Williams, J.P., Sime, P.J., 2006. Oropharyngeal aspiration of a silica suspension produces a superior model of silicosis in the mouse when compared to intratracheal instillation. Exp. Lung Res. 32, 181–199. ˜ J.C., Hutt, J.A., Sopori, M.L., 2010. GranuLangley, R.J., Mishra, N.C., Pena-Philippides, loma formation induced by low-dose chronic silica inhalation is associated with an anti-apoptotic response in Lewis rats. J. Toxicol. Environ. Health 73, 669–683. Lardot, C.G., Huaux, F.A., Broeckaert, F.R., Declerck, P.J., Delos, M., Fubini, B., Lison, D.F., 1998. Role of urokinase in the fibrogenic response of the lung to mineral particles. Am. J. Respir. Crit. Care Med. 157, 617–628.
23
Lassance, R.M., Prota, L.F., Maron-Gutierrez, T., Garcia, C.S., Abreu, S.C., Pássaro, C.P., Xisto, D.G., Castiglione, R.C., Carreira Jr., H., Ornellas, D.S., Santana, M.C., Souza, S.A., Gutfilen, B., Fonseca, L.M., Rocco, P.R., Morales, M.M., 2009. Intratracheal instillation of boné marrow-derived cell in na experimental modelo f silicosis. Respir. Physiol. Neurobiol. 169, 227–233. Mannetje, A., Steenland, K., Attfield, M., Boffetta, P., Checkoway, H., DeKlerk, N., Koskela, R.S., 2002. Exposure-response analysis and risk assessment for silica and silicosis mortality in apooled analysis of six cohorts. Occup. Environ. Med. 59, 721–722. Martínez, C., Prieto, A., García, L., Quero, A., González, S., Casan, P., 2010. Silicosis, una enfermedad con presente activo. Arch. Bronconeumol. 46, 97–100. Mazzoli-Rocha, F., Dos Santos, A.N., Fernandes, S., Ferreira Normando, V.M., Malm, O., Nascimento Saldiva, P.H., Wanderley Picanc¸o-Diniz, D.L., Faffe, D.S., Zin, W.A., 2010. Pulmonary function and histological impairment in mice after acute exposure to aluminum dust. Inhal. Toxicol. 22, 861–867. Molinelli, A.R., Madden, M.C., McGee, J.K., Stonehuerner, J.G., Ghio, A.J., 2002. Effect of metal removal on the toxicity of airborne particulate matter from the Utah Valley. Inhal. Toxicol. 14, 1069–1086. Moore, M., 1999. Crystalline silica: occurrence and use. Indoor Built Environ. 8, 82–88. Moore, B.B., Hogaboam, C.M., 2008. Murine models of pulmonary fibrosis. Am. J. Physiol. Cell Mol. Physiol. 294, L152–L160. Mossman, B.T., Churg, A., 1998. Mechanisms in the pathogenesis of asbestosis and silicosis. Am. J. Respir. Crit. Care Med. 157, 1666–1680. Munger, J.S., Huang, X., Kawakatsu, H., Griffiths, M.J., Dalton, S.L., Wu, J., Pittet, J.F., Kaminski, N., Garat, C., Matthay, M.A., Rifkin, D.B., Sheppard, D., 1999. The integrin alpha v beta 6 binds and activates latent TGF beta 1: a mechanism for regulating pulmonary inflammation and fibrosis. Cell 96, 319–328. Nagase, T., Lei, M., Robatto, F.M., Eidelman, D.H., Ludwig, M.S., 1992. Tissue viscance during induced constriction in rabbit lung: morphological–physiological correlation. J. Appl. Physiol. 73, 1900–1907. NIOSH: National Institute for Occupational Safety and Health. Silica, Crystal, http://www.cdc.gov/niosh/pel88/14808-60.html (2011, accessed 01.4.13). Ortiz, L.A., Lasky, J., Gozal, E., Ruiz, V., Lungarella, G., Cavarra, E., Brody, A.R., Friedman, M., Pardo, A., Selman, M., 2001. Tumor necrosis factor receptor deficiency alters matrix metalloproteinase 13/tissue inhibitor of metalloproteinase 1 expression in murine silicosis. Am. J. Respir. Crit. Care Med. 163, 244–252. OSHA: Occupational Safety and Health Administration. Silica Crystalline-Tripoli, 1989, www.cdc.gov/niosh/pel88/1317-95.html (2011, accessed 01.4.13). Park, E.J., Roh, J., Kim, Y., Choi, K., 2011. A single instillation of amorphous silica nanoparticles induced inflammatory responses and tissue damage until day 28 after exposure. J. Health Sci. 57, 60–71. Peters, A., Wichmann, H.E., Tuch, T., Heinrich, J., Heyder, J., 1997. Respiratory effects are associated with the number of ultrafine particles. Am. J. Respir. Crit. Care Med. 155, 1376–1383. Piguet, P.F., Collart, M.A., Grau, G.E., Sappino, A.P., Vassalli, P., 1990. Requirement of tumour necrosis factor for development of silica-induced pulmonary fibrosis. Nature 344, 245–247. Rimal, B., Greenberg, A.K., Rom, W.N., 2005. Basic pathogenic mechanisms in silicosis: current understanding. Curr. Opin. Pulm. Med. 11, 169–173. Sakamoto, H., Zhao, L.H., Jain, F., Kradin, R., 2002. IL-12p40(−/−) mice treated with intratracheal bleomycin exhibit decreased pulmonary inflammation and increased fibrosis. Exp. Mol. Pathol. 72, 1–9. Saldiva, P.H.N., Zin, W.A., Santos, R.L.B., Eidelman, D.H., Milic-Emili, J., 1992. Alveolar pressure measurement in open-chest rats. J. Appl. Physiol. 72, 302– 306. Scabilloni, J.F., Wang, L., Antonini, J.M., Roberts, J.R., Castranova, V., Mercer, R.R., 2005. Matrix metalloproteinase induction in fibrosis and fibrotic nodule formation due to silica inhalation. Am. J. Physiol. Lung Cell. Mol. Physiol. 288, L709–L717. Sirajuddin, A., Kanne, J.P., 2009. Occupational lung disease. J. Thorac. Imaging 24, 310–320. Soukup, J.M., Ghio, A.J., Becker, S., 2000. Soluble components of Utah Valley particulate pollution alter alveolar macrophage function in vivo and in vitro. Inhal. Toxicol. 12, 401–414. Srivastava, K.D., Rom, W.N., Jagirdar, J., Yie, T.A., Gordon, T., Tchou-Wong, K.M., 2002. Crucial role of interleukin-1beta and nitric oxide synthase in silica-induced inflammation and apoptosis in mice. Am. J. Respir. Crit. Care Med. 165, 527– 533. Steenland, K., Mannetje, A., Boffetta, P., 2001. Pooled exposure-response analyses and risk assessment for lung cancer in 10 cohorts of silica-exposed workers: an IARC multicentre study. Can. Caus. Cont. 12, 773–784. Takayoshi, K., Ogami, A., Yamato, H., Oyabu, T., Mormoto, Y., Tanaka, I., 2007. Effect of particle size of intratracheally instilled crystallina silica on pulmonary inflammation. J. Occup. Health 49, 88–94. Tanino, Y., Makita, H., Miyamoto, K., Betsuyaku, T., Ohtsuka, Y., Nishihira, J., Nishimura, M., 2002. Role of macrophage migration inhibitory factor in bleomycin-induced lung injury and fibrosis in mice. Am. J. Physiol. Lung Cell Mol. Physiol. 283, L156–L162. Tao, F., Gonzalez-Flecha, B., Kobzik, L., 2003. Reactive oxygen species in pulmonary inflammation by ambient particulates. Free Radic. Biol. Med. 35, 327– 340. Thakur, S.A., Beamer, C.A., Migliaccio, C.T., Holian, A., 2009. Critical role of MARCO in crystalline silica–induced pulmonary inflammation. Toxicol. Sci. 108, 462– 471.
24
M.N. Machado et al. / Respiratory Physiology & Neurobiology 196 (2014) 17–24
Wiessner, J.H., Mandel, N.S., Sohnle, P.G., Mandel, G.S., 1989. Effect of particle size on quartz-induced hemolysis and on lung inflammation and fibrosis. Exp. Lung Res. 15, 801–812. Wright, J.L., Harrison, N., Wiggs, B., Churg, A., 1988. Quartz but not iron oxide causes air-flow obstruction, emphysema, and small airways lesions in the rat. Am. Rev. Respir. Dis. 138, 129–135.
Zhang, Y., Lee, T.C., Guillemin, B., Yu, M.C., Rom, W.N., 1993. Enhanced IL-1 beta and tumor necrosis factor-alpha release and messenger RNA expression in macrophages from idiopathic pulmonary fibrosis or after asbestos exposure. J. Immunol. 50, 4188–4196.