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Respiratory Physiology & Neurobiology 196 (2014) 17–24

Contents lists available at ScienceDirect

Respiratory Physiology & Neurobiology

jou rn al h om epa ge: www.elsev ier .com/ locate / resphys io l

ulmonary functional and morphological damage after exposure toripoli dust

ariana Nascimento Machadoa, Aline Cunha Schmidta,aulo Hilário Nascimento Saldivab, Débora Souza Faffea, Walter Araujo Zina,∗

Universidade Federal do Rio de Janeiro, Instituto de Biofísica Carlos Chagas Filho, Rio de Janeiro, BrazilUniversidade de São Paulo, Faculdade de Medicina, São Paulo, Brazil

r t i c l e i n f o

rticle history:eceived 22 November 2013eceived in revised form 13 February 2014ccepted 13 February 2014

eywords:ilicosis

a b s t r a c t

Tripoli is a microcrystalline siliceous rock used to polish metals and precious stones. Its inhalation hasbeen associated with increased prevalence of breathing complaints and pneumoconiosis. However, itsacute human exposure has not been so far studied. We aimed at evaluating the putative mechanical, mor-phological, biochemical and inflammatory lung damage in mice acutely exposed to Tripoli dust. BALB/cmice 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

nflammationranulomaripoli dustung mechanics

of saline i.t. The experiments were done 15 days later. TRIP mice showed higher pulmonary mechanicalimpedance, polymorphonuclear cells, TNF-�, IL1-� and IL-6 than CTRL. TRIP presented granulomatousnodules containing collagenous fibers that occupied 35% of the lung tissue area. In conclusion, acuteexposure to Tripoli dust triggered important lung damage in mice lungs that if found in human workerscould trigger severe illness.

© 2014 Elsevier B.V. All rights reserved.

ntroduction

The chronic inhalation of crystalline silicon dioxide (SiO2) isssociated with the occurrence of silicosis. Despite being one of therstly recognized occupational lung diseases, silicosis remains an

mportant cause of morbidity and mortality worldwide (Martínezt al., 2010). This pneumoconiosis displays persistent inflamma-ion, fibroblast proliferation, and excessive collagen depositionThakur et al., 2009). Furthermore, the cytotoxic effects of silican lung tissue yield macrophage death, subsequent release ofnflammatory cytokines, such as TNF-�, IL-1� and IL-6, and manyther substances. As a net result fibrosis (Piguet et al., 1990; Davist al., 1998; Mossman and Churg, 1998; Srivastava et al., 2002;imal et al., 2005; Hamilton et al., 2008; Sirajuddin and Kanne,

009) and apoptosis (Borges et al., 2002; Srivastava et al., 2002;angley et al., 2010) ensue. The continuous recruitment and acti-ation of macrophages and granulocytes contributes to the chronic

∗ Corresponding author at: Laboratório de Fisiologia da Respirac ão, Instituto deiofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Av Carlos Cha-as Filho 373, CCS, Rm G2-042, Ilha do Fundão, 21941-902 - Rio de Janeiro, RJ, Brazil.el.: +55 21 2562 6557; fax: +55 21 2280 8193.

E-mail addresses: walter zin@hotmail.com, wazin@biof.ufrj.br (W.A. Zin).

ttp://dx.doi.org/10.1016/j.resp.2014.02.007569-9048/© 2014 Elsevier B.V. All rights reserved.

inflammatory process and, thus, to tissue remodeling (Scabilloniet al., 2005; Delgado et al., 2006). As part of the fibrotic processsilicotic nodules or granulomas (Scabilloni et al., 2005) are formed.Increased pulmonary mechanical impedance represents the func-tional 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 sand-stone, granite and silex and presents polymorphisms, the principalnaturally occurring crystalline silica being quartz (Moore, 1999).Cristobalite, tridymite and Tripoli constitute the three other formsof crystalline silica. Tripoli presents unique applications as an abra-sive owing to its hardness and because its grain structure lacksdistinct edges and corners. It is a mild abrasive, making it suitablefor use in toothpaste and tooth polishing compounds, industrialsoaps, metal/jewelry polishing mixtures, resins, ceramics, paints,rubber, and cement (Keller, 1978). Along the processing and use ofTripoli powder the dust generated can be inhaled by human beings,not only workers but the general population as well, and may causean inflammatory lung disease. It should be noted that Tripoli dustcontains more components than SiO2, what could induce a different

harmful outcome. However, no study on the detailed acute func-tional respiratory impairment secondary to exposure to Tripoli dusthas been reported so far either in human beings or in experimentalanimals. Furthermore, no epidemiological work on the prevalence

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r incidence of Tripoli-triggered disease has been published. Inther words, Tripoli powder may be generating more harm thano far recognized by physicians and public health authorities.

The aim of the present study was to describe the physical andhemical characteristics of the Tripoli dust used, and to verifyhether the exposure to Tripoli dust induces lung morphological,

nflammatory and mechanical burdens that could resemble those ofhronic silicosis, thus calling physicians and environmental healthfficers’ attention to the Tripoli issue.

ethods

nimal and experimental protocol

Twenty-one female BALB/c mice (20–25 g) were randomlyivided into 2 groups. In control group (CTRL, n = 6) mice were intra-racheally (i.t.) instilled with 0.05 mL of sterile saline solution (0.9%aCl), whereas Tripoli-administered animals (TRIP, n = 15) received

ntratracheally 20 mg of Tripoli dust vortexed in 0.05 mL of saline,espectively, as previously described in murine models of acuteilicosis (Faffe et al., 2001; Borges et al., 2001, 2002). Before thedministration of the latter suspension, a stock solution of 600 mgf Tripoli was placed in a 2-mL Eppendorf tube and saline solutionas added to reach a final volume of 1.5 mL. The suspension was

ortex-mixed for 10 min and 0.05 mL of it was then collected with ailson precision micropipette (Gilson, Inc., Middleton, WI, USA) and

mmediately given to the animal. The vortexing was repeated forvery TRIP animal. Fifteen days after saline or Tripoli administrationhe animals were analyzed.

article analysis

The dust sample was kindly provided by a gemstone-polishingompany in São Lourenc o, Brazil. It was taken from the same supplyeing at the polishers’ disposal. It was dried in an oven at 50 ◦C untilompletely de-hydrated. Elements were determined by an energyispersive X-ray fluorescence spectrometer (EDX 700HS, Shimadzuorp, Analytical Instruments Division, Kyoto, Japan). AluminumAl), cobalt (Co), iron (Fe), potassium (K), magnesium (Mg), sodiumNa), sulfur (S), silicon (Si), aluminum oxide (Al2O3), calcium oxideCaO), red iron oxide (Fe2O3), potassium oxide (K2O), magnesiumxide (MgO), manganese oxide (MnO), sodium oxide (Na2O), tita-ium dioxide (TiO2) and silicon dioxide (SiO2) were determinednd the results expressed as percent composition (wt %) of par-icles. The trace elements bromine (Br), copper (Cu), germaniumGe), lutecium (Lu), manganese (Mn), nickel (Ni), rubidium (Rb),elenium (Se), tin (Sn), strontium (Sr), titanium (Ti), zinc (Zn), andirconium (Zr) were measured and the results expressed as parti-les per million (ppm) of particles. Three independent samples ofhe particulate matter were analyzed for this purpose.

The distribution of particle sizes, as measured by their volumend surface, and the diameters encompassing 90%, 50% and 10% ofhe particulate matter were determined by laser diffraction (Longench Mastersizer S, Malvern Instruments Ltd, Malvern, Worces-ershire, UK). The particulate matter was visualized by scanninglectron microscopy (JEOL 5310, Tokyo, Japan).

ulmonary mechanics

Fifteen days after saline or Tripoli dust administration, the ani-als were sedated with diazepam (1 mg i.p.) and anesthetized

ith pentobarbital sodium (20 mg kg body weight−1 i.p.), paralyzedith pancuronium bromide (0.1 mg kg body weight−1 i.v.), andechanically ventilated (Samay VR15, Universidad de la Repub-

ica, Montevideo, Uruguay) with a frequency of 100 breaths min−1,

y & Neurobiology 196 (2014) 17–24

tidal volume of 0.2 mL, flow equal to 1 mL s−1, and positive end-expiratory pressure amounting to 2 cmH2O. The anterior chestwall was surgically removed. A pneumotachograph (1.5-mm ID,length = 4.2 cm, distance between side ports = 2.1 cm) was con-nected to the tracheal cannula for the measurement of airflow (V′).Changes in lung volume were obtained by flow signal digital inte-gration. The pressure gradient across the pneumotachograph wasdetermined by means of a Validyne MP45-2 differential pressuretransducer (Engineering Corp, Northridge, CA, USA). Equipmentresistive pressure (=Req.V′) was subtracted from pulmonary resis-tive pressure so that the present results represent intrinsic values.Transpulmonary pressure was measured with a Validyne MP-45differential pressure transducer (Engineering Corp, Northridge,CA, USA). Briefly, we determined lung resistive (�P1) and vis-coelastic/inhomogeneous (�P2) pressures, static elastance (Est),and viscoelastic component of elastance (�E) by the end-inflationocclusion method (Bates et al., 1985). �P1 selectively reflects air-way resistance, and �P2 represents stress relaxation or viscoelasticproperties and mechanical heterogeneities of the lung (Bates et al.,1989; Saldiva et al., 1992). Lung mechanics were measured 10–15times in each animal.

Histological study

Heparin (1000 IU) was intravenously injected immediately afterthe determination of respiratory mechanics. The trachea wasclamped at end expiration, and the abdominal aorta and venacava were sectioned, yielding a massive hemorrhage that quicklyeuthanized the mice. The right lungs were removed en bloc andquick-frozen by immersion in liquid nitrogen and fixed withCarnoy’s solution (Nagase et al., 1992). After fixation, the tissue wasembedded in paraffin. Four-�m-thick slices were cut and stainedwith hematoxylin-eosin or picrosirius red.

Morphometric analysis was performed with an integrating eye-piece 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 deter-mined in each sample by the point-counting technique (Gundersenet al., 1988) across 10 random non-overlapping microscopic fieldsat ×400 magnification. The total amount of points also includedthose falling on tissue, airways and other non-alveolar struc-tures.

The number of mononuclear (MN) and polymorphonuclear(PMN) cells in the pulmonary tissue was counted in each animalacross 10 random non-overlapping microscopic fields at ×1000magnification in a 10,000 �m2 granuloma free area; in the samefield 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 thepoint-counting technique across 20 random non-coincident micro-scopic fields per animal at a magnification of ×200. Percentage oflung tissue occupied by granulomatous nodules was scored as fol-lowing: phase 1, nodules present only in the lung parenchyma;phase 2, nodules around the airways; phase 3, nodules obstructingthe airway; and, phase 4, lung nodules in various structures.

Analysis of cytokines

Samples of lung cytosol were analyzed by ELISA for the detectionof the inflammatory cytokines TNF-�, IL-1�, IL-6 (ELISA kits, R&DSystems 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 frequencyd ).

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Table 1Mechanics, histology and inflammation markers in lung parenchyma.

CTRL TRIP

Mechanics�P1 (cmH2O) 0.40 (0.38–0.50) 0.52 (0.44–0.80)�P2 (cmH2O) 1.22 (1.06–1.48) 1.66 (1.29–2.22)*

Est (cmH2O/mL) 24.04 ± 1.60 33.07 ± 2.64*

�E (cmH2O/mL) 6.17 (5.18–7.46) 8.26 (6.35–11.17)*

HistologyNormal area (%) 70.00 ± 0.80 11.00 ± 1.10*

Alveolar collapse (%) 14.00 ± 0.50 37.00 ± 0.70*

PMN (cel × 10−3/�m2) 4.00 ± 0.01 6.00 ± 0.01*

MN (cel × 10−3/�m2) 11.00 ± 1.00 12.00 ± 1.00

CytokinesTNF-� (pg/mL) 79.98 ± 2.67 115.08 ± 2.41*

IL-1� (pg/mL) 129.71 ± 11.27 609.09 ± 22.98*

IL-6 (pg/mL) 55.59 ± 1.90 81.90 ± 4.87*

Values are mean ± SEM or median (25–75%). Control mice (CTRL, n = 6) and thoseinstilled with 20 mg of Tripoli dust/50 �L saline (TRIP, n = 15); �P1 and �P2, resistiveand viscoelastic/inhomogeneous pressures, respectively; Est, static elastance; �E,viscoelastic component of elastance; PMN and MN, polymorpho- and mononuclear

istribution of particle diameters (columns) and accumulated frequency (solid line

tatistical analysis

SigmaPlot 11 statistical package (Systat Software, Inc., Chicago,L, USA) was used. The normality of the data (Kolmogorov–Smirnovest with Lilliefors’ correction) and the homogeneity of variancesLevene median test) were tested. When both conditions were sat-sfied Student’s t-test was used. Otherwise Mann-Whitney test wasmployed. The significance level was set at 5%.

esults

article analysis

Scanning electron micrographs of particles are shown in Fig. 1A.ripoli dust particles were not spherical in shape, displaying anrregular form and aggregates. Fig. 1B depicts the frequency dis-ribution of particle diameters. Ninety percent of the particlesresented a diameter below 17.28 �m, being 50% below 5.09 �mnd 10% below 1.54 �m. The average sizes of the particles accordingo their volume and surface were 7.57 and 3.47 �m, respec-ively.

Metals and oxides in the Tripoli dust are presented in Fig. 2; aigh concentration of the element Si was found as SiO2.

ulmonary mechanics

TRIP group presented significantly higher lung mechanicalarameters than CTRL, excluding �P1 that was not differentetween the groups (Table 1).

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 intratra-cheally instilled with 20 mg of Tripoli (Panels B–F) are depicted

in Fig. 3. In panel B phase 1 granuloma shows accumulation ofmacrophages containing dust particles, eliciting a slight fibroticresponse (more intense red staining); in panel C (phase 2), the

20 M.N. Machado et al. / Respiratory Physiolog

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 (Al2O3), calcium oxide (CaO), red iron oxide (Fe2O3), potas-sium oxide (K2O), magnesium oxide (MgO), manganese oxide (MnO), sodium oxide(Na2O), titanium dioxide (TiO2) and silicon dioxide (SiO2) were measured and theresults expressed as WT (%) of particles. Trace elements bromine (Br), copper (Cu),germanium (Ge), lutecium (Lu), manganese (Mn), nickel (Ni), rubidium (Rb), sele-nium (Se), tin (Sn), strontium (Sr), titanium (Ti), zinc (Zn), and zirconium (Zr) weremm

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easured and the results expressed as ppm of particles or WT (%). ppm, part perillion; WT, percent composition by weight.

lveoli around the granuloma exhibit an acute inflammatoryesponse and contain dust particles within macrophages; inanel D (phase 3), obliterative bronchiolitis shows a fibrotic

nflammatory polip protruding into the bronchiolar lumen; and inanel E (phase 4), multiple dust nodules and inflammatory reac-ion composed by dust particles, large number of macrophages,arenchymal collapse and fibrosis are present in the gas exchang-

ng territory. Panel F displays lung parenchyma under polarizedight showing the birefringent particles of Tripoli dust as brightpots. The overall area fraction of granulomatous nodules was5.10 ± 0.05% in TRIP group. The percentages of nodules in lung

issue found in phases 1, 2, 3, and 4 were 38.7 ± 16.7%, 20.0 ± 9.7%,.0 ± 6.1%, and 35.4 ± 16.7% (mean ± SD), respectively.

TRIP animals presented increased alveolar collapse (Table 1)ith polymorphonuclear (PMN) and total cells influx in lung

y & Neurobiology 196 (2014) 17–24

parenchyma (Table 1). Spearman correlation was used to evaluatethe association between static elastance and percentage of col-lapsed air spaces. Significant correlation between alveolar collapseand 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 Tripoli-instilled mice than in control group (Table 1).

Discussion

Our results demonstrated that acute exposure to Tripoli dustwas done with particles presenting various shapes with a meanaerodynamic diameter of about 5 �m. Due to their elevated contentof silica and silicon dioxide these particles triggered lung mor-phological changes similar to classical acute silicosis. Intratrachealinstillation of 20 mg of Tripoli induced pulmonary influx of inflam-matory cells, increased release of pro-inflammatory cytokines suchas TNF-�, IL-1� and IL-6, collapsed alveoli, and granulomas withexpression of collagen fibers in the lung parenchyma. As a resultpulmonary mechanical impedance was impaired mainly at tissuelevel, turning the lung stiffer and offering a higher resistance tomovement.

Once inhaled, silica particles are deposited in different partsof the respiratory system, according to their size and initiate thepathological process. Toxicity varies according to particle diameter.In humans toxicity becomes very important when the aerody-namic diameter of the particles is less than 10 �m, which enablesthem to reach the pulmonary alveoli. In rats and mice this valueapproximates 2 �m for intratracheally instilled silica (Wiessneret 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 theadministered Tripoli dust reached the lung periphery. It should bestressed that the fine and ultrafine particles are known as “breath-able” and are able to penetrate the airways, reaching the alveoli(Dusseldorp et al., 1995; Peters et al., 1997; Brown et al., 2002; Taoet al., 2003). Otherwise, they can be eliminated by the mucociliarysystem or, when deposited in the alveolar regions, can be phago-cytosed by macrophages. It is believed that these fine and ultrafineparticles can still penetrate into the lung parenchyma and reachthe bloodstream (Donaldson et al., 2001a, 2001b; Donaldson andStone, 2003).

As depicted in Fig. 2 our particulate matter showed a very highconcentration of the element silicon and silicon dioxide, as well asother elements and oxides. Thus, Tripoli dust is not pure silicondioxide, the silica dust most frequently used to produce exper-imental models of silicosis. Metal containing particles inducedpulmonary changes, reinforcing their potential role in determin-ing particle toxicity (Soukup et al., 2000; Dye et al., 2001; Molinelliet al., 2002). Mazzoli-Rocha et al. (2010) demonstrated that a sin-gle aerosolization of small quantities of particles containing mainlyaluminum induced acute respiratory inflammation, as suggestedby 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 pulmonarymechanics.

Kim et al. (2010) assessed and compared the in vitro toxicity offour different oxide nanoparticles (Al2O3, CeO2, TiO2 and ZnO) to

human lung epithelial cells. Among four tested nanoparticles, ZnOexhibited the highest cytotoxicity in terms of cell proliferation,cell viability, membrane integrity, and colony formation. Al2O3,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 mgof 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 theairways; 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 withp Tripolm s 100 �

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olarized light (panel F) shows scattered interstitial Tripoli particles as bright spots.

icroscopic fields were evaluated per animal at a magnification of ×200. Bar equal

nd cell viability. However, TiO2 induced oxidative stress in aoncentration- and time-dependent manner. Al2O3 seems to beess toxic than the other nanoparticles even after long time expo-ure. These findings suggest the relevance of chemical componentsn the toxicity of Tripoli powder. However, it is not possible toxclude the contribution of non-determined constituents of thearticle composition.

As observed in other murine models of fibrosis, the responses strain-dependent, being BALB/c mice more resistant to silicanstillation (Moore and Hogaboam, 2008). In this context, 2.5 mgf silica in/60 �L of saline were enough to trigger lung inflamma-ion and fibrosis in C57BL/6 mice (Giordano et al., 2010), whereasur animals were exposed to 20 mg of Tripoli dust in 50 �L of salinenstilled directly into the trachea, a dose very close to those reportedy others, who used silicon dioxide in mice and rats (Gross et al.,984; Wright et al., 1988; Borges et al., 2001; Faffe et al., 2001;orges et al., 2002; Lassance et al., 2009). However, considering thatnly circa 50% of our dust presented a diameter less than 5 �m, ourffective dose of PM5 was half theirs. Indeed Tripoli dust presentsarticle diameters larger than those found in commercially avail-ble silicon dioxide (1–5 �m).

A limit exposure to Tripoli dust has not been described yet,ut the permissible exposure limit for dust containing respirablerystalline silica is 0.1 mg/m3 (NIOSH, 2011). The Occupationalafety and Health Administration (OSHA) has determined that thexposure limit of quartz from 0.08 to 0.1 mg/m3 can be used as

basis to limit exposure to the Tripoli particles (NIOSH, 2011).owever, even these levels may not be low enough to preventhronic disease. In this line, Greaves (2000) concluded that the.1 mg/m3 recommended exposure limit of the NIOSH might note sufficiently protective for a substantial proportion of workers.dditionally, Steenland et al. (2001) described that the estimated

xcess lifetime risk (through age 75) of lung cancer for a workerxposed from age 20 to 65 at 0.1 mg/m3 respirable crystalline silicaas 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

i particles can be seen in the nodules in panels B–E. Twenty random non-coincidentm.

workers to a limit exposure of 0.1 mg/m3 of respirable silica in theenvironment. Therefore we used a dose of 20 mg of Tripoli to eval-uate possible pulmonary complications owing to exposure to thisdust.

The time lag between exposure and measurement representsanother factor involved in the response. Although silicosis developsover years in humans exposed to silica dust, experimental stud-ies in BALB/c mice showed that even a single exposure leads tofunctional and histological impairment (Borges et al., 2001; Faffeet al., 2001; Borges et al., 2002). Some studies report the devel-opment of fibrosis within the first month after exposure (Lardotet al., 1998; Borges et al., 2001; Faffe et al., 2001; Lakatos et al.,2006; Borges et al., 2002), or even after longer periods (Barbarinet al., 2005; Giordano et al., 2010). To the best of our knowledge,we report for the first time the acute effects of the exposure toTripoli dust.

Exposure to Tripoli dust resulted in polymorphonuclear cellinflux into the lung parenchyma as previously reported with silica(Faffe et al., 2001). These authors showed progressive histologicalchanges in a murine model of silicosis. We observed that TNF-�,IL-1� and IL-6 levels in the lung tissue increased significantly afteracute exposure to Tripoli (Table 1). In a murine model of silicosis,IL-1� was associated with mononuclear cell-related inflammationand collagen deposition (Zhang et al., 1993). We might advance thatthe cytotoxic effects of Tripoli particles in the lung could be relatedto the following cascade, similar to that of silica: death of alve-olar macrophages, subsequent release of inflammatory cytokines(TNF-�, IL-1� and IL-6 among others), stimulation of fibroblastsactivity, development of granulomas and subsequent pulmonaryfibrosis (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 bronchoalveolarlavage fluid. These mediators seem to be involved mainly in earlydisease, since they precede inflammation and fibrosis (Driscoll

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t al., 1995). However, the alveolar macrophages are related tohe maintenance of silicotic inflammation in a vicious cycle: theyhagocytize the silica particles, are activated and damaged, releas-

ng the silica particle back into the lung environment. Then anotheracrophage engulfs the same particle and perpetuates the process

Greenberg et al., 2007; Gilberti et al., 2008). Despite the impor-ant role of inflammation in fibrogenesis, some evidence suggestshat inflammation is not necessarily related to the fibrotic responsend that additional pathogenic pathways may be responsible forevelopment of the fibrotic response in the lung. Some studieshow that lung inflammation is not always followed by fibroticisease (Adamson et al., 1992; Huaux et al., 1998; Munger et al.,999), whereas other works reveal that control of inflammation isot always associated with reduced fibrosis (Tanino et al., 2002;akamoto et al., 2002).

Chronic exposure to silica particles is histologically character-zed by the presence of granulomas, alveolar septal thickeningnd accumulation of inflammatory cells such as macrophagesDelgado et al., 2006). Ortiz et al. (2001) intratracheally instilled

ice with silica (0.2 g/kg, average of 5 mg of silica per animal,articles presenting an average diameter of 1 �m) and observedranulomas in the lung parenchyma 28 days after the challenge,ut qualitatively these nodules were not as important as thoseresented in our study. Park et al. (2011) evaluated changes in

ung tissue 1, 7, 14 and 28 days after a single intratracheal instil-ation of silica nanoparticles (1 mg/kg) and found inflammatoryell migration to the alveolar space and infiltration in alveolarall. In the present Tripoli-exposed lungs there were granulomas

ontaining large numbers of mononuclear cells, fibroblasts and col-agen fibers arranged in a circular orientation, showing featuresf immature silicotic nodules (Fig. 3). More advanced stages ofissue remodelling display the classical formation of fibrotic nod-les with collagen fibers concentrically aligned, a central hyalineore, and lumping and agglomeration of small silicotic nodulesCastranova and Vallyathan, 2000; Ding et al., 2002; Rimal et al.,005). The fraction area of pulmonary tissue occupied by granu-

omas in the TRIP group was 35.1%. In our experimental modelhese granulomas were present in four phases. Since 80% of the

acrophages die within 12 h after exposure to silica and silicanternalization by macrophages occurs even faster (Gilberti et al.,008), our results may suggest a still active inflammatory pro-ess as indicated by the high percentage of phase 1 granulomasogether with well established ones. This hypothesis is supportedy our findings of elevated levels of pro-inflammatory cytokinesTable 1).

The aforementioned histological and immunologicalhenomena brought about significant changes in mechanicalarameters (Table 1). Our results demonstrated that intratracheal

nstillation of Tripoli dust induced lung injury and increased lungmpedance determined on the 15th day after exposure. In accor-ance with our results Borges et al. (2001) intratracheally instilledALB/c mice with silica dust and reported an elastic componentf pulmonary impedance similar to ours. Control groups in bothtudies presented similar results. We did not find changes in theressure spent against central airway resistance (Table 1); in thisontext, Hnizdo et al. (1994) reported that large airway diseaseas not positively associated with silicosis in 242 miners thatever smoked. Central airway resistance can increase in silicosishen lymph nodes and/or granulomas protrude into or compress

he airways, which seems to be of minor importance in the presenttudy, since we found only 6% phase 3 granulomatous nodules.orges et al. (2001) reported an increased �P1 in mice, but they

id not evaluate the categories of granulomas and found intra-ronchial cellular infiltration obstructing the lumen. We foundhat both elastic and viscoelastic components of lung mechanicsere increased in TRIP mice (Table 1); supporting our results, Faffe

y & Neurobiology 196 (2014) 17–24

et al. (2001) measured lung tissue mechanics in silica-exposedanimals and found a similar result.

Our study presents some limitations: (1) it should be stressedthat a large part of non-respirable particles was detected in theTripoli dust (more than 50% with a diameter above 5 �m). In micemost particles larger than 2 �m would not reach the lung peripheryand could lead to an overload of particles possibly overwhelmingthe physiological clearance mechanisms. To prove this point lungburden analyses are required; (2) we did not study a dose-responsecurve and used a single dosis according with our previous studieson models of silicosis; (3) ours is a study focused on the acute out-comes of the exposure to Tripoli dust and did not address chroniceffects; (4) no animals exposed to pure silicon were included as agroup.

Conclusions

We demonstrated for the first time that exposure to Tripoli dust,a powder different from pure silicon dioxide and commonly used inindustry and households, acutely damaged mice lungs, as suggestedby an increased presence of inflammatory cells and mediators inthe lung, collapse of airspaces, and remodeling consisting of gran-uloma formation and collagen fibers deposition. Altogether theseresponses impaired pulmonary elastic and viscoelastic mechanicalproperties. The disease profile is similar to that of chronic silicosis,but it should be stressed that Tripoli powder can be more frequentlyfound 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 Car-los de Souza Quaresma and Mr. João Luiz Coelho Rosas Alves fortheir skillful technical assistance and to Julyana Costa Vieira for herparticipation in gathering respiratory mechanics data.

This study was supported by: The Centers of Excellence Pro-gram (PRONEX-MCT/FAPERJ), The Brazilian Council for Scientificand Technological Development (CNPq/MCT), The Carlos ChagasFilho 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 Useof Animals, Health Sciences Center, Federal University of Rio deJaneiro (protocol no. IBCCF 046). All animals received humane carein compliance with the “Principles of Laboratory Animal Care” for-mulated by the National Society for Medical Research and with the“Guide for the Care and Use of Laboratory Animals” prepared by theNational Academy of Sciences, USA.

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