RESEARCH Open Access Role Of Metal Oxide Nanoparticles In .
Andujar et al. Particle and Fibre Toxicology 2014, tent/11/1/23RESEARCHOpen AccessRole of metal oxide nanoparticles inhistopathological changes observed in the lungof weldersPascal Andujar1,2,3†, Angélique Simon-Deckers2,4†, Françoise Galateau-Sallé5,6, Barbara Fayard4, Gregory Beaune7,8,9,Bénédicte Clin6,10, Marie-Annick Billon-Galland11, Olivier Durupthy7,8,9, Jean-Claude Pairon1,2,3, Jean Doucet4,Jorge Boczkowski2,3,12 and Sophie Lanone1,2,3*AbstractBackground: Although major concerns exist regarding the potential consequences of human exposure tonanoparticles (NP), no human toxicological data is currently available. To address this issue, we took welders, whopresent various adverse respiratory outcomes, as a model population of occupational exposure to NP.The aim of this study was to evaluate if welding fume-issued NP could be responsible, at least partially, in the lungalterations observed in welders.Methods: A combination of imaging and material science techniques including ((scanning) transmission electronmicroscopy ((S)TEM), energy dispersive X-ray (EDX), and X-ray microfluorescence (μXRF)), was used to characterizeNP content in lung tissue from 21 welders and 21 matched control patients. Representative NP were synthesized,and their effects on macrophage inflammatory secretome and migration were evaluated, together with the effectof this macrophage inflammatory secretome on human lung primary fibroblasts differentiation.Results: Welding-related NP (Fe, Mn, Cr oxides essentially) were identified in lung tissue sections from welders, inmacrophages present in the alveolar lumen and in fibrous regions. In vitro macrophage exposure to representativeNP (Fe2O3, Fe3O4, MnFe2O4 and CrOOH) induced the production of a pro-inflammatory secretome (increasedproduction of CXCL-8, IL-1ß, TNF-α, CCL-2, 3, 4, and to a lesser extent IL-6, CCL-7 and 22), and all but Fe3O4 NPinduce an increased migration of macrophages (Boyden chamber). There was no effect of NP-exposed macrophagesecretome on human primary lung fibroblasts differentiation.Conclusions: Altogether, the data reported here strongly suggest that welding-related NP could be responsible, atleast in part, for the pulmonary inflammation observed in welders. These results provide therefore the first evidenceof a link between human exposure to NP and long-term pulmonary effects.Keywords: Welding, Occupational exposure, Metal oxide nanoparticle, Inflammation, LungBackgroundNanotechnologies represent a major stake of the 21rst century. They are aimed at conceiving, characterizing and producing materials at the scale of the billionth meter; thenanometer. These nanomaterials (among which are particleswith a diameter lesser than 100 nm - nanoparticles, NP)* Correspondence: Sophie.lanone@inserm.fr†Equal contributors1Centre Hospitalier Intercommunal de Créteil, Service de Pneumologie et dePathologie Professionnelle, 94000 Créteil, France2INSERM, U955, Equipe 4, 94000 Créteil, FranceFull list of author information is available at the end of the articlepresent new properties that make them particularly interesting to industrialists. The list of actual uses and applicationsfor nanomaterials is already substantial, and will certainlybecome exponential in the next future. Such developments,in spite of potential benefits in numerous domains, i.e. inthe field of nanomedicine, are accompanied by concernsregarding the potential consequences of human exposure(in the general population or in an occupational context),with particular threat at the respiratory level [1-3]. Theneed for toxicity evaluation is motivated by numerousstudies already available in the literature demonstrating 2014 Andujar et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the CreativeCommons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, andreproduction in any medium, provided the original work is properly credited. The Creative Commons Public DomainDedication waiver ) applies to the data made available in this article,unless otherwise stated.
Andujar et al. Particle and Fibre Toxicology 2014, tent/11/1/23that animal exposure to engineered NP is followed, amongother pathological manifestations, by the induction of a pulmonary inflammation, associated with lung remodelling[4-8]. However, so far, there is almost no study assessingthe consequences of human exposure to manufactured NP.This point is critical since human exposure studies constitute the cornerstone of risk evaluation for health after(chronic) exposure to environmental agents, especiallyin the occupational context.During their everyday occupation, welders are exposed toa complex aerosol of gases (e.g. carbon monoxide, ozone),and hazardous metal fumes composed of chain-likeagglomerates of particles, with a primary size in thenanometer size range [9]. Indeed, up to 11% of the totalmass, and 80% of the total number of particles emitted inwelding fumes are NP [10,11]. Various adverse respiratoryoutcomes have been described in welders, among whichinflammation and lung remodeling are largely described[12-16]. However, the underlying responsible mechanismsremain to be elucidated.We therefore made the hypothesis that these abnormalities could result, at least partly, from an inflammatory effect directly induced by welding fume-issued NPpresent in the pulmonary tissue. Such hypothesis hasnever been studied in the literature so far, probablydue to the technical challenges related to the analysisand characterization of NP tissular content in biologicalsamples in situ. We therefore setup an original interdisciplinary study, combining the expertise of material sciencephysicists, physico-chemists, biologists and medical doctors.A broad range of imaging and material science techniques(scanning/transmission electron microscopy (S/TEM), energy dispersive X-ray (EDX), and X-ray microfluorescence(μXRF)), was used to characterize the association betweenlung histological modifications and the presence of NP in21 well-documented arc welders and matched unexposedindividuals. Since this analysis revealed the presence of NPin macrophages in the alveolar lumen and in areas of fibrous tissue in welders, our physico-chemist partner chemically synthesized NP representative of those identified inpulmonary tissue (Fe2O3, Fe3O4, MnFe2O4 and CrOOH),to investigate their effects (in combination or not withexposure to cigarette smoke, as the majority of patientswere smokers) on human macrophages. These exposureswere performed in combination or not with exposure tocigarette smoke, as the majority of patients were smokers.Our results show that exposure to welding-related NPinduce the production of a pro-inflammatory secretrome (increased production of CXCL-8, IL-1ß, TNF-α,CCL-2, 3, 4, and to a lesser extent IL-6, CCL-7 and 22).Moreover, all but Fe3O4 NP induce an increased migrationof macrophages.Finally, there was no effect of macrophagepro-inflammatory secretome on human primary lung fibroblasts differentiation. These results provide therefore thePage 2 of 13first evidence of a link between human exposure to NPand long-term pulmonary effects.ResultsClinical and histological characterization of patientsThe clinical characteristics of the 42 patients included inour study are given in Table 1. Consistent with the population studied, female were significantly less representedin the welder group as compared to controls (p 0.038).Otherwise, consistent with the selective matching of controls with welders, the two populations were similar forage and tobacco history.CD68 staining revealed the presence of a higher number of macrophages in lung tissue sections from weldersas compared to control patients, localized in the alveolarlumen as well as in the fibrous tissue (Figure 1). Importantly, black spots corresponding to aggregates of particleswere essentially colocalized with CD68 staining suggestinga macrophagic internalization of these aggregates. Theiron load, as revealed by Perls staining (Figure 1) and thequantification of siderophages (iron-laden macrophages)and ferruginous bodies (formed by macrophages) (Table 2),were significantly higher in welders as compared to control patients. Finally, lung fibrotic lesions were identifiedTable 1 Clinicopathological characteristics of control andwelder patients.Mean age (years SD)Controls(n 21)Welders(n 21)P value58.6 10.858.5 10.7nsGenderFemale6 (28.6%)1 (4.8%)0.038Male15 (71.4%)20 (95.2%)nsTobacco smokingSmoking statusnsCurrent smokers9 (43.0%)6 (28.6%)Former smokers9 (43.0%)12 (57.1%)Never smokersCumulative consumption (P-Y SD)3 (14.0%)3 (14.3%)24.6 29.022.2 15.2nsAge at onset (years SD)16.8 3.418.3 4.2nsAge at cessation (years SD)51.6 14.048.8 11.2nsDuration (years SD)32.6 16.733.7 10.3ns5.7 9.38.2 10.4nsPositive occupational questionnaire021Age at onset (years SD)-19.9 7.6Age at cessation (years SD)-48.2 9.9Duration (years SD)-27.0 9.2Duration since cessation (years SD)-9.2 10.4Duration since cessation (years SD)Welding fumes exposurens: not significant; Max: maximum; Min: minimum; P-Y: packs-years; SD:standard deviation.-
Andujar et al. Particle and Fibre Toxicology 2014, tent/11/1/23Page 3 of 13Figure 1 Histological observation of lung tissue sections. Representative optical microscopy images of lung tissue sections from a Controland a Welder. Serial sections were stained with hematoxylin-eosin-saffron (HES), CD68 or Perls. Scale bar: 100 μm. Inset: higher magnification ofthe welder sample.in both control and welders, but were significantly moresevere in welders as compared to control patients irrespective of the histological localization (Figure 1 and Table 2).There was no difference for other respiratory lesionssuch as respiratory bronchiolectasis, bronchiolar dysplasia or metaplasia, or bronchiolitis between the twogroups (Table 2).Patients’ particle burden characterizationElemental mapping in lung tissue sectionsWe first wanted to get a general overview of the elementalmapping at the global level of the tissue. To that matter, weused synchrotron-based X-ray microfluorescence (μXRF)and determined the spatial distribution of elements performing 100 100 μm maps. Figure 2A shows typicalelemental μXRF maps obtained for sulfur (S), iron (Fe),and manganese (Mn) in control patients and welders.Since S represents a characteristic element of biologicalmaterials, the shape of the tissue can be drawn by the Smap [17]. Using this approach, we observed a significantoverload of Fe, Mn, chromium (Cr) essentially and to alesser extent for titanium (Ti) in welders as compared tocontrol patients (Figure 2B). This signal was essentiallylocalized in the fibrous tissue (Figure 2), and to a lesserextent in the alveolar lumen (data not shown). Remarkably, we could observe that iron-rich zones were oftenco-localized with other element-rich zones, particularlyMn and Cr (Figure 2 and data not shown).NP identification in lung tissue homogenatesTo determine individual exposure to asbestos bodies andnon fibrous mineral particles, a mineralogical analysis oflung tissue homogenates was performed. These experiments confirmed the metallic overload in welder’s lungand identified a significantly higher mass concentration oftotal mineral and especially metallic particles in welders ascompared to control patients (Table 3). Consistent withthe initial selective matching of controls with welders,the two populations were similar for asbestos exposurecharacteristics. Finally, in order to characterize the sizeand chemical nature of the particles present in thepulmonary samples, we used a combined STEM-EDX(Scanning Transmission Electron Microscopy (STEM)Energy Dispersive X-Ray (EDX)) analysis of lung tissuehomogenates, and demonstrated that the particles are ofnanometric size (20–25 nm individual diameter - STEManalysis Figure 3A), and confirmed the chemical nature(Fe, Mn, Cr essentially) and the co-localization of thechemical signals (EDX analysis, Figure 3B).NP identification in lung tissue sectionsFollowing the detection of NP in the lung tissue homogenates, we questioned the identification of these NP in situat the nanometer scale and characterized their localizationin lung tissue sections from patients, especially in areas oflung remodeling. We therefore proceeded to a combinedTEM-EDX analysis of lung tissue sections and observed
Andujar et al. Particle and Fibre Toxicology 2014, tent/11/1/23Page 4 of 13Table 2 Histological analysis of lung tissue in control andwelder patientsControls(n 21)Welders(n 21)P valueSynthesis of representative NPSiderophagesPresence (n patients)10170.0240.71 0.901.57 1.170.0196160.0020.43 0.811.81 8Paraseptal810nsAlveolar35nsDiffuse interstitial65nsPleural42ns2.29 1.593.91 1.510.003Number (Mean semi-quantitativescore SD)bFerruginous bodies (n patients)Presence (n patients)Number (Mean semi-quantitativescore SD)bFibrotic lesions(Number of patients)Severity of fibrosisa(Mean semi-quantitative score SD)GlobalMn NP in situ in lung tissue from welders, especiallyin macrophages.Peribronchiolar1.10 0.771.86 0.850.005Perivascular1.05 0.921.81 0.810.010Alveolar and peribronchiolar1.57 1.122.67 1.160.004Bronchiolectasis1613nsBronchiolar metaplasia20nsOther respiratory lesionsBronchiolar dysplasia15nsRespiratory Bronchiolitis – IntertitialLung Disease1010nsDesquamative intertitial pneumonitis129nsEndogenous Lipoid Pneumonitis13nsns: not significant; SD: standard deviation.aSemi-quantitative score [4 classes 0: absent; 1: rare; 2: frequent; 3:important]. Adapted from Roggli classification [54].bSemi-quantitative score [5 classes 0: 0/slide; 1: 5/slide; 2: 5 to 10/slide; 3:10 to 50/slide ; 4: 50/slide].that, in comparison with control patients, large quantities ofNP were localized in macrophages residues, and was presentin the fibrous tissue of welders (Figure 4). In accordancewith the results obtained in lung tissue homogenates, theseNP presented a spherical shape, and a diameter centeredaround 20–25 nm. The chemical nature of the complexmetal oxide NP in the welders lungs were mainly Fe, Crand/or Mn (and aluminium (Al), silicium (Si), and nickel(Ni) to a lesser extent), whereas it was essentially Al andSi NP in the control patients (data not shown).Overall, these different material science techniquesallow us to demonstrate the presence of Fe, Cr and/orIn order to answer the question whether the metal NPidentified above have a causal role in the development ofpulmonary alterations observed in welders, we next chemically synthesized 4 NP representative of those found inlung tissue samples; Fe2O3, Fe3O4, MnFe2O4 and CrOOH.As shown in Figure 5, all NP were spherical and presentedan average diameter of 25 nm (Fe2O3, Fe3O4, MnFe2O4)or 15 nm (CrOOH) (Figure 5A-D). The NP nature wasverified by X-ray diffraction spectra (Figure 5E-H).The in vitro effect of the synthesized NP on themacrophage secretomeBecause NP identified in lungs from welders were inmacrophages, we first evaluated the effects of humanmacrophage exposure to the chemically synthesizedrepresentative NP in terms of inflammatory secretome.These experiments revealed the secretion of significantlyincreased amounts of IL-1ß, TNF-α, CCL-2, 3, 4,and lesser levels of IL-6, CCL-7 and 22 in macrophagesexposed to 25 μg/cm2 (a concentration representative ofoccupational exposure [18]) of all but Fe3O4 NP as compared to unexposed cells (Figure 6). Moreover, CXCL-8production was also significantly higher in macrophagesexposed to all but Fe3O4 NP at the dose of 5 μg/cm2, andto all NP when used at 25 μg/cm2 (Figure 6). No modification of the secretion of IL-4, 5, 10, 12, 13, PDGFAA, PDGF-BB, or TGF-ß was detected, whatever the NPutilized (data not shown). MMP-1 and MMP-7 levels incell culture supernatants were significantly increased inMnFe2O4-treated macrophages as compared to unexposed cells (25 μg/cm2, Additional file 1: Figure S1).Moreover, exposure to Fe2O3 NP at 25 μg/cm2 induced anincreased production of MMP-7 and TIMP-2 as comparedto unexposed cells (Additional file 1: Figure S1). Finally, nomodification of the expression of MMP-2, 9, 10, TIMP1, 3, and 4 was observed irrespective of NP exposure(data not shown).Since welding fumes contain a mixture of NP, we nextquestioned whether a mix of the most representative NPcould have a synergistic effect on the macrophage secretome. These experiments showed that exposure of macrophages to the (1:1:1:1) combination of Fe2O3, Fe3O4,MnFe2O4 and CrOOH NP didn’t produce any synergistic effect in terms of cytokine or MMP/TIMP secretion(Figure 6 and Additional file 1: Figure S1). Finally, asthe majority of the welders were smokers, we analyzedif the combined exposure to NP and cigarette smokecould have a synergistic pro-inflammatory effects. Theseexposures did not demonstrate any synergistic effect of
Andujar et al. Particle and Fibre Toxicology 2014, tent/11/1/23Page 5 of 13Figure 2 Elemental mapping of lung tissue sections. A: Representative elemental map obtained by X-ray micro-fluorescence (μXRF) in aControl and a Welder. Signal of sulphur: green. Signal of iron: blue. Signal of manganese: red. Scale bar: 100 μm. For each element, fluorescenceintensities are proportional to the total amount of element. B: Quantification of the signal obtained for each element, given as a ratio to that ofsulphur. *: p 0.05 vs Control, **: p 0.01 vs Control, n 6-8 per group. Open circles: Controls (grey horizontal bar: mean value). Black squares:Welders (black horizontal bar: mean value).cigarette smoke in macrophages response to NP exposure(Additional file 2: Figure S2 and Additional file 3: Figure S3).Effect of macrophage secretome on macrophage migrationFollowing the identification of the increased number ofmacrophages in welders lung, the consequence of anautocrine effect of macrophage secretome on the migration capacity of these cells was investigated. Concurrentlywith our previous data, the secretome obtained from allexposures with the exception of the Fe3O4 NP-exposedmacrophages was able to activate macrophage migration(Additional file 4: Figure S4).Expression of pro-inflammatory cytokines in lungtissue sectionsTo further scrutinize the clinical relevance of the macrophage secretome induction by NP exposure, a series ofimmuno-histochemistry experiments were performedin lung tissue sections from control and welders, for asubset of cytokines that were highly produced by NPexposed macrophages. As shown in Figure 7 and quantified in Table 4, the expression of IL-1ß, TNF-α andCCL-3 was significantly higher in lung sections fromwelders as compared to controls, whereas no differencewas observed for CCL-2 expression. For the 3 positive
Andujar et al. Particle and Fibre Toxicology 2014, tent/11/1/23Page 6 of 13Table 3 Mineralogical analysis and asbestos exposure quantification of lung tissue homogenates in control andwelder patientsControls(n 21)Welders(n 21)P value24.8 7.621.4 8.0nsTotal70.2 84.3155.9 148.80.012Metallic17.3 22.773.9 66.00.0005Non metallic48.8 61.180.9 105.1ns88Dry lung tissue weight (mg) (Mean SD)7Mineral particle concentration in lung tissue (number of particles x 10 per g; Mean SD)Asbestos exposurePositive occupational questionnaireAB/g dry lung tissue (median)[Min–Max] 1000 AB/g dry lung tissue sAB: asbestos bodies; SD: standard deviation.cytokines, and in accord
RESEARCH Open Access Role of metal oxide nanoparticles in histopathological changes observed in the lung of welders Pascal Andujar1,2,3†, Angélique Simon-Deckers2,4†, Françoise Galateau-Sallé5,6, Barbara Fayard4, Gregory Beaune7,8,9, Bénédicte Clin6,10, Marie-Annick Billon-Galland11, Olivier Durupthy7,8,9, Jean-Claude Pairon1,2,3, Jean Doucet4,
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