IL-1α induces CD11b(low) alveolar macrophage proliferation and maturation during granuloma formation.

Macrophages play a central role in immune and tissue responses of granulomatous lung diseases induced by pathogens and foreign bodies. Circulating monocytes are generally viewed as central precursors of these tissue effector macrophages. Here, we provide evidence that granulomas derive from alveolar macrophages serving as a local reservoir for the expansion of activated phagocytic macrophages. By exploring lung granulomatous responses to silica particles in IL-1-deficient mice, we found that the absence of IL-1α, but not IL-1ß, was associated with reduced CD11b(high) phagocytic macrophage accumulation and fewer granulomas. This defect was associated with impaired alveolar clearance and resulted in the development of pulmonary alveolar proteinosis (PAP). Reconstitution of IL-1α(-/-) mice with recombinant IL-1α restored lung clearance functions and the pulmonary accumulation of CD11b(high) phagocytic macrophages. Mechanistically, IL-1α induced the proliferation of CD11b(low) alveolar macrophages and differentiated these cells into CD11b(high) macrophages which perform critical phagocytic functions and organize granuloma. We newly discovered here that IL-1α triggers lung responses requiring macrophage proliferation and maturation from tissue-resident macrophages.

The complex cascade of cellular events governing inflammasome activation and IL-1ß processing in response to inhaled particles.

The innate immune system is the first line of defense against inhaled particles. Macrophages serve important roles in particle clearance and inflammatory reactions. Following recognition and internalization by phagocytes, particles are taken up in vesicular phagolysosomes. Intracellular phagosomal leakage, redox unbalance and ionic movements induced by toxic particles result in pro-IL-1ß expression, inflammasome complex engagement, caspase-1 activation, pro-IL-1ß cleavage, biologically-active IL-1ß release and finally inflammatory cell death termed pyroptosis. In this review, we summarize the emerging signals and pathways involved in the expression, maturation and secretion of IL-1ß during these responses to particles. We also highlight physicochemical characteristics of particles (size, surface and shape) which determine their capacity to induce inflammasome activation and IL-1ß processing.

Critical role of aquaporins in interleukin 1ß (IL-1ß)-induced inflammation.

Rapid changes in cell volume characterize macrophage activation, but the role of water channels in inflammation remains unclear. We show here that, in vitro, aquaporin (AQP) blockade or deficiency results in reduced IL-1ß release by macrophages activated with a variety of NLRP3 activators. Inhibition of AQP specifically during the regulatory volume decrease process is sufficient to limit IL-1ß release by macrophages through the NLRP3 inflammasome axis. The immune-related activity of AQP was confirmed in vivo in a model of acute lung inflammation induced by crystals. AQP1 deficiency is associated with a marked reduction of both lung IL-1ß release and neutrophilic inflammation. We conclude that AQP-mediated water transport in macrophages constitutes a general danger signal required for NLRP3-related inflammation. Our findings reveal a new function of AQP in the inflammatory process and suggest a novel therapeutic target for anti-inflammatory therapy.

Why does the hemolytic activity of silica predict its pro-inflammatory activity?

BACKGROUND: The hemolytic activity of inhaled particles such as silica has been widely investigated in the past and represents a usual toxicological endpoint to characterize particle reactivity despite the fact that red blood cells (RBCs) are not involved in the pathogenesis of pulmonary inflammation or fibrosis caused by some inhaled particles. The inflammatory process induced by silica starts with the activation of the inflammasome, which leads to the release of mature IL-1ß. One of the upstream mechanisms causing activation of the inflammasome is the labilization of the phagolysosomal membrane after particle phagocytosis. Considering RBC lysis as a model of membrane damage, we evaluated the relationship between hemolytic activity and inflammasome-dependent release of IL-1ß for a panel of selected silica particles, in search of the toxicological significance of the hemolytic activity of an inhaled particle. METHODS: Well-characterized silica particles, including four quartz samples and a vitreous silica, with different surface properties and hemolytic potential were tested for their capacity to induce inflammasome-dependent release of IL-1ß in LPS-primed primary murine peritoneal macrophages by ELISA and Western blot analysis. The mechanisms of IL-1ß maturation and release were clarified by using ASC-deficient cells and inhibitors of phagocytosis and cathepsin B. RESULTS: The silica samples induced dose-dependent hemolysis and IL-1ß release of different amplitudes. A significant correlation between IL-1ß release and hemolytic activity was evidenced (r = 0.827) by linear regression analysis. IL-1ß release was completely abolished in ASC-deficient cells and reduced by inhibitors, confirming the involvement of the inflammasome and the requirement of phagocytosis and cathepsin B for activation. CONCLUSIONS: The same physico-chemical properties of silica particles which are relevant for the lysis of the RBC membrane also appear implicated in the labilization of the phagolysosome, leading to inflammasome activation and release of the pro-inflammatory cytokine IL-1ß. These findings strengthen the relevance of the hemolysis assay to predict the pro-inflammatory activity of silica dusts.

The alarmin IL-1α is a master cytokine in acute lung inflammation induced by silica micro- and nanoparticles.

BACKGROUND: Inflammasome-activated IL-1ß plays a major role in lung neutrophilic inflammation induced by inhaled silica. However, the exact mechanisms that contribute to the initial production of precursor IL-1ß (pro-IL-1ß) are still unclear. Here, we assessed the implication of alarmins (IL-1α, IL-33 and HMGB1) in the lung response to silica particles and found that IL-1α is a master cytokine that regulates IL-1ß expression. METHODS: Pro- and mature IL-1ß as well as alarmins were assessed by ELISA, Western Blot or qRT-PCR in macrophage cultures and in mouse lung following nano- and micrometric silica exposure. Implication of these immune mediators in the establishment of lung inflammatory responses to silica was investigated in knock-out mice or after antibody blockade by evaluating pulmonary neutrophil counts, CXCR2 expression and degree of histological injury. RESULTS: We found that the early release of IL-1α and IL-33, but not HMGB1 in alveolar space preceded the lung expression of pro-IL-1ß and neutrophilic inflammation in silica-treated mice. In vitro, the production of pro-IL-1ß by alveolar macrophages was significantly induced by recombinant IL-1α but not by IL-33. Neutralization or deletion of IL-1α reduced IL-1ß production and neutrophil accumulation after silica in mice. Finally, IL-1α released by J774 macrophages after in vitro exposure to a range of micro- and nanoparticles of silica was correlated with the degree of lung inflammation induced in vivo by these particles. CONCLUSIONS: We demonstrated that in response to silica exposure, IL-1α is rapidly released from pre-existing stocks in alveolar macrophages and promotes subsequent lung inflammation through the stimulation of IL-1ß production. Moreover, we demonstrated that in vitro IL-1α release from macrophages can be used to predict the acute inflammogenic activity of silica micro- and nanoparticles.

In search of the chemical basis of the hemolytic potential of silicas.

The membranolytic activity of silica particles toward red blood cells (RBCs) has been known for a long time and is sometimes associated with silica pathogenicity. However, the molecular mechanism and the reasons why hemolysis differs according to the silica form are still obscure. A panel of 15 crystalline (pure and commercial) and amorphous (pyrogenic, precipitated from aqueous solutions, vitreous) silica samples differing in size, origin, morphology, and surface chemical composition were selected and specifically prepared. Silica particles were grouped into six groups to compare their potential in disrupting RBC membranes so that one single property differed in each group, while other features were constant. Free radical production and crystallinity were not strict determinants of hemolytic activity. Particle curvature and morphology modulated the hemolytic effect, but silanols and siloxane bridges at the surface were the main actors. Hemolysis was unrelated to the overall concentration of silanols as fully rehydrated surfaces (such as those obtained from aqueous solution) were inert, and one pyrogenic silica also lost its membranolytic potential upon progressive dehydration. Overall results are consistent with a model whereby hemolysis is determined by a defined surface distribution of dissociated/undissociated silanols and siloxane groups strongly interacting with specific epitopes on the RBC membrane.

Oxidative stress induced by pure and iron-doped amorphous silica nanoparticles in subtoxic conditions.

Amorphous silica nanoparticles (SiO2-NPs) have found broad applications in industry and are currently intensively studied for potential uses in medical and biomedical fields. Several studies have reported cytotoxic and inflammatory responses induced by SiO2-NPs in different cell types. The present study was designed to examine the association of oxidative stress markers with SiO2-NP induced cytotoxicity in human endothelial cells. We used pure monodisperse amorphous silica nanoparticles of two sizes (16 and 60 nm; S16 and S60) and a positive control, iron-doped nanosilica (16 nm; SFe), to study the generation of hydroxyl radicals (HO·) in cellular-free conditions and oxidative stress in cellular systems. We investigated whether SiO2-NPs could influence intracellular reduced glutathione (GSH) and oxidized glutathione (GSSG) levels, increase lipid peroxidation (malondialdehyde (MDA) and 4-hydroxyalkenal (HAE) concentrations), and up-regulate heme oxygenase-1 (HO-1) mRNA expression in the studied cells. None of the particles, except SFe, produced ROS in cell-free systems. We found significant modifications for all parameters in cells treated with SFe nanoparticles. At cytotoxic doses of S16 (40-50 µg/mL), we detected weak alterations of intracellular glutathione (4 h) and a marked induction of HO-1 mRNA (6 h). Cytotoxic doses of S60 elicited similar responses. Preincubation of cells being exposed to SiO2-NPs with an antioxidant (5 mM N-acetylcysteine, NAC) significantly reduced the cytotoxic activity of S16 and SFe (when exposed up to 25 and 50 µg/mL, respectively) but did not protect cells treated with S60. Preincubation with NAC significantly reduced HO-1 mRNA expression in cells treated with SFe but did not have any effect on HO-1 mRNA level in cell exposed to S16 and S60. Our study demonstrates that the chemical composition of the silica nanoparticles is a dominant factor in inducing oxidative stress.

Model system to study the influence of aggregation on the hemolytic potential of silica nanoparticles.

A well-defined silica nanoparticle model system was developed to study the effect of the size and structure of aggregates on their membranolytic activity. The aggregates were stable and characterized using transmission electron microscopy, dynamic light scattering, nitrogen adsorption, small-angle X-ray scattering, infrared spectroscopy, and electron paramagnetic resonance. Human red blood cells were used for assessing the membranolytic activity of aggregates. We found a decreasing hemolytic activity for increasing hydrodynamic diameter of the nanoparticle aggregates, in contrast to trends observed for isolated particles. We propose here a qualitative model that considers the fractal structure of the aggregates and its influence on membrane deformation to explain these observations. The open structure of the aggregates means that only a limited number of primary particles, from which the aggregates are built up, are in contact with the cell membrane. The adhesion energy is thus expected to decrease resulting in an overall lowered driving force for membrane deformation. Hence, the hemolytic activity of aggregates, following an excessive deformation of the cell membrane, decreases as the aggregate size increases. Our results indicate that the aggregate size and structure determine the hemolytic activity of silica nanoparticle aggregates.

Restoration of T-cell Effector Function, Depletion of Tregs, and Direct Killing of Tumor Cells: The Multiple Mechanisms of Action of a-TIGIT Antagonist Antibodies.

TIGIT is an immune checkpoint inhibitor expressed by effector CD4+ and CD8+ T cells, NK cells, and regulatory T cells (Tregs). Inhibition of TIGIT-ligand binding using antagonistic anti-TIGIT mAbs has shown in vitro potential to restore T-cell function and therapeutic efficacy in murine tumor models when combined with an anti-PD(L)-1 antibody. In the current work, we demonstrate broader TIGIT expression than previously reported in healthy donors and patients with cancer with expression on γδ T cells, particularly in CMV-seropositive donors, and on tumor cells from hematologic malignancies. Quantification of TIGIT density revealed tumor-infiltrating Tregs as the population expressing the highest receptor density. Consequently, the therapeutic potential of anti-TIGIT mAbs might be wider than the previously described anti-PD(L)-1-like restoration of αß T-cell function. CD155 also mediated inhibition of γδ T cells, an immune population not previously described to be sensitive to TIGIT inhibition, which could be fully prevented via use of an antagonistic anti-TIGIT mAb (EOS-448). In PBMCs from patients with cancer, as well as in tumor-infiltrating lymphocytes from mice, the higher TIGIT expression in Tregs correlated with strong antibody-dependent killing and preferential depletion of this highly immunosuppressive population. Accordingly, the ADCC/ADCP-enabling format of the anti-TIGIT mAb had superior antitumor activity, which was dependent upon Fcγ receptor engagement. In addition, the anti-TIGIT mAb was able to induce direct killing of TIGIT-expressing tumor cells both in human patient material and in animal models, providing strong rationale for therapeutic intervention in hematologic malignancies. These findings reveal multiple therapeutic opportunities for anti-TIGIT mAbs in cancer therapeutics.

Synthesis and characterization of stable monodisperse silica nanoparticle sols for in vitro cytotoxicity testing.

For the investigation of the interaction of nanoparticles with biomolecules, cells, organs, and animal models there is a need for well-characterized nanoparticle suspensions. In this paper we report the preparation of monodisperse dense amorphous silica nanoparticles (SNP) suspended in physiological media that are sterile and sufficiently stable against aggregation. SNP sols with various particle sizes (2-335 nm) were prepared via base-catalyzed hydrolysis and polymerization of tetraethyl orthosilicate under sterile conditions using either ammonia (Stober process (1) ) or lysine catalyst (Lys-Sil process (2) ). The series was complemented with commercial silica sols (Ludox). Silica nanoparticle suspensions were purified by dialysis and dispersed without using any dispersing agent into cell culture media (Dulbecco's Modified Eagle's medium) containing antibiotics. Particle sizes were determined by dynamic light scattering. SNP morphology, surface area, and porosity were characterized using electron microscopy and nitrogen adsorption. The SNP sols in cell culture medium were stable for several days. The catalytic activity of the SNP in the conversion of hydrogen peroxide into hydroxyl radicals was investigated using electron paramagnetic resonance. The catalytic activity per square meter of exposed silica surface area was found to be independent of particle size and preparation method. Using this unique series of nanoparticle suspensions, the relationship between cytotoxicity and particle size was investigated using human endothelial and mouse monocyte-macrophage cells. The cytotoxicity of the SNP was strongly dependent on particle size and cell type. This unique methodology and the collection of well-characterized SNP will be useful for further in vitro studies exploring the physicochemical determinants of nanoparticle toxicity.

Size-dependent cytotoxicity of monodisperse silica nanoparticles in human endothelial cells.

The effect that monodisperse amorphous spherical silica particles of different sizes have on the viability of endothelial cells (EAHY926 cell line) is investigated. The results indicate that exposure to silica nanoparticles causes cytotoxic damage (as indicated by lactate dehydrogenase (LDH) release) and a decrease in cell survival (as determined by the tetrazolium reduction, MTT, assay) in the EAHY926 cell line in a dose-related manner. Concentrations leading to a 50% reduction in cell viability (TC(50)) for the smallest particles tested (14-, 15-, and 16-nm diameter) ranging from 33 to 47 microg cm(-2) of cell culture differ significantly from values assessed for the bigger nanoparticles: 89 and 254 microg cm(-2) (diameter of 19 and 60 nm, respectively). Two fine silica particles with diameters of 104 and 335 nm show very low cytotoxic response compared to nanometer-sized particles with TC(50) values of 1095 and 1087 microg cm(-2), respectively. The smaller particles also appear to affect the exposed cells faster with cell death (by necrosis) being observed within just a few hours. The surface area of the tested particles is an important parameter in determining the toxicity of monodisperse amorphous silica nanoparticles.

Characterization of the Selective Indoleamine 2,3-Dioxygenase-1 (IDO1) Catalytic Inhibitor EOS200271/PF-06840003 Supports IDO1 as a Critical Resistance Mechanism to PD-(L)1 Blockade Therapy.

Tumors use indoleamine 2,3-dioxygenase-1 (IDO1) as a major mechanism to induce an immunosuppressive microenvironment. IDO1 expression is upregulated in many cancers and considered to be a resistance mechanism to immune checkpoint therapies. IDO1 is induced in response to inflammatory stimuli such as IFNγ and promotes immune tolerance by depleting tryptophan and producing tryptophan catabolites, including kynurenine, in the tumor microenvironment. This leads to effector T-cell anergy and enhanced Treg function through upregulation of FoxP3. As a nexus for the induction of key immunosuppressive mechanisms, IDO1 represents an important immunotherapeutic target in oncology. Here, we report the identification and characterization of the novel selective, orally bioavailable IDO1 inhibitor EOS200271/PF-06840003. It reversed IDO1-induced T-cell anergy in vitro In mice carrying syngeneic tumor grafts, PF-06840003 reduced intratumoral kynurenine levels by over 80% and inhibited tumor growth both in monotherapy and, with an increased efficacy, in combination with antibodies blocking the immune checkpoint ligand PD-L1. We demonstrate that anti-PD-L1 therapy results in increased IDO1 metabolic activity thereby providing additional mechanistic rationale for combining PD-(L)1 blockade with IDO1 inhibition in cancer immunotherapies. Supported by these preclinical data and favorable predicted human pharmacokinetic properties of PF-06840003, a phase I open-label, multicenter clinical study (NCT02764151) has been initiated.

Regulation of macrophage motility by the water channel aquaporin-1: crucial role of M0/M2 phenotype switch.

The water channel aquaporin-1 (AQP1) promotes migration of many cell types. Although AQP1 is expressed in macrophages, its potential role in macrophage motility, particularly in relation with phenotype polarization, remains unknown. We here addressed these issues in peritoneal macrophages isolated from AQP1-deficient mice, either undifferentiated (M0) or stimulated with LPS to orientate towards pro-inflammatory phenotype (classical macrophage activation; M1). In non-stimulated macrophages, ablation of AQP1 (like inhibition by HgCl2) increased by 2-3 fold spontaneous migration in a Src/PI3K/Rac-dependent manner. This correlated with cell elongation and formation of lamellipodia/ruffles, resulting in membrane lipid and F4/80 recruitment to the leading edge. This indicated that AQP1 normally suppresses migration of resting macrophages, as opposed to other cell types. Resting Aqp1-/- macrophages exhibited CD206 redistribution into ruffles and increased arginase activity like IL4/IL13 (alternative macrophage activation; M2), indicating a M0-M2 shift. In contrast, upon M1 orientation by LPS in vitro or peritoneal inflammation in vivo, migration of Aqp1-/- macrophages was reduced. Taken together, these data indicate that AQP1 oppositely regulates macrophage migration, depending on stimulation or not by LPS, and that macrophage phenotypic and migratory changes may be regulated independently of external cues.

The cytotoxic activity of amorphous silica nanoparticles is mainly influenced by surface area and not by aggregation.

The aggregation state of NP has been a significant source of difficulty for assessing their toxic activity and great efforts have been done to reduce aggregation of and/or to disperse NP in experimental systems. The exact impact of aggregation on toxicity has, however, not been adequately assessed. Here we compared in vitro the cytotoxic activity of stable monodisperse and aggregated silicon-based nanoparticles (SNP) without introducing a dispersing agent that may affect NP properties. SNP aggregates (180 nm) were produced by controlled electrostatic aggregation through addition of KCl to a Ludox SM sol (25 nm) followed by stabilization and extensive dialysis. The size of the preparations was characterized by TEM and DLS; specific surface area and porosity were derived from N(2) sorption measurements. Macrophage (J774) and fibroblast (3T3) cell lines were exposed to monodisperse or aggregate-enriched suspensions of SNP in DMEM in absence of serum. The cytotoxic activity of the different preparations was assessed by the WST1 assay after 24h of exposure. Parameters that determined the cytotoxic activity were traced by comparing the doses of the different preparations that induced half a maximal reduction in WST1 activity (ED(50)) in both cell lines. We found that ED(50) (6-9 µg/ml and 15-22 µg/ml, in J774 and 3T3, respectively) were hardly affected upon aggregation, which was consistent with the fact that the specific surface area of the SNP, a significant determinant of their cytotoxic activity, was unaffected upon aggregation (283-331 m(2)/g). Thus studying small aggregated NP could be as relevant as studying disperse primary NP, when aggregates keep the characteristics of NP, i.e. a high specific surface area and a nanosize dimension. This conclusion does, however, not necessarily hold true for other toxicity endpoints for which the determinants may be different and possibly modified by the aggregation process.

Lung fibrosis induced by crystalline silica particles is uncoupled from lung inflammation in NMRI mice.

Previous studies in rats have suggested a causal relationship between progressive pulmonary inflammation and lung fibrosis induced by crystalline silica particles. We report here that, in NMRI mice, the lung response to silica particles is accompanied by a mild and non progressive pulmonary inflammation which is dispensable for the development of lung fibrosis. We found that glucocorticoid (dexamethasone) dramatically reduced lung injury, cellular inflammation and pro-inflammatory cytokine expression (TNF-α, IL-1ß and KC) but had no significant effect on silica-induced lung fibrosis and expression of the fibrogenic and suppressive cytokines TGF-ß and IL-10 in mice. Other anti-inflammatory molecules such as the COX inhibitor piroxicam or the phosphodiesterase 5 inhibitor sildenafil also reduced lung inflammation without modifying collagen, TGF-ß or IL-10 lung content. Our findings indicate that the development of lung fibrosis in silica-treated NMRI mice is not driven by inflammatory lung responses and suggest that suppressive cytokines may represent critical fibrotic factors and potential therapeutic targets in silicosis.

Influence of size, surface area and microporosity on the in vitro cytotoxic activity of amorphous silica nanoparticles in different cell types.

Identifying the physico-chemical characteristics of nanoparticles (NPs) that drive their toxic activity is the key to conducting hazard assessment and guiding the design of safer nanomaterials. Here we used a set of 17 stable suspensions of monodisperse amorphous silica nanoparticles (SNPs) with selected variations in size (diameter, 2-335 nm), surface area (BET, 16-422 m(2)/g) and microporosity (micropore volume, 0-71 microl/g) to assess with multiple regression analysis the physico-chemical determinants of the cytotoxic activity in four different cell types (J774 macrophages, EAHY926 endothelial cells, 3T3 fibroblasts and human erythrocytes). We found that the response to these SNPs is governed by different physico-chemical parameters which vary with cell type: In J774 macrophages, the cytotoxic activity (WST1 assay) increased with external surface area (alphas method) and decreased with micropore volume (r(2) of the model, 0.797); in EAHY926 and 3T3 cells, the cytotoxic activity of the SNPs (MTT and WST1 assay, respectively) increased with surface roughness and small diameter (r(2), 0.740 and 0.872, respectively); in erythrocytes, the hemolytic activity increased with the diameter of the SNP (r(2), 0.860). We conclude that it is possible to predict with good accuracy the in vitro cytotoxic potential of SNPs on the basis of their physico-chemical characteristics. These determinants are, however, complex and vary with cell type, reflecting the pleiotropic interactions of nanoparticles with biological systems.

Exploring the aneugenic and clastogenic potential in the nanosize range: A549 human lung carcinoma cells and amorphous monodisperse silica nanoparticles as models.

We explored how to assess the genotoxic potential of nanosize particles with a well validated assay, the in vitro cytochalasin-B micronucleus assay, detecting both clastogens and aneugens. Monodisperse Stöber amorphous silica nanoparticles (SNPs) of three different sizes (16, 60 and 104 nm) and A549 lung carcinoma cells were selected as models. Cellular uptake of silica was monitored by ICP-MS. At non-cytotoxic doses the smallest particles showed a slightly higher fold induction of micronuclei (MNBN). When considering the three SNPs together, particle number and total surface area appeared to account for MNBN induction as they both correlated significantly with the amplitude of the effect. Using nominal or cellular dose did not show statistically significant differences. Likewise, alkaline comet assay and FISH-centromeric probing of MNBN indicated a weak and not statistically significant induction of oxidative DNA damage, chromosome breakage and chromosome loss. This line of investigation will contribute to adequately design and interpret nanogenotoxicity assays.

Absence of carcinogenic response to multiwall carbon nanotubes in a 2-year bioassay in the peritoneal cavity of the rat.

Toxicological investigations of carbon nanotubes have shown that they can induce pulmonary toxicity, and similarities with asbestos fibers have been suggested. We previously reported that multiwall carbon nanotubes (MWCNT) induced lung inflammation, granulomas and fibrotic reactions. The same MWCNT also caused mutations in epithelial cells in vitro and in vivo. These inflammatory and genotoxic activities were related to the presence of defects in the structure of the nanotubes. In view of the strong links between inflammation, mutations and cancer, these observations prompted us to explore the carcinogenic potential of these MWCNT in the peritoneal cavity of rats. The incidence of mesothelioma and other tumors was recorded in three groups of 50 male Wistar rats injected intraperitoneally with a single dose of MWCNT with defects (2 or 20 mg/animal) and MWCNT without defects (20 mg/animal). Two additional groups of 26 rats were used as positive (2 mg UICC crocidolite/animal) and vehicle controls. After 24 months, although crocidolite induced a clear carcinogenic response (34.6% animals with mesothelioma vs. 3.8% in vehicle controls), MWCNT with or without structural defects did not induce mesothelioma in this bioassay (4, 0, or 6%, respectively). The incidence of tumors other than mesothelioma was not significantly increased across the groups. The initial hypothesis of a contrasting carcinogenic activity between MWCNT with and without defects could not be verified in this bioassay. We discuss the possible reasons for this absence of carcinogenic response, including the length of the MWCNT tested (< 1 mum on average), the absence of a sustained inflammatory reaction to MWCNT, and the capacity of these MWCNT to quench free radicals.

Sintered indium-tin-oxide (ITO) particles: a new pneumotoxic entity.

Indium-Tin-Oxide (ITO) is a sintered mixture of indium- (In(2)O(3)) and tin-oxide (SnO(2)) in a ratio of 90:10 (wt:wt) that is used for the manufacture of LCD screens and related high technology applications. Interstitial pulmonary diseases have recently been reported in workers from ITO producing plants. The present study was conducted to identify experimentally the exact chemical component responsible for this toxicity and to address possible mechanisms of action. The reactivity of respirable ITO particles was compared with that of its single components alone or their unsintered 90:10 mixture (MIX) both in vivo and in vitro. For all endpoints considered, ITO particles behaved as a specific toxic entity. In vivo, after a single pharyngeal administration (2-20 mg per rat), ITO particles induced a strong inflammatory reaction. At day 3, the inflammatory reaction (cell accumulation, LDH and protein in bronchoalveolar lavage fluid) appeared more marked with ITO particles than with each oxide separately or the MIX. This inflammatory reaction persisted and even worsened after 15 days. After 60 days, this inflammation was still present but no significant fibrotic response was observed. The cytotoxicity of ITO was assessed in vitro in lung epithelial cells (RLE) and macrophages (NR8383 cell line). While ITO particles (up to 200 microg/ml) did not affect epithelial cell integrity (LDH release), a strong cytotoxic response was found in macrophages exposed to ITO, but not to its components alone or mixed. ITO particles also induced an increased frequency of micronuclei in type II pneumocytes in vivo but not in RLE in vitro, suggesting the preponderance of a secondary genotoxic mechanism. To address the possible mechanism of ITO toxicity, reactive oxygen species production was assessed by electron paramagnetic resonance spectrometry in an acellular system. Carbon centered radicals (COO-.) and Fenton-like activity were detected in the presence of ITO particles, not with In(2)O(3), SnO(2) alone, or the MIX. Because the unsintered mixture of SnO(2) and In(2)O(3) particles was unable to reproduce the reactivity/toxicity of ITO particles, the sintering process through which SnO(2) molecules are introduced within the crystal structure of In(2)O(3) appears critical to explain the unique toxicological properties of ITO. The inflammatory and genotoxic activities of ITO dust indicate that a strict control of exposure is needed in industrial settings.