A tiered approach to investigate the inhalation toxicity of cobalt substances. Tier 2a: Grouping cobalt compounds based on their capacity to stabilize HIF-1α in human alveolar epithelial cells in vitro.

Excessive inhalation of cobalt (Co) dust can have harmful effects on the respiratory tract, yet all cobalt substances do not have the same potential for inducing toxicity. The prevalent hypothesis is that the potential of Co substances to release Co2+ ions in the organism and in cells drives their toxicity profile. Here, we explored the possibility of grouping Co substances for predicting inhalation toxicity based on in vitro data using the stabilization of hypoxia-inducible factor (HIF)-1α as a read out for intracellular Co ion content. We evaluated the potential of 11 inorganic Co compounds and two Co metal powder samples to stabilize intracellular HIF-1α in alveolar epithelial cells (A549) after 24 h exposure to 250-1000 µM Co equivalents. Cytotoxic activity of the substances was assessed in parallel after 72 h at the same doses. Two groups were identified: (1) substances with high intracellular bioavailability (n=9), causing cytotoxicity and stabilizing HIF-1α and (2) substances with low intracellular bioavailability (n = 4), and not inducing these effects. This study provides a link between screening-level data (solubility in artificial lung fluids, Tier 1) and hypothesized biological key events.

Monocytic Ontogeny of Regenerated Macrophages Characterizes the Mesotheliomagenic Responses to Carbon Nanotubes.

Macrophages are not only derived from circulating blood monocytes or embryonic precursors but also expand by proliferation. The origin determines macrophage fate and functions in steady state and pathological conditions. Macrophages predominantly infiltrate fibre-induced mesothelioma tumors and contribute to cancer development. Here, we revealed their ontogeny by comparing the response to needle-like mesotheliomagenic carbon nanotubes (CNT-7) with tangled-like non-mesotheliomagenic CNT-T. In a rat peritoneal cavity model of mesothelioma, both CNT induced a rapid macrophage disappearance reaction (MDR) of MHCIIlow resident macrophages generating an empty niche available for macrophage repopulation. Macrophage depletion after mesotheliomagenic CNT-7 was followed by a substantial inflammatory reaction, and macrophage replenishment completed after 7 days. Thirty days after non-mesotheliomagenic CNT-T, macrophage repopulation was still incomplete and accompanied by a limited inflammatory reaction. Cell depletion experiments, flow cytometry and RNA-seq analysis demonstrated that, after mesotheliomagenic CNT-7 exposure, resident macrophages were mainly replaced by an influx of monocytes, which differentiated locally into MHCIIhigh inflammatory macrophages. In contrast, the low inflammatory response induced by CNT-T was associated by the accumulation of self-renewing MHCIIlow macrophages that initially derive from monocytes. In conclusion, the mesotheliomagenic response to CNT specifically relies on macrophage niche recolonization by monocyte-derived inflammatory macrophages. In contrast, the apparent homeostasis after non-mesotheliomagenic CNT treatment involves a macrophage regeneration by proliferation. Macrophage depletion and repopulation are thus decisive events characterizing the carcinogenic activity of particles and fibres.

Dietary nanoparticles alter the composition and function of the gut microbiota in mice at dose levels relevant for human exposure.

BACKGROUND: Nanotechnologies provide new opportunities for improving the safety, quality, shelf life, flavor and appearance of foods. The most common nanoparticles (NPs) in human diet are silver metal, mainly present in food packaging and appliances, and silicon and titanium dioxides used as additives. The rapid development and commercialization of consumer products containing these engineered NPs is, however, not well supported by appropriate toxicological studies and risk assessment. Local and systemic toxicity and/or disruption of the gut microbiota (GM) have already been observed after oral administration of NPs in experimental animals, but results are not consistent and doses used were often much higher than the estimated human intakes. In view of the strong evidence linking alterations of the GM to cardiometabolic (CM) diseases, we hypothesized that dietary NPs might disturb this GM-CM axis. MATERIALS AND METHODS: We exposed male C57BL/6JRj mice (n = 13 per dose group) to dietary NPs mixed in food pellets at doses relevant for human exposure: Ag (0, 4, 40 or 400 µg/kg pellet), SiO2 (0, 0.8, 8 and 80 mg/kg pellet) or TiO2 (0, 0.4, 4 or 40 mg/kg pellet). After 24 weeks of exposure, we assessed effects on the GM and CM health (n = 8 per dose group). The reversibility of the effects was examined after 8 additional weeks without NPs exposure (recovery period, n ≤ 5 per dose group). RESULTS: No overt toxicity was recorded. The GM ß-diversity was dose-dependently disrupted by the three NPs, and the bacterial short chain fatty acids (SCFAs) were dose-dependently reduced after the administration of SiO2 and TiO2 NPs. These effects disappeared completely or partly after the recovery period, strengthening the association with dietary NPs. We did not observe atheromatous disease or glucose intolerance after NP exposure. Instead, dose-dependent decreases in the expression of IL-6 in the liver, circulating triglycerides (TG) and urea nitrogen (BUN) were recorded after administration of the NPs. CONCLUSION: We found that long-term oral exposure to dietary NPs at doses relevant for estimated human intakes disrupts the GM composition and function. These modifications did not appear associated with atheromatous or deleterious metabolic outcomes.

Systemic effects and impact on the gut microbiota upon subacute oral exposure to silver acetate in rats.

CONTEXT: The addition of silver (Ag) to food items, and its migration from food packaging and appliances results in a dietary exposure in humans, estimated to 70-90 µg Ag/day. In view of the well-known bactericidal activity of Ag ions, concerns arise about a possible impact of dietary Ag on the gut microbiota (GM), which is a master determinant of human health and diseases. Repeated oral administration of Ag acetate (AgAc) can also cause systemic toxicity in rats with reported NOAELs of 4 mg AgAc/b.w./d for impaired fertility and 0.4 mg AgAc/b.w./d for developmental toxicity. OBJECTIVE: The objective of this study was to investigate whether oral exposure to AgAc can induce GM alterations at doses causing reproductive toxicity in rats. METHODS: Male and female Wistar rats were exposed during 10 weeks to AgAc incorporated into food (0, 0.4, 4 or 40 mg/kg b.w./d), and we analyzed the composition of the GM (α- and ß-diversity). We documented bacterial function by measuring short-chain fatty acid (SCFA) production in cecal content. Ferroxidase activity, a biomarker of systemic Ag toxicity, was measured in serum. RESULTS AND CONCLUSIONS: From 4 mg/kg b.w./d onwards, we recorded systemic toxicity, as indicated by the reduction of serum ferroxidase activity, as well as serum Cu and Se concentrations. This systemic toxic response to AgAc might contribute to explain reprotoxic manifestations. We observed a dose-dependent modification of the GM composition in male rats exposed to AgAc. No impact of AgAc exposure on the production of bacterial SCFA was recorded. The limited GM changes recorded in this study do not appear related to a reprotoxicity outcome.

Femtosecond pulsed laser microscopy: a new tool to assess the in vitro delivered dose of carbon nanotubes in cell culture experiments.

BACKGROUND: In vitro models are widely used in nanotoxicology. In these assays, a careful documentation of the fraction of nanomaterials that reaches the cells, i.e. the in vitro delivered dose, is a critical element for the interpretation of the data. The in vitro delivered dose can be measured by quantifying the amount of material in contact with the cells, or can be estimated by applying particokinetic models. For carbon nanotubes (CNTs), the determination of the in vitro delivered dose is not evident because their quantification in biological matrices is difficult, and particokinetic models are not adapted to high aspect ratio materials. Here, we applied a rapid and direct approach, based on femtosecond pulsed laser microscopy (FPLM), to assess the in vitro delivered dose of multi-walled CNTs (MWCNTs). METHODS AND RESULTS: We incubated mouse lung fibroblasts (MLg) and differentiated human monocytic cells (THP-1) in 96-well plates for 24 h with a set of different MWCNTs. The cytotoxic response to the MWCNTs was evaluated using the WST-1 assay in both cell lines, and the pro-inflammatory response was determined by measuring the release of IL-1ß by THP-1 cells. Contrasting cell responses were observed across the MWCNTs. The sedimentation rate of the different MWCNTs was assessed by monitoring turbidity decay with time in cell culture medium. These turbidity measurements revealed some differences among the MWCNT samples which, however, did not parallel the contrasting cell responses. FPLM measurements in cell culture wells revealed that the in vitro delivered MWCNT dose did not parallel sedimentation data, and suggested that cultured cells contributed to set up the delivered dose. The FPLM data allowed, for each MWCNT sample, an adjustment of the measured cytotoxicity and IL-1ß responses to the delivered doses. This adjusted in vitro activity led to another toxicity ranking of the MWCNT samples as compared to the unadjusted activities. In macrophages, this adjusted ranking was consistent with existing knowledge on the impact of surface MWCNT functionalization on cytotoxicity, and might better reflect the intrinsic activity of the MWCNT samples. CONCLUSION: The present study further highlights the need to estimate the in vitro delivered dose in cell culture experiments with nanomaterials. The FPLM measurement of the in vitro delivered dose of MWCNTs can enrich experimental results, and may refine our understanding of their interactions with cells.

Cationic Nanoliposomes Are Efficiently Taken up by Alveolar Macrophages but Have Little Access to Dendritic Cells and Interstitial Macrophages in the Normal and CpG-Stimulated Lungs.

The purpose of this study was to assess whether cationic nanoliposomes could address tumor vaccines to dendritic cells in the lungs in vivo. Nanoliposomes were prepared using a cationic lipid, dimethylaminoethanecarbamoyl-cholesterol (DC-cholesterol) or dioleoyltrimethylammoniumpropane (DOTAP), and dipalmitoylphosphatidylcholine (DPPC), the most abundant phospholipid in lung surfactant. The liposomes presented a size below 175 nm and they effectively entrapped tumor antigens, an oligodeoxynucletotide containing CpG motifs (CpG) and the fluorescent dye calcein used as a tracer. Although the liposomes could permanently entrap a large fraction of the actives, they could not sustain their release in vitro. Liposomes made of DOTAP were safe to respiratory cells in vitro, while liposomes composed of DC-cholesterol were cytotoxic. DOTAP nanoliposomes were mainly taken up by alveolar macrophages following delivery to the lungs in mice. Few dendritic cells took up the liposomes, and interstitial macrophages did not take up liposomal calcein more than they took up soluble calcein. Stimulation of the innate immune system using liposomal CpG strongly enhanced uptake of calcein liposomes by all phagocytes in the lungs. Although a small percentage of dendritic cells took up the nanoliposomes, alveolar macrophages represented a major barrier to dendritic cell access in the lungs.

Respiratory hazard of Li-ion battery components: elective toxicity of lithium cobalt oxide (LiCoO2) particles in a mouse bioassay.

Rechargeable Li-ion batteries (LIB) are increasingly produced and used worldwide. LIB electrodes are made of micrometric and low solubility particles, consisting of toxicologically relevant elements. The health hazard of these materials is not known. Here, we investigated the respiratory hazard of three leading LIB components (LiFePO4 or LFP, Li4Ti5O12 or LTO, and LiCoO2 or LCO) and their mechanisms of action. Particles were characterized physico-chemically and elemental bioaccessibility was documented. Lung inflammation and fibrotic responses, as well as particle persistence and ion bioavailability, were assessed in mice after aspiration of LIB particles (0.5 or 2 mg); crystalline silica (2 mg) was used as reference. Acute inflammatory lung responses were recorded with the 3 LIB particles and silica, LCO being the most potent. Inflammation persisted 2 m after LFP, LCO and silica, in association with fibrosis in LCO and silica lungs. LIB particles persisted in the lungs after 2 m. Endogenous iron co-localized with cobalt in LCO lungs, indicating the formation of ferruginous bodies. Fe and Co ions were detected in the broncho-alveolar lavage fluids of LFP and LCO lungs, respectively. Hypoxia-inducible factor (HIF) -1α, a marker of fibrosis and of the biological activity of Co ions, was upregulated in LCO and silica lungs. This study identified, for the first time, the respiratory hazard of LIB particles. LCO was at least as potent as crystalline silica to induce lung inflammation and fibrosis. Iron and cobalt, but not lithium, ions appear to contribute to LFP and LCO toxicity, respectively.

New interplay between interstitial and alveolar macrophages explains pulmonary alveolar proteinosis (PAP) induced by indium tin oxide particles.

Occupational exposure to indium tin oxide (ITO) particles has been associated with the development of severe lung diseases, including pulmonary alveolar proteinosis (PAP). The mechanisms of this lung toxicity remain unknown. Here, we reveal the respective roles of resident alveolar (Siglec-Fhigh AM) and recruited interstitial (Siglec-Flow IM) macrophages contributing in concert to the development of PAP. In mice treated with ITO particles, PAP is specifically associated with IL-1α (not GM-CSF) deficiency and Siglec-Fhigh AM (not Siglec-Flow IM) depletion. Mechanistically, ITO particles are preferentially phagocytosed and dissolved to soluble In3+ by Siglec-Flow IM. In contrast, Siglec-Fhigh AM weakly phagocytose or dissolve ITO particles, but are sensitive to released In3+ through the expression of the transferrin receptor-1 (TfR1). Blocking pulmonary Siglec-Flow IM recruitment in CCR2-deficient mice reduces ITO particle dissolution, In3+ release, Siglec-Fhigh AM depletion, and PAP formation. Restoration of IL-1-related Siglec-Fhigh AM also prevented ITO-induced PAP. We identified a new mechanism of secondary PAP development according to which metal ions released from inhaled particles by phagocytic IM disturb IL-1α-dependent AM self-maintenance and, in turn, alveolar clearance.

CCR2+ monocytic myeloid-derived suppressor cells (M-MDSCs) inhibit collagen degradation and promote lung fibrosis by producing transforming growth factor-ß1.

Monocytes infiltrating scar tissue are predominantly viewed as progenitor cells. Here, we show that tissue CCR2+ monocytes have specific immunosuppressive and profibrotic functions. CCR2+ monocytic cells are acutely recruited to the lung before the onset of silica-induced fibrosis in mice. These tissue monocytes are defined as monocytic myeloid-derived suppressor cells (M-MDSCs) because they significantly suppress T-lymphocyte proliferation in vitro. M-MDSCs collected from silica-treated mice also express transforming growth factor (TGF)-ß1, which stimulates lung fibroblasts to release tissue inhibitor of metalloproteinase (TIMP)-1, an inhibitor of metalloproteinase collagenolytic activity. By using LysMCreCCR2loxP/loxP mice, we show that limiting CCR2+ M-MDSC accumulation reduces the pulmonary contents of TGF-ß1, TIMP-1 and collagen after silica treatment. M-MDSCs do not differentiate into lung macrophages, granulocytes or fibrocytes during pulmonary fibrogenesis. Collectively, our data indicate that M-MDSCs contribute to lung fibrosis by specifically promoting a non-degrading collagen microenvironment. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Mesothelioma response to carbon nanotubes is associated with an early and selective accumulation of immunosuppressive monocytic cells.

BACKGROUND: The asbestos-like toxicity of some engineered carbon nanotubes (CNT), notably their capacity to induce mesothelioma, is a serious cause of concern for public health. Here we show that carcinogenic CNT induce an early and sustained immunosuppressive response characterized by the accumulation of monocytic Myeloid Derived Suppressor Cells (M-MDSC) that counteract effective immune surveillance of tumor cells. METHODS: Wistar rats and C57BL/6 mice were intraperitoneally injected with carcinogenic multi-walled Mitsui-7 CNT (CNT-7) or crocidolite asbestos. Peritoneal mesothelioma development and immune cell accumulation were assessed until 12 months. Leukocyte sub-populations were identified by recording expression of CD11b/c and His48 by flow cytometry. The immunosuppressive activity on T lymphocytes of purified peritoneal leukocytes was assessed in a co-culture assay with activated spleen cells. RESULTS: We demonstrate that long and short mesotheliomagenic CNT-7 injected in the peritoneal cavity of rats induced, like asbestos, an early and selective accumulation of monocytic cells (CD11b/c(int) and His48(hi)) which possess the ability to suppress polyclonal activation of T lymphocytes and correspond to M-MDSC. Peritoneal M-MDSC persisted during the development of peritoneal mesothelioma in CNT-7-treated rats but were only transiently recruited after non-carcinogenic CNT (CNT-M, CNT-T) injection. Peritoneal M-MDSC did not accumulate in mice which are resistant to mesothelioma development. CONCLUSIONS: Our data provide new insights into the initial pathogenic events induced by CNT, adding a new component to the adverse outcome pathway leading to mesothelioma development. The specificity of the M-MDSC response after carcinogenic CNT exposure highlights the interest of this response for detecting the ability of new nanomaterials to cause cancer.

Towards predicting the lung fibrogenic activity of MWCNT: Key role of endocytosis, kinase receptors and ERK 1/2 signaling.

Carbon nanotubes (CNT) have been reported to induce lung inflammation and fibrosis in rodents. We investigated the direct and indirect cellular mechanisms mediating the fibrogenic activity of multi-wall (MW) CNT on fibroblasts. We showed that MWCNT indirectly stimulate lung fibroblast (MLg) differentiation, via epithelial cells and macrophages, whereas no direct effect of MWCNT on fibroblast differentiation or collagen production was detected. MWCNT directly stimulated the proliferation of fibroblasts primed with low concentrations of growth factors, such as PDGF, TGF-ß or EGF. MWCNT prolonged ERK 1/2 phosphorylation induced by low concentrations of PDGF or TGF-ß in fibroblasts. This phenomenon and the proliferative activity of MWCNT on fibroblasts was abrogated by the inhibitors of ERK 1/2, PDGF-, TGF-ß- and EGF-receptors. This activity was also reduced by amiloride, an endocytosis inhibitor. Finally, the lung fibrotic response to several MWCNT samples (different in length and diameter) correlated with their in vitro capacity to stimulate the proliferation of fibroblasts and to prolong ERK 1/2 signaling in these cells. Our findings point to a crosstalk between MWCNT, kinase receptors, ERK 1/2 signaling and endocytosis which stimulates the proliferation of fibroblasts. The mechanisms of action identified in this study contribute to predict the fibrogenic potential of MWCNT.

Does carbonation of steel slag particles reduce their toxicity? An in vitro approach.

Mineral carbonation can stabilize industrial residues and, in the steel industry, may contribute to simultaneously valorize CO2 emissions and slag. We hypothesized that, by restricting the leaching of metals of toxicological concern such as Cr and V, carbonation can suppress the toxicity of these materials. The cytotoxic activity (WST1 assay) of slag dusts collected from a stainless and a Linz-Donawitz (LD) steel plant, before and after carbonation, was examined in J774 macrophages. The release of Cr, V, Fe, Mn and Ni was measured after incubation in artificial lung fluids mimicking the extracellular and phagolysosomal milieu to which particles are confronted after inhalation. LD slag had the higher Fe, Mn and V content, and was more cytotoxic than stainless steel slag. The cytotoxic activity of LD but not of stainless dusts was reduced after carbonation. The cytotoxic activity of the dusts toward J774 macrophages necessitated a direct contact with the cells and was reduced in the presence of inhibitors of phagocytosis (cytochalasin D) or phagolysosome acidification (bafilomycin), pointing to a key role of metallic constituents released in phagolysosomes. This in vitro study supports a limited reduction of the cytotoxic activity of LD, but not of stainless, steel dusts upon carbonation.

Nanometer-long Ge-imogolite nanotubes cause sustained lung inflammation and fibrosis in rats.

BACKGROUND: Ge-imogolites are short aluminogermanate tubular nanomaterials with attractive prospected industrial applications. In view of their nano-scale dimensions and high aspect ratio, they should be examined for their potential to cause respiratory toxicity. Here, we evaluated the respiratory biopersistence and lung toxicity of 2 samples of nanometer-long Ge-imogolites. METHODS: Rats were intra-tracheally instilled with single wall (SW, 70 nm length) or double wall (DW, 62 nm length) Ge-imogolites (0.02-2 mg/rat), as well as with crocidolite and the hard metal particles WC-Co, as positive controls. The biopersistence of Ge-imogolites and their localization in the lung were assessed by ICP-MS, X-ray fluorescence, absorption spectroscopy and computed micro-tomography. Acute inflammation and genotoxicity (micronuclei in isolated type II pneumocytes) was assessed 3 d post-exposure; chronic inflammation and fibrosis after 2 m. RESULTS: Cytotoxic and inflammatory responses were shown in bronchoalveolar lavage 3 d after instillation with Ge-imogolites. Sixty days after exposure, a persistent dose-dependent inflammation was still observed. Total lung collagen, reflected by hydroxyproline lung content, was increased after SW and DW Ge-imogolites. Histology revealed lung fibre reorganization and accumulation in granulomas with epithelioid cells and foamy macrophages and thickening of the alveolar walls. Overall, the inflammatory and fibrotic responses induced by SW and DW Ge-imogolites were more severe (on a mass dose basis) than those induced by crocidolite. A persistent fraction of Ge-imogolites (15% of initial dose) was mostly detected as intact structures in rat lungs 2 m after instillation and was localized in fibrotic alveolar areas. In vivo induction of micronuclei was significantly increased 3 d after SW and DW Ge-imogolite instillation at non-inflammatory doses, indicating the contribution of primary genotoxicity. CONCLUSIONS: We showed that nm-long Ge-imogolites persist in the lung and promote genotoxicity, sustained inflammation and fibrosis, indicating that short high aspect ratio nanomaterials should not be considered as innocuous materials. Our data also suggest that Ge-imogolite structure and external surface determine their toxic activity.

Lung inflammation and thymic atrophy after bleomycin are controlled by the prostaglandin D2 receptor DP1.

Acute lung injury (ALI) can be accompanied by secondary systemic manifestations. In a model of ALI induced by bleomycin (bleo), we examined the response of D prostanoid receptor 1 (DP1)-deficient mice (DP1(-/-)) to better understand these processes. DP1 deficiency aggravated the toxicity of bleo as indicated by enhanced body weight loss, mortality, and lung inflammation including bronchoalveolar permeability and neutrophilia. Thymic atrophy was also observed after bleo and was strongly exacerbated in DP1(-/-) mice. This resulted from the enhanced depletion of immature T lymphocytes in the thymus of DP1(-/-) mice, a phenomenon usually related to increased glucocorticoid release in blood. Serum corticosterone was more elevated in DP1(-/-) mice after bleo than in wild-type (wt) mice. Thymocytes of DP1(-/-) mice were not more sensitive to dexamethasone in vitro, and systemic delivery of dexamethasone or peritoneal inflammation after LPS induced a similar thymic atrophy in wt and DP1(-/-) mice, indicating that pulmonary DP1 was critical to the control of thymic atrophy after bleo. DP1(-/-) mice showed increased lung and/or blood mediators involved in neutrophil recruitment and/or glucocorticoid production/thymic atrophy (osteopontin, leukemia inhibitory factor, and keratinocyte-derived chemokine) after bleo. Finally, local pulmonary DP1 activation or inhibition in wt mice abrogated or amplified thymic atrophy after bleo, respectively. Altogether, our data reveal that ALI can perturb the systemic T-cell pool by inducing thymic atrophy and that both pathological processes are controlled by the pulmonary DP1 receptor. This new pathway represents a potential therapeutic target in ALI.

Towards predicting the lung fibrogenic activity of nanomaterials: experimental validation of an in vitro fibroblast proliferation assay.

BACKGROUND: Carbon nanotubes (CNT) can induce lung inflammation and fibrosis in rodents. Several studies have identified the capacity of CNT to stimulate the proliferation of fibroblasts. We developed and validated experimentally here a simple and rapid in vitro assay to evaluate the capacity of a nanomaterial to exert a direct pro-fibrotic effect on fibroblasts. METHODS: The activity of several multi-wall (MW)CNT samples (NM400, the crushed form of NM400 named NM400c, NM402 and MWCNTg 2400) and asbestos (crocidolite) was investigated in vitro and in vivo. The proliferative response to MWCNT was assessed on mouse primary lung fibroblasts, human fetal lung fibroblasts (HFL-1), mouse embryonic fibroblasts (BALB-3T3) and mouse lung fibroblasts (MLg) by using different assays (cell counting, WST-1 assay and propidium iodide PI staining) and dispersion media (fetal bovine serum, FBS and bovine serum albumin, BSA). C57BL/6 mice were pharyngeally aspirated with the same materials and lung fibrosis was assessed after 2 months by histopathology, quantification of total collagen lung content and pro-fibrotic cytokines in broncho-alveolar lavage fluid (BALF). RESULTS: MWCNT (NM400 and NM402) directly stimulated fibroblast proliferation in vitro in a dose-dependent manner and induced lung fibrosis in vivo. NM400 stimulated the proliferation of all tested fibroblast types, independently of FBS- or BSA- dispersion. Results obtained by WST1 cell activity were confirmed with cell counting and cell cycle (PI staining) assays. Crocidolite also stimulated fibroblast proliferation and induced pulmonary fibrosis, although to a lesser extent than NM400 and NM402. In contrast, shorter CNT (NM400c and MWCNTg 2400) did not induce any fibroblast proliferation or collagen accumulation in vivo, supporting the idea that CNT structure is an important parameter for inducing lung fibrosis. CONCLUSIONS: In this study, an optimized proliferation assay using BSA as a dispersant, MLg cells as targets and an adaptation of WST-1 as readout was developed. The activity of MWCNT in this test strongly reflects their fibrotic activity in vivo, supporting the predictive value of this in vitro assay in terms of lung fibrosis potential.