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BackgroundTitanium dioxide (TiO2) nanomaterials have considerable beneficial uses as photocatalysts and solar cells. It has been established for many years that pigment-grade TiO2 (200 nm sphere) is relatively inert when internalized into a biological model system (in vivo or in vitro). For this reason, TiO2 nanomaterials are considered an attractive alternative in applications where biological exposures will occur. Unfortunately, metal oxides on the nanoscale (one dimension < 100 nm) may or may not exhibit the same toxic potential as the original material. A further complicating issue is the effect of modifying or engineering of the nanomaterial to be structurally and geometrically different from the original material.ResultsTiO2 nanospheres, short (< 5 μm) and long (> 15 μm) nanobelts were synthesized, characterized and tested for biological activity using primary murine alveolar macrophages and in vivo in mice. This study demonstrates that alteration of anatase TiO2 nanomaterial into a fibre structure of greater than 15 μm creates a highly toxic particle and initiates an inflammatory response by alveolar macrophages. These fibre-shaped nanomaterials induced inflammasome activation and release of inflammatory cytokines through a cathepsin B-mediated mechanism. Consequently, long TiO2 nanobelts interact with lung macrophages in a manner very similar to asbestos or silica.ConclusionsThese observations suggest that any modification of a nanomaterial, resulting in a wire, fibre, belt or tube, be tested for pathogenic potential. As this study demonstrates, toxicity and pathogenic potential change dramatically as the shape of the material is altered into one that a phagocytic cell has difficulty processing, resulting in lysosomal disruption.

BackgroundPrior studies have demonstrated a rapid and progressive acute phase response to bolus aspiration of multi-walled carbon nanotubes (MWCNTs). In this study we sought to test the hypothesis that inhalation exposure to MWCNT produces a fibrotic response and that the response is chronically persistent. To address the hypothesis that inhaled MWCNTs cause persistent morphologic changes, male C57BL/6 J mice were exposed in a whole-body inhalation system to a MWCNT aerosol and the fibrotic response in the alveolar region examined at up to 336 days after termination of exposure.MethodsInhalation exposure was to a 5 mg/m3 MWCNT aerosol for 5 hours/day for 12 days (4 times/week for 3 weeks). At the end of inhalation exposures, lungs were either lavaged for analysis of bronchoalveolar lavage (BAL) or preserved by vascular perfusion of fixative while inflated with air at 1, 14, 84, 168 and 336 days post inhalation exposure. Separate, clean-air control groups were also studied. Light microscopy, enhanced darkfield microscopy and field emission electron microscopy (FESEM) of tissue sections were used to analyze the distribution of lung burden following inhalation exposure. Morphometric measurements of Sirius Red staining for fibrillar collagen were used to assess the connective tissue response. Serial section analysis of enhanced darkfield microscope images was used to examine the redistribution of MWCNT fibers within the lungs during the post-exposure period.ResultsAt day 1 post-exposure 84 ± 3 and 16 ± 2 percent of the lung burden (Mean ± S.E., N = 5) were in the alveolar and airway regions, respectively. Initial distribution within the alveolar region was 56 ± 5, 7 ± 4 and 20 ± 3 percent of lung burden in alveolar macrophages, alveolar airspaces and alveolar tissue, respectively. Clearance reduced the alveolar macrophage burden of MWCNTs by 35 percent between 1 and 168 days post-exposure, while the content of MWCNTs in the alveolar tissue increased by 63 percent. Large MWCNT structures containing greater than 4 fibers were 53.6 percent of the initial lung burden and accounted for the majority of the decline with clearance, while lung burden of singlet MWCNT was essentially unchanged. The mean linear intercept of alveolar airspace, a measure of the expansion of the lungs, was not significantly different between groups. Pulmonary inflammation and damage, measured as the number of polymorphnuclear leukocytes (PMNs) or lactate dehydrogenase activity (LDH) and albumin in BAL, increased rapidly (1 day post) after inhalation of MWCNTs and declined slowly with time post-exposure. The fibrillar collagen in the alveolar region of MWCNT-exposed mice demonstrated a progressive increase in thickness over time (0.17 ± 0.02, 0.22 ± 0.02, 0.26 ± 0.03, 0.25 ± 0.02 and 0.29 ± 0.01 microns for 1, 14, 84, 168 and 336 days post-exposure) and was significantly different from clean-air controls (0.16 ± 0.02) at 84 and (0.15 ± 0.02) at 336 days post-exposure.ConclusionsDespite the relatively low fraction of the lung burden being delivered ...

This study investigated the in vivo pulmonary toxicity of inhaled multi-walled carbon nanotubes (MWCNT). Mice-inhaled aerosolized MWCNT (10 mg/m³, 5 h/day) for 2, 4, 8 or 12 days. MWCNT lung burden was linearly related to exposure duration. MWCNT-induced pulmonary inflammation was assessed by determining whole lung lavage (WLL) polymorphonuclear leukocytes (PMN). Lung cytotoxicity was assessed by WLL fluid LDH activities. WLL fluid albumin concentrations were determined as a marker of alveolar air-blood barrier integrity. These parameters significantly increased in MWCNT-exposed mice versus controls and were dose-dependent. Histopathologic alterations identified in the lung included (1) bronciolocentric inflammation, (2) bronchiolar epithelial hyperplasia and hypertrophy, (3) fibrosis, (4) vascular changes and (5) rare pleural penetration. MWCNT translocated to the lymph node where the deep paracortex was expanded after 8 or 12 days. Acute inhalation of MWCNT induced dose-dependent pulmonary inflammation and damage with rapid development of pulmonary fibrosis, and also demonstrated that MWCNT can reach the pleura after inhalation exposure.

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Multi-walled carbon nanotubes (MWCNT) are new materials with a wide range of industrial and commercial applications. However, their nano-scaled size and fiber-like shape render them respirable and potentially fibrogenic if inhaled into the lungs. To understand MWCNT fibrogenesis, we analyzed the pathologic and molecular aspects of the early phase response to MWCNT in mouse lungs. MWCNT induced rapid and pronounced lesions in the lungs characterized by increased cellularity and formation of fibrotic foci, most notably near where MWCNT deposited, within 14 days post-exposure. Deposition of collagen fibers was markedly increased in the alveolar septa and fibrotic foci, accompanied by elevated expression of fibrotic genes Col1a1, Col1a2, and Fn1 at both mRNA and protein levels. Fibrosis was induced rapidly at 40 μg, wherein fibrotic changes were detected on day 1 and reached a maximal intensity on day 7 through day 14. Induction of fibrosis was dose-dependent at the dose range of 5-40 μg, 7 days post-exposure. MWCNT elicited rapid and prominent infiltrations of neutrophils and macrophages alongside fibrosis implicating acute inflammation in the fibrotic response. At the molecular level, MWCNT induced elevated expression of proinflammatory cytokines TNFα, IL1α, IL1β, IL6, and CCL2 in lung tissues as well as the bronchoalveolar lavage fluid, in a dose- and time-dependent manner. MWCNT also increased the expression of fibrogenic growth factors TGF-β1 and PDGF-A in the lungs significantly. These findings underscore the interplay between acute inflammation and the early fibrotic response in the initiation and propagation of pulmonary fibrosis induced by MWCNT.

The current study tests the hypothesis that multi-walled carbon nanotubes (MWCNT) with different surface chemistries exhibit different bioactivity profiles in vivo. In addition, the study examined the potential contribution of the NLRP3 inflammasome in MWCNT-induced lung pathology. Unmodified (BMWCNT) and MWCNT that were surface functionalised with -COOH (FMWCNT), were instilled into C57BL/6 mice. The mice were then examined for biomarkers of inflammation and injury, as well as examined histologically for development of pulmonary disease as a function of dose and time. Biomarkers for pulmonary inflammation included cytokines, mediators and the presence of inflammatory cells (IL-1β, IL-18, IL-33, cathepsin B and neutrophils) and markers of injury (albumin and lactate dehydrogenase). The results show that surface modification by the addition of the -COOH group to the MWCNT, significantly reduced the bioactivity and pathogenicity. The results of this study also suggest that in vivo pathogenicity of the BMWCNT and FMWCNT correlates with activation of the NLRP3 inflammasome in the lung.

Concerns over the potential for multi-walled carbon nanotubes (MWCNT) to induce lung carcinogenesis have emerged. This study sought to (1) identify gene expression signatures in the mouse lungs following pharyngeal aspiration of well-dispersed MWCNT and (2) determine if these genes were associated with human lung cancer risk and progression. Genome-wide mRNA expression profiles were analyzed in mouse lungs (n=160) exposed to 0, 10, 20, 40, or 80 µg of MWCNT by pharyngeal aspiration at 1, 7, 28, and 56 days post-exposure. By using pairwise-Statistical Analysis of Microarray (SAM) and linear modeling, 24 genes were selected, which have significant changes in at least two time points, have a more than 1.5 fold change at all doses, and are significant in the linear model for the dose or the interaction of time and dose. Additionally, a 38-gene set was identified as related to cancer from 330 genes differentially expressed at day 56 post-exposure in functional pathway analysis. Using the expression profiles of the cancer-related gene set in 8 mice at day 56 post-exposure to 10 µg of MWCNT, a nearest centroid classification accurately predicts human lung cancer survival with a significant hazard ratio in training set (n=256) and test set (n=186). Furthermore, both gene signatures were associated with human lung cancer risk (n=164) with significant odds ratios. These results may lead to development of a surveillance approach for early detection of lung cancer and prognosis associated with MWCNT in the workplace.