Long-term effects of carbon containing engineered nanomaterials and asbestos in the lung: one year postexposure comparisons.

The hallmark geometric feature of single-walled carbon nanotubes (SWCNT) and carbon nanofibers (CNF), high length to width ratio, makes them similar to a hazardous agent, asbestos. Very limited data are available concerning long-term effects of pulmonary exposure to SWCNT or CNF. Here, we compared inflammatory, fibrogenic, and genotoxic effects of CNF, SWCNT, or asbestos in mice 1 yr after pharyngeal aspiration. In addition, we compared pulmonary responses to SWCNT by bolus dosing through pharyngeal aspiration and inhalation 5 h/day for 4 days, to evaluate the effect of dose rate. The aspiration studies showed that these particles can be visualized in the lung at 1 yr postexposure, whereas some translocate to lymphatics. All these particles induced chronic bronchopneumonia and lymphadenitis, accompanied by pulmonary fibrosis. CNF and asbestos were found to promote the greatest degree of inflammation, followed by SWCNT, whereas SWCNT were the most fibrogenic of these three particles. Furthermore, SWCNT induced cytogenetic alterations seen as micronuclei formation and nuclear protrusions in vivo. Importantly, inhalation exposure to SWCNT showed significantly greater inflammatory, fibrotic, and genotoxic effects than bolus pharyngeal aspiration. Finally, SWCNT and CNF, but not asbestos exposures, increased the incidence of K-ras oncogene mutations in the lung. No increased lung tumor incidence occurred after 1 yr postexposure to SWCNT, CNF, and asbestos. Overall, our data suggest that long-term pulmonary toxicity of SWCNT, CNF, and asbestos is defined, not only by their chemical composition, but also by the specific surface area and type of exposure.

Dual acute proinflammatory and antifibrotic pulmonary effects of short palate, lung, and nasal epithelium clone-1 after exposure to carbon nanotubes.

Carbon nanotubes (CNTs; allotropes of carbon with a cylindrical nanostructure) have emerged as one of the most commonly used types of nanomaterials, with numerous applications in industry and biomedicine. However, the inhalation of CNTs has been shown to elicit pulmonary toxicity, accompanied by a robust inflammatory response with an early-onset fibrotic phase. Epithelial host-defense proteins represent an important component of the pulmonary innate immune response to foreign inhalants such as particles and bacteria. The short palate, lung, and nasal epithelium clone-1 (SPLUNC1) protein, a member of the bactericidal/permeability-increasing-fold (BPIF)-containing protein family, is a 25-kD secretory protein that is expressed in nasal, oropharyngeal, and lung epithelia, and has been shown to have multiple functions, including antimicrobial and chemotactic activities, as well as surfactant properties. This study sought to assess the importance of SPLUNC1-mediated pulmonary responses in airway epithelial secretions, and to explore the biological relevance of SPLUNC1 to inhaled particles in a single-walled carbon nanotube (SWCNT) model. Using Scgb1a1-hSPLUNC1 transgenic mice, we observed that SPLUNC1 significantly modified host inflammatory responses by increasing leukocyte recruitment and enhancing phagocytic activity. Furthermore, we found that transgenic mice were more susceptible to SWCNT exposure at the acute phase, but showed resistance against lung fibrogenesis through pathological changes in the long term. The binding of SPLUNC1 also attenuated SWCNT-induced TNF-α secretion by RAW 264.7 macrophages. Taken together, our data indicate that SPLUNC1 is an important component of mucosal innate immune defense against pulmonary inhaled particles.

Carbon nanotubes enhance metastatic growth of lung carcinoma via up-regulation of myeloid-derived suppressor cells.

Metastatic establishment and growth of Lewis lung carcinoma is promoted by single-walled carbon nanotubes (SWCNT) in C57BL6/J mice. The effect is mediated by increased local and systemic accumulation of myeloid-derived suppressor cells (MDSC), as their depletion abrogated pro-tumor activity in vivo. These data are important for the design of novel theranostics platforms with modules capable of depleting or functionally suppressing MDSC to ensure effective immunosurveillance in the tumor microenvironment.

Graphene oxide, but not fullerenes, targets immunoproteasomes and suppresses antigen presentation by dendritic cells.

Graphene oxide (GO) and C60 - or C60 -TRIS fullerenes, internalized by murine dendritic cells (DCs), differently affect their abilities to present antigens to T-cells. While C60 -fullerenes stimulate the ovalbumin-specific MHC class I-restricted T-cell response, GO impairs the stimulatory potential of DCs. In contrast to C60 -fullerenes, GO decreases the intracellular levels of LMP7 immunoproteasome subunits required for processing of protein antigens. This is important for the development of DC-based vaccines.

Oxidative stress, inflammatory biomarkers, and toxicity in mouse lung and liver after inhalation exposure to 100% biodiesel or petroleum diesel emissions.

Over the past decade, soy biodiesel (BD) has become a first alternative energy source that is economically viable and meets requirements of the Clean Air Act. Due to lower mass emissions and reduced hazardous compounds compared to diesel combustion emissions (CE), BD exposure is proposed to produce fewer adverse health effects. However, considering the broad use of BD and its blends in different industries, this assertion needs to be supported and validated by mechanistic and toxicological data. Here, adverse effects were compared in lungs and liver of BALB/cJ mice after inhalation exposure (0, 50, 150, or 500 µg/m3; 4 h/d, 5 d/wk, for 4 wk) to CE from 100% biodiesel (B100) and diesel (D100). Compared to D100, B100 CE produced a significant accumulation of oxidatively modified proteins (carbonyls), an increase in 4-hydroxynonenal (4-HNE), a reduction of protein thiols, a depletion of antioxidant gluthatione (GSH), a dose-related rise in the levels of biomarkers of tissue damage (lactate dehydrogenase, LDH) in lungs, and inflammation (myeloperoxidase, MPO) in both lungs and liver. Significant differences in the levels of inflammatory cytokines interleukin (IL)-6, IL-10, IL-12p70, monocyte chemoattractant protein (MCP)-1, interferon (IFN) γ, and tumor necrosis factor (TNF)-α were detected in lungs and liver upon B100 and D100 CE exposures. Overall, the tissue damage, oxidative stress, inflammation, and cytokine response were more pronounced in mice exposed to BD CE. Further studies are required to understand what combustion products in BD CE accelerate oxidative and inflammatory responses.

Factoring-in agglomeration of carbon nanotubes and nanofibers for better prediction of their toxicity versus asbestos.

BACKGROUND: Carbon nanotubes (CNT) and carbon nanofibers (CNF) are allotropes of carbon featuring fibrous morphology. The dimensions and high aspect ratio of CNT and CNF have prompted the comparison with naturally occurring asbestos fibers which are known to be extremely pathogenic. While the toxicity and hazardous outcomes elicited by airborne exposure to single-walled CNT or asbestos have been widely reported, very limited data are currently available describing adverse effects of respirable CNF. RESULTS: Here, we assessed pulmonary inflammation, fibrosis, oxidative stress markers and systemic immune responses to respirable CNF in comparison to single-walled CNT (SWCNT) and asbestos. Pulmonary inflammatory and fibrogenic responses to CNF, SWCNT and asbestos varied depending upon the agglomeration state of the particles/fibers. Foci of granulomatous lesions and collagen deposition were associated with dense particle-like SWCNT agglomerates, while no granuloma formation was found following exposure to fiber-like CNF or asbestos. The average thickness of the alveolar connective tissue--a marker of interstitial fibrosis--was increased 28 days post SWCNT, CNF or asbestos exposure. Exposure to SWCNT, CNF or asbestos resulted in oxidative stress evidenced by accumulations of 4-HNE and carbonylated proteins in the lung tissues. Additionally, local inflammatory and fibrogenic responses were accompanied by modified systemic immunity, as documented by decreased proliferation of splenic T cells ex vivo on day 28 post exposure. The accuracies of assessments of effective surface area for asbestos, SWCNT and CNF (based on geometrical analysis of their agglomeration) versus estimates of mass dose and number of particles were compared as predictors of toxicological outcomes. CONCLUSIONS: We provide evidence that effective surface area along with mass dose rather than specific surface area or particle number are significantly correlated with toxicological responses to carbonaceous fibrous nanoparticles. Therefore, they could be useful dose metrics for risk assessment and management.

Pulmonary exposure to single-walled carbon nanotubes does not affect the early immune response against Toxoplasma gondii.

BACKGROUND: Single-walled carbon nanotubes (SWCNT) trigger pronounced inflammation and fibrosis in the lungs of mice following administration via pharyngeal aspiration or inhalation. Human exposure to SWCNT in an occupational setting may occur in conjunction with infections and this could yield enhanced or suppressed responses to the offending agent. Here, we studied whether the sequential exposure to SWCNT via pharyngeal aspiration and infection of mice with the ubiquitous intracellular parasite Toxoplasma gondii would impact on the immune response of the host against the parasite. METHODS: C57BL/6 mice were pre-exposed by pharyngeal administration of SWCNT (80 + 80 µg/mouse) for two consecutive days followed by intravenous injection with either 1x103 or 1x104 green fluorescence protein and luciferase-expressing T. gondii tachyzoites. The dissemination of T. gondii was monitored by in vivo bioluminescence imaging in real time for 7 days and by plaque formation. The inflammatory response was analysed in bronchoalveolar lavage (BAL) fluid, and by assessment of morphological changes and immune responses in lung and spleen. RESULTS: There were no differences in parasite distribution between mice only inoculated with T. gondii or those mice pre-exposed for 2 days to SWCNT before parasite inoculum. Lung and spleen histology and inflammation markers in BAL fluid reflected the effects of SWCNT exposure and T. gondii injection, respectively. We also noted that CD11c positive dendritic cells but not F4/80 positive macrophages retained SWCNT in the lungs 9 days after pharyngeal aspiration. However, co-localization of T. gondii with CD11c or F4/80 positive cells could not be observed in lungs or spleen. Pre-exposure to SWCNT did not affect the splenocyte response to T. gondii. CONCLUSIONS: Taken together, our data indicate that pre-exposure to SWCNT does not enhance or suppress the early immune response to T. gondii in mice.

MDSC and TGFß Are Required for Facilitation of Tumor Growth in the Lungs of Mice Exposed to Carbon Nanotubes.

During the last decades, changes have been observed in the frequency of different histologic subtypes of lung cancer, one of the most common causes of morbidity and mortality, with a declining proportion of squamous cell carcinomas and an increasing proportion of adenocarcinomas, particularly in developed countries. This suggests the emergence of new etiologic factors and mechanisms, including those defining the lung microenvironment, promoting tumor growth. Assuming that the lung is the main portal of entry for broadly used nanomaterials and their established proinflammatory propensities, we hypothesized that nanomaterials may contribute to changes facilitating tumor growth. Here, we report that an acute exposure to single-walled carbon nanotubes (SWCNT) induces recruitment and accumulation of lung-associated myeloid-derived suppressor cells (MDSC) and MDSC-derived production of TGFß, resulting in upregulated tumor burden in the lung. The production of TGFß by MDSC requires their interaction with both SWCNT and tumor cells. We conclude that pulmonary exposure to SWCNT favors the formation of a niche that supports ingrowth of lung carcinoma in vivo via activation of TGFß production by SWCNT-attracted and -presensitized MDSC.

Graphene oxide attenuates Th2-type immune responses, but augments airway remodeling and hyperresponsiveness in a murine model of asthma.

Several lines of evidence indicate that exposure to nanoparticles (NPs) is able to modify airway immune responses, thus facilitating the development of respiratory diseases. Graphene oxide (GO) is a promising carbonaceous nanomaterial with unique physicochemical properties, envisioned for a multitude of medical and industrial applications. In this paper, we determined how exposure to GO modulates the allergic pulmonary response. Using a murine model of ovalbumin (OVA)-induced asthma, we revealed that GO, given at the sensitization stage, augmented airway hyperresponsiveness and airway remodeling in the form of goblet cell hyperplasia and smooth muscle hypertrophy. At the same time, the levels of the cytokines IL-4, IL-5, and IL-13 were reduced in broncho-alveolar lavage (BAL) fluid in GO-exposed mice. Exposure to GO during sensitization with OVA decreased eosinophil accumulation and increased recruitment of macrophages in BAL fluid. In line with the cytokine profiles, sensitization with OVA in the presence of GO stimulated the production of OVA-specific IgG2a and down-regulated the levels of IgE and IgG1. Moreover, exposure to GO increased the macrophage production of the mammalian chitinases, CHI3L1 and AMCase, whose expression is associated with asthma. Finally, molecular modeling has suggested that GO may directly interact with chitinase, affecting AMCase activity, which has been directly proven in our studies. Thus, these data show that GO exposure attenuates Th2 immune response in a model of OVA-induced asthma, but leads to potentiation of airway remodeling and hyperresponsiveness, with the induction of mammalian chitinases.

Citrullination of proteins: a common post-translational modification pathway induced by different nanoparticles in vitro and in vivo.

AIM: Rapidly expanding manufacture and use of nanomaterials emphasize the requirements for thorough assessment of health outcomes associated with novel applications. Post-translational protein modifications catalyzed by Ca(2+)-dependent peptidylargininedeiminases have been shown to trigger immune responses including autoantibody generation, a hallmark of immune complexes deposition in rheumatoid arthritis. Therefore, the aim of the study was to assess if nanoparticles are able to promote protein citrullination. MATERIALS & METHODS: Human A549 and THP-1 cells were exposed to silicon dioxide, carbon black or single-walled carbon nanotubes. C57BL/6 mice were exposed to respirable single-walled carbon nanotubes. Protein citrullination, peptidylargininedeiminases activity and target proteins were evaluated. RESULTS: The studied nanoparticles induced protein citrullination both in cultured human cells and mouse lung tissues. Citrullination occurred via the peptidylargininedeiminase-dependent mechanism. Cytokeratines 7, 8, 18 and plectins were identified as intracellular citrullination targets. CONCLUSION: Nanoparticle exposure facilitated post-translational citrullination of proteins.

Direct effects of carbon nanotubes on dendritic cells induce immune suppression upon pulmonary exposure.

Pharyngeal aspiration of single-walled carbon nanotubes (SWCNTs) caused inflammation, pulmonary damage, and an altered cytokine network in the lung. Local inflammatory response in vivo was accompanied by modified systemic immunity as documented by decreased proliferation of splenic T cells. Preincubation of naïve T cells in vitro with SWCNT-treated dendritic cells reduced proliferation of T cells. Our data suggest that in vivo exposure to SWCNT modifies systemic immunity by modulating dendritic cell function.