Multi-walled carbon nanotubes elicit concordant changes in DNA methylation and gene expression following long-term pulmonary exposure in mice.

Pulmonary exposure to multi-walled carbon nanotubes (MWCNTs) causes inflammation and fibrosis. Our previous work has shown that industrially produced MWCNTs trigger specific changes in gene expression in the lungs of exposed animals. To elucidate whether epigenetic effects play a role for these gene expression changes, we performed whole genome bisulphite sequencing to assess DNA methylation patterns in the lungs 56 days after exposure to MWCNTs. Lung tissues were also evaluated with respect to histopathological changes and cytokine profiling of bronchoalveolar lavage (BAL) fluid was conducted using a multi-plex array. Integrated analysis of transcriptomics data and DNA methylation data revealed concordant changes in gene expression. Functional analysis showed that the muscle contraction, immune system/inflammation, and extracellular matrix pathways were the most affected pathways. Taken together, the present study revealed that MWCNTs exert epigenetic effects in the lungs of exposed animals, potentially driving the subsequent gene expression changes.

Comparative analysis of lung and blood transcriptomes in mice exposed to multi-walled carbon nanotubes.

Pulmonary exposure to multi-walled carbon nanotubes (MWCNT) causes inflammation, fibroproliferation, immunotoxicity, and systemic responses in rodents. However, the search for representative biomarkers of exposure is an ongoing endeavor. Whole blood gene expression profiling is a promising new approach for the identification of novel disease biomarkers. We asked if the whole blood transcriptome reflects pathology-specific changes in lung gene expression caused by MWCNT. To answer this question, we performed mRNA sequencing analysis of the whole blood and lung in mice administered MWCNT or vehicle solution via pharyngeal aspiration and sacrificed 56 days later. The pattern of lung mRNA expression as determined using Ingenuity Pathway Analysis (IPA) was indicative of continued inflammation, immune cell trafficking, phagocytosis, and adaptive immune responses. Simultaneously, innate immunity-related transcripts (Plunc, Bpifb1, Reg3g) and cancer-related pathways were downregulated. IPA analysis of the differentially expressed genes in the whole blood suggested increased hematopoiesis, predicted activation of cancer/tumor development pathways, and atopy. There were several common upregulated genes between whole blood and lungs, important for adaptive immune responses: Cxcr1, Cd72, Sharpin, and Slc11a1. Trim24, important for TH2 cell effector function, was downregulated in both datasets. Hla-dqa1 mRNA was upregulated in the lungs and downregulated in the blood, as was Lilrb4, which controls the reactivity of immune response. "Cancer" disease category had opposing activation status in the two datasets, while the only commonality was "Hypersensitivity". Transcriptome changes occurring in the lungs did not produce a completely replicable pattern in whole blood; however, specific systemic responses may be shared between transcriptomic profiles.

Comparative cytotoxicity of respirable surface-treated/untreated calcium carbonate rock dust particles in vitro.

Calcium carbonate rock dust (RD) is used in mining to reduce the explosivity of aerosolized coal. During the dusting procedures, potential for human exposure occurs, raising health concerns. To improve RD aerosolization, several types of anti-caking surface treatments exist. The aim of the study was to evaluate cytotoxicity of four respirable RD samples: untreated/treated limestone (UL/TL), untreated/treated marble (UM/TM), and crystalline silica (SiO2) as a positive control in A549 and THP-1 transformed human cell lines. Respirable fractions were generated and collected using FSP10 high flow-rate cyclone samplers. THP-1 cells were differentiated with phorbol-12-myristate-13-acetate (20 ng/ml, 48 h). Cells were exposed to seven different concentrations of RD and SiO2 (0-0.2 mg/ml). RD caused a slight decrease in viability at 24 or 72 h post-exposure and were able to induce inflammatory cytokine production in A549 cells, however, with considerably less potency than SiO2. In THP-1 cells at 24 h, there was significant dose-dependent lactate dehydrogenase, inflammatory cytokine and chemokine release. Caspase-1 activity was increased in SiO2- and, on a lesser scale, in TM- exposed cells. To test if the increased toxicity of TM was uptake-related, THP-1 cells were pretreated with Cytochalasin D (CytD) or Bafilomycin A (BafA), followed by exposure to RD or SiO2 for 6 h. CytD blocked the uptake and significantly decreased cytotoxicity of all particles, while BafA prevented caspase-1 activation but not cytotoxic effects of TM. Only TM was able to induce an inflammatory response in THP-1 cells, however it was much less pronounced compared to silica.

Mediation of the single-walled carbon nanotubes induced pulmonary fibrogenic response by osteopontin and TGF-ß1.

PURPOSE OF THE STUDY: A number of in vivo studies have shown that pulmonary exposure to carbon nanotubes (CNTs) may lead to an acute local inflammatory response, pulmonary fibrosis, and granulomatous lesions. Among the factors that play direct roles in initiation and progression of fibrotic processes are epithelial-mesenchymal transition and myofibroblasts recruitment/differentiation, both mediated by transforming growth factor-ß1 (TGF-ß1). Yet, other contributors to TGF-ß1 associated signaling, such as osteopontin (OPN) has not been fully investigated. MATERIALS AND METHODS: OPN-knockout female mice (OPN-KO) along with their wild-type (WT) counterparts were exposed to single-walled carbon nanotubes (SWCNT) (40 µg/mouse) via pharyngeal aspiration and fibrotic response was assessed 1, 7, and 28 days post-exposure. Simultaneously, RAW 264.7 and MLE-15 cells were treated with SWCNT (24 hours, 6 µg/cm2 to 48 µg/cm2) or bleomycin (0.1 µg/ml) in the presence of OPN-blocking antibody or isotype control, and TGF-ß1 was measured in supernatants. RESULTS AND CONCLUSIONS: Diminished lactate dehydrogenase activity at all time points, along with less pronounced neutrophil influx 24 h post-exposure, were measured in broncho-alveolar lavage (BAL) of OPN-KO mice compared to WT. Pro-inflammatory cytokine release (IL-6, TNF-α, MCP-1) was reduced. A significant two-fold increase of TGF-ß1 was found in BAL of WT mice at 7 days, while TGF-ß1 levels in OPN-KO animals remained unaltered. Histological examination revealed marked decrease in granuloma formation and less collagen deposition in the lungs of OPN-KO mice compared to WT. RAW 264.7 but not MLE-15 cells exposed to SWCNT and bleomycin had significantly less TGF-ß1 released in the presence of OPN-blocking antibody. We believe that OPN is important in initiating the cellular mechanisms that produce an overall pathological response to SWCNT and it may act upstream of TGF-ß1. Further investigation to understand the mechanistic details of such interactions is critical to predict outcomes of pulmonary exposure to CNT.

Fibrosis biomarkers in workers exposed to MWCNTs.

Multi-walled carbon nanotubes (MWCNT) with their unique physico-chemical properties offer numerous technological advantages and are projected to drive the next generation of manufacturing growth. As MWCNT have already found utility in different industries including construction, engineering, energy production, space exploration and biomedicine, large quantities of MWCNT may reach the environment and inadvertently lead to human exposure. This necessitates the urgent assessment of their potential health effects in humans. The current study was carried out at NanotechCenter Ltd. Enterprise (Tambov, Russia) where large-scale manufacturing of MWCNT along with relatively high occupational exposure levels was reported. The goal of this small cross-sectional study was to evaluate potential biomarkers during occupational exposure to MWCNT. All air samples were collected at the workplaces from both specific areas and personal breathing zones using filter-based devices to quantitate elemental carbon and perform particle analysis by TEM. Biological fluids of nasal lavage, induced sputum and blood serum were obtained from MWCNT-exposed and non-exposed workers for assessment of inflammatory and fibrotic markers. It was found that exposure to MWCNTs caused significant increase in IL-1ß, IL6, TNF-α, inflammatory cytokines and KL-6, a serological biomarker for interstitial lung disease in collected sputum samples. Moreover, the level of TGF-ß1 was increased in serum obtained from young exposed workers. Overall, the results from this study revealed accumulation of inflammatory and fibrotic biomarkers in biofluids of workers manufacturing MWCNTs. Therefore, the biomarkers analyzed should be considered for the assessment of health effects of occupational exposure to MWCNT in cross-sectional epidemiological studies.

In Vitro Toxicity Evaluation of Lignin-(Un)coated Cellulose Based Nanomaterials on Human A549 and THP-1 Cells.

A significant amount of research toward commercial development of cellulose based nanomaterials (CNM) is now in progress with some potential applications. Using human A549 and THP-1 cells, we evaluated the biological responses of various CNMs, made out of similar material but with functional and morphological variations. While A549 cells displayed minimal or no cytotoxic responses following exposure to CNMs, THP-1 cells were more susceptible to cytotoxicity, cellular damage and inflammatory responses. Further analysis of these biological responses evaluated using hierarchical clustering approaches was effective in discriminating (dis)-similarities of various CNMs studied and identified potential inflammatory factors contributing to cytotoxicity. No correlation between cytotoxicity and surface properties of CNMs was found. This study clearly highlights that, in addition to the source and characteristics of CNMs, cell type-specific differences in the recognition/uptake of CNMs along with their inherent capability to respond to external stimuli are crucial for assessing the toxicity of CNMs.

Fibrous nanocellulose, crystalline nanocellulose, carbon nanotubes, and crocidolite asbestos elicit disparate immune responses upon pharyngeal aspiration in mice.

With the rapid development of synthetic alternatives to mineral fibers, their possible effects on the environment and human health have become recognized as important issues worldwide. This study investigated effects of four fibrous materials, i.e. nanofibrillar/nanocrystalline celluloses (NCF and CNC), single-walled carbon nanotubes (CNTs), and crocidolite asbestos (ASB), on pulmonary inflammation and immune responses found in the lungs, as well as the effects on spleen and peripheral blood immune cell subsets. BALB/c mice were given NCF, CNC, CNT, and ASB on Day 1 by oropharyngeal aspiration. At 14 days post-exposure, the animals were evaluated. Total cell number, mononuclear phagocytes, polymorphonuclear leukocytes, lymphocytes, and LDH levels were significantly increased in ASB and CNT-exposed mice. Expression of cytokines and chemokines in bronchoalveolar lavage (BAL) was quite different in mice exposed to four particle types, as well as expression of antigen presentation-related surface proteins on BAL cells. The results revealed that pulmonary exposure to fibrous materials led to discrete local immune cell polarization patterns with a TH2-like response caused by ASB and TH1-like immune reaction to NCF, while CNT and CNC caused non-classical or non-uniform responses. These alterations in immune response following pulmonary exposure should be taken into account when testing the applicability of new nanosized materials with fibrous morphology.