Single-walled Carbon Nanotubes Exposure Research Articles

Impaired autophagy-accelerated senescence of alveolar type II epithelial cells drives pulmonary fibrosis induced by single-walled carbon nanotubes

BackgroundThe rapid increase in production and application of carbon nanotubes (CNTs) has led to wide public concerns in their potential risks to human health. Single-walled CNTs (SWCNTs), as an extensively applied type of CNTs, have shown strong capacity to induce pulmonary fibrosis in animal models, however, the intrinsic mechanisms remain uncertain.ResultsIn vivo experiments, we showed that accelerated senescence of alveolar type II epithelial cells (AECIIs) was associated with pulmonary fibrosis in SWCNTs-exposed mice, as well as SWCNTs-induced fibrotic lungs exhibited impaired autophagic flux in AECIIs in a time dependent manner. In vitro, SWCNTs exposure resulted in profound dysfunctions of MLE-12 cells, characterized by impaired autophagic flux and accelerated cellular senescence. Furthermore, the conditioned medium from SWCNTs-exposed MLE-12 cells promoted fibroblast-myofibroblast transdifferentiation (FMT). Additionally, restoration of autophagy flux with rapamycin significantly alleviated SWCNTs-triggered senescence and subsequent FMT whereas inhibiting autophagy using 3-MA aggravated SWCNTs-triggered senescence in MLE-12 cells and FMT.ConclusionSWCNTs trigger senescence of AECIIs by impairing autophagic flux mediated pulmonary fibrosis. The findings raise the possibility of senescence-related cytokines as potential biomarkers for the hazard of CNTs exposure and regulating autophagy as an appealing target to halt CNTs-induced development of pulmonary fibrosis.Graphical

Trophic Transfer of Single-Walled Carbon Nanotubes at the Base of the Food Chain and Toxicological Response.

The potential for trophic transfer of single-walled carbon nanotubes (SWCNTs) was assessed using the green algae Tetraselmis suecica and the blue mussel Mytilus edulis in a series of laboratory experiments. Swanee River Natural Organic Matter (SRNOM)-dispersed SWCNTs were introduced into growing algal cultures. Light microscopical observations, confirmed by scanning electronic microscopy (SEM) and Raman spectroscopy, showed that SWCNT agglomerates adhered to the external algal cell walls and transmission electronic microscopy (TEM) results suggested internalization. A direct effect of SWCNT exposure on the algae was a significant decrease in growth, expressed as chlorophyll a concentration and cell viability. Mussels, fed with algae in the presence of SWCNTs, led to significantly increased pseudofaeces production, indicating selective feeding. Nevertheless, histological sections of the mussel digestive gland following exposure showed evidence of SWCNT-containing algae. Furthermore, DNA damage and oxidative stress biomarker responses in the mussel haemocytes and gill tissue were significantly altered from baseline values and were consistent with previously observed responses to SWCNT exposure. In conclusion, the observed SWCNT-algal interaction demonstrated the potential for SWCNT entrance at the base of the food chain, which may facilitate their trophic transfer with potential consequences for human exposure and health.

Exposure of carbon nanotubes affects testis and brain of common carp

Carbon nanotubes production has been rapidly increasing for many potential applications, however, the environmental impact of this nanomaterial needs to be comprehended. The present work focused on unraveling the effects of single-walled carbon nanotubes (SWCNT) in the common carp, Cyprinus carpio. The physicochemical properties of SWCNT were analyzed with X-ray diffraction, Fourier transforms infra-red, UV-Vis absorption, transmission electron microscopy (TEM), and Raman spectroscopy before testing for exposure impact. The effects of SWCNT, were investigated by exposing to two doses viz., 10 and 50 μg/L, for 7 days in adult common carp, in vivo. Expression of key steroidogenic and transcription factor genes related to testis and brain were downregulated after the treatment. The concomitant decreases in serum testosterone and 11-ketotestosterone levels revealed the impact of SWCNT after exposure. Further, SWCNT exposure induced antioxidant enzymes namely glutathione-S-transferases, superoxide dismutase, and catalase in both testis and brain. Concurrently, histological and TEM analysis of testis revealed structural disarray. In addition, SWCNT treatment, in testicular and brain primary cell cultures decreased cell viability with an increase of reactive oxygen species levels, leading to a significant elevation of apoptotic cells. In line with this, low mitochondrial membrane potential and DNA damage were also observed during post SWCNT treatment. Taken together, transient exposure of SWCNT causes toxic effects and alters testicular and brain function in the common carp. Thus, the discharge of carbon nanotubes poses a greater risk to the aquatic environment warranting regulatory measures.

Carbon Nanotubes Induce Metabolomic Profile Disturbances in Zebrafish: NMR-Based Metabolomics Platform.

The present study aims to investigate the metabolic effects of single-walled carbon nanotubes (SWCNT) on zebrafish (Danio rerio) using 1H nuclear magnetic resonance (1H-NMR) spectroscopy. However, there is no significant information available regarding the characterization of organic molecules, and metabolites with SWCNT exposure. Noninvasive biofluid methods have improved our understanding of SWCNT metabolism in zebrafish in recent years. Here, we used targeted metabolomics to quantify a set of metabolites within biological systems. SWCNT at various concentrations was given to zebrafish, and the metabolites were extracted using two immiscible solvent systems, methanol and chloroform. Metabolomics profiling was used in association with univariate and multivariate data analysis to determine metabolomic phenotyping. The metabolites, malate, oxalacetate, phenylaniline, taurine, sn-glycero-3-phosphate, glycine, N-acetyl mate, lactate, ATP, AMP, valine, pyruvate, ADP, serine, niacinamide are significantly impacted. The metabolism of amino acids, energy and nucleotides are influenced by SWCNT which might indicate a disturbance in metabolic reaction networks. In conclusion, using high-throughput analytical methods, we provide a perspective of metabolic impacts and the underlying associated metabolic pathways.

Postchronic Single-Walled Carbon Nanotube Exposure Causes Irreversible Malignant Transformation of Human Bronchial Epithelial Cells through DNA Methylation Changes.

As environmental pollutants and possible carcinogens, carbon nanotubes (CNTs) have recently been found to induce carcinogenesis and tumor metastasis after long-term pulmonary exposure. However, whether CNT-induced carcinogenesis can be inherited and last for generations remains unclear. Herein, postchronic single-walled carbon nanotubes (SWCNTs) exposed human lung cell model (BEAS-2B cells) are established to investigate SWCNT-induced carcinogenesis. At a tolerated sublethal dose level, postchronic SWCNT exposure significantly increases the migration and invasion abilities of BEAS-2B cells, leading to malignant cell transformation. Notably, the malignant transformation of BEAS-2B cells is irreversible within a 60 day recovery period after SWCNT exposure, and the malignant transformation activities of cells gradually increase during the recovery period. Moreover, these transformed cells promote carcinogenesis in vivo, accompanied by a raised level of biomarkers of lung adenocarcinoma. Further mechanism analyses reveal that postchronic exposure to SWCNTs causes substantial DNA methylation and transcriptome dysregulation of BEAS-2B cells. Subsequent enrichment and clinical database analyses reveal that differentially expressed/methylated genes of BEAS-2B cells are enriched in cancer-related biological pathways. These results not only demonstrate that postchronic SWCNT-exposure-induced carcinogenesis is heritable but also uncover a mechanism from the perspective of DNA methylation.

Single-Walled Carbon Nanotubes Attenuate Cytotoxic and Oxidative Stress Response of Pb in Human Lung Epithelial (A549) Cells.

Combined exposure of single-walled carbon nanotubes (SWCNTs) and trace metal lead (Pb) in ambient air is unavoidable. Most of the previous studies on the toxicity of SWCNTs and Pb have been conducted individually. There is a scarcity of information on the combined toxicity of SWCNTs and Pb in human cells. This work was designed to explore the combined effects of SWCNTs and Pb in human lung epithelial (A549) cells. SWCNTs were prepared through the plasma-enhanced vapor deposition technique. Prepared SWCNTs were characterized by x-ray diffraction, x-ray photoelectron spectroscopy, scanning electron microscopy, and dynamic light scattering. We observed that SWCNTs up to a concentration of 100 µg/mL was safe, while Pb induced dose-dependent (5–100 µg/mL) cytotoxicity in A549 cells. Importantly, cytotoxicity, cell cycle arrest, mitochondrial membrane potential depletion, lipid peroxidation, and induction of caspase-3 and -9 enzymes following Pb exposure (50 µg/mL for 24 h) were efficiently attenuated by the co-exposure of SWCNTs (10 µg/mL for 24 h). Furthermore, generation of Pb-induced pro-oxidants (reactive oxygen species and hydrogen peroxide) and the reduction of antioxidants (antioxidant enzymes and glutathione) were also mitigated by the co-exposure of SWCNTs. Inductively coupled plasma-mass spectrometry results suggest that the adsorption of Pb on the surface of SWCNTs could attenuate the bioavailability and toxicity of Pb in A549 cells. Our data warrant further research on the combined effects of SWCNTs and Pb in animal models.

More serious autophagy can be induced by ZnO nanoparticles than single-walled carbon nanotubes in rat tracheal epithelial cells.

Metal oxide nanoparticles and carbon nanoparticles, as common nanoparticles (NPs), can cause autophagy in certain cells, which will lead to biohealth risk issues. This study determined the difference in autophagy induced by zinc oxide nanoparticles (ZnO NPs) and single-walled carbon nanotubes (SWCNTs) in respiratory epithelial cells. ICP-OES results showed that NPs uptake as well as the intercellular contents of particles affected cytotoxicity in a dose-dependent manner. ZnO NPs-30 nm had a distinct green dot structure representing autophagy, the SWCNTs exposure group had a few green light spots at a concentration of 10 μg/L. The ROS content of the ZnO NP-30 nm exposure group had the greatest increase at a concentration of 1000 μg/L, which was 2.5 times higher than that of the control, the SWCNTs exposure group showed a 2.2-fold increase. A slight downregulation of p-mTOR was detected, and the ZnO NPs-30 nm treatment group had the significant downregulation rate. The gene and protein expression levels of Beclin-1 and LC3B were upregulated as the exposure concentration increased. The protein expression of Beclin-1 and LC3B in the 1000 μg/L ZnO NPs-30 nm exposure group were 5.21 times and 4.12 times that of the control, respectively. The mRNA expression of Beclin-1 and LC3B in the 1000 μg/L ZnO NPs-30 nm exposure group were 5.04 times and 3.61 times that of the control, respectively. At any concentration, the effect of ZnO NPs-30 nm was greater than that of the SWCNTs. Interaction and crosstalk analysis showed that exposure to ZnO NPs-30 nm caused autophagy through the aggregation of undegraded autophagosomes, whereas SWCNTs exposure induced diminished intercellular oxidative stress to inhibit autophagy. Therefore, this study demonstrated that the effects of autophagy induced by ZnO NPs-30 nm and SWCNTs were different. The health risks of ZnO-30 nm NPs are higher than those of SWCNTs.

Favorable influence of ssDNA-functionalized SWCNT on the navigation pattern of C. elegans

Single-Walled Carbon Nanotubes (SWCNTs) are widely used as potential carriers in drug delivery systems. The objective of this work was to observe the effects of pristine, and single-stranded DNA oligonucleotide functionalized SWCNT over the locomotion pattern in the nematode model, Caenorhabditis elegans. The implication of ssDNA in the as-modified SWCNT has been understood in terms of lowering the toxic effects of the pristine SWCNT and thereby normalizing the altered navigation pattern of the worms. Chemotaxis assay displayed an increased migration of the worms towards pristine and ssDNA functionalized SWCNT in comparison to ultrapure water as control. Additionally, paralytic movement was observed with SWCNT exposure in contrast to ssDNA-functionalized SWCNT-exposed animals. High doses of pristine SWCNT further displayed an increased expression of specific proteins obtained from the total protein extracts of the animals. Thus, the alteration in the navigation pattern of the worms, treated with pristine SWCNT, might be ascribed as a result of differentially expressed muscle proteins. Moreover, it is interesting to note that the modified SWCNT was effective in alleviating the atypical form of the inherent movements of the worm. Future investigation will further reveal the protein targets responsible for the altered navigation pattern of the worms.

(Amazon Catalyst at ECS Grant Winner) Carbon Catalysts for Cost-Efficient Hydrogen Production in PEM Electrolyzers

Exposure of single-walled carbon nanotubes (CNTs), as well as other lower cost layered graphitic carbons (LGCs), to acidic intercalants, in combination with low voltage electrochemical cycling, induces in these materials hydrogen evolution activity that initiates at near zero overpotential and is on par with that of platinum catalysts.1 These results encouraged the further development and testing of the CNT catalyst in a PEM water splitting electrolyzer prototype. Once activated, the CNT cathode demonstrated 1 A/cm2 of HER current at 1.64 V, comparable to a similarly tested commercial Pt-loaded cathode. While carbon nanotubes are not intrinsically scarce, the single wall nanotubes used in these electrode tests remain relatively expensive. Commercial significance would benefit from a demonstration of comparable performance from activated lower cost layered graphitic carbons in a PEM electrolyzer prototype. The following LGC materials, having a combination of high electrical conductivity, large pore volumes, and high specific surface areas, were tested in terms of their ability to achieve low HER overpotential during the activation process: microcrystalline graphitic particles, Vulcan XC-72 carbon, highly graphitized stacked-cup tubular carbon nanofibers (CNFs), and carbon needle felt. CNF electrodes demonstrated slightly higher mass basis activity than that of the other tested LGCs in acid electrolyte in a standard three-terminal electrochemical cell. Here we report excellent performance from activated CNF cathodes, prepared by a scalable spray-coating method, in our PEM electrolyzer prototype (IrRuOx as anode catalyst). As anticipated, a control MEA, based on the non-activated CNF cathode and tested in PEM electrolyzer under identical conditions, showed poor performance, indicating a low electrocatalytic HER activity of the electrode. The activated CNF cathode-based PEM electrolyzer achieved 1 A/cm2 at 1.78 V, while a control commercial MEA with Pt-loaded cathode/IrRuOx anode (a high Pt catalyst loading of 3 mg/cm2 cathode, Fuel Cells Etc) tested in the same PEM electrolyzer cell demonstrated 1 A/cm2 at 1.7 V. Acknowledgements. This work is supported by Amazon through ECS Amazon Catalyst Program.

Ecological Effects of Single-Walled Carbon Nanotubes on Soil Microbial Communities and Soil Fertility

The manufacturers of single-walled carbon nanotubes (SWCNTs) are continuously expanding their manufacturing and commercial markets, indicating that the environmental release and accumulation of SWCNTs in soil is inevitable. However, little is known about the effects of SWCNTs on soil physicochemical properties and soil microbial communities. Our results showed that treatment with SWCNTs resulted in an enhancement of microorganism metabolism related to soil organic compound degradation and a change in the structure of soil microbial communities, but the diversity of soil microorganisms was not significantly affected. The decrease in soil urease activity and the increase in the relative abundance of Nitrospirae after SWCNTs exposure might be relevant to the induction of soil nitrification. The relative abundances of phosphate-solubilizing microorganisms increased after exposure to SWCNTs, which was beneficial for phosphorus bioavailability in the soil. Our current study highlights that exposure to SWCNTs at concentrations of 3 and 10µg/g can change the composition of soil microorganism communities, promote soil organic degradation and improve soil fertility by enhancing N and P availability in a short time.

The viability of nerve cells exposed to SWCNTs used in sport equipment and the protection effect of vitamin C

ABSTRACTTo explore the viability of the nerve response model cell (mouse N2a) induced by the exposure of single-walled carbon nanotubes (SWCNTs). N2a cells were exposed to SWCNTs suspension at different concentrations (0, 12. 5, 25, 50 μg·ml−1) for 24 h. MTT assay was used to analyze the viability of N2a cells. Additionally, reactive oxygen species (ROS) and glutathione (GSH) levels were measured to determine the oxidative stress. It was demonstrated that as the exposure concentration to SWNTs increased, the vitality of N2a cells decreased, ROS content increased and GSH content decreased. In existence of vitamin C (Vit C), cell damage in the experimental group treated with SWNTs of 50 μg·ml−1, the highest exposure concentration, was alleviated to the maximum extent. Results suggested that exposure to SWCNTs could induce decreased viability in N2a cells to certain extent via oxidative stress pathway. However, as antioxidant, Vit C could protect N2a cells by reducing the level of oxidative damage.

Differences in MWCNT- and SWCNT-induced DNA methylation alterations in association with the nuclear deposition

BackgroundSubtle DNA methylation alterations mediated by carbon nanotubes (CNTs) exposure might contribute to pathogenesis and disease susceptibility. It is known that both multi-walled carbon nanotubes (MWCNTs) and single-walled carbon nanotubes (SWCNTs) interact with nucleus. Such, nuclear-CNT interaction may affect the DNA methylation effects.In order to understand the epigenetic toxicity, in particular DNA methylation alterations, of SWCNTs and short MWCNTs, we performed global/genome-wide, gene-specific DNA methylation and RNA-expression analyses after exposing human bronchial epithelial cells (16HBE14o- cell line). In addition, the presence of CNTs on/in the cell nucleus was evaluated in a label-free way using femtosecond pulsed laser microscopy.ResultsGenerally, a higher number of SWCNTs, compared to MWCNTs, was deposited at both the cellular and nuclear level after exposure. Nonetheless, both CNT types were in physical contact with the nuclei. While particle type dependency was noticed for the identified genome-wide and gene-specific alterations, no global DNA methylation alteration on 5-methylcytosine (5-mC) sites was observed for both CNTs. After exposure to MWCNTs, 2398 genes were hypomethylated (at gene promoters), and after exposure to SWCNTs, 589 CpG sites (located on 501 genes) were either hypo- (N = 493 CpG sites) or hypermethylated (N = 96 CpG sites).Cells exposed to MWCNTs exhibited a better correlation between gene promoter methylation and gene expression alterations. Differentially methylated and expressed genes induced changes (MWCNTs > SWCNTs) at different cellular pathways, such as p53 signalling, DNA damage repair and cell cycle. On the other hand, SWCNT exposure showed hypermethylation on functionally important genes, such as SKI proto-oncogene (SKI), glutathione S-transferase pi 1 (GTSP1) and shroom family member 2 (SHROOM2) and neurofibromatosis type I (NF1), which the latter is both hypermethylated and downregulated.ConclusionAfter exposure to both types of CNTs, epigenetic alterations may contribute to toxic or repair response. Moreover, our results suggest that the observed differences in the epigenetic response depend on particle type and differential CNT-nucleus interactions.

Carbon nanotubes physicochemical properties influence the overall cellular behavior and fate

The unique properties of single walled carbon nanotubes (SWCNTs) make them viable candidates for versatile implementation in the next generation of biomedical devices for targeted delivery of chemotherapeutic agents or cellular-sensing probes. Such implementation requires user-tailored changes in SWCNT's physicochemical characteristics to allow for efficient cellular integration while maintaining nanotubes’ functionality. However, isolated reports showed that user-tailoring could induce deleterious effects in exposed cells, from decrease in cellular proliferation, to changes in cellular adhesion, generation of reactive oxygen species or phenotypical variations, just to name a few. Before full implementation of SWCNTs is achieved, their toxicological profiles need to be mechanistically correlated with their physicochemical properties to determine how the induced cellular fate is related to the exposure conditions or samples' characteristics. Our study provides a comprehensive analysis of the synergistic cyto- and genotoxic effects resulted from short-term exposure of human lung epithelial cells to pristine (as manufactured) and user-tailored SWCNTs, as a function of their physicochemical properties. Specifically, through a systematic approach we are correlating the nanotube uptake and nanotube-induced cellular changes to the sample's physicochemical characteristics (e.g., metal impurities, length, agglomerate size, surface area, dispersion, and surface functionalization). By identifying changes in active hallmarks involved in cell-cell connections and maintaining epithelial layer integrity, we also determine the role that short-term exposure to SWCNTs plays in the overall cellular fate and cellular transformation. Lastly, we assess cellular structure-function relationships to identify non-apoptotic pathways induced by SWCNTs exposure that could however lead to changes in cellular behavior and cellular transformation. Our results show that the degree of cell transformation is a function of the physicochemical properties of the SWCNT, with the nanotube with higher length, higher metal content and larger agglomerate size reducing cell viability to a larger extent. Such changes in cell viability are also complemented by changes in cell structure, cycle and cell-cell interactions, all responsible for maintaining cell fate.

Modelling the quantitative aspects of AOPs

The unique properties of single walled carbon nanotubes (SWCNTs) make them viable candidates for versatile implementation in the next generation of biomedical devices for targeted delivery of chemotherapeutic agents or cellular-sensing probes. Such implementation requires user-tailored changes in SWCNT's physicochemical characteristics to allow for efficient cellular integration while maintaining nanotubes’ functionality. However, isolated reports showed that user-tailoring could induce deleterious effects in exposed cells, from decrease in cellular proliferation, to changes in cellular adhesion, generation of reactive oxygen species or phenotypical variations, just to name a few. Before full implementation of SWCNTs is achieved, their toxicological profiles need to be mechanistically correlated with their physicochemical properties to determine how the induced cellular fate is related to the exposure conditions or samples' characteristics. Our study provides a comprehensive analysis of the synergistic cyto- and genotoxic effects resulted from short-term exposure of human lung epithelial cells to pristine (as manufactured) and user-tailored SWCNTs, as a function of their physicochemical properties. Specifically, through a systematic approach we are correlating the nanotube uptake and nanotube-induced cellular changes to the sample's physicochemical characteristics (e.g., metal impurities, length, agglomerate size, surface area, dispersion, and surface functionalization). By identifying changes in active hallmarks involved in cell-cell connections and maintaining epithelial layer integrity, we also determine the role that short-term exposure to SWCNTs plays in the overall cellular fate and cellular transformation. Lastly, we assess cellular structure-function relationships to identify non-apoptotic pathways induced by SWCNTs exposure that could however lead to changes in cellular behavior and cellular transformation. Our results show that the degree of cell transformation is a function of the physicochemical properties of the SWCNT, with the nanotube with higher length, higher metal content and larger agglomerate size reducing cell viability to a larger extent. Such changes in cell viability are also complemented by changes in cell structure, cycle and cell-cell interactions, all responsible for maintaining cell fate.

Single-walled carbon nanotubes (SWCNTs) inhibit heat shock protein 90 (HSP90) signaling in human lung fibroblasts and keratinocytes

Single-walled carbon nanotubes (SWCNTs) are carbon-based nanomaterials that possess immense industrial potential. Despite accumulating evidence that exposure to SWCNTs might be toxic to humans, our understanding of the mechanisms for cellular toxicity of SWCNTs remain limited. Here, we demonstrated that acute exposure of short (1–3μm) and regular-length (5–30μm) pristine, carboxylated or hydroxylated SWCNTs inhibited cell proliferation in human somatic and human stem cells in a cell type-dependent manner. The toxicity of regular-length pristine SWCNT was most evidenced in NP69>CYT00086>MCF-10A>MRC-5>HaCaT > HEK-293T>HepG2. In contrast, the short pristine SWCNTs were relatively less toxic in most of the cells being tested, except for NP69 which is more sensitive to short pristine SWCNTs as compared to regular-length pristine SWCNTs. Interestingly, carboxylation and hydroxylation of regular-length SWCNTs, but not the short SWCNTs, significantly reduced the cytotoxicity. Exposure of SWCNTs also induced caspase 3 and 9 activities, mitochondrial membrane depolarization, and significant apoptosis and necrosis in MRC-5 embryonic lung fibroblasts. In contrast, SWCNTs inhibited the proliferation of HaCaT human keratinocytes without inducing cell death. Further analyses by gene expression profiling and Connectivity Map analysis showed that SWCNTs induced a gene expression signature characteristic of heat shock protein 90 (HSP90) inhibition in MRC-5 cells, suggesting that SWCNTs may inhibit the HSP90 signaling pathway. Indeed, exposure of MRC-5 cells to SWCNTs results in a dose-dependent decrease in HSP90 client proteins (AKT, CDK4 and BCL2) and a concomitant increase in HSP70 expression. In addition, SWCNTs also significantly inhibited HSP90-dependent protein refolding. Finally, we showed that ectopic expression of HSP90, but not HSP40 or HSP70, completely abrogated the cytotoxic effects of SWCNTs, suggesting that SWCNT-induced cellular toxicity is HSP90 dependent. In summary, our findings suggest that the toxic effects of SWCNTs are mediated through inhibition of HSP90 in human lung fibroblasts and keratinocytes.

Acute Nanoparticle Exposure to Vocal Folds: A Laboratory Study

Airway exposure to nanoparticles is common in occupational settings. Inhaled nanoparticles have toxic effects on respiratory tissue. Vocal folds are also at direct risk from inhaled nanoparticles. This study investigated the effects of single-walled carbon nanotubes (SWCNT), a type of nanoparticle, on vocal fold epithelium and fibroblasts. These cell types were selected for study as the epithelium is the outer layer of the vocal folds and fibroblasts are the most common cell type in connective tissue underlying the epithelium. Native porcine vocal fold epithelium and cultured human vocal fold fibroblasts were exposed to SWCNTs (100 ng/mL) and control (no SWCNT) in vitro. Epithelial and fibroblast viability was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Epithelial barrier integrity was assessed with transepithelial resistance and sodium fluorescein permeability. Epithelial tight junctional protein occludin expression was measured with Western blot. Gene expressions of the fibroblast-specific protein 1 (FSP-1), α-smooth muscle actin (α-SMA), and collagen III (Col-III) were assessed using quantitative polymerase chain reaction. Transcriptional expression of genes encoding FSP-1 and Col-III was increased significantly following SWCNT exposure. There were no significant differences between control and SWCNT groups on any of the other measures. SWCNT exposure induces vocal fold fibroblasts to a fibrotic phenotype. These data help us understand vocal fold defense mechanisms and lay the groundwork for studying the physiological effects of nanoparticle exposure in vivo.

(Invited) Organelle-Specific Targeting and Localization of Single Wall Carbon Nanotubes

Subcellular interaction and targeting of nanomaterials is important both to determine toxicity and for potential subcellular therapies of medical nanotechnology. Our collaborative research groups have investigated the intracellular targeting of single wall carbon nanotubes (SWCNTs) to different organelles based on tube varying length and surface coatings. SWCNTs longer than 1 µm do not localize within cells to any appreciable amount except for professional phagosomes such as macrophages. Purified, well-dispersed SWCNTs near 150 nm can be delivered at extremely high levels to numerous cell types, primarily dependent on cell metabolism and SWCNT surface functionalization. We have found that SWCNTs can be readily non-covalently dispersed with different bioactive macromolecules including (but not limited to) PEG triblock PF127 and bovine serum albumin (BSA) protein. SWCNT-PF127 are membrane active, escape endosomes and localize to actin inside the cell wherein many are retained inside of cells. SWCNT-BSA localize with endoplasmic reticulum (ER) and lipid vesicles along the metabolic pathway within cells. SWCNTs-BSA are then lost from cells via exocytosis unless endosomes are disrupted inside cells. Unless specifically targeted, we find that SWCNTs do not localize to the nucleus or mitochondria of cells. Thus, long-term cellular vitality and genetic stability of cells does not appear to be altered with SWCNT exposure. Specifically, mitochondria structure and function are not altered with high levels of SWCNT treatment (either SWCNT-BSA or SWCNT-PF127) including ATP production or any other aspects of long-term cellular toxicity. Generally in these cellular studies, there are unique challenges in merging nanomaterials characterization with cellular science and engineering. Most imaging technologies of cellular structure and function requires optical microscopy including immunocytochemistry, expression of fluorescent-tagged proteins and assays with fluorescent readouts. However, SWCNTs are optically active and quench fluorescence intensity obscuring intensity-based signals, which limits imaging capabilities and alters accuracy and precision of cellular assays. We have utilized the optical activity of the SWCNT as a marker for colocalization by quantifying changes in fluorescence lifetime of the fluorophore within the Förster radius of the SWCNT. This and other complications of nano-cell interactions necessitates the need for numerous complementary techniques when assessing structural and functional association between SWCNTs and cellular organelles and compartments.

Rheological alteration of erythrocytes exposed to carbon nanotubes.

Single-walled carbon nanotubes (SWNTs) have been increasingly used in a variety of biomedical applications, such as in vivo delivery of drugs and tumor imaging. Potential exposure of SWNTs to human red blood cells (RBCs) may cause serious toxicity including alteration of mechanical properties of cells. The present study investigated the cellular response to exposure of SWNTs with measuring rheological characteristics of RBCs, including hemolysis, deformability, aggregation, and morphological changes. RBCs were exposed to two different dispersion-state samples (i.e. individual SWNTs and bundled SWNTs) in chitosan hydroxyphenyl acetamide (CHPA) solutions. The concentrations of SWNTs were carefully chosen to avoid any hemorheological alterations due to hemolysis. Rheological characteristics were measured using microfluidic-laser diffractometry and aggregometry. Our results show that the bundled SWNTs had higher hemolytic activity than did the individual SWNTs. RBC aggregation apparently decreased as the concentration of SWNTs or incubation time increased. Additionally, bundled SWNTs caused significant alterations in the shape and fusion of RBCs. In conclusion, bundled SWNTs were found to be more toxic than individual SWNTs. These results provide important insights into the interactions between RBCs and SWNTs and will facilitate assessment of the risk of nanomaterial toxicity of blood.

Exposure Assessment in a Single-Walled Carbon Nanotube Primary Manufacturer.

This study was aimed at documenting and characterizing occupational exposure to single-walled carbon nanotubes (SWCNTs) generated in a primary manufacturing plant. It also compared various strategies of exposure monitoring. A 6-day measurement protocol was scheduled (D1-D6) including both (i) quasi-personal monitoring with an array of direct reading instruments (DRIs) and (ii) offline electron microscopy analyses of surface and breathing zone filter-based samples. The first step (D1 and D2) consisted of contamination screenings resulting from the various SWCNT production tasks using a multimetric approach. Surface sampling was also carried out to assess workplace cross-contamination. The second step (D3-D6) focused on the exposure monitoring during recovery/cleaning task, by comparing three personal elemental carbon (EC) measurements [respirable EC using a cyclone following the NIOSH 5040 method (REC-CYC), respirable and thoracic EC using parallel particle impactors [REC-PPI and TEC-PPI, respectively)] and gravimetric mass concentration measurements. DustTrak DRX and electrical low-pressure impactor measurements indicated that particles were released during weighing, transferring, and recovery/cleaning tasks of the manufacturing process. Electron microscopy revealed the presence of agglomerated SWCNTs only during the recovery/cleaning task. REC-CYC concentrations remained under the limits of quantification; REC-PPI showed levels up to 58 µg m-3; and TEC-PPI ranged from 40 to 70 µg m-3. Ratios calculated between gravimetric measurements and estimated DustTrak mass concentrations ranged from 2.8 to 4.9. Cross-contamination appeared to be limited since SWCNTs was only found on surface samples collected close to the reactor in the production room. This case study showed that the DustTrak DRX should be the preferred device among DRIs to identify potential exposure to SWCNTs. However, there is a risk of false positive since it is a non-specific instrument; therefore, the actual release of SWCNTs must be confirmed with scanning electron microscopy/transmission electron microscopy analyses. Besides, using EC measurements as a proxy for SWCNT exposure assessments, as suggested by the NIOSH, is still challenging since interferences can occur with other EC sources such as carbon black, which is also present in the workplace.

Insight into the Mechanisms of Combined Toxicity of Single-Walled Carbon Nanotubes and Nickel Ions in Macrophages: Role of P2X7 Receptor.

Coexistence of nanomaterials and environmental pollutants requires in-depth understanding of combined toxicity and underlying mechanism. In this work, we found that coexposure to the mixture of noncytotoxic level of single-walled carbon nanotubes (SWCNTs) (10 μg/mL) and Ni2+ (20 μM) induced significant cytotoxicity in macrophages. However, almost equal amount of intracellular Ni2+ was detected after Ni2+/SWCNT coexposure or Ni2+ single exposure, indicating no enhanced cellular uptake of Ni2+ occurred. SDS-PAGE analysis revealed 50% more SWCNTs retained in Ni2+/SWCNT exposed cells than that with SWCNT exposure alone, regardless of the exposure sequence (coexposure, Ni2+ pre- or post-treatment), suggesting inhibited SWCNT exocytosis by Ni2+. The increased cellular dose of SWCNTs could quantitatively account for the elevated toxicity of Ni2+/SWCNT mixture to cells. It was then found that agonist (ATP) and antagonist (o-ATP) of P2X7R could regulate intracellular SWCNT amount and the cytotoxicity accordingly. In addition, inhibition of P2X7R by P2X7-targeting siRNA diminished the inhibitory effect of Ni2+. It was therefore concluded that Ni2+ impeded SWCNT exocytosis by inhibiting P2X7R, leading to higher intracellular retention of SWCNTs and elevated cytotoxicity. Our work identified exocytosis inhibition as an important mechanism for SWCNT/Ni2+ toxicity, and revealed the crucial role of P2X7R in mediating such inhibitory effect.