Multi-walled carbon nanotubes induce human microvascular endothelial cellular effects in an alveolar-capillary co-culture with small airway epithelial cells

Background Nanotechnology, particularly the use of multi-walled carbon nanotubes (MWCNT), is a rapidly growing discipline with implications for advancement in a variety of fields. A major route of exposure to MWCNT during both occupational and environmental contact is inhalation. While many studies showed adverse effects to the vascular endothelium upon MWCNT exposure, in vitro results often do not correlate with in vivo effects. This study aimed to determine if an alveolar-capillary co-culture model could determine changes in the vascular endothelium after epithelial exposure to MWCNT. Methods A co-culture system in which both human small airway epithelial cells and human microvascular endothelial cells were separated by a Transwell membrane so as to resemble an alveolar-capillary interaction was used. Following exposure of the epithelial layer to MWCNT, the effects to the endothelial barrier were determined. Results Exposure of the epithelial layer to MWCNT induced multiple changes in the endothelial cell barrier, including an increase in reactive oxygen species, actin rearrangement, loss of VE-cadherin at the cell surface, and an increase in endothelial angiogenic ability. Overall increases in secreted VEGFA, sICAM-1, and sVCAM-1 protein levels, as well as increases in intracellular phospho-NF-κB, phospho-Stat3, and phospho-p38 MAPK, were also noted in HMVEC after epithelial exposure. Conclusion The co-culture system identified that alveolar-capillary exposure to MWCNT induced multiple changes to the underlying endothelium, potentially through cell signaling mediators derived from MWCNT-exposed epithelial cells. Therefore, the co-culture system appears to be a relevant in vitromethod to study the pulmonary toxicity of MWCNT

Dispersion of single-walled carbon nanotubes by a natural lung surfactant for pulmonary in vitro and in vivo toxicity studies

<p>Abstract</p> <p>Background</p> <p>Accumulating evidence indicate that the degree of dispersion of nanoparticles has a strong influence on their biological activities. The aims of this study were to develop a simple and rapid method of nanoparticle dispersion using a natural lung surfactant and to evaluate the effect of dispersion status of SWCNT on cytotoxicity and fibrogenicity <it>in vitro </it>and <it>in vivo</it>.</p> <p>Results</p> <p>The natural lung surfactant Survanta^®was used to disperse single-walled carbon nanotubes (SWCNT) in a biological medium. At physiologically relevant concentrations, Survanta^®produced well dispersed SWCNT without causing a cytotoxic or fibrogenic effect. <it>In vitro </it>studies show that Survanta^®-dispersed SWCNT (SD-SWCNT) stimulated proliferation of lung epithelial cells at low doses (0.04-0.12 μg/ml or 0.02-0.06 μg/cm²exposed surface area) but had a suppressive effect at high doses. Non-dispersed SWCNT (ND-SWCNT) did not exhibit these effects, suggesting the importance of dispersion status of SWCNT on bioactivities. Studies using cultured human lung fibroblasts show that SD-SWCNT stimulated collagen production of the cells. This result is supported by a similar observation using Acetone/sonication dispersed SWCNT (AD-SWCNT), suggesting that Survanta^®did not mask the bioactivity of SWCNT. Likewise, <it>in vivo </it>studies show that both SD-SWCNT and AD-SWCNT induced lung fibrosis in mice, whereas the dispersing agent Survanta^®alone or Survanta^®-dispersed control ultrafine carbon black had no effect.</p> <p>Conclusions</p> <p>The results indicate that Survanta^®was effective in dispersing SWCNT in biological media without causing cytotoxic effects at the test concentrations used in this study. SD-SWCNT stimulated collagen production of lung fibroblasts <it>in vitro </it>and induced lung fibrosis <it>in vivo</it>. Similar results were observed with AD-SWCNT, supporting the conclusion that Survanta^®did not mask the bioactivities of SWCNT and thus can be used as an effective dispersing agent. Since excessive collagen production is a hallmark of lung fibrosis, the results of this study suggest that the <it>in vitro </it>model using lung fibroblasts may be an effective and rapid screening tool for prediction of the fibrogenic potential of SWCNT <it>in vivo</it>.</p

Comparison of stainless and mild steel welding fumes in generation of reactive oxygen species

<p>Abstract</p> <p>Background</p> <p>Welding fumes consist of a wide range of complex metal oxide particles which can be deposited in all regions of the respiratory tract. The welding aerosol is not homogeneous and is generated mostly from the electrode/wire. Over 390,000 welders were reported in the U.S. in 2008 while over 1 million full-time welders were working worldwide. Many health effects are presently under investigation from exposure to welding fumes. Welding fume pulmonary effects have been associated with bronchitis, metal fume fever, cancer and functional changes in the lung. Our investigation focused on the generation of free radicals and reactive oxygen species from stainless and mild steel welding fumes generated by a gas metal arc robotic welder. An inhalation exposure chamber located at NIOSH was used to collect the welding fume particles.</p> <p>Results</p> <p>Our results show that hydroxyl radicals (^.OH) were generated from reactions with H₂O₂and after exposure to cells. Catalase reduced the generation of <b>^.</b>OH from exposed cells indicating the involvement of H₂O₂. The welding fume suspension also showed the ability to cause lipid peroxidation, effect O₂consumption, induce H₂O₂generation in cells, and cause DNA damage.</p> <p>Conclusion</p> <p>Increase in oxidative damage observed in the cellular exposures correlated well with <b>^.</b>OH generation in size and type of welding fumes, indicating the influence of metal type and transition state on radical production as well as associated damage. Our results demonstrate that both types of welding fumes are able to generate ROS and ROS-related damage over a range of particle sizes; however, the stainless steel fumes consistently showed a significantly higher reactivity and radical generation capacity. The chemical composition of the steel had a significant impact on the ROS generation capacity with the stainless steel containing Cr and Ni causing more damage than the mild steel. Our results suggest that welding fumes may cause acute lung injury. Since type of fume generated, particle size, and elapsed time after generation of the welding exposure are significant factors in radical generation and particle deposition these factors should be considered when developing protective strategies.</p

Distribution and persistence of pleural penetrations by multi-walled carbon nanotubes

<p>Abstract</p> <p>Background</p> <p>Multi-walled carbon nanotubes (MWCNT) are new manufactured nanomaterials with a wide spectrum of commercial applications. The durability and fiber-like dimensions (mean length 3.9 μm long × 49 nm diameter) of MWCNT suggest that these fibers may migrate to and have toxicity within the pleural region. To address whether the pleura received a significant and persistent exposure, C57BL/6J mice were exposed by pharyngeal aspiration to 10, 20, 40 and 80 μg MWCNT or vehicle and the distribution of MWCNT penetrations determined at 1, 7, 28 and 56 days after exposure. Following lung fixation and sectioning, morphometric methods were used to determine the distribution of MWCNT and the number of MWCNT fiber penetrations of three barriers: alveolar epithelium (alveolar penetrations), the alveolar epithelium immediately adjacent to the pleura (subpleural tissue), and visceral pleural surface (intrapleural space).</p> <p>Results</p> <p>At 1 day 18%, 81.6% and 0.6% of the MWCNT lung burden was in the airway, the alveolar, and the subpleural regions, respectively. There was an initial, high density of penetrations into the subpleural tissue and the intrapleural space one day following aspiration which appeared to decrease due to clearance by alveolar macrophages and/or lymphatics by day 7. However, the density of penetrations increased to steady state levels in the subpleural tissue and intrapleural from day 28 - 56. At day 56 approximately 1 in every 400 fiber penetrations was in either the subpleural tissue or intrapleural space. Numerous penetrations into macrophages in the alveolar airspaces throughout the lungs were demonstrated at all times but are not included in the counts presented.</p> <p>Conclusions</p> <p>The results document that MWCNT penetrations of alveolar macrophages, the alveolar wall, and visceral pleura are both frequent and sustained. In addition, the findings demonstrate the need to investigate the chronic toxicity of MWCNT at these sites.</p

The streptococcal collagen-like protein-1 (Scl1) is a significant determinant for biofilm formation by group a Streptococcus

<p>Abstract</p> <p>Background</p> <p>Group A <it>Streptococcus </it>(GAS) is a human-specific pathogen responsible for a number of diseases characterized by a wide range of clinical manifestations. During host colonization GAS-cell aggregates or microcolonies are observed in tissues. GAS biofilm, which is an <it>in vitro </it>equivalent of tissue microcolony, has only recently been studied and little is known about the specific surface determinants that aid biofilm formation. In this study, we demonstrate that surface-associated streptococcal collagen-like protein-1 (Scl1) plays an important role in GAS biofilm formation.</p> <p>Results</p> <p>Biofilm formation by M1-, M3-, M28-, and M41-type GAS strains, representing an intraspecies breadth, were analyzed spectrophotometrically following crystal violet staining, and characterized using confocal and field emission scanning electron microscopy. The M41-type strain formed the most robust biofilm under static conditions, followed by M28- and M1-type strains, while the M3-type strains analyzed here did not form biofilm under the same experimental conditions. Differences in architecture and cell-surface morphology were observed in biofilms formed by the M1- and M41-wild-type strains, accompanied by varying amounts of deposited extracellular matrix and differences in cell-to-cell junctions within each biofilm. Importantly, all Scl1-negative mutants examined showed significantly decreased ability to form biofilm <it>in vitro</it>. Furthermore, the Scl1 protein expressed on the surface of a heterologous host, <it>Lactococcus lactis</it>, was sufficient to induce biofilm formation by this organism.</p> <p>Conclusions</p> <p>Overall, this work (i) identifies variations in biofilm formation capacity among pathogenically different GAS strains, (ii) identifies GAS surface properties that may aid in biofilm stability and, (iii) establishes that the Scl1 surface protein is an important determinant of GAS biofilm, which is sufficient to enable biofilm formation in the heterologous host <it>Lactococcus</it>. In summary, the GAS surface adhesin Scl1 may have an important role in biofilm-associated pathogenicity.</p

Iron oxide nanoparticles induce human microvascular endothelial cell permeability through reactive oxygen species production and microtubule remodeling

<p>Abstract</p> <p>Background</p> <p>Engineered iron nanoparticles are being explored for the development of biomedical applications and many other industry purposes. However, to date little is known concerning the precise mechanisms of translocation of iron nanoparticles into targeted tissues and organs from blood circulation, as well as the underlying implications of potential harmful health effects in human.</p> <p>Results</p> <p>The confocal microscopy imaging analysis demonstrates that exposure to engineered iron nanoparticles induces an increase in cell permeability in human microvascular endothelial cells. Our studies further reveal iron nanoparticles enhance the permeability through the production of reactive oxygen species (ROS) and the stabilization of microtubules. We also showed Akt/GSK-3β signaling pathways are involved in iron nanoparticle-induced cell permeability. The inhibition of ROS demonstrate ROS play a major role in regulating Akt/GSK-3β – mediated cell permeability upon iron nanoparticle exposure. These results provide new insights into the bioreactivity of engineered iron nanoparticles which can inform potential applications in medical imaging or drug delivery.</p> <p>Conclusion</p> <p>Our results indicate that exposure to iron nanoparticles induces an increase in endothelial cell permeability through ROS oxidative stress-modulated microtubule remodeling. The findings from this study provide new understandings on the effects of nanoparticles on vascular transport of macromolecules and drugs.</p

Raw Single-Wall Carbon Nanotubes Induce Oxidative Stress and Activate MAPKs, AP-1, NF-κB, and Akt in Normal and Malignant Human Mesothelial Cells

Background Single-wall carbon nanotubes (SWCNTs), with their unique physicochemical and mechanical properties, have many potential new applications in medicine and industry. There has been great concern subsequent to preliminary investigations of the toxicity, biopersistence, pathogenicity, and ability of SWCNTs to translocate to subpleural areas. These results compel studies of potential interactions of SWCNTs with mesothelial cells. Objective Exposure to asbestos is the primary cause of malignant mesothelioma in 80–90% of individuals who develop the disease. Because the mesothelial cells are the primary target cells of asbestos-induced molecular changes mediated through an oxidant-linked mechanism, we used normal mesothelial and malignant mesothelial cells to investigate alterations in molecular signaling in response to a commercially manufactured SWCNT. Methods In the present study, we exposed mesothelial cells to SWCNTs and investigated reactive oxygen species (ROS) generation, cell viability, DNA damage, histone H2AX phosphorylation, activation of poly(ADP-ribose) polymerase 1 (PARP-1), stimulation of extracellular signal-regulated kinase (ERKs), Jun N-terminal kinases (JNKs), protein p38, and activation of activator protein-1 (AP-1), nuclear factor κB (NF-κB), and protein serine-threonine kinase (Akt). Results Exposure to SWCNTs induced ROS generation, increased cell death, enhanced DNA damage and H2AX phosphorylation, and activated PARP, AP-1, NF-κB, p38, and Akt in a dose-dependent manner. These events recapitulate some of the key molecular events involved in mesothelioma development associated with asbestos exposure. Conclusions The cellular and molecular findings reported here do suggest that SWCNTs can cause potentially adverse cellular responses in mesothelial cells through activation of molecular signaling associated with oxidative stress, which is of sufficient significance to warrant in vivo animal exposure studies

Characterization of silver particles in the stratum corneum of healthy subjects and atopic dermatitis patients dermally exposed to a silver-containing garment

Silver is increasingly being used in garments to exploit its antibacterial properties. Information on the presence of silver nanoparticles (AgNPs) in garments and their in vivo penetration across healthy and impaired skin from use is limited. We investigated the presence of AgNPs in a silver containing garment and in the stratum corneum (SC) of healthy subjects (CTRLs) and individuals with atopic dermatitis (AD). Seven CTRLs and seven AD patients wore a silver sleeve (13% Ag w/w) 8 h/day for five days on a forearm and a placebo sleeve on the other forearm. After five days, the layers of the SC were collected by adhesive tapes. The silver particles in the garment and SC were characterized by scanning electron microscopy with energy dispersive X-ray analysis (SEM-EDX) and atomic force microscopy (AFM). AFM and SEM revealed the presence of sub-micrometre particles having a broad range of sizes (30\u2013500 nm) on the surface of the garment that were identified as silver. On the SC tapes collected from different depths, aggregates with a wide range of sizes (150 nm\u20132 \u3bcm) and morphologies were found. Most aggregates contained primarily silver, although some also contained chlorine and sulfur. There was no clear difference in the number or size of the aggregates observed in SC between healthy and AD subjects. After use, AgNPs and their aggregates were present in the SC at different depths of both healthy subjects and AD patients. Their micrometre size suggests that aggregation likely occurred in the SC

Differential Pulmonary Effects of CoO and La2O3 Metal Oxide Nanoparticle Responses During Aerosolized Inhalation in Mice

Background: Although classified as metal oxides, cobalt monoxide (CoO) and lanthanum oxide (La2O3) nanoparticles, as representative transition and rare earth oxides, exhibit distinct material properties that may result in different hazardous potential in the lung. The current study was undertaken to compare the pulmonary effects of aerosolized whole body inhalation of these nanoparticles in mice. Results: Mice were exposed to filtered air (control) and 10 or 30 mg/m3 of each particle type for 4 days and then examined at 1 h, 1, 7 and 56 days post-exposure. The whole lung burden 1 h after the 4 day inhalation of CoO nanoparticles was 25 % of that for La2O3 nanoparticles. At 56 days post exposure, \u3c 1 % of CoO nanoparticles remained in the lungs; however, 22–50 % of the La2O3 nanoparticles lung burden 1 h post exposure was retained at 56 days post exposure for low and high exposures. Significant accumulation of La2O3 nanoparticles in the tracheobronchial lymph nodes was noted at 56 days post exposure. When exposed to phagolysosomal simulated fluid, La nanoparticles formed urchin-shaped LaPO4 structures, suggesting that retention of this rare earth oxide nanoparticle may be due to complexation of cellular phosphates within lysosomes. CoO nanoparticles caused greater lactate dehydrogenase release in the bronchoalveolar fluid (BALF) compared to La2O3 nanoparticles at 1 day post exposure, while BAL cell differentials indicate that La2O3 nanoparticles generated more inflammatory cell infiltration at all doses and exposure points. Histopathological analysis showed acute inflammatory changes at 1 day after inhalation of either CoO or La2O3 nanoparticles. Only the 30 mg/m3 La2O3 nanoparticles exposure caused chronic inflammatory changes and minimal fibrosis at day 56 post exposure. This is in agreement with activation of the NRLP3 inflammasome after in vitro exposure of differentiated THP-1 macrophages to La2O3 but not after CoO nanoparticles exposure. Conclusion: Taken together, the inhalation studies confirmed the trend of our previous sub-acute aspiration study, which reported that CoO nanoparticles induced more acute pulmonary toxicity, while La2O3 nanoparticles caused chronic inflammatory changes and minimal fibrosis

Evaluation of pulmonary and systemic toxicity following lung exposure to graphite nanoplates: a member of the graphene-based nanomaterial family

Background: Graphene, a monolayer of carbon, is an engineered nanomaterial (ENM) with physical and chemical properties that may offer application advantages over other carbonaceous ENMs, such as carbon nanotubes (CNT). The goal of this study was to comparatively assess pulmonary and systemic toxicity of graphite nanoplates, a member of the graphene-based nanomaterial family, with respect to nanoplate size. Methods: Three sizes of graphite nanoplates [20 μm lateral (Gr20), 5 μm lateral (Gr5), and \u3c2 \u3eμm lateral (Gr1)] ranging from 8–25 nm in thickness were characterized for difference in surface area, structure,, zeta potential, and agglomeration in dispersion medium, the vehicle for in vivo studies. Mice were exposed by pharyngeal aspiration to these 3 sizes of graphite nanoplates at doses of 4 or 40 μg/mouse, or to carbon black (CB) as a carbonaceous control material. At 4 h, 1 day, 7 days, 1 month, and 2 months post-exposure, bronchoalveolar lavage was performed to collect fluid and cells for analysis of lung injury and inflammation. Particle clearance, histopathology and gene expression in lung tissue were evaluated. In addition, protein levels and gene expression were measured in blood, heart, aorta and liver to assess systemic responses. Results: All Gr samples were found to be similarly composed of two graphite structures and agglomerated to varying degrees in DM in proportion to the lateral dimension. Surface area for Gr1 was approximately 7-fold greater than Gr5 and Gr20, but was less reactive reactive per m2 . At the low dose, none of the Gr materials induced toxicity. At the high dose, Gr20 and Gr5 exposure increased indices of lung inflammation and injury in lavage fluid and tissue gene expression to a greater degree and duration than Gr1 and CB. Gr5 and Gr20 showed no or minimal lung epithelial hypertrophy and hyperplasia, and no development of fibrosis by 2 months post-exposure. In addition, the aorta and liver inflammatory and acute phase genes were transiently elevated in Gr5 and Gr20, relative to Gr1. Conclusions: Pulmonary and systemic toxicity of graphite nanoplates may be dependent on lateral size and/or surface reactivity, with the graphite nanoplates \u3e 5 μm laterally inducing greater toxicity which peaked at the early time points post-exposure relative to the 1–2 μm graphite nanoplate