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

A comparison of cytotoxicity and oxidative stress from welding fumes generated with a new nickel-, copper-based consumable versus mild and stainless steel-based welding in RAW 264.7 mouse macrophages.

Welding processes that generate fumes containing toxic metals, such as hexavalent chromium (Cr(VI)), manganese (Mn), and nickel (Ni), have been implicated in lung injury, inflammation, and lung tumor promotion in animal models. While federal regulations have reduced permissible worker exposure limits to Cr(VI), this is not always practical considering that welders may work in confined spaces and exhaust ventilation may be ineffective. Thus, there has been a recent initiative to minimize the potentially hazardous components in welding materials by developing new consumables containing much less Cr(VI) and Mn. A new nickel (Ni) and copper (Cu)-based material (Ni-Cu WF) is being suggested as a safer alternative to stainless steel consumables; however, its adverse cellular effects have not been studied. This study compared the cytotoxic effects of the newly developed Ni-Cu WF with two well-characterized welding fumes, collected from gas metal arc welding using mild steel (GMA-MS) or stainless steel (GMA-SS) electrodes. RAW 264.7 mouse macrophages were exposed to the three welding fumes at two doses (50 µg/ml and 250 µg/ml) for up to 24 hours. Cell viability, reactive oxygen species (ROS) production, phagocytic function, and cytokine production were examined. The GMA-MS and GMA-SS samples were found to be more reactive in terms of ROS production compared to the Ni-Cu WF. However, the fumes from this new material were more cytotoxic, inducing cell death and mitochondrial dysfunction at a lower dose. Additionally, pre-treatment with Ni-Cu WF particles impaired the ability of cells to phagocytize E. coli, suggesting macrophage dysfunction. Thus, the toxic cellular responses to welding fumes are largely due to the metal composition. The results also suggest that reducing Cr(VI) and Mn in the generated fume by increasing the concentration of other metals (e.g., Ni, Cu) may not necessarily improve welder safety

Pre-incubation with Ni-Cu WF impairs phagocytosis.

<p>RAW 264.7 cells were treated with 50 µg/ml welding fume suspensions for 3 or 6 h or cytochalasin D (Cyto D) for 3 h. Cells were washed, pHrodo Red <i>E. coli</i> BioParticles were added for 2 h, and plates were read to measure changes in fluorescence. Error bars represent the mean ± SD (n = 4). *, p<0.05 compared to PBS controls.</p

Metal content analysis of the welding fumes.

a<p>Relative to all metals analyzed; <b>bold</b> used to emphasize elements with the most toxic potential based on previous studies of welding fumes; SE, standard error.</p

Welding fumes do not induce pro-inflammatory cytokine production.

<p>RAW 264.7 cells were treated with welding fume suspensions or 1 µg/ml LPS for 24 h. Media were collected, and ELISAs were run to measure levels of TNFα (<b>A</b>), IL-6 (<b>B</b>) and IL-1β (<b>C</b>). Error bars represent the mean ± SD (n = 3). *, p<0.05 compared to PBS controls.</p

Reduced viability is not due to excessive ROS production.

<p>RAW 264.7 cells were pre-treated with and without <i>N</i>-acetyl-L-cysteine (NAC) for 1 h and then for 24 h with suspensions of welding fumes. MultiTox-Fluor Reagent (Promega) was added to all wells for 1 h at 37°C, and plates were read at 400ex/505em to determine the fluorescence signal from live cells. Error bars represent the mean ± SD (n = 3). There was no significant difference between − and + NAC for treatment groups. *, p<0.05 compared to PBS control.</p

Generation of Reactive Oxygen Species from Silicon Nanowires

Processing and synthesis of purified nanomaterials of diverse composition, size, and properties is an evolving process. Studies have demonstrated that some nanomaterials have potential toxic effects and have led to toxicity research focusing on nanotoxicology. About two million workers will be employed in the field of nanotechnology over the next 10 years. The unknown effects of nanomaterials create a need for research and development of techniques to identify possible toxicity. Through a cooperative effort between National Institute for Occupational Safety and Health and IBM to address possible occupational exposures, silicon-based nanowires (SiNWs) were obtained for our study. These SiNWs are anisotropic filamentary crystals of silicon, synthesized by the vapor-liquid-solid method and used in bio-sensors, gas sensors, and field effect transistors. Reactive oxygen species (ROS) can be generated when organisms are exposed to a material causing cellular responses, such as lipid peroxidation, H 2 O 2 production, and DNA damage. SiNWs were assessed using three different in vitro environments (H 2 O 2 , RAW 264.7 cells, and rat alveolar macrophages) for ROS generation and possible toxicity identification. We used electron spin resonance, analysis of lipid peroxidation, measurement of H 2 O 2 production, and the comet assay to assess generation of ROS from SiNW and define possible mechanisms. Our results demonstrate that SiNWs do not appear to be significant generators of free radicals

Welding fumes reduce RAW 264.7 cell viability.

<p>Cells were treated with suspensions of welding fumes for 24(<b>A</b>) and percentage of viable cells (<b>B</b>) within each sample. Error bars represent the mean ± SD (n = 4). *, p<0.05 compared to PBS control. MS; GMA-MS, SS; GMA-SS, Ni; Ni-Cu WF.</p

Electron spin resonance measurement of •OH radicals following reaction of welding fumes with H₂O₂ or RAW 264.7 cells.

<p><b>A.</b> An acellular mixture of welding fume suspensions (1 mg/ml), H₂O₂ (1 mM), and spin trap DMPO (100 mM) in PBS were incubated in test tubes for 3 min at room temperature, and scanned via ESR. Signal intensity (peak height) is used to measure the relative amount of hydroxyl radicals and error bars represent the mean ± SD (n = 3). Inset: representative GMA-MS spectra. <b>B.</b> The same as in <b>A</b>, except without H₂O₂ but including 2×10⁶ RAW 264.7 cells/ml. Samples were incubated at 37°C for 5 min prior to measurements. (n = 5–6). Insets: representative GMA-MS spectra and with catalase treatment (+Cat) for cellular ESR. *, p<0.05.</p

Sintered indium-tin oxide particles induce pro-inflammatory responses in vitro, in part through inflammasome activation.

Indium-tin oxide (ITO) is used to make transparent conductive coatings for touch-screen and liquid crystal display electronics. As the demand for consumer electronics continues to increase, so does the concern for occupational exposures to particles containing these potentially toxic metal oxides. Indium-containing particles have been shown to be cytotoxic in cultured cells and pro-inflammatory in pulmonary animal models. In humans, pulmonary alveolar proteinosis and fibrotic interstitial lung disease have been observed in ITO facility workers. However, which ITO production materials may be the most toxic to workers and how they initiate pulmonary inflammation remain poorly understood. Here we examined four different particle samples collected from an ITO production facility for their ability to induce pro-inflammatory responses in vitro. Tin oxide, sintered ITO (SITO), and ventilation dust particles activated nuclear factor kappa B (NFκB) within 3 h of treatment. However, only SITO induced robust cytokine production (IL-1β, IL-6, TNFα, and IL-8) within 24 h in both RAW 264.7 mouse macrophages and BEAS-2B human bronchial epithelial cells. Our lab and others have previously demonstrated SITO-induced cytotoxicity as well. These findings suggest that SITO particles activate the NLRP3 inflammasome, which has been implicated in several immune-mediated diseases via its ability to induce IL-1β release and cause subsequent cell death. Inflammasome activation by SITO was confirmed, but it required the presence of endotoxin. Further, a phagocytosis assay revealed that pre-uptake of SITO or ventilation dust impaired proper macrophage phagocytosis of E. coli. Our results suggest that adverse inflammatory responses to SITO particles by both macrophage and epithelial cells may initiate and propagate indium lung disease. These findings will provide a better understanding of the molecular mechanisms behind an emerging occupational health issue