Graphene and related materials (GRMs) have unique optical and thermal characteristics and are expected to be adopted for industrial applications. However, there are concerns with respect to their safety to human health. To conduct cytotoxicity and mutagenicity assessments, exfoliated graphene (EGr) dispersed in Tween-20® was diluted in cell culture medium. Rat alveolar macrophage viability significantly decreased after 24 h exposure to 1 and 10 μg/mL EGr. No significant levels of intracellular reactive oxygen species were detected in the 2',7'-dichlorodihydrofluorescin diacetate assay after 24 h of exposure to EGr. The levels of the pro-inflammatory cytokines macrophage inflammatory protein-1α, interleukin (IL)-1β, IL-18, macrophage chemoattractant protein-1, and tumor necrosis factor α were significantly higher in cells treated with 10 μg/mL EGr for 24 h than in untreated controls. Transmission electron microscopy confirmed that EGr was present in the cytoplasm of the cells. Many genes were upregulated by EGr treatment, and significantly overrepresented gene ontology categories included the biological processes "response to external stimulus", "response to stress", "cell-cell signaling", "biological adhesion", and "cell proliferation". EGr did not induce genetic mutations in E. coli or cause micronucleus induction in mouse bone marrow cells. The results suggest that EGr cytotoxicity should be carefully considered.
Multi-walled carbon nanotubes (MWCNTs) have industrial applications in the nanotechnology field. The physico-chemical properties of MWCNTs vary greatly depending on MWCNT manufacture and application. It has been pointed out that their needle shape and high durability are important factors that determine the biopersistence of fibers and can lead to inhalation toxicity or cytotoxicity. In this study, we prepared six suspensions of MWCNTs differing in diameter and length, and performed in vitro cell-based assays for 24 h using NR8383 rat alveolar macrophages. Rigid, needle-shaped MWCNTs with a large diameter (>50 mm) penetrated the cytoplasm and decreased cell survival without generating intracellular reactive oxygen species (ROS), significantly up-regulated many genes involved in inflammatory responses, response to oxidative stress and apoptosis, and extracellular matrix degradation. Bent MWCNTs with a small diameter (<20 mm) were phagocytosed in vacuole-like cellular compartments and decreased cell survival along with intracellular ROS generation. Straight, thin MWCNTs with a small diameter (<20 mm) caused a slight intracellular ROS generation but no decrease in cell viability. Some straight, long, and thin MWCNTs were found in the mitochondria and near the nuclei; however, no mutagenesis was observed. The in vitro cell-based assays showed high cytotoxicity of MWCNTs with a large diameter (>50 mm), moderate and low cytotoxicity of MWCNTs with a small diameter (<20 mm). These results suggested that the diameter of MWCNTs considerably contributes to their cytotoxicity.
Concern over the influence of carbon nanotubes (CNTs) on human health has arisen due to advances; however, little is known about the potential toxicity of CNTs. In this study, impurity-free single-wall carbon nanotubes (SWCNTs), with different physical properties in cell culture medium, were prepared by a novel dispersion procedure. SWCNTs with small bundles (short linear shape) and SWCNTs with large bundles (long linear shape) did not cause a significant inhibition of cell proliferation, induction of apoptosis or arrest of cell cycle progression in A549 alveolar epithelial cells. Expression of many genes involved in the inflammatory response, apoptosis, response to oxidative stress and degradation of the extracellular matrix were not markedly upregulated or downregulated. However, SWCNTs with relatively large bundles significantly increased the level of intracellular reactive oxygen species (ROS) in a dose-dependent manner, and the levels of these ROS were higher than those of SWCNTs with relatively small bundles or commercial SWCNTs with residual metals. Transmission electron microscopy (TEM) revealed that impurity-free SWCNTs were observed in the cytoplasm and vacuoles of cells after 24 h. These results suggested that the physical properties, especially the size and length of the bundles of the SWCNTs dispersed in cell culture medium, contributed to a change in intracellular ROS generation, even for the same bulk SWCNTs. Additionally, the residual metals associated with the manufacturing of SWCNTs may not be a definitive parameter for intracellular ROS generation in A549 cells.
Safety assessment of cellulose nanofibrils (CNFs) is required to accelerate the utilization of these materials in industrial applications. The present study aimed to characterize the effects on rat pulmonary inflammation over a period of 90 days following intratracheal instillation of three types of CNFs or multi-walled carbon nanotubes (MWCNTs) at doses of 0.5, 1.0, or 2.0 mg/kg. The pulmonary inflammatory responses induced by phosphorylated CNFs (CNF1), 2,2,6,6-tetramethylpiperidine-1-oxyl radical (TEMPO)-oxidized CNFs (CNF2), CNFs produced via mechanical defibrillation (CNF3), and MWCNTs were investigated using bronchoalveolar lavage fluid analysis, histopathological findings, and comprehensive gene expression profiling of rat lungs. CNF1 and CNF2 with approximately equal diameter (7.0–8.0 nm) and length (0.8–1.0 µm) distributions induced inflammation after dosing, which was attenuated 90 days post-instillation. CNF3 of relatively greater thickness (21.2 nm) and longer length (1.7 μm) deposited around the terminal bronchioles were observed after instillation. Acute inflammatory responses in the alveoli induced by CNF3 were mild compared with those induced by other materials and attenuated 90 days post-instillation. MWCNTs induced severe pulmonary inflammatory responses that continued during the test period. The inflammation failed to resolve within 90 days post-instillation. A hierarchical cluster analysis revealed comparable gene expression profiles for CNF1, CNF2, and CNF3, whereas profiles of MWCNTs were different from those of other test substances. This study suggests that pulmonary inflammation is associated with the diameter and length distributions of CNFs and that the pulmonary inflammation caused by CNFs is mild compared with that caused by MWCNTs. Graphic abstract
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