Effects of advanced nanomaterials on the respiratory system of a murine COPD model.

This study assessed the effects of cellulose nanofibrils (CNFs) and multi-walled carbon nanotubes (MWCNTs) on lung inflammation in a cigarette smoke-induced chronic obstructive pulmonary disease (COPD) mouse model. Prior to instillation, COPD model mice displayed distinctive cellular compositions and elevated cytokine levels in bronchoalveolar lavage fluid (BALF). After intratracheal instillation of 80 µg CNFs, no significant histopathological changes, BALF composition alterations, or cytokine level shifts were observed on day 28. This suggests minimal lung impact and no interference with reducing smoke-induced inflammation. In contrast, the instillation of 80 µg MWCNTs resulted in significant histopathological changes, increased cellular composition, and elevated cytokine levels in BALF on day 28. These findings indicate that CNF exposure had little effect on the lungs and did not impede the reduction of smoke-induced inflammation, while MWCNT exposure hindered the attenuation of pulmonary inflammatory response. The study emphasizes the importance of considering diverse cases, including individuals with pre-existing respiratory conditions, when assessing occupational safety and health risks associated with advanced nanomaterial exposure.

Contaminant microorganisms in the in vitro evaluation of cellular responses of cellulose nanofibers and their microbial inactivation using gamma irradiation.

Cellulose nanofibers (CNFs) are fibrous nanomaterials produced from plants. Since some nanomaterials are toxic, toxicity evaluation, including in vitro examinations using cultured cells, is essential for the effective use of CNFs. On the other hand, microorganisms in the environment can contaminate CNF suspensions. The contamination of CNF samples and the effects of contaminating microorganisms on in vitro examinations were investigated in this study. Microorganism contamination in CNF samples was examined, and microbial inactivation of CNF suspensions using gamma irradiation was evaluated. After gamma-ray irradiation at absorbed doses of 0.5, 1, 5, and 10 kGy, the cellular effects of CNF suspensions were examined using 6 types of cultured cell, HaCaT, A549, Caco-2, MeT-5A, THP-1, and NR8383 cells. CNF samples were contaminated with bacteria and CNF suspensions exhibited endotoxin activity. Gamma irradiation effectively inactivated the microorganisms contained in the CNF suspensions. When the absorbed dose was 10 kGy, the fiber length of CNF was shortened, but the effect on CNF was small at 1.0 kGy or less. CNF suspensions showed lipopolysaccharides (LPS)-like cellular responses and strongly induced interleukin-8, especially in macrophages. Absorbed doses of at least 10 kGy did not affect the LPS-like activity. In this study, it was shown that the CNF suspension may be contaminated with microorganisms. Gamma irradiation was effective for microbial inactivation of suspension for invitor toxicity evaluation of CNF. In vitro evaluation of CNFs requires attention to the effects of contaminants such as LPS.

Genotoxicity assessment of cellulose nanofibrils using a standard battery of in vitro and in vivo assays.

Cellulose nanofibrils (CNFs) are identified as novel nanomaterials with many potential applications. Since CNFs are fibrous manufactured nanomaterials, their potential carcinogenic effects and mesothelial toxicity raise some concerns. In this study, we conducted a standard battery of in vitro and in vivo assays to evaluate the genotoxicity of two CNF types using different manufacturing methods and physicochemical properties. Namely, one was CNF produced via chemical modification by TEMPO (2,2,6,6-tetramethylpiperidine-1-oxyl radical)-mediated oxidation, while the other was CNF produced via mechanical defibrillation using needle bleached kraft pulp. A bacterial reverse mutation test and a mouse lymphoma TK assay revealed that CNFs at 100 µg/mL did not induce bacterial reverse mutations and in vitro mammalian cell gene mutation. Further, in vitro chromosomal aberration tests demonstrated that CNFs at 100 µg/mL did not induce chromosomal aberration in Chinese hamster lung fibroblasts. From the mammalian erythrocyte micronucleus test, no statistically significant increase was observed in the proportion of micronucleated polychromatic erythrocytes in the bone marrow cells of rats intratracheally instilled with any concentration of CNFs (0.25-1.0 mg/kg) compared with values from respective negative control groups. Therefore, this battery of in vitro and in vivo assays illustrated that the CNFs examined in this study did not induce genotoxicity, suggesting our results provide valuable insight on the future use of these materials in various industrial applications.

Screening of preservatives and evaluation of sterilized cellulose nanofibers for toxicity studies.

OBJECTIVES: The aim of this study is to establish a sterilization method for cellulose nanofibers (CNFs) dispersions that uses multiple preservatives with different hydrophilicities without affecting the physical and chemical properties of CNFs, and to provide useful information for sample preparation in future toxicity study of CNFs. METHODS: Various preservatives were added to the phosphorylated CNF dispersions, endotoxin level and the numbers of bacteria and fungi in the CNF dispersion were analyzed. The pH values and viscosity of sterilized CNF dispersions were compared with those of control and autoclaved CNF dispersions. RESULTS: Phosphorylated CNF dispersions at a concentration of 2.0 mg/mL or lower and the addition of 10 µg/mL benzalkonium chloride alone or 250 µg/mL methyl parahydroxybenzoate and 250 µg/mL propyl parahydroxybenzoate in combination can sterilize CNF dispersions without changing the physical and chemical properties of CNFs. CONCLUSIONS: We developed sterilization method for CNF dispersions that uses multiple preservatives with different hydrophilicities without affecting the physical and chemical properties of CNFs. This sterilization method for CNFs dispersions can be applied to the safety assessment of CNF with different physicochemical properties in the future.

Cytotoxicity profiles of multi-walled carbon nanotubes with different physico-chemical properties.

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 µm) 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 µm) were phagocytosed in vacuole-like cellular compartments and decreased cell survival along with intracellular ROS generation. Straight, thin MWCNTs with a small diameter (<20 µm) 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 µm), moderate and low cytotoxicity of MWCNTs with a small diameter (<20 µm). These results suggested that the diameter of MWCNTs considerably contributes to their cytotoxicity.

Assessment of cytotoxicity and mutagenicity of exfoliated graphene.

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)-1ß, 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.

Basic study of intratracheal instillation study of nanomaterials for the estimation of the hazards of nanomaterials.

In order to examine the usefulness of intratracheal instillation of nanoparticles for the screening of the harmful effects of nanoparticles, we performed intratracheal instillation studies of nanomaterials on rats using different delivery devices and postures as a basic study. Multiwall carbon nanotubes (MWCNTs) with a geometric mean length and secondary diameter of 2.16 µm and 752 nm, respectively, were used as the nanomaterials. Male F344 rats were intratracheally exposed to 0.04 or 0.2 mg/rat of MWCNT, were dissected at 1 d and 3 d, and cell analyses of the bronchoalveolar lavage fluid (BALF) were analyzed. Two delivery devices were used for the intratracheal instillation of the MWCNTs: a gavage needle and a microsprayer aerolizer. Both induced neutrophil influx in the lung at 1 and 3 d, and there were no significant differences in neutrophil inflammation between the two delivery devices. The main distribution of pulmonary inflammation by both delivery devices was in the centrilobular spaces in the lung. Two postures were used: an angle of approximately 45 degrees and a standing posture on a board, both of which also induced pulmonary influx in BALF and pulmonary inflammation mainly in the centrilobular spaces, with no large difference in pulmonary inflammation between the two postures. Taken together, the differences in the delivery devices and postures of the rats in the intratracheal instillation did not affect the acute pulmonary toxicity of the nanomaterials.

Pulmonary and pleural inflammation after intratracheal instillation of short single-walled and multi-walled carbon nanotubes.

Relationships between the physical properties of carbon nanotubes (CNTs) and their toxicities have been studied. However, little research has been conducted to investigate the pulmonary and pleural inflammation caused by short-fiber single-walled CNTs (SWCNTs) and multi-walled CNTs (MWCNTs). This study was performed to characterize differences in rat pulmonary and pleural inflammation caused by intratracheal instillation with doses of 0.15 or 1.5mg/kg of either short-sized SWCNTs or MWCNTs. Data from bronchoalveolar lavage fluid analysis, histopathological findings, and transcriptional profiling of rat lungs obtained over a 90-day period indicated that short SWCNTs caused persistent pulmonary inflammation. In addition, the short MWCNTs markedly impacted alveoli immediately after instillation, with the levels of pulmonary inflammation following MWCNT instillation being reduced in a time-dependent manner. MWCNT instillation induced greater levels of pleural inflammation than did short SWCNTs. SWCNTs and MWCNTs translocated in mediastinal lymph nodes were observed, suggesting that SWCNTs and MWCNTs underwent lymphatic drainage to the mediastinal lymph nodes after pleural penetration. Our results suggest that short SWCNTs and MWCNTs induced pulmonary and pleural inflammation and that they might be transported throughout the body after intratracheal instillation. The extent of changes in inflammation differed following SWCNT and MWCNT instillation in a time-dependent manner.

The induction of lipid peroxidation during the acute oxidative stress response induced by intratracheal instillation of fine crystalline silica particles in rats.

Crystalline silica (SiO2) is an important material for industry but is considered potentially carcinogenic. Inhalation of a crystalline SiO2 aerosol may contribute to serious lung diseases. Crystalline SiO2 particles are commonly used as a positive control in toxicity assays of particulate materials (e.g. nanoparticles). Crystalline SiO2 induces oxidative stress resulting in lipid peroxidation, but the acute oxidative stress response in the lung is not well understood. Lipid peroxidation during the acute stage of oxidative stress after instillation of crystalline SiO2 into rats was examined by bronchoalveolar lavage fluid (BALF) analysis. The levels of 8-iso-prostaglandin F2α and hydroxyoctadecadienoic acid (HODE) in the BALF were measured using liquid chromatography coupled to quadrupole mass spectrometry. The concentration of the antioxidant protein heme oxygenase-1 (HO-1) in the BALF was determined using enzyme-linked immunosorbent assay. Intratracheal instillation of crystalline SiO2 increased the level of HODE and HO-1 in BALF at 24 h after administration. The levels of HODE and HO-1 returned to baseline at 72 h after instillation. Lactate dehydrogenase leakage was observed only after 1 h instillation. These results suggest that the contribution of oxidative stress to the pulmonary toxicity of crystalline SiO2 is minimal in the early acute stage after exposure.

Risk Assessment of the Carbon Nanotube Group.

This study assessed the health risks via inhalation and derived the occupational exposure limit (OEL) for the carbon nanotube (CNT) group rather than individual CNT material. We devised two methods: the integration of the intratracheal instillation (IT) data with the inhalation (IH) data, and the "biaxial approach." A four-week IH test and IT test were performed in rats exposed to representative materials to obtain the no observed adverse effect level, based on which the OEL was derived. We used the biaxial approach to conduct a relative toxicity assessment of six types of CNTs. An OEL of 0.03 mg/m(3) was selected as the criterion for the CNT group. We proposed that the OEL be limited to 15 years. We adopted adaptive management, in which the values are reviewed whenever new data are obtained. The toxicity level was found to be correlated with the Brunauer-Emmett-Teller (BET)-specific surface area (BET-SSA) of CNT, suggesting the BET-SSA to have potential for use in toxicity estimation. We used the published exposure data and measurement results of dustiness tests to compute the risk in relation to particle size at the workplace and showed that controlling micron-sized respirable particles was of utmost importance. Our genotoxicity studies indicated that CNT did not directly interact with genetic materials. They supported the concept that, even if CNT is genotoxic, it is secondary genotoxicity mediated via a pathway of genotoxic damage resulting from oxidative DNA attack by free radicals generated during CNT-elicited inflammation. Secondary genotoxicity appears to involve a threshold.

Size effects of single-walled carbon nanotubes on in vivo and in vitro pulmonary toxicity.

To elucidate the effect of size on the pulmonary toxicity of single-wall carbon nanotubes (SWCNTs), we prepared two types of dispersed SWCNTs, namely relatively thin bundles with short linear shapes (CNT-1) and thick bundles with long linear shapes (CNT-2), and conducted rat intratracheal instillation tests and in vitro cell-based assays using NR8383 rat alveolar macrophages. Total protein levels, MIP-1α expression, cell counts in BALF, and histopathological examinations revealed that CNT-1 caused pulmonary inflammation and slower recovery and that CNT-2 elicited acute lung inflammation shortly after their instillation. Comprehensive gene expression analysis confirmed that CNT-1-induced genes were strongly associated with inflammatory responses, cell proliferation, and immune system processes at 7 or 30 d post-instillation. Numerous genes were significantly upregulated or downregulated by CNT-2 at 1 d post-instillation. In vitro assays demonstrated that CNT-1 and CNT-2 SWCNTs were phagocytized by NR8383 cells. CNT-2 treatment induced cell growth inhibition, reactive oxygen species production, MIP-1α expression, and several genes involved in response to stimulus, whereas CNT-1 treatment did not exert a significant impact in these regards. These results suggest that SWCNTs formed as relatively thin bundles with short linear shapes elicited delayed pulmonary inflammation with slower recovery. In contrast, SWCNTs with a relatively thick bundle and long linear shapes sensitively induced cellular responses in alveolar macrophages and elicited acute lung inflammation shortly after inhalation. We conclude that the pulmonary toxicity of SWCNTs is closely associated with the size of the bundles. These physical parameters are useful for risk assessment and management of SWCNTs.

Ascorbic acid attenuates acute pulmonary oxidative stress and inflammation caused by zinc oxide nanoparticles.

OBJECTIVES: It is known that inhalation of zinc oxide nanoparticles (ZnO NPs) induces acute pulmonary dysfunction, including oxidative stress, inflammation, and injury, but there are no reports on how to prevent these adverse effects. We have previously reported that the pulmonary symptoms caused by ZnO NPs were associated with oxidative stress; in the present study, we therefore investigated the use of ascorbic acid (AA), which is known as vitamin C, to prevent these toxic effects. METHODS: A ZnO NP dispersion was introduced into rat lungs by intratracheal injection, and thereafter a 1% aqueous AA solution was given as drinking water. Bronchoalveolar lavage fluid was collected at 1 day and 1 week after injection, and lactate dehydrogenase (LDH) activity, heme oxygenase-1 (HO-1), and interleukin-6 (IL-6) levels were measured. In addition, expression of the chemokine cytokine-induced neutrophil chemoattractants (CINCs), HO-1, and metallothionein-1 (MT-1) genes in the lungs were determined. RESULTS: Acute oxidative stress induced by ZnO NPs was suppressed by supplying AA. Increases in LDH activity and IL-6 concentration were also suppressed by AA, as was the expression of the CINC-1, CINC-3, and HO-1 genes. CONCLUSIONS: Oral intake of AA prevents acute pulmonary oxidative stress and inflammation caused by ZnO NPs. Intake of AA after unanticipated exposure to ZnO NPs is possibly the first effective treatment for the acute pulmonary dysfunction they cause.

Intratracheal instillation of single-wall carbon nanotubes in the rat lung induces time-dependent changes in gene expression.

The use of carbon nanotubes in the industry has grown; however, little is known about their toxicological mechanism of action. Single-wall carbon nanotube (SWCNT) suspensions were administered by single intratracheal instillation in rats. Persistence of alveolar macrophage-containing granuloma was observed around the sites of SWCNT aggregation at 90 days post-instillation in 0.2-mg- or 0.4-mg-injected doses per rat. Meanwhile, gene expression profiling revealed that a large number of genes involved in the inflammatory response were markedly upregulated until 90 days or 180 days post-instillation. Subsequently, gene expression patterns were dramatically altered at 365 days post-instillation, and the number of upregulated genes involved in the inflammatory response was reduced. These results suggested that alveolar macrophage-containing granuloma reflected a characteristic of the histopathological transition period from the acute-phase to the subchronic-phase of inflammation, as well as pulmonary acute phase response persistence up to 90 or 180 days after intratracheal instillation in this experimental setting. The expression levels of the genes Ctsk, Gcgr, Gpnmb, Lilrb4, Marco, Mreg, Mt3, Padi1, Slc26a4, Spp1, Tnfsf4 and Trem2 were persistently upregulated in a dose-dependent manner until 365 days post-instillation. In addition, the expression levels of Atp6v0d2, Lpo, Mmp7, Mmp12 and Rnase9 were significantly upregulated until 754 days post-instillation. We propose that these persistently upregulated genes in the chronic-phase response following the acute-phase response act as potential biomarkers in lung tissue after SWCNT instillation. This study provides further insight into the time-dependent changes in genomic expression associated with the pulmonary toxicity of SWCNTs.

Cellular effects of industrial metal nanoparticles and hydrophilic carbon black dispersion.

The effects of five types of metal nanoparticles, gold (Au), silver (Ag), platinum (Pt), Au-polyvinylpyrrolidone (PVP) colloid, and Pt-PVP colloid, and two types of hydrophilic carbon black on cell behavior were examined. Stable nanoparticle dispersions were prepared and applied to the culture medium of human keratinocyte (HaCaT) and human lung carcinoma (A549) cells for 6 and 24 hr. Then, the mitochondrial activity (MTT assay) and the induction of cellular oxidative stress were examined. The exposure to Au and Ag decreased mitochondrial activity. The exposure to Pt nanoparticles induced an increase in the intracellular reactive oxygen species (ROS) level. In contrast, Au-PVP, Pt-PVP, and hydrophilic carbon black did not exhibit any effects. The observed increase in the ROS level induced by the Pt nanoparticles in this study contradicted our previous findings, in which Pt did not produce chemically reactive molecules. Some nanoparticle dispersions included chemicals as the dispersant, which is used in industrial applications. In some cases, the dispersing agent may have caused some cellular effects. Adsorption of agents on the surface of the nanoparticles may be an important factor here. Hence, the cellular effects of industrial nanoparticles should be evaluated carefully.

Evaluation of particles released from single-wall carbon nanotube/polymer composites with or without thermal aging by an accelerated abrasion test.

To provide data required for assessing the environmental health and safety risks of nanocomposites, abrasion-induced particle release from single-wall carbon nanotube (SWCNT)/polymer composites with or without thermal aging were evaluated by a shot blast system. First, overall composite weight loss (i.e., overall particle release) as a result of shot blasting was measured. Incorporating 5 wt% SWCNTs in polystyrene (PS) matrix was observed to reduce overall particle release by approximately 30% compared with pure PS. Heat treatment of the 5 wt% SWCNT/PS composites at 100°C for 10 days induced very slight change in overall particle release due to shot blasting. However, heat treatment at 350°C for 1 hr greatly deteriorated the abrasion resistance of the composites, enhancing overall particle release. Second, to verify the existence and form of SWCNTs released from the composites, released particles were observed by electron microscopy. Micron-sized particles with protruding SWCNTs and submicron-sized SWCNT clusters were observed in the particles released from the composites. Heat treatment of the composites at 350°C for 1 hr enhanced SWCNT release, which mainly formed clusters or rope-like bundles.

Evaluation of cellular influences caused by calcium carbonate nanoparticles.

The cellular effects of calcium carbonate (CaCO3) nanoparticles were evaluated. Three kinds of CaCO3 nanoparticles were employed in our examinations. One of the types of CaCO3 nanoparticles was highly soluble. And solubility of another type of CaCO3 nanoparticle was lower. A stable CaCO3 nanoparticle medium dispersion was prepared and applied to human lung carcinoma A549 cells and human keratinocyte HaCaT cells. Then, mitochondrial activity, cell membrane damage, colony formation ability, DNA injury, induction of oxidative stress, and apoptosis were evaluated. Although the influences of CaCO3 nanoparticles on mitochondrial activity and cell membrane damage were small, "soluble" CaCO3 nanoparticles exerted some cellular influences. Soluble CaCO3 nanoparticles also induced a cell morphological change. Colony formation was inhibited by CaCO3 nanoparticle exposure. In particular, soluble CaCO3 nanoparticles completely inhibited colony formation. The influence on intracellular the reactive oxygen species (ROS) level was small. Soluble CaCO3 nanoparticles caused an increase in C/EBP-homologous protein (CHOP) expression and the activation of caspase-3. Moreover, CaCO3 exposure increased intracellular the Ca²âº level and activated calpain. These results suggest that cellular the influences of CaCO3 nanoparticles are mainly caused by intracellular calcium release and subsequently disrupt the effect of calcium signaling. In conclusion, there is possibility that soluble CaCO3 nanoparticles induce cellular influences such as a cell morphological change. Cellular influence of CaCO3 nanoparticles is caused by intracellular calcium release. If inhaled CaCO3 nanoparticles have the potential to influence cellular events. However, the effect might be not severe because calcium is omnipresent element in cell.

Evaluation of cellular effects of silicon dioxide nanoparticles.

Silica nanoparticles (nSiO2s) are an important type of manufactured nanoparticles. Although there are some reports about the cytotoxicity of nSiO2, the association between physical and chemical properties of nSiO2s and their cellular effects is still unclear. In this study, we examined the correlation between the physiochemical properties and cellular effects of three kinds of amorphous nSiO2s; sub-micro-scale amorphous SiO2, and micro-scale amorphous and crystalline SiO2 particles. The SiO2 particles were dispersed in culture medium and applied to HaCaT human keratinocytes and A549 human lung carcinoma cells. nSiO2s showed stronger protein adsorption than larger SiO2 particles. Moreover, the cellular effects of SiO2 particles were independent of the particle size and crystalline phase. The extent of cell membrane damage and intracellular ROS levels were different among nSiO2s. Upon exposure to nSiO2s, some cells released lactate dehydrogenase (LDH), whereas another nSiO2 did not induce LDH release. nSiO2s caused a slight increase in intracellular ROS levels. These cellular effects were independent of the specific surface area and primary particle size of the nSiO2s. Additionally, association of solubility and protein adsorption ability of nSiO2 to its cellular effects seemed to be small. Taken together, our data suggest that nSiO2s do not exert potent cytotoxic effects on cells in culture, especially compared to the effects of micro-scale SiO2 particles. Further studies are needed to address the role of surface properties of nSiO2s on cellular processes and cytotoxicity.

Analysis of pulmonary surfactant in rat lungs after intratracheal instillation of short and long multi-walled carbon nanotubes.

Multi-walled carbon nanotubes (MWCNTs) are interesting new materials, but there is some concern about their harmfulness due to their fibrous nature. To determine the difference in the biological effects of MWCMTs by fiber length, we prepared two MWCNT samples from one bulk sample. One consisted of cut up short fibers (Short; average length=0.94 µm) and the other was just dispersed (Long; average length=3.4 µm). The samples were administered to male Wistar rats by intratracheal instillation at doses of 0.2 mg and 1 mg/animal (Short) and 0.2 mg and 0.6 mg/animal (Long). The animals were sacrificed at time points from 3 d to 12 months after administration. Bronchoalveolar lavage fluid (BALF) was taken from the lungs and pathological specimens were prepared. The concentrations of phospholipids, total protein and surfactant protein D (SP-D) in the pulmonary surfactant of the BALF were determined, the surface tension of BALF was measured, and the inflammation score was determined by the point-counting method to assess pulmonary tissue inflammation. The present study suggests that inflammatory response in the lung was slightly higher for long MWCNTs than for short MWCNTs when compared at the same mass dose. The correlation between pulmonary surfactant components and BALF surface tension was also evaluated. The Spearman's rank correlation coefficients obtained for the phospholipid, total protein and SP-D concentrations were -0.068 (p=0.605), -0.360 (p=0.005) and -0.673 (p=0.000), respectively. Surface tension, measured by a simple method, should be reflected in the change of a surfactant protein, such as SP-D.

In vitro evaluation of cellular influences induced by stable fullerene C70 medium dispersion: induction of cellular oxidative stress.

Fullerene is one of the nanocarbons that is expected to have applications to life science, such as nanomedicines. An understanding of the cellular influences of fullerene is essential for its application to life science. Although C60 and C70 are both known as major fullerenes, most previous reports about the cellular influences of fullerene are about C60. Thus we evaluated the cellular influences caused by C70. A stable and uniform C70-medium dispersion was prepared. The dispersion was stable for the experimental period. Mitochondrial activity (MTT assay), colony forming ability (clonogenic assay), induction of oxidative stress (intracellular ROS and lipid peroxidation levels) and cellular uptake (TEM observation) in human keratinocyte HaCaT and lung carcinoma A549 cells exposed to C70 were examined. C70 did not influence mitochondrial activity. On the other hand, C70 dispersion inhibited colony formation at the concentration of 25.2 µg mL(-1). Exposure to C70 dispersion caused an increase in intracellular ROS and lipid peroxidation levels. The induction of intracellular ROS level was inhibited by pre-treatment of the cells by antioxidants. TEM observations of C70 exposed cells showed cellular uptake of C70. These results were similar to the cellular influences caused by C60 which were reported by us previously. Although C70 did not cause cell death, it caused the induction of intracellular oxidative stress.

Physical properties of single-wall carbon nanotubes in cell culture and their dispersal due to alveolar epithelial cell response.

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.