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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.

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.

Inhalation studies and intratracheal instillation studies using laboratory animals are commonly conducted for pulmonary toxicity tests of nanomaterials. In our study, male Wister rats were exposed to nickel oxide (NiO) particles including a nano-scale, even for aerosols and suspensions, in a 4-week inhalation and intratracheal instillation. Using polymorphonuclear neutrophils (PMNs) in bronchoalveolar lavage fluid as a biomarker of inflammation, we attempted to quantify the relationship between responses to inhalation and intratracheal instillation of the nanoparticles, based on surface area doses. Four kinds of NiO suspension samples with different specific surface areas were singly injected via the tracheas of the rats. The relationship between the instilled doses and PMN production was examined 3 days and 1 month after the instillation. In parallel, 4-week inhalation studies, using two of the suspensions, were conducted for aerosols generated by a pressurized nebulizer. NiO samples induced PMN responses 3 days after instillation according to the surface area doses, but not the mass doses, as has been reported in many studies. When the same NiO samples were tested in a 4-week inhalation and intratracheal instillation, the amount of pulmonary deposition of the sample after the 4-week inhalation, and an intratracheally instilled dose about ten-times higher, induced similar PMN responses 3 days after termination of inhalation and instillation. Using the relationship between these responses to 4-week inhalation and intratracheal instillation, it may be possible to estimate what aerosol concentrations of other nanomaterials might cause the same responses of PMN production as intratracheal instillation tests.

The aim of the present study is to understand the association between metal ion release from nickel oxide (NiO) nanoparticles and induction of oxidative stress in the lung. NiO nanoparticles have cytotoxic activity through nickel ion release and subsequent oxidative stress. However, the interaction of oxidative stress and nickel ion release in vivo is still unclear. In the present study, we examined the effect of metal ion release on oxidative stress induced by NiO nanoparticles. Additionally, nano and fine TiO(2) particles as insoluble particles were also examined. Rat lung was exposed to NiO and TiO(2) nanoparticles by intratracheal instillation. The NiO nanoparticles released Ni(2+) in dispersion. Bronchoalveolar lavage fluid (BALF) was collected at 1, 24, 72 h and 1 week after instillation. The lactate dehydrogenase (LDH) and HO-1 levels were elevated at 24 and 72 h after instillation in the animals exposed to the NiO nanoparticles. On the other hand, total hydroxyoctadecadienoic acid (tHODE), which is an oxidative product of linoleic acid, as well as SP-D and α-tochopherol levels were increased at 72 h and 1 week after instillation. Fine NiO particles, and nano and fine TiO(2) particles did not show lung injury or oxidative stress from 1 h to 1 week after instillation. These results suggest that Ni(2+) release is involved in the induction of oxidative stress by NiO nanoparticles in the lung. Ni(2+) release from NiO nanoparticles is an important factor inoxidative stress-related toxicity, not only in vitro but also in vivo.

It is important to conduct a risk assessment that includes hazard assessment and exposure assessment for the safe production and handling of newly developed nanomaterials. We conducted an inhalation study of a multi-wall carbon nanotube (MWCNT) as a hazard assessment. Male Wistar rats were exposed to well-dispersed MWCNT for 4 weeks by whole body inhalation. The exposure concentration in the chamber was 0.37 ± 0.18 mg/m³. About 70% of the MWCNTs in the chamber were single fiber. The geometric mean diameter (geometric standard deviation, GSD) and geometric mean length (GSD) of the aerosolized MWCNTs in the chamber were 63 nm (1.5) and 1.1 μm (2.7), respectively. The amounts of MWCNT deposited in the rat lungs were determined by the X-ray diffraction method and elemental carbon analysis. The average deposited amounts at 3 days after the inhalation were 68 μg/lung by the X-ray diffraction method and 76 μg/lung by elemental carbon analysis. The calculated deposition fractions were 18% and 20% in each analysis. The amount of retained MWCNT in the lungs until 3 months after the inhalation decreased exponentially and the calculated biological half times of MWCNT were 51 days and 54 days, respectively. The clearance was not delayed, but a slight increase in lung weight at 3 days after the inhalation was observed.

We evaluated the pulmonary pathological features of rats that received a single intratracheal instillation and a 4-week inhalation of a fullerene. We used fullerene C(60) (nanom purple; Frontier Carbon Co. Ltd, Japan) in this study. Male Wistar rats received intratracheal dose of 0.1, 0.2, or 1 mg of C(60), and were sacrificed at 3 days, 1 week, 1 month, 3 months, 6 months, and 12 months. In the inhalation study, Wistar rats received C(60) or nickel oxide by whole-body inhalation for 6 h/day, 5 days/week, 4 weeks, and were sacrificed at 3 days, 1 month, and 3 months after the end of exposure. During the observation period, no tumors or granulomas were observed in either study. Histopathological evaluation by the point counting method (PCM) showed that a high dose of C(60) (1 mg) instillation led to a significant increase of areas of inflammation in the early phase (until 1 week). In the inhalation study of the C(60)-exposed group, PCM evaluation showed significant changes in the C(60)-exposed group only at 3 days after exposure; after 1 month, no significant changes were observed. The present study demonstrated that the pulmonary inflammation pattern after exposure to well-characterized C(60) via both intratracheal and inhalation instillation was slight and transient. These results support our previous studies that showed C(60) has no significant adverse effects in intratracheal and inhalation instillation studies.