Masami Hirohashi

Pulmonary toxicity of well-dispersed multi-wall carbon nanotubes following inhalation and intratracheal instillation

Abstract Multi-walled carbon nanotubes (MWCNTs), dispersed in suspensions consisting mainly of individual tubes, were used for intratracheal instillation and inhalation studies. Rats intratracheally received a dose of 0.2 mg, or 1 mg of MWCNTs and were sacrificed from 3 days to 6 months. MWCNTs induced a pulmonary inflammation, as evidenced by a transient neutrophil response in the low-dose groups, and presence of small granulomatous lesion and persistent neutrophil infiltration in the high-dose groups. In the inhalation study, rats were exposed to 0.37 mg/m3 aerosols of well-dispersed MWCNTs (>70% of MWCNTs were individual fibers) for 4 weeks, and were sacrificed at 3 days, 1 month, and 3 months after the end of exposure. The inhalation exposures delivered less amounts of MWCNTs into the lungs, and therefore less pulmonary inflammation responses was observed, as compared to intratracheal instillation. The results of our study show that well-dispersed MWCNT can produce pulmonary lesions, including inflammation.

Inflammogenic effect of well-characterized fullerenes in inhalation and intratracheal instillation studies

BackgroundWe used fullerenes, whose dispersion at the nano-level was stabilized by grinding in nitrogen gas in an agitation mill, to conduct an intratracheal instillation study and an inhalation exposure study. Fullerenes were individually dispersed in distilled water including 0.1% Tween 80, and the diameter of the fullerenes was 33 nm. These suspensions were directly injected as a solution in the intratracheal instillation study. The reference material was nickel oxide in distilled water. Wistar male rats intratracheally received a dose of 0.1 mg, 0.2 mg, or 1 mg of fullerenes and were sacrificed after 3 days, 1 week, 1 month, 3 months, and 6 months. In the inhalation study, Wistar rats were exposed to fullerene agglomerates (diameter: 96 ± 5 nm; 0.12 ± 0.03 mg/m3; 6 hours/days for 5 days/week) for 4 weeks and were sacrificed at 3 days, 1 month, and 3 months after the end of exposure. The inflammatory responses and gene expression of cytokine-induced neutrophil chemoattractants (CINCs) were examined in rat lungs in both studies.ResultsIn the intratracheal instillation study, both the 0.1 mg and 0.2 mg fullerene groups did not show a significant increase of the total cell and neutrophil count in BALF or in the expression of CINC-1,-2αβ and-3 in the lung, while the high-dose, 1 mg group only showed a transient significant increase of neutrophils and expression of CINC-1,-2αβ and -3. In the inhalation study, there were no increases of total cell and neutrophil count in BALF, CINC-1,-2αβ and-3 in the fullerene group.ConclusionThese data in intratracheal instillation and inhalation studies suggested that well-dispersed fullerenes do not have strong potential of neutrophil inflammation.

Expression of cytokine-induced neutrophil chemoattractant in rat lungs by intratracheal instillation of nickel oxide nanoparticles

Since nanoparticles easily agglomerate to form larger particles, it is important to maintain the size of their agglomerates at the nano-level to evaluate the harmful effect of the nanoparticles. We prevented agglomeration of nickel oxide nanoparticles by ultrasound diffusion and filtration, established an acute exposure model using animals, and examined inflammation and chemokine expression. The mass median diameter of nickel oxide nanoparticle agglomerates suspended in distilled water for intratracheal instillation was 26 nm (8.41 nm weighted average surface primary diameter). Male Wistar rats received intratracheal instillation of nickel oxide nanoparticles at 0.1 mg (0.33 mg/kg) or 0.2 mg (0.66 mg/kg), and were dissected 3 days, 1 week, 1 month, 3 months, and 6 months after the instillation. The control group received intratracheal instillation of distilled water. Three chemokines (cytokine-induced neutrophil chemoattractant-1 (CINC-1), CINC-2αβ, and CINC-3) in the lung tissue and bronchoalveolar lavage fluid (BALF) were determined by quantitative measurement of protein by ELISA. Both CINC-1 and CINC-2αβ concentration was elevated from day 3 to 3 months in lung tissue and from day 3 to 6 months in BALF. On the other hand, CINC-3 was elevated on day 3 in both lung tissue and BALF, and then decreased. The total cell and neutrophil counts in BALF were increased from day 3 to 3 months. In lung tissue, infiltration of mainly neutrophils and alveolar macrophages was observed from day 3 to 6 months in alveoli. These results suggest that CINC was involved in lung injury by nickel oxide nanoparticles.

Expression of inflammation-related cytokines following intratracheal instillation of nickel oxide nanoparticles

Abstract The objective of this study was to examine what kinds of cytokines are related to lung disorder by well-dispersed nanoparticles. The mass median diameter of nickel oxide in distilled water was 26 nm. Rats intratracheally received 0.2 mg of nickel oxide suspended in distilled water, and were sacrificed from three days to six months. The concentrations of 21 cytokines including inflammation, fibrosis and allergy-related ones were measured in the lung. Infiltration of alveolar macrophages was observed persistently in the nickel oxide-exposed group. Expression of macrophage inflammatory protein-1α showed a continued increase in lung tissue and broncho-alveolar lavage fluid (BALF) while interleukin-1α (IL-1α), IL-1β in lung tissue and monocyte chemotactic protein-1 in BALF showed transient increases. Taken together, it was suggested that nano-agglomerates of nickel oxide nanoparticles have a persistent inflammatory effect, and the transient increase in cytokine expression and persistent increases in CC chemokine were involved in the persistent pulmonary inflammation.

Pulmonary toxicity of well-dispersed single-wall carbon nanotubes after inhalation

Abstract Single-wall carbon nanotubes (SWCNTs) were well-dispersed by ultrasonication to conduct an inhalation study. SWCNTs were generated using a pressurised nebuliser with liquid suspension of SWCNTs. Wistar rats were exposed to the well-dispersed SWCNT (diameter of bundle: 0.2 μm; length of bundle: 0.7 μm) for 4 weeks. The low and high mass concentrations of SWCNTs were 0.03 ± 0.003 and 0.13 ± 0.03 mg/m3, respectively. The rats were sacrificed at 3 days, 1 month, and 3 months after the end of exposure. There were no increases of total cell or neutrophil counts in the bronchoalveolar lavage fluid (BALF), or the concentration of cytokine-induced neutrophil chemoattractant in the lungs or BALF in both the high and low concentration-exposed groups. Pulmonary infiltration of neutrophils was not observed in either exposed group throughout the observation period. Well-dispersed SWCNT did not induce neutrophil inflammation in the lung under the conditions in the present study.

Pulmonary Toxicity Following an Intratracheal Instillation of Nickel Oxide Nanoparticle Agglomerates

Pulmonary Toxicity Following an Intratracheal Instillation of Nickel Oxide Nanoparticle Agglomerates: Yasuo Morimoto, et al. Institute of Industrial Ecological Sciences, University of Occupational and Environmental Health, Japan—

Pathological features of rat lung following inhalation and intratracheal instillation of C(60) fullerene.

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 C60 (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 C60, and were sacrificed at 3 days, 1 week, 1 month, 3 months, 6 months, and 12 months. In the inhalation study, Wistar rats received C60 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 C60 (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 C60-exposed group, PCM evaluation showed significant changes in the C60-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 C60 via both intratracheal and inhalation instillation was slight and transient. These results support our previous studies that showed C60 has no significant adverse effects in intratracheal and inhalation instillation studies.

Expression of Heme Oxygenase-1 in the Lungs of Rats Exposed to Crystalline Silica

Expression of Heme Oxygenase‐1 in the Lungs of Rats Exposed to Crystalline Silica: Hiroko Nagatomo, et al. Department of Occupational Pneumology, Institute of Industrial and Ecological Sciences, University of Occupational and Environmental Health—Oxidative stress is thought to be the pathogenesis of pulmonary fibrosis induced by particles, and heme oxygenase‐1 (HO‐1) protects lung tissue against oxidative stress. We hypothesized that HO‐1 is also associated with oxidative lung injury caused by exposure to particles. The present study was conducted to investigate the time course of HO‐1 expression of lungs exposed to crystalline silica in vivo. Male Wistar rats were administered 1 mg or 2 mg of crystalline silica suspended in saline by a single intratracheal instillation and were sacrificed at 3 d, 1 wk, 1 month, 3 months and 6 months of recovery time. The expression of HO‐1 was observed by western blot analysis and immunostaining. Protein levels of HO‐1 were increased compared to the controls at 3 d, and from 1 month to 6 months following intratracheal instillation of 2 mg of crystalline silica. The levels of HO‐1 were increased compared to the controls from 1 month to 6 months following intratracheal instillation of 1 mg of crystalline silica. Many HO‐1 positive cells were found particularly in the alveolar macrophages during immunostaining. These findings suggest that HO‐1 is related to lung injury arising from exposure to crystalline silica.

Expression of Heme Oxygenase-1 in the Lungs of Rats Exposed to Crocidolite Asbestos

Abstract Oxidative stress is thought to be the pathogenesis of pulmonary fibrosis induced by asbestos, and heme oxygenase-1 (HO-1) protects lung tissue against oxidative stress. We hypothesized that HO-1 is associated with oxidative lung injury caused by exposure to asbestos. This study was conducted to investigate the time course of HO-1 expression of lungs exposed to crocidolite asbestos in vivo. Male Wistar rats were administered 1 mg or 2 mg crocidolite asbestos suspended in saline by a single intratracheal instillation and were sacrificed at 3 d, 1 wk, 1 mo, 3 mo, and 6 mo of recovery time. The expression of HO-1 was observed by Western blot analysis and immunostaining. Protein levels of HO-1 increased at from 3 d to 6 mo following intratracheal instillation of 2 mg crocidolite asbestos. The levels of HO-1 increased at 1 wk and 1 mo following intratracheal instillation of 1 mg crocidolite asbestos. Many HO-1-positive cells were found, particularly in the alveolar macrophages, during immunostaining. These findings suggest that HO-1 may be related to lung disorder induced by dust and therefore can act as a biomarker of lung injury due to dust exposure.

Biopersistence of inhaled MWCNT in rat lungs in a 4-week well-characterized exposure

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.