Akira Ogami

Gene expression profiles in rat lung after inhalation exposure to C60 fullerene particles.

Concern over the influence of nanoparticles on human health has risen due to advances in the development of nanotechnology. We are interested in the influence of nanoparticles on the pulmonary system at a molecular level. In this study, gene expression profiling of the rat lung after whole-body inhalation exposure to C(60) fullerene (0.12mg/m(3); 4.1x10(4) particles/cm(3), 96nm diameter) and ultrafine nickel oxide (Uf-NiO) particles (0.2mg/m(3); 9.2x10(4) particles/cm(3), 59nm diameter) as a positive control were employed to gain insights into these molecular events. In response to C(60) fullerene exposure for 6h a day, for 4 weeks (5 days a week), C(60) fullerene particles were located in alveolar epithelial cells at 3 days post-exposure and engulfed by macrophages at both 3 days and 1 month post-exposures. Gene expression profiles revealed that few genes involved in the inflammatory response, oxidative stress, apoptosis, and metalloendopeptidase activity were up-regulated at both 3 days and 1 month post-exposure. Only some genes associated with the immune system process, including major histocompatibility complex (MHC)-mediated immunity were up-regulated. These results were significantly different from those of Uf-NiO particles which induced high expression of genes associated with chemokines, oxidative stress, and matrix metalloproteinase 12 (Mmp12), suggesting that Uf-NiO particles lead to acute inflammation for the inhalation exposure period, and the damaged tissues were repaired in the post-exposure period. We suggest that C(60) fullerene might not have a severe pulmonary toxicity under the inhalation exposure condition.

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.

Pathological features of different sizes of nickel oxide following intratracheal instillation in rats

Focusing on the “size” impact of particles, the objective of this study was to analyze morphological and qualitative changes over time in the development of inflammation and collagen deposition in lung tissue after intratracheal instillation of two sizes of nickel oxide in rats, in comparison with the results of instillation of crystalline silica and titanium dioxide. The fine-sized nickel oxide sample (nNiOm: median diameter of agglomerated particles 0.8 μm) was prepared from crude particles of nickel oxide (median diameter of primary particle 27 nm) by liquid-phase separation. Another samples of micrometer-sized nickel oxide (NiO: median diameter of particles 4.8 μm), crystalline silica (Min-U-SIL-5; geometric mean diameter 1.6 μm, geometric standard deviation [GSD] 2.0), and TiO2 (geometric mean diameter 1.5 μm, GSD 1.8) were also used. Well-sonicated samples of 2 mg per 0.4 ml saline or saline alone (control) were intratracheally instilled into Wistar rats (males, 10 wk old). Bronchoalveolar lavage fluid (BAL)F and lung tissue were examined at 3 days, 1 wk, 1 mo, 3 mo, and 6 mo after instillation, from 5 rats of each group. Histopathological findings showed that the infiltration of macrophages or polymorphonuclear cells and the alveolitis in rats treated with nNiOm were remarkable over time and similar to the effects of crystalline silica. The numbers of total cells in BALF and the percentage of plymorphonuclear leukocytes (PMNs) also increased in the nNiOm group and silica group. The point counting method (PCM) showed a significant increase of inflammatory area, with the peak at 3 mo after instillation in the nNiOm group. In contrast, NiO treatment showed only a slight inflammatory change. Collagen deposition in two regions in the lung tissue (alveolar duct and pleura) showed an increasing collagen deposition rate in nNiOm at 6 mo. Our results suggest that submicrometer nano-nickel oxide is associated with greater toxicity, as for crystalline silica, than micrometer-sized nickel oxide. Biological effects of factors of particle size reduction, when dealing with finer particles such as nanoparticles, were reconfirmed to be important in the evaluation of respirable particle toxicity.

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.

Effect of Particle Size of Intratracheally Instilled Crystalline Silica on Pulmonary Inflammation

Effect of Particle Size of Intratracheally Instilled Crystalline Silica on Pulmonary Inflammation: Takayoshi Kajiwara, et al. Department of Environmental Health Engineering, University of Occupational and Environmental Health—Crystalline silica, known as a causal substance of silicosis, has been carefully evaluated for its carcinogenicity and fibrogenicity. In this study, we instilled crystalline silica of two different size (S1.8 :1.80 µm (S.D. 2.0), S0.7 :0.74 µm (S.D. 1.5)) into the trachea of rats to evaluate the size effects of the particles on pulmonary inflammation. S1.8 and S0.7 samples were administered to rats by a single intratracheal instillation (2 mg/ 0.4 ml saline). At three days, 1 wk and 1, 3 and 6 months after the instillation, the blood, bronchoalveolar lavage fluid (BALF), and pulmonary tissues were analyzed. Six images per HE‐stained section were digitally captured and examined by the point counting method (PCM). Polymorphonuclear leukocyte (PMN)‐in‐blood specimens and cytospin specimens from BALF were stained immunohistochemically with BrdU. At six months after the instillation, the effects on inflammatory cells in the pulmonary tissues and BALF tended to be more marked in the rats instilled with S1.8 than those instilled with S0.7. Particularly, clear differences were observed in the number of inflammatory cells in BALF. Even if the particles are of the same chemical composition, the results suggest that, their biological effects vary depending on their particle size. Therefore, when such particles are used in workplaces, strict control systems should be established according to the risks present by different sizes of particles.

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.

Biopersistence of inhaled nickel oxide nanoparticles in rat lung

In order to investigate whether or not airborne nanoparticles with a minimum agglomeration could be used for exposure tests on animals, we developed a nanoparticle generation system and examined the biological effects of the particles in an inhalation study. The generation system was composed of an ultrasonic nebulizer and diffusion dryers, and 30 Wistar male rats were exposed to nickel oxide (NiO) nanoparticles for 4 wk (6 h/day). The geometric mean diameter of the particles and the daily average exposure concentration determined by a combination of a differential mobility analyzer and a condensation nucleus counter in the exposure chamber were 139 ± 12 nm and 1.0 ± 0.5 × 105 particles/cm3, respectively. At 4 days and 1 and 3 mo after the inhalation, each group of 10 rats were sacrificed and NiO nanoparticles deposited in the lung were determined by chemical analysis and the biopersistence (biological half time) was calculated. The deposited amount of NiO nanoparticles in the rat lungs at 4 days after the inhalation was 29 ± 4 μg. The retained particle amount in the rat lungs after the inhalation exponentially decreased and the calculated biological half time was 62 days. The histopathological change was not severe just after the inhalation nor throughout the observation time. We concluded that nanoparticles with a minimum agglomeration were dispersed stably in the chamber and exposed to rats for 4 wk and that deposited amounts in the rat lungs and the biopersistence of the particles and the biological response in lung were detected.

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—