Chikara Kadoya

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.

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.

Histopathological changes in rat lung following intratracheal instillation of silicon carbide whiskers and potassium octatitanate whiskers.

Abstract We evaluated the pattern of pulmonary inflammation for the assessment of the biological hazards of two man-made mineral fibers. Rats were exposed by intratracheal instillation to a 2 mg dose of each of two kinds of man-made mineral fibers (PT1, potassium octatitanate whisker; and SiCW, silicon carbide whisker), or three kinds of comparable respirable particles (crystalline silica, crocidolite asbestos, and titanium dioxide, TiO2). The lung tissue was evaluated at 3 day, 1 wk, and 1, 3 and 6 mo after exposure. Digital images taken of the lung sections were examined by morphometric point counting method (PCM). PT1 and SiCW showed a similar inflammatory pattern, which contains temporal inflammation such as moderate alveolitis within 1 wk after the exposure, and in later phase aggregation foci of instilled fibers. Differences in repair patterns of these two man-made mineral fibers showed that the toxicity of these two fibers is less toxic than for crocidolite or crystalline silica. Although SiCW showed a higher inflammation score than TiO2 within 1 mo after instillation, the inflammation scores and fibrotic changes of PT1 and SiCW were not significant as TiO2 at 3 mo and 6 mo in this study. Careful use should be recommended when these materials are used in the workplace.

Comparison of dose-response relations between 4-week inhalation and intratracheal instillation of NiO nanoparticles using polimorphonuclear neutrophils in bronchoalveolar lavage fluid as a biomarker of pulmonary inflammation

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.

Effect of polymerized toner on rat lung in chronic inhalation study.

In order to evaluate the chronic effect of polymerized toner particles on the lung, inflammation- and fibrosis-related genes were analyzed and 8-hydroxydeoxyguanosine (8-OHdG) was examined by using the lung tissue of rats subjected to 24 months of toner inhalation exposure. Wistar female rats were divided into four groups (5 weeks old, 30 rats in each): the high concentration exposure group (16.3 ± 0.6 mg/m3), the medium concentration exposure group (4.4 ± 0.3 mg/m3), the low concentration exposure group (1.6 ± 0.2 mg/m3), and the control group (clean air). The material used was black toner, and its aerodynamic diameter in the exposure chamber was 3.0 μm. The rats were exposed to the material for 24 months (6 hours/day, 5 days/week) and dissected after the exposure period. RNA was extracted from one lung and the gene expression related to inflammation and fibrosis. Matrix metalloproteinase-2 (MMP-2), tissue inhibitor of metalloproteinase-2 (TIMP-2), and type I collagen were analyzed according to the ratio of each gene/β-actin. Also, 8-OHdG level in the lung tissue was measured by HPLC with an electrochemical detector. Small fibrotic foci were found in the toner exposed groups; however, progressive or irreversible fibrosis was not found. The incidence of small fibrotic foci and cell aggregation increased in a dose-dependent manner. There were no significant differences of expression of MMP-2, TIMP-2, and type I collagen between the control group and each exposed group. Lung tumors did not develop in each group. A significant production of 8-OHdG was not observed in the toner exposed groups. In conclusion, toner produced by polymerization was not associated with evidence of carcinogenesis in this experiment.

Analysis of pulmonary surfactant in rat lungs after inhalation of nanomaterials: Fullerenes, nickel oxide and multi-walled carbon nanotubes

Abstract The health risks of inhalation exposure to engineered nanomaterials in the workplace are a major concern in recent years, and hazard assessments of these materials are being conducted. The pulmonary surfactant of lung alveoli is the first biological entity to have contact with airborne nanomaterials in inhaled air. In this study, we retrospectively evaluated the pulmonary surfactant components of rat lungs after a 4-week inhalation exposure to three different nanomaterials: fullerenes, nickel oxide (NiO) nanoparticles and multi-walled carbon nanotubes (MWCNT), with similar levels of average aerosol concentration (0.13–0.37 mg/m3). Bronchoalveolar lavage fluid (BALF) of the rat lungs stored after previous inhalation studies was analyzed, focusing on total protein and the surfactant components, such as phospholipids and surfactant-specific SP-D (surfactant protein D) and the BALF surface tension, which is affected by SP-B and SP-C. Compared with a control group, significant changes in the BALF surface tension and the concentrations of phospholipids, total protein and SP-D were observed in rats exposed to NiO nanoparticles, but not in those exposed to fullerenes. Surface tension and the levels of surfactant phospholipids and proteins were also significantly different in rats exposed to MWCNTs. The concentrations of phospholipids, total protein and SP-D and BALF surface tension were correlated significantly with the polymorphonuclear neutrophil counts in the BALF. These results suggest that pulmonary surfactant components can be used as measures of lung inflammation.

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

Abstract 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.