Junko Maru

Comparative pulmonary toxicity study of nano-TiO2 particles of different sizes and agglomerations in rats: Different short- and long-term post-instillation results

Two intratracheal instillation experiments with nano-size titanium dioxide (TiO(2)) particles of different sizes and agglomerations were conducted in rats to compare the biological responses induced by the different particles. In experiment 1, 5 mg/kg of nano-TiO(2) particles of different primary sizes was intratracheally instilled in rats. In experiment 2, a similar procedure was followed with 5 mg/kg of nano-TiO(2) particles of the same primary sizes but different agglomerations in liquid. Following the instillations, body and lung weight measurements, bronchoalveolar fluid (BALF) cells and inflammatory biomarkers assessment, and histopathological evaluations of the lungs and other tissues were conducted. Pulmonary inflammatory responses until 1 week post-instillation differed among the TiO(2) particle-exposed groups: that is, smaller particles induced greater inflammation in the short-term observations. With regard to the long-term effects (>1 week post-instillation), however, pulmonary inflammation remarkably recovered in all the TiO(2) particle-exposed groups, with no differences between the groups regardless of particle size. On the other hand, no clear relationship was observed between the TiO(2) particle-exposed groups with different agglomerations but the same primary size. These findings suggest that different evaluations can be derived on the basis of the observations up to 1 week post-instillation and those after 1 month post-instillation. In most of the current studies, the relationship between pulmonary responses and instilled particle sizes has been discussed only on the basis of the 24 h post-instillation results, which could be a misleading evaluation. Consequently, our findings indicate that both short- and long-term effects should be evaluated when assessing the toxicity of nanoparticles based on the results of intratracheal instillation studies.

Association of zinc ion release and oxidative stress induced by intratracheal instillation of ZnO nanoparticles to rat lung

Zinc oxide (ZnO) nanoparticles are one of the important industrial nanoparticles. The production of ZnO nanoparticles is increasing every year. On the other hand, it is known that ZnO nanoparticles have strong cytotoxicity. In vitro studies using culture cells revealed that ZnO nanoparticles induce severe oxidative stress. However, the in vivo influence of ZnO nanoparticles is still unclear. In the present study, rat lung was exposed to ZnO nanoparticles by intratracheal instillation, and the influences of ZnO nanoparticles to the lung in the acute phase, particularly oxidative stress, were examined. Additionally, in vitro cellular influences of ZnO nanoparticles were examined using lung carcinoma A549 cells and compared to in vivo examinations. The ZnO nanoparticles used in this study released zinc ion in both dispersions. In the in vivo examinations, ZnO dispersion induced strong oxidative stress in the lung in the acute phase. The oxidative stress induced by the ZnO nanoparticles was stronger than that of a ZnCl(2) solution. Intratracheal instillation of ZnO nanoparticles induced an increase of lipid peroxide, HO-1 and alpha-tocopherol in the lung. The ZnO nanoparticles also induced strong oxidative stress and cell death in culture cells. Intracellular zinc level and reactive oxygen species were increased. These results suggest that ZnO nanoparticles induce oxidative stress in the lung in the acute phase. Intracellular ROS level had a high correlation with intracellular Zn(2+) level. ZnO nanoparticles will stay in the lung and continually release zinc ion, and thus stronger oxidative stress is induced.

In vitro and in vivo genotoxicity tests on fullerene C60 nanoparticles

There are several conflicting reports on the genotoxicity of fullerene C(60) in the literature. To determine the genotoxic potential of C(60) nanoparticles, we prepared stable nano-sized C(60) suspensions using 0.1% carboxymethylcellulose sodium (CMC-Na) or 0.1% Tween 80 aqueous solution. We conducted a bacterial reverse mutation test with Ames Salmonella typhimurium TA98, TA100, TA1535, and TA1537 strains and Escherichia coli strain and a chromosomal aberration test with cultured Chinese hamster CHL/IU cells in the presence and absence of metabolic activation under dark conditions and visible light irradiation using a stable C(60) nanoparticle suspension with CMC-Na. In addition, we performed a bone marrow micronucleus test using a stable C(60) nanoparticle suspension with Tween 80 on ICR mice. C(60) nanoparticles did not show a positive mutagenic response up to the maximum dose of 1000 microg/plate with any tester strain in the bacterial reverse mutation test regardless of metabolic activation and irradiation, although a slight but not significant increase in the number of revertants was observed in TA100 and WP2 uvrA/pKM101. No increase in the incidence of chromosomal aberrations was observed at any C(60) nanoparticle dose regardless of metabolic activation and irradiation in the chromosomal aberration test up to the maximum doses of 100 and 200 microg/mL. In addition, the micronucleus test showed that the in vivo clastogenic ability of the C(60) nanoparticles was negative up to the maximum dose of 88 mg/kg x 2. Therefore, we concluded that the stable and well-characterized C(60) nanoparticles did not have genotoxic ability in the bacterial reverse mutation assay, in vitro chromosome aberration assay, nor in vivo micronucleus assay.

Biological response and morphological assessment of individually dispersed multi-wall carbon nanotubes in the lung after intratracheal instillation in rats

Biological responses of multi-wall carbon nanotubes (MWCNTs) were assessed after a single intratracheal instillation in rats. The diameter and median length of the MWCNTs used in this study were approximately 60 nm and 1.5 μm, respectively. Groups of male Sprague-Dawley rats were intratracheally instilled with 0.04, 0.2, or 1 mg/kg of the individually dispersed MWCNT suspension. After instillation, the bronchoalveolar lavage fluid was assessed for inflammatory cells and markers, and the lung, liver, kidney, spleen, and cerebrum were histopathologically evaluated at 3-day, 1-week, 1-month, 3-month, and 6-month post-exposure. Transient pulmonary inflammatory responses were observed only in the lungs of the group of rats exposed to 1 mg/kg of MWCNTs. Morphology of the instilled MWCNTs in the lungs of rats was assessed using light microscopy and transmission electron microscopy (TEM). Light microscopy examination revealed that MWCNTs deposited in the lungs of the rats were typically phagocytosed by the alveolar macrophages and these macrophages were consequently accumulated in the alveoli until 6-month post-exposure. The 400 TEM images obtained showed that all MWCNTs were located in the alveolar macrophages or macrophages in the interstitial tissues, and MWCNTs were not located in the cells of the interstitial tissues. There was no evidence of chronic inflammation, such as angiogenesis or fibrosis, induced by MWCNT instillation. These results suggest that MWCNTs were being processed and cleared by alveolar macrophages.

Comparison of acute oxidative stress on rat lung induced by nano and fine-scale, soluble and insoluble metal oxide particles: NiO and TiO2

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 TiO2 particles as insoluble particles were also examined. Rat lung was exposed to NiO and TiO2 nanoparticles by intratracheal instillation. The NiO nanoparticles released Ni2+ 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 TiO2 particles did not show lung injury or oxidative stress from 1 h to 1 week after instillation. These results suggest that Ni2+ release is involved in the induction of oxidative stress by NiO nanoparticles in the lung. Ni2+ release from NiO nanoparticles is an important factor inoxidative stress-related toxicity, not only in vitro but also in vivo.

Genotoxicity evaluation of fullerene C60 nanoparticles in a comet assay using lung cells of intratracheally instilled rats.

The genotoxicity of fullerene C(60) nanoparticles was evaluated in vivo with comet assays using the lung cells of rats given C(60) nanoparticles. The C(60) nanoparticles were intratracheally instilled as a single dose at 0.5 or 2.5mg/kg or repeated dose at 0.1 or 0.5mg/kg, once a week for 5 weeks, to male rats. The lungs were obtained 3 or 24h after a single instillation and 3h after repeated instillation. Inflammatory responses were observed in the lungs obtained 24h after a single instillation at 2.5mg/kg and repeated instillation at 0.5mg/kg. Histopathological examinations revealed that C(60) nanoparticles caused slight changes including hemorrhages in alveoli and the cellular infiltration of macrophages and neutrophils in alveoli. In comet assays using rat lung cells, no increase in % Tail DNA was found in any group given C(60) nanoparticles. These findings indicate that C(60) nanoparticles had no potential for DNA damage in comet assays using the lungs cells of rats given C(60) even at doses causing inflammation.

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

Abstract 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.

In vivo genotoxicity study of single-wall carbon nanotubes using comet assay following intratracheal instillation in rats.

The genotoxicity of single-wall carbon nanotubes (SWCNTs) was evaluated in vivo using the comet assay after intratracheal instillation in rats. The SWCNTs were instilled at a dosage of 0.2 or 1.0mg/kg body weight (single instillation group) and 0.04 or 0.2mg/kg body weight once a week for 5weeks (repeated instillation group). As a negative control, 1% Tween 80 was instilled in a similar manner. As a positive control, ethyl methanesulfonate (EMS) at 500mg/kg was administered once orally 3h prior to dissection. Histopathologically, inflammation in the lung was observed for all the SWCNTs in both single and repeated groups. In the comet assay, there was no increase in% tail DNA in any of the SWCNT-treated groups. In the EMS-treated groups, there was a significant increase in% tail DNA compared with the negative control group. The present study indicated that a single intratracheal instillation of SWCNTs (1.0mg/kg) or repeated intratracheal instillation (0.2mg/kg) once a week for five weeks induced a clear inflammatory response (hemorrhage in the alveolus, infiltration of alveolar macrophages and neutrophiles), but no DNA damage, in the lungs in rats. Under the conditions of the test, SWCNTs were not genotoxic in the comet assay following intratracheal instillation in rats.

In vivo comet assay of multi‐walled carbon nanotubes using lung cells of rats intratracheally instilled

The genotoxicity of multi‐walled carbon nanotubes (MWCNTs) was evaluated in vivo with comet assays using the lung cells of rats given MWCNTs. The MWCNTs were intratracheally instilled as a single dose at 0.2 or 1.0 mg kg–1 or a repeated dose at 0.04 or 0.2 mg kg–1, once a week for 5 weeks, to male rats. The rats were sacrificed 3 or 24 h after the single instillation and were sacrificed 3 h after the last instillation in the repeated instillation groups. Histopathological examinations of the lungs revealed that MWCNTs caused inflammatory changes including the infiltration of macrophages and neutrophils after a single instillation and repeated instillation at both doses. In comet assays using rat lung cells, no changes in % Tail DNA were found in any group given MWCNTs. These findings indicate that MWCNTs do not have the potential to cause DNA damage in comet assays using the lung cells of rats given MWCNTs at doses causing inflammatory responses. Copyright

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.