Effects of advanced nanomaterials on the respiratory system of a murine COPD model.

This study assessed the effects of cellulose nanofibrils (CNFs) and multi-walled carbon nanotubes (MWCNTs) on lung inflammation in a cigarette smoke-induced chronic obstructive pulmonary disease (COPD) mouse model. Prior to instillation, COPD model mice displayed distinctive cellular compositions and elevated cytokine levels in bronchoalveolar lavage fluid (BALF). After intratracheal instillation of 80 µg CNFs, no significant histopathological changes, BALF composition alterations, or cytokine level shifts were observed on day 28. This suggests minimal lung impact and no interference with reducing smoke-induced inflammation. In contrast, the instillation of 80 µg MWCNTs resulted in significant histopathological changes, increased cellular composition, and elevated cytokine levels in BALF on day 28. These findings indicate that CNF exposure had little effect on the lungs and did not impede the reduction of smoke-induced inflammation, while MWCNT exposure hindered the attenuation of pulmonary inflammatory response. The study emphasizes the importance of considering diverse cases, including individuals with pre-existing respiratory conditions, when assessing occupational safety and health risks associated with advanced nanomaterial exposure.

Contaminant microorganisms in the in vitro evaluation of cellular responses of cellulose nanofibers and their microbial inactivation using gamma irradiation.

Cellulose nanofibers (CNFs) are fibrous nanomaterials produced from plants. Since some nanomaterials are toxic, toxicity evaluation, including in vitro examinations using cultured cells, is essential for the effective use of CNFs. On the other hand, microorganisms in the environment can contaminate CNF suspensions. The contamination of CNF samples and the effects of contaminating microorganisms on in vitro examinations were investigated in this study. Microorganism contamination in CNF samples was examined, and microbial inactivation of CNF suspensions using gamma irradiation was evaluated. After gamma-ray irradiation at absorbed doses of 0.5, 1, 5, and 10 kGy, the cellular effects of CNF suspensions were examined using 6 types of cultured cell, HaCaT, A549, Caco-2, MeT-5A, THP-1, and NR8383 cells. CNF samples were contaminated with bacteria and CNF suspensions exhibited endotoxin activity. Gamma irradiation effectively inactivated the microorganisms contained in the CNF suspensions. When the absorbed dose was 10 kGy, the fiber length of CNF was shortened, but the effect on CNF was small at 1.0 kGy or less. CNF suspensions showed lipopolysaccharides (LPS)-like cellular responses and strongly induced interleukin-8, especially in macrophages. Absorbed doses of at least 10 kGy did not affect the LPS-like activity. In this study, it was shown that the CNF suspension may be contaminated with microorganisms. Gamma irradiation was effective for microbial inactivation of suspension for invitor toxicity evaluation of CNF. In vitro evaluation of CNFs requires attention to the effects of contaminants such as LPS.

Genotoxicity assessment of cellulose nanofibrils using a standard battery of in vitro and in vivo assays.

Cellulose nanofibrils (CNFs) are identified as novel nanomaterials with many potential applications. Since CNFs are fibrous manufactured nanomaterials, their potential carcinogenic effects and mesothelial toxicity raise some concerns. In this study, we conducted a standard battery of in vitro and in vivo assays to evaluate the genotoxicity of two CNF types using different manufacturing methods and physicochemical properties. Namely, one was CNF produced via chemical modification by TEMPO (2,2,6,6-tetramethylpiperidine-1-oxyl radical)-mediated oxidation, while the other was CNF produced via mechanical defibrillation using needle bleached kraft pulp. A bacterial reverse mutation test and a mouse lymphoma TK assay revealed that CNFs at 100 µg/mL did not induce bacterial reverse mutations and in vitro mammalian cell gene mutation. Further, in vitro chromosomal aberration tests demonstrated that CNFs at 100 µg/mL did not induce chromosomal aberration in Chinese hamster lung fibroblasts. From the mammalian erythrocyte micronucleus test, no statistically significant increase was observed in the proportion of micronucleated polychromatic erythrocytes in the bone marrow cells of rats intratracheally instilled with any concentration of CNFs (0.25-1.0 mg/kg) compared with values from respective negative control groups. Therefore, this battery of in vitro and in vivo assays illustrated that the CNFs examined in this study did not induce genotoxicity, suggesting our results provide valuable insight on the future use of these materials in various industrial applications.

Pulmonary toxicity, cytotoxicity, and genotoxicity of submicron-diameter carbon fibers with different diameters and lengths.

Submicron-diameter carbon fibers (SCFs) are a type of fine-diameter fibrous carbon material that can be used in various applications. To accelerate their practical application, a hazard assessment of SCFs must be undertaken. This study demonstrated the pulmonary toxicity, cytotoxicity, and genotoxicity of three types of SCFs with different diameters and lengths. The average diameter and length of SCFs were 259.2 nm and 11.7 µm in SCF1 suspensions, 248.5 nm and 6.7 µm in SCF2 suspensions, and 183.0 nm and 13.7 µm in SCF3 suspensions, respectively. The results of pulmonary inflammation and recovery following intratracheal instillation with SCFs at doses of 0.25, 0.5, or 1.0 mg/kg showed that the pulmonary toxicity of SCFs was SCF3 > SCF1 > SCF2. These results suggest that SCF diameter and length are most likely important contributing factors associated with lung SCF clearance, pulmonary inflammation, and recovery. Furthermore, SCFs are less pulmonary toxic than bent multi-walled carbon nanotubes. Cell viability, pro-inflammatory cytokine and intracellular reactive oxygen species productions, morphological changes, gene expression profiling in NR8383 rat alveolar macrophage cells showed that the cytotoxic potency of SCFs is: SCF3 > SCF1 > SCF2. These results showed that SCFs with small diameters had high cytotoxicity, and SCFs with short lengths had low cytotoxicity. We conclude that pulmonary toxicity and cytotoxicity are associated with the diameter and length distributions of SCFs. In addition, a standard battery for genotoxicity testing, namely the Ames test, an in vitro chromosomal aberration test, and a mammalian erythrocyte micronucleus test, demonstrated that the three types of SCFs did not induce genotoxicity. Our findings provide new evidence for evaluating the potential toxicity of not only SCFs used in this study but also various SCFs which differ depending on the manufacturing processes or physicochemical properties.

Screening of preservatives and evaluation of sterilized cellulose nanofibers for toxicity studies.

OBJECTIVES: The aim of this study is to establish a sterilization method for cellulose nanofibers (CNFs) dispersions that uses multiple preservatives with different hydrophilicities without affecting the physical and chemical properties of CNFs, and to provide useful information for sample preparation in future toxicity study of CNFs. METHODS: Various preservatives were added to the phosphorylated CNF dispersions, endotoxin level and the numbers of bacteria and fungi in the CNF dispersion were analyzed. The pH values and viscosity of sterilized CNF dispersions were compared with those of control and autoclaved CNF dispersions. RESULTS: Phosphorylated CNF dispersions at a concentration of 2.0 mg/mL or lower and the addition of 10 µg/mL benzalkonium chloride alone or 250 µg/mL methyl parahydroxybenzoate and 250 µg/mL propyl parahydroxybenzoate in combination can sterilize CNF dispersions without changing the physical and chemical properties of CNFs. CONCLUSIONS: We developed sterilization method for CNF dispersions that uses multiple preservatives with different hydrophilicities without affecting the physical and chemical properties of CNFs. This sterilization method for CNFs dispersions can be applied to the safety assessment of CNF with different physicochemical properties in the future.

Cytotoxicity profiles of multi-walled carbon nanotubes with different physico-chemical properties.

Multi-walled carbon nanotubes (MWCNTs) have industrial applications in the nanotechnology field. The physico-chemical properties of MWCNTs vary greatly depending on MWCNT manufacture and application. It has been pointed out that their needle shape and high durability are important factors that determine the biopersistence of fibers and can lead to inhalation toxicity or cytotoxicity. In this study, we prepared six suspensions of MWCNTs differing in diameter and length, and performed in vitro cell-based assays for 24 h using NR8383 rat alveolar macrophages. Rigid, needle-shaped MWCNTs with a large diameter (>50 µm) penetrated the cytoplasm and decreased cell survival without generating intracellular reactive oxygen species (ROS), significantly up-regulated many genes involved in inflammatory responses, response to oxidative stress and apoptosis, and extracellular matrix degradation. Bent MWCNTs with a small diameter (<20 µm) were phagocytosed in vacuole-like cellular compartments and decreased cell survival along with intracellular ROS generation. Straight, thin MWCNTs with a small diameter (<20 µm) caused a slight intracellular ROS generation but no decrease in cell viability. Some straight, long, and thin MWCNTs were found in the mitochondria and near the nuclei; however, no mutagenesis was observed. The in vitro cell-based assays showed high cytotoxicity of MWCNTs with a large diameter (>50 µm), moderate and low cytotoxicity of MWCNTs with a small diameter (<20 µm). These results suggested that the diameter of MWCNTs considerably contributes to their cytotoxicity.

Assessment of cytotoxicity and mutagenicity of exfoliated graphene.

Graphene and related materials (GRMs) have unique optical and thermal characteristics and are expected to be adopted for industrial applications. However, there are concerns with respect to their safety to human health. To conduct cytotoxicity and mutagenicity assessments, exfoliated graphene (EGr) dispersed in Tween-20® was diluted in cell culture medium. Rat alveolar macrophage viability significantly decreased after 24 h exposure to 1 and 10 µg/mL EGr. No significant levels of intracellular reactive oxygen species were detected in the 2',7'-dichlorodihydrofluorescin diacetate assay after 24 h of exposure to EGr. The levels of the pro-inflammatory cytokines macrophage inflammatory protein-1α, interleukin (IL)-1ß, IL-18, macrophage chemoattractant protein-1, and tumor necrosis factor α were significantly higher in cells treated with 10 µg/mL EGr for 24 h than in untreated controls. Transmission electron microscopy confirmed that EGr was present in the cytoplasm of the cells. Many genes were upregulated by EGr treatment, and significantly overrepresented gene ontology categories included the biological processes "response to external stimulus", "response to stress", "cell-cell signaling", "biological adhesion", and "cell proliferation". EGr did not induce genetic mutations in E. coli or cause micronucleus induction in mouse bone marrow cells. The results suggest that EGr cytotoxicity should be carefully considered.

Basic study of intratracheal instillation study of nanomaterials for the estimation of the hazards of nanomaterials.

In order to examine the usefulness of intratracheal instillation of nanoparticles for the screening of the harmful effects of nanoparticles, we performed intratracheal instillation studies of nanomaterials on rats using different delivery devices and postures as a basic study. Multiwall carbon nanotubes (MWCNTs) with a geometric mean length and secondary diameter of 2.16 µm and 752 nm, respectively, were used as the nanomaterials. Male F344 rats were intratracheally exposed to 0.04 or 0.2 mg/rat of MWCNT, were dissected at 1 d and 3 d, and cell analyses of the bronchoalveolar lavage fluid (BALF) were analyzed. Two delivery devices were used for the intratracheal instillation of the MWCNTs: a gavage needle and a microsprayer aerolizer. Both induced neutrophil influx in the lung at 1 and 3 d, and there were no significant differences in neutrophil inflammation between the two delivery devices. The main distribution of pulmonary inflammation by both delivery devices was in the centrilobular spaces in the lung. Two postures were used: an angle of approximately 45 degrees and a standing posture on a board, both of which also induced pulmonary influx in BALF and pulmonary inflammation mainly in the centrilobular spaces, with no large difference in pulmonary inflammation between the two postures. Taken together, the differences in the delivery devices and postures of the rats in the intratracheal instillation did not affect the acute pulmonary toxicity of the nanomaterials.

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.

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

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.

Physical properties of single-wall carbon nanotubes in cell culture and their dispersal due to alveolar epithelial cell response.

Concern over the influence of carbon nanotubes (CNTs) on human health has arisen due to advances; however, little is known about the potential toxicity of CNTs. In this study, impurity-free single-wall carbon nanotubes (SWCNTs), with different physical properties in cell culture medium, were prepared by a novel dispersion procedure. SWCNTs with small bundles (short linear shape) and SWCNTs with large bundles (long linear shape) did not cause a significant inhibition of cell proliferation, induction of apoptosis or arrest of cell cycle progression in A549 alveolar epithelial cells. Expression of many genes involved in the inflammatory response, apoptosis, response to oxidative stress and degradation of the extracellular matrix were not markedly upregulated or downregulated. However, SWCNTs with relatively large bundles significantly increased the level of intracellular reactive oxygen species (ROS) in a dose-dependent manner, and the levels of these ROS were higher than those of SWCNTs with relatively small bundles or commercial SWCNTs with residual metals. Transmission electron microscopy (TEM) revealed that impurity-free SWCNTs were observed in the cytoplasm and vacuoles of cells after 24 h. These results suggested that the physical properties, especially the size and length of the bundles of the SWCNTs dispersed in cell culture medium, contributed to a change in intracellular ROS generation, even for the same bulk SWCNTs. Additionally, the residual metals associated with the manufacturing of SWCNTs may not be a definitive parameter for intracellular ROS generation in A549 cells.

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.

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.

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.

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 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.

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.

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.

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.

Comparative pulmonary toxicity study of nano-TiO(2) 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.