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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 SCF3SCF1SCF2. 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: SCF3SCF1SCF2. 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.

Graphical abstract, Highlights • CNFs did not induce bacterial reverse and in vitro mammalian cell gene mutation. • CNFs did not induce chromosomal aberration in CHL/IU cells. • CNFs did not increase the proportion of micronucleated polychromatic erythrocytes in rat bone marrow. • Three types of CNFs with different manufacturing methods exhibited no genotoxicity., 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.

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 ( 50 µm), moderate and low cytotoxicity of MWCNTs with a small diameter (

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)-1β, 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.

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.

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.

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.

Single-wall carbon nanotubes are a major type of nano-objects that have industrial applications such as fuel cells. In this study, four types of single-wall carbon nanotubes and their abilities to aggravate allergic reactions were examined: those containing Fe, those containing Ni, and the corresponding purified metal-free ones. These were administered to mice via pharyngeal aspiration. Subsequently, the mice inhaled ovalbumin a total of eight times over three weeks. After inhalation of ovalbumin, the concentrations of total immunoglobulin E and ovalbumin-specific immunoglobulin E in serum increased in mice treated with purified metal-free and Fe containing single-wall carbon nanotubes while those containing Ni did not affect total and ovalbumin-specific immunoglobulin E levels. Additionally, the purified metal-free and Fe containing nanotubes caused the gene expression of heme oxygenase-1, chemokine (C-X-C motif) ligand 2, and tumor necrosis factor-α, suggesting that some kinds of single-wall car...

The effects of iron content, fiber length, and stability of carbon nanotube (CNT) suspension on cells were examined. Five kinds of single-wall carbon nanotube (SWCNT) suspensions were prepared: with catalytic iron, without iron, long SWCNTs (stable), short SWCNTs (stable), and short SWCNT (unstable). These suspensions were applied to A549, THP-1, and mouse peritoneal macrophage cells. After a 24-h exposure, the mitochondrial activity, cell membrane damage, intracellular oxidative stress, and expression of cytokine genes were determined. Among these properties of SWCNTs, stability of CNT suspension had the most influence on the cells, whereas the effects of iron content and fiber length were small. The unstable SWCNT suspension caused a substantial increase in intracellular ROS levels. Additionally, the cellular effects of stable multi-wall carbon nanotubes (MWCNTs) were examined. The MWCNT suspension did not show any cellular effects. Overall, influences of CNT suspension on mitochondrial activity and cell membrane damage were small. These results suggest that the physical properties of CNT suspension are important factors for their cellular effects. Thus, CNT suspensions prepared with the same material but having different physical properties would differ in the cellular effects they exert, including cytotoxicity. Therefore, physical characterization of CNT suspensions is essential to the evaluation of CNT toxicity. In particular, stability of CNT suspension notably influenced the intracellular ROS level.

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.

We examined the induction of oxidative stress by carbon nanohorns (CNHs) in vitro and in vivo. For the in vitro examination, a stable CNH dispersion was prepared and applied to human lung carcinoma A549 cells and human keratinocyte HaCaT cells. CNH was taken up into the cells by endocytosis. CNHs did not influence cell viability or colony forming ability. The intracellular reactive oxygen species level was increased in CNH exposed cells at a concentration of 1.0 mg/ml, but intracellular lipid peroxidation level was not affected by CNH exposure. The pulmonary oxidative stress induction of CNH were also examined by intratracheal instillation in rats, and showed a transient oxidative stress in the lung at a concentration of 1.0 mg/animal. Although CNH did not show any significant cytotoxicity and short term pulmonary toxicity including oxidative stress, chronic toxicity of CNH is still not clear. Therefore, for efficient application of CNH, further examinations of biological influence of CNH are necessary.

The pulmonary toxicity of multi-wall carbon nanotubes (MWCNT) were examined by intratracheal instillation. We prepared a well-dispersed MWCNT dispersion including MWCNTs of 3.71 µm geometric average length. The fiber length of most of the MWCNTs in the dispersion was 10 µm or less. The MWCNT dispersion was administered to rat lung by single intratracheal instillation at doses of 0.2 mg and 0.6 mg/rat. Bronchoalveolar lavage fluid (BALF) was collected at 3 days, 1 week, 1 month, 3 months, and 6 months after instillation. The influences of the longer MWCNTs on the induction of inflammation and oxidative stress were examined by the number of neutrophils, cytokine induced neutrophil chemoattractant-1 (CINC-1), CINC-2, CINC-3 and HO-1 in the BALF. Additionally, ho-1 gene expression in the lung was examined. The intratracheal instillation of MWCNT induced transient inflammation dose dependently in the lung. The number of neutrophils was highest at 3 days after instillation and then decreased. However, the neutrophils in the MWCNT administered animals tended to be higher than in the control group until 3 months after instillation. The CINC-1 and CINC-2 concentrations in the BALF increased at 1 month after instillation. There were no significant differences in CINC-3 and HO-1 between the MWCNT administered animals and the control animals. These results revealed that the MWCNTs of 1–10 µm in length induced persistent inflammation in rat lung. There were no remarkable differences between the MWCNTs in the present study and previously reported, shorter MWCNTs prepared from “the same” raw MWCNT material.

The pulmonary toxicity of multi-wall carbon nanotubes (MWCNT) were examined by intratracheal instillation. We prepared a well-dispersed MWCNT dispersion including MWCNTs of 3.71 µm geometric average length. The fiber length of most of the MWCNTs in the dispersion was 10 µm or less. The MWCNT dispersion was administered to rat lung by single intratracheal instillation at doses of 0.2 mg and 0.6 mg/rat. Bronchoalveolar lavage fluid (BALF) was collected at 3 days, 1 week, 1 month, 3 months, and 6 months after instillation. The influences of the longer MWCNTs on the induction of inflammation and oxidative stress were examined by the number of neutrophils, cytokine induced neutrophil chemoattractant-1 (CINC-1), CINC-2, CINC-3 and HO-1 in the BALF. Additionally, ho-1 gene expression in the lung was examined. The intratracheal instillation of MWCNT induced transient inflammation dose dependently in the lung. The number of neutrophils was highest at 3 days after instillation and then decreased. However, the neutrophils in the MWCNT administered animals tended to be higher than in the control group until 3 months after instillation. The CINC-1 and CINC-2 concentrations in the BALF increased at 1 month after instillation. There were no significant differences in CINC-3 and HO-1 between the MWCNT administered animals and the control animals. These results revealed that the MWCNTs of 1–10 µm in length induced persistent inflammation in rat lung. There were no remarkable differences between the MWCNTs in the present study and previously reported, shorter MWCNTs prepared from “the same” raw MWCNT material.

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 © 2012 John Wiley & Sons, Ltd.

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.

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 60nm and 1.5μm, respectively. Groups of male Sprague–Dawley rats were intratracheally instilled with 0.04, 0.2, or 1mg/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 1mg/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.

Multi-wall carbon nanotubes (MWNTs) were dispersed into water by ultrasonication in order to prepare the suspension used in the in vivo toxicity tests. The MWNTs were suspended in the aqueous solutions of Tween80 containing as a dispersant, and the suspensions were sonicated using a bath-type ultrasonicator. The particle size distribution and the dispersion state of the MWNTs in aqueous solution was examined under several conditions concerning the ultrasonic dispersion, i.e., the mass ratio of Tween80 to MWNTs, the sonication time, and the height of water and the temperature in the ultrasonic bath. The MWNTs of a fiber length less than 20 μm were well dispersed in the aqueous system when the height of water was controlled at an adequate level, where the suspension resonated.The amount of Tween80 adsorbed by MWNTs was examined by the thermogravimetric analysis of the residue of the ultrafiltration assisted by ultracentrifugation of the MWNTs suspension. When the MWNTs were well dispersed by ultrasonication, the mass ratio of Tween80 adsorbed by MWNTs was considerably low, compared with the charged mass ratio of Tween80 to MWNTs.

A dispersion method using a bead mill was developed for preparing suspensions used in hazard testing of nanosize fullerenes. Fullerene crystals (C 60 ) of several μm to 100 μm in size were wet ground using a bead mill and dispersed in pure water containing Tween 80 as a dispersant. The particle size, dispersing condition, crystallization and fullerene oxide in the suspension prepared by bead milling were evaluated. As a result, the conditions of bead milling where the sizes of fullerene crystals were reduced to 100 nm or less were found, which made it possible to prepare a fullerene aqueous dispersion system using Tween 80. Furthermore, by centrifugation, it became possible to prepare fullerene suspensions in which the particle size of the majority of the suspended particles was 100 nm or less. The prepared suspensions remained stable for almost two months. Controlling milling conditions and milling time made it possible to achieve dispersion while maintaining the crystallized state of the fullerenes, and operating in anaerobic conditions under shade effectively suppressed the production of oxidized fullerenes. The fullerene suspensions prepared by bead milling and centrifugation were suitable for endotracheal administration tests and inhalation tests using rats.

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.

Applying S. Taylor's approach (1986), we make an extensive analysis on the Japanese stock market, foreign exchange market and the Japanese Government Bond Futures market. The purpose of this paper is to empirically reveal the structure of the Japanese markets via Taylor's model rather than to propose a new model. For this reason, we include a variety of analyzed data particularly for the Japanese stock market and the foreign exchange market because the results can be used in a different manner. The paper consists of three parts. But each part can be read separately.

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.

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.

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.

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 μg/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 μg/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 × 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.

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.

Although the Japanese economy has experienced a remarkable growth in the postwar period, it would not be an overstatement to say that there has been no definitive analysis done on the functions performed by the capital market, particularly the stock market, and thus we have no common understanding of it. One of the possible reasons for this state is that industrial funds have mainly been supplied through the financial market centering around banks, whereas the stock market has played only a minor role. As a result of this, even in the few analyses that have been carried out, certain special characteristics of the Japanese stock market (the so-called backwardness compared to that of the United States, for example) have been emphasized, and even the viability of its functioning has been questioned.

The large volume of government bonds issued since 1975 has been a strong driving force for structural change in the Japanese bond markets during the last decade. The increasing amount of outstanding government bonds has made the trading volume in the secondary market increase rapidly, accompanied with the development of interdealer trading. This interdealer trading system is an interesting and impressive phenomena, one that has developed as the Japanese bond market changed, and is important analytically as well. The development of this institution gives researchers an op? portunity to measure the increased efficiency of the developing market. This pa? per attempts to describe the development of these markets, point out the role that interdealer markets have played in improving the transactional efficiency of these markets, and document the benefits?in the form of lowered commission spreads (lowered liquidity cost)?that have accrued to the users of financial mar? kets in Japan as they pay less for the liquidity services provided by dealers. We will define transactional efficiency in this single dimension, lowered bid-ask spreads. Prior research in this area [4] has examined the conditions under which interdealer trading occurs in securities markets and pointed out that such trading may impose limits on the divergence of inventories among dealers that would decrease the market bid and ask spreads. In this paper, interdealer trading means the transactions of bonds among securities companies functioning as a dealer in the over-the-counter market (OTC market). To put it another way, the interdealer market could be regarded as a wholesale market, and the OTC market as a retail one. At present, trading is put into practice through three kinds of channels: the direct transactions among secu? rities companies, the Nihon Sohgo Shohken or bond broker (called BB), and the block trade of government bonds (BTGB) administrated by the Securities Ex? change. In the case of direct transactions, a dealer must seek out a compatible trading partner by himself. As discussed later, however, those transactions in the