Difference in carcinogenicities of two different vapor grown carbon fibers with different physicochemical characteristics induced by intratracheal instillation in rats.

BACKGROUND: Carbon fibers are high aspect ratio structures with diameters on the submicron scale. Vapor grown carbon fibers are contained within multi-walled carbon tubes, with VGCF™-H commonly applied as a conductive additive in lithium-ion batteries. However, several multi-walled carbon fibers, including MWNT-7, have been reported to induce lung carcinogenicity in rats. This study investigated the carcinogenic potential of VGCF™-H fibers in F344 rats of both sexes with the vapor grown carbon fibers VGCF™-H and MWNT-7 over 2 years. The carbon fibers were administered to rats by intratracheal instillation at doses of 0, 0.016, 0.08, and 0.4 mg/kg (total doses of 0, 0.128, 0.64, and 3.2 mg/kg) once per week for eight weeks and the rats were observed for up to 2 years after the first instillation. RESULTS: Histopathological examination showed the induction of malignant mesothelioma on the pleural cavity with dose-dependent increases observed at 0, 0.128, 0.64, and 3.2 mg/kg in rats of both sexes that were exposed to MWNT-7. On the other hand, only two cases of pleural malignant mesothelioma were observed in the VGCF™-H groups; both rats that received 3.2 mg/kg in male. The animals in the MWNT-7 groups either died or became moribund earlier than those in the VGCF™-H groups, which is thought related to the development of malignant mesothelioma. The survival rates were higher in the VGCF™-H group, and more carbon fibers were observed in the pleural lavage fluid (PLF) of the MWNT-7 groups. These results suggest that malignant mesothelioma is related to the transfer of carbon fibers into the pleural cavity. CONCLUSIONS: The intratracheal instillation of MWNT-7 clearly led to carcinogenicity in both male and female rats at all doses. The equivocal evidence for carcinogenic potential that was observed in male rats exposed to VGCF™-H was not seen in the females. The differences in the carcinogenicities of the two types of carbon fibers are thought due to differences in the number of carbon fibers reaching the pleural cavity. The results indicate that the carcinogenic activity of VGCF™-H is lower than that of MWNT-7.

Lung toxicity of a vapor-grown carbon fiber in comparison with a multi-walled carbon nanotube in F344 rats.

Carbon fibers have excellent physicochemical and electrical properties. Vapor-grown carbon fibers are a type of carbon fibers that have a multi-walled carbon tube structure with a high aspect ratio. The representative vapor-grown carbon fiber, VGCFTM-H, is extremely strong and stable and has superior thermal and electrical conductivity. Because some high-aspect-ratio multi-walled carbon nanotubes (MWCNTs) have been reported to have toxic and carcinogenic effects in the lungs of rodents, we performed a 13-week lung toxicity study using VGCFTM-H in comparison with one of MWCNTs, MWNT-7, in rats. Male and female F344 rats were intratracheally administered VGCFTM-H at doses of 0.2, 0.4, and 0.8 mg/kg bw or MWNT-7 at doses of 0.4 and 0.8 mg/kg bw once a week for 8 weeks and then up to week 13 without treatment. The lung burden was equivalent in the VGCFTM-H and MWNT-7 groups; however, the lung weight had increased and the inflammatory and biochemical parameters in the broncho-alveolar lavage fluid and histopathological parameters, including inflammatory cell infiltration, alveolar type II cells proliferation, alveolar fibrosis, pleural fibrosis, lung mesothelium proliferation, and diaphragm fibrosis, were milder in the VGCFTM-H group than in the MWNT-7 group. In addition, the proliferating cell nuclear antigen (PCNA)-positive index in the visceral and pleural mesothelium was significantly higher in the MWNT-7 group than in the controls, but not in the VGCFTM-H group. Thus, the results of this study indicate that the lung and pleural toxicities of VGCFTM-H were less than those of MWNT-7.

Comparison of the effects of multiwall carbon nanotubes on the epithelial cells and macrophages.

Effects of two kinds of multiwall carbon nanotubes (MWCNTs) on cells were examined. The effects of MWNT-7, which has been reported to be carcinogenic, and MWCNT-B, whose toxicity is unclear, were examined in both epithelial cells and macrophages. Human lung carcinoma A549 cells were used as representative epithelial cells and differentiated human monocyte THP-1 cells, as well as rat pulmonary macrophages NR8383, were employed to examine possible harmful effects of the MWCNTs. The MWCNTs induced the production of chemokines such as interleukin-8 (IL-8). MWCNTs were found to more strongly affect macrophages than epithelial cells. In addition, the toxicity was more pronounced in the MWNT-7 exposed cells than in those exposed to MWCNT-B. Cytochalasin D and amiloride treatment of differentiated THP-1 cells reduced cell-associated MWCNTs and IL-8 induction. To confirm these cellular influences in vivo, intratracheal administration of each type of MWCNT was performed by pharyngeal aspiration in the mouse lung. Analysis of bronchoalveolar lavage fluid (BALF) showed increase of inflammatory monocyte in MWNT-7 exposed animals at 1week after. In addition, neutrophils in the BALF were also significantly increased MWNT-7 exposed animals at 1 week and 1 month after. Aspiration of MWNT-7 caused formation of granulomas in the lung. Formation of the granulomas was not observed in the case of MWCNT-B. These results suggest that cellular uptake of the MWCNTs by phagocytosis and chemokine induction is important aspects of their toxicity.

Effect of iron overload from multi walled carbon nanotubes on neutrophil-like differentiated HL-60 cells.

Multi walled carbon nanotubes (MWCNTs) are one of the most intensively explored nanomaterials because of their unique physical and chemical properties. Due to the widespread use of MWCNTs, it is important to investigate their effects on human health. The precise mechanism of MWCNT toxicity has not been fully elucidated. The present study was designed to examine the mechanisms of MWCNT toxicity toward human promyelocytic leukemia HL-60 cells. First, we found that MWCNTs decreased the viability of neutrophil-like differentiated HL-60 cells but not undifferentiated HL-60 cells. Because neutrophil-like differentiated HL-60 cells exhibit enhanced phagocytic activity, the cytotoxicity of MWCNTs is dependent on the intracellularly localized MWCNTs. Next, we revealed that the cytotoxicity of MWCNTs is correlated with the intracellular accumulation of iron that is released from the engulfed MWCNTs in an acidic lysosomal environment. The intracellular accumulation of iron was repressed by treatment with cytochalasin D, a phagocytosis inhibitor. In addition, our results indicated that iron overload enhanced the release of interleukin-8 (IL-8), a chemokine that activates neutrophils, and subsequently elevated intracellular calcium concentration ([Ca2+]i). Finally, we found that the sustained [Ca2+]i elevation resulted in the loss of mitochondrial membrane potential and the increase of caspase-3 activity, thereby inducing apoptotic cell death. These findings suggest that the iron overload caused by engulfed MWCNTs results in the increase of IL-8 production and the elevation of [Ca2+]i, thereby activating the mitochondria-mediated apoptotic pathway.

Comprehensive Tool to Assess Oral Feeding Support for Functional Recovery in Post-acute Rehabilitation.

OBJECTIVE: To determine the influence of the Kuchi-kara Taberu (KT) index on rehabilitation outcomes during hospitalized convalescent rehabilitation. DESIGN: A historical controlled study. SETTING AND PARTICIPANTS: A rehabilitation hospital. PARTICIPANTS: Patients who were admitted to a convalescent rehabilitation ward from June 2014 to May 2017. MEASURES: Patients' background characteristics included age, sex, nutritional status, activities of daily living (ADL) assessed using the Functional Impedance Measure (FIM), dysphagia assessed using the Functional Oral Intake Scale (FOIS), and reasons for rehabilitation. The following values before (control group) and after initiation of the KT index intervention period (intervention group) were compared: gain of FIM, length of stay, accumulated rehabilitation time, discharge destination, gain of FOIS, gain of body weight (BW), and nutritional intake (energy and protein). RESULTS: Mean age was 76.4 ± 12.3 years (n = 233). There were no significant differences in the baseline characteristics of the patients at admission between the control and intervention groups, except for reason of rehabilitation. The intervention group demonstrated statistically higher values for the total (P = .004) and motor FIM gain (P = .003), total (P = .018) and motor FIM efficiency (P = .016), and FOIS gain (P < .001), compared with values in the control group. The proportion of patients returning home was statistically more frequent in the intervention group compared with that in the control group (73.4% vs 85.5%, odds ratio 2.135, 95% confidence interval [CI] 1.108-4.113, P = .022). Multivariate analyses indicated that intervention using the KT index was a significant independent factor for increased FIM gain (ß coefficient = 0.163, 95% CI 1.379-8.329, P = .006) and returning home (adjusted odds ratio 2.570, 95% CI 1.154-5.724, P = .021). CONCLUSIONS/IMPLICATIONS: A rehabilitation program using the KT index may lead to improvement of inpatient outcomes in post-acute care. Further prospective research is warranted to confirm the efficacy of this program.

Ascorbic acid prevents zinc oxide nanoparticle-induced intracellular oxidative stress and inflammatory responses.

Exposure to zinc oxide nanoparticles (ZnO NPs) promotes acute pulmonary toxicity through oxidative stress and inflammation. Furthermore, dissolved zinc from ZnO NPs induces the formation of intracellular reactive oxygen species (ROS). We previously reported that supplemental ascorbic acid (AA) inhibits ZnO NP-induced acute pulmonary toxicity in a rat model; however, the mechanism of this action remains unclear. Therefore, we investigated the effects of AA on ZnO NP-induced cytotoxicity in human lung carcinoma A549 cells. AA was found to suppress intracellular production of ROS, and thus reduce the subsequent inflammation of ZnO NPs. However, intracellular Zn2+ concentrations were higher in AA-treated cells than in AA-untreated cells. AA was found to react with Zn2+ but not with the ZnO NPs themselves. These results suggest the possibility that AA-chelated extracellular Zn2+ and the Zn-AA complex was readily taken up into cell. Even if the intracellular Zn2+ level was high, cytotoxicity might be reduced because the Zn-AA complex was stable. Co-treatment of AA to A549 inhibited ROS production and subsequent intracellular inflammatory responses. These results are consistent with those previously reported from an in vivo model. Thus, two possibilities can be considered about the cytotoxicity-reducing the effect of AA: antioxidant efficacy and chelating effect.

The induction of lipid peroxidation during the acute oxidative stress response induced by intratracheal instillation of fine crystalline silica particles in rats.

Crystalline silica (SiO2) is an important material for industry but is considered potentially carcinogenic. Inhalation of a crystalline SiO2 aerosol may contribute to serious lung diseases. Crystalline SiO2 particles are commonly used as a positive control in toxicity assays of particulate materials (e.g. nanoparticles). Crystalline SiO2 induces oxidative stress resulting in lipid peroxidation, but the acute oxidative stress response in the lung is not well understood. Lipid peroxidation during the acute stage of oxidative stress after instillation of crystalline SiO2 into rats was examined by bronchoalveolar lavage fluid (BALF) analysis. The levels of 8-iso-prostaglandin F2α and hydroxyoctadecadienoic acid (HODE) in the BALF were measured using liquid chromatography coupled to quadrupole mass spectrometry. The concentration of the antioxidant protein heme oxygenase-1 (HO-1) in the BALF was determined using enzyme-linked immunosorbent assay. Intratracheal instillation of crystalline SiO2 increased the level of HODE and HO-1 in BALF at 24 h after administration. The levels of HODE and HO-1 returned to baseline at 72 h after instillation. Lactate dehydrogenase leakage was observed only after 1 h instillation. These results suggest that the contribution of oxidative stress to the pulmonary toxicity of crystalline SiO2 is minimal in the early acute stage after exposure.

Ascorbic acid attenuates acute pulmonary oxidative stress and inflammation caused by zinc oxide nanoparticles.

OBJECTIVES: It is known that inhalation of zinc oxide nanoparticles (ZnO NPs) induces acute pulmonary dysfunction, including oxidative stress, inflammation, and injury, but there are no reports on how to prevent these adverse effects. We have previously reported that the pulmonary symptoms caused by ZnO NPs were associated with oxidative stress; in the present study, we therefore investigated the use of ascorbic acid (AA), which is known as vitamin C, to prevent these toxic effects. METHODS: A ZnO NP dispersion was introduced into rat lungs by intratracheal injection, and thereafter a 1% aqueous AA solution was given as drinking water. Bronchoalveolar lavage fluid was collected at 1 day and 1 week after injection, and lactate dehydrogenase (LDH) activity, heme oxygenase-1 (HO-1), and interleukin-6 (IL-6) levels were measured. In addition, expression of the chemokine cytokine-induced neutrophil chemoattractants (CINCs), HO-1, and metallothionein-1 (MT-1) genes in the lungs were determined. RESULTS: Acute oxidative stress induced by ZnO NPs was suppressed by supplying AA. Increases in LDH activity and IL-6 concentration were also suppressed by AA, as was the expression of the CINC-1, CINC-3, and HO-1 genes. CONCLUSIONS: Oral intake of AA prevents acute pulmonary oxidative stress and inflammation caused by ZnO NPs. Intake of AA after unanticipated exposure to ZnO NPs is possibly the first effective treatment for the acute pulmonary dysfunction they cause.

Intratracheal instillation of single-wall carbon nanotubes in the rat lung induces time-dependent changes in gene expression.

The use of carbon nanotubes in the industry has grown; however, little is known about their toxicological mechanism of action. Single-wall carbon nanotube (SWCNT) suspensions were administered by single intratracheal instillation in rats. Persistence of alveolar macrophage-containing granuloma was observed around the sites of SWCNT aggregation at 90 days post-instillation in 0.2-mg- or 0.4-mg-injected doses per rat. Meanwhile, gene expression profiling revealed that a large number of genes involved in the inflammatory response were markedly upregulated until 90 days or 180 days post-instillation. Subsequently, gene expression patterns were dramatically altered at 365 days post-instillation, and the number of upregulated genes involved in the inflammatory response was reduced. These results suggested that alveolar macrophage-containing granuloma reflected a characteristic of the histopathological transition period from the acute-phase to the subchronic-phase of inflammation, as well as pulmonary acute phase response persistence up to 90 or 180 days after intratracheal instillation in this experimental setting. The expression levels of the genes Ctsk, Gcgr, Gpnmb, Lilrb4, Marco, Mreg, Mt3, Padi1, Slc26a4, Spp1, Tnfsf4 and Trem2 were persistently upregulated in a dose-dependent manner until 365 days post-instillation. In addition, the expression levels of Atp6v0d2, Lpo, Mmp7, Mmp12 and Rnase9 were significantly upregulated until 754 days post-instillation. We propose that these persistently upregulated genes in the chronic-phase response following the acute-phase response act as potential biomarkers in lung tissue after SWCNT instillation. This study provides further insight into the time-dependent changes in genomic expression associated with the pulmonary toxicity of SWCNTs.

Dose-dependent clearance kinetics of intratracheally administered titanium dioxide nanoparticles in rat lung.

AEROSIL(®) P25 titanium dioxide (TiO2) nanoparticles dispersed in 0.2% disodium phosphate solution were intratracheally administered to male F344 rats at doses of 0 (control), 0.375, 0.75, 1.5, 3.0, and 6.0 mg/kg. The rats were sacrificed under anesthesia at 1 day, 3 days, 7 days, 4 weeks, 13 weeks, and 26 weeks after administration. Ti levels in various pulmonary and extrapulmonary organs were determined using sensitive inductively coupled plasma sector field mass spectrometry. One day after administration, the lungs contained 62-83% of TiO2 administered dose. Twenty-six weeks after administration, the lungs retained 6.6-8.9% of the TiO2 administered at the 0.375, 0.75, and 1.5 mg/kg doses, and 13% and 31% of the TiO2 administered at the 3.0 and 6.0 mg/kg doses, respectively. The pulmonary clearance rate constants from compartment 1, k1, were estimated using a 2-compartment model and were found to be higher for the 0.375 and 0.75 mg/kg doses of TiO2 (0.030/day for both) than for TiO2 doses of 1.5-6.0 mg/kg (0.014-0.022/day). The translocation rate constants from compartment 1 to 2, k12, were estimated to be 0.015 and 0.018/day for the 0.375 and 0.75 mg/kg doses, and 0.0025-0.0092/day for doses of 1.5-6.0mg/kg. The pulmonary clearance rate constants from compartment 2, k2, were estimated to be 0.0086 and 0.0093/day for doses of 0.375 and 0.75 mg/kg, and 0-0.00082/day for 1.5-6.0 mg/kg doses. Translocation of TiO2 from the lungs to the thoracic lymph nodes increased in a time- and dose-dependent manner, accounting for 0.10-3.4% of the administered dose at 26 weeks. The measured thoracic lymph node burdens were a much better fit to the thoracic lymph node burdens estimated assuming translocation from compartment 1 to the thoracic lymph nodes, rather than those estimated assuming translocation from compartment 2 to the thoracic lymph nodes. The translocation rate constants from the lungs to the thoracic lymph nodes, kLung→Lym, were 0.000037-0.00081/day, and these also increased with increasing doses of TiO2. Although a small amount of TiO2 had translocated to the liver by 3 days after the administration (0.0023-0.012% of the highest dose administered, 6.0 mg/kg), translocation to the other extrapulmonary organs was not detected.

Long-term variability of urinary salt excretion and blood pressure in hypertensive patients.

We investigated the long-term trend and variability of urinary salt (sodium chloride) excretion in hypertensive patients. Subjects included 186 hypertensive patients (103 women and 83 men, mean age: 58.5±10.5 years) who underwent 10 successful 24-h home urine collections over a mean observation period of 7.7 years. We measured 24-h urinary salt excretion and blood pressure (BP) sequentially at the time of each collection and monitored the long-term trend and variability of urinary salt excretion. BP significantly decreased from 145±16/85±11 mm Hg to 130±12/70±11 mm Hg and was associated with an increased use of antihypertensive drugs. The 24-h urinary salt excretion also decreased from 9.5±3.6 g per day at the first measurement to 8.5±3.2 g per day at the 10th measurement. Urinary salt excretion during the observation period ranged from a minimum value of 5.2±1.8 g per day to a maximum value of 13.4±3.6 g per day with a coefficient of variation of 29.2±8.1%. When subjects were assigned to a low, medium and high salt group based on the tertiles of the first measurement of urinary salt excretion and the tertiles based on the mean value of 10 measurements during the observation period, only 56.2% remained in the same category, suggesting that a single measurement of urinary salt excretion can only predict long-term urinary salt excretion in approximately half of the individuals. In conclusion, urinary salt excretion shows large variability such that a single measurement may not be sufficient to assess salt intake in individuals.

Tissue distribution and clearance of intravenously administered titanium dioxide (TiO2) nanoparticles.

The organ-tissue distribution and clearance of Degussa P25 TiO2 nanoparticles were determined after intravenous administration to rats (0.95 mg/kg bodyweight) using an inductively coupled plasma sector field mass spectrometer. The detection limits of Ti analysis, 0.54 and 1.4 ng/mL for blood and urine and 0.35-2.0 ng/g tissue for several organ tissues, enabled determination of tissue distribution and clearance for organs in which Ti content could not be previously determined due to low concentrations. Blood concentrations of TiO2 were 420 and 19 ng/mL at 5 and 15 min after administration, which were equivalent of only 2.8% and 0.13% of the administration dose, respectively. At 6 h, 94%, 2.0%, 0.17%, 0.023%, 0.014% and 0.026% of administered TiO2 was found in the liver, spleen, lung, kidney, heart and blood, respectively. Liver and spleen TiO2 burden was significantly higher in the administration than control group (p < 0.01) and did not decrease up to 30 days after administration, while TiO2 burden in the lung, kidney, heart and blood decreased over time. A two-step decay model was more suitable than a one-step decay model for the decay curves of pulmonary TiO2 burden but did not improve fitting to the decay curves of kidney TiO2 burden. No translocation to the brain was confirmed at a lower detection limit than was applied in previous studies. Ti content in faeces and urine in the TiO2 administration group did not differ from that in the control group.

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.

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.

Evaluation of cellular influences of platinum nanoparticles by stable medium dispersion.

Platinum nanoparticles have industrial application, for example in catalysis, and are used in consumer products such as cosmetics and supplements. Therefore, among the many nanoparticles, platinum is one of the more accessible nanoparticles for consumers. Most platinum nanoparticles that are used in cosmetics and supplements which have an anti-oxidant activity are modified particles. However, the cellular influences of pristine platinum nanoparticles are still unclear, although it has been reported that platinum nanoparticles induce oxidative stress. In this study, we investigated the cellular influences induced by pure pristine platinum nanoparticles. Platinum nanoparticles of 100% purity were dispersed in a cell culture medium and stable medium dispersion was obtained. The platinum nanoparticle medium dispersion was applied to two kinds of cultured cells, A549 and HaCaT cells, and the cellular influences were examined. Cell viability (MTT assay), cell proliferation (clonogenic assay), apoptosis induction (caspase-3 activity), intracellular ROS level (DCFH assay), and lipid peroxidation level (DPPP assay) were measured as markers of cellular influences. Transmission electron microscope observation showed cellular uptake of platinum nanoparticles. However, the platinum nanoparticles did not drive any markers. It is known that some metal oxide nanoparticles such as NiO and CuO show severe cytotoxicity via metal ion release. Compared with these toxic nanoparticles, the platinum nanoparticles used in this study did not release platinum ions into the culture media. These results suggest that the physically and chemically inactive cellular influences of platinum nanoparticles are small.

A determination method of pristine multiwall carbon nanotubes in rat lungs after intratracheal instillation exposure by combustive oxidation-nondispersive infrared analysis.

This paper describes a method for determination of multiwall carbon nanotubes (MWCNTs) in rat lungs after intratracheal instillation exposure. The MWCNTs were quantitatively decomposed to CO(2) by combustive oxidation and were then determined by non-dispersive infrared analysis. Samples were pretreated by acid digestion, muffle ashing and in situ preheating to remove interferences due to coexisting biological carbon from the lung tissue sample, while preserving the MWCNTs as in its their original form. The preservation was confirmed by transmission electron microscopic observation of the pretreated samples of exposed lung tissues and by the fact that the recoveries of MWCNTs spiked to the lung tissues were close to 100%. The detection limit for MWCNTs obtained by the proposed method was 0.30 µg and the repeatability as expressed by the relative standard deviation was 5.6% (n=4). The method was sufficiently sensitive and precise to apply to real samples of rat lung to investigate the in vivo persistence of intratracheally instilled MWCNTs. To our knowledge, this is the first report of this type of sample pretreatment and direct determination of pristine MWCNTs without modification or tagging. Conventional indirect methods use tagging with other compounds or metal impurities in the CNTs for detection, and the detachment of these tags can increase uncertainties in the determination of the CNTs. The tags can also change how the CNTs persist in vivo, which can lead to an incorrect understanding of the persistence of pristine CNTs in vivo.

Evaluation of acute oxidative stress induced by NiO nanoparticles in vivo and in vitro.

OBJECTIVES: Nickel oxide (NiO) is an important industrial material, and it is also a harmful agent. The toxicity of NiO is size-related: nanoparticles are more toxic than fine-particles. The toxic mechanism induced by NiO nanoparticles remains unexplained, and the relationship between in vitro and in vivo NiO toxicity results is unclear. In the present study, we focused on the oxidative stress caused by NiO nanoparticles by examining and comparing in vitro and in vivo acute responses induced by NiO nanoparticles. METHODS: Cellular responses induced by black NiO nanoparticles with a primary particle size of 20 nm, were examined in human lung carcinoma A549 cells. In vivo responses were examined by instillation of NiO nanoparticles into rat trachea. Bronchoalveolar lavage fluid (BALF) was collected after intratracheal instillation at different time points, and concentrations of lipid peroxide heme oxygenase-1 (HO-1), surfactant protein-D (SP-D) and lactate dehydrogenase (LDH) in BALF were measured. RESULTS: The levels of intracellular reactive oxygen species and lipid peroxidation in A549 cells increased with increasing exposure to NiO nanoparticles, and increases in gene expressions of HO-1 and SP-D were observed in A549 cells. The lipid peroxide level in BALF significantly increased after 24 h instillation but decreased three days later. LDH leakage was also observed three days later. CONCLUSIONS: NiO nanoparticles induce oxidative stress-related lung injury. In vivo and in vitro oxidative stress was induced resulting in activation of antioxidant systems. Based on these responses, we conclude that the results of the in vivo and in vitro studies tend to correspond.

Clearance kinetics of fullerene C60 nanoparticles from rat lungs after intratracheal C60 instillation and inhalation C60 exposure.

Fullerene (carbon sixty [C(60)]) has potential industrial and medical applications. In the future, people working in or residing near manufacturing facilities may be exposed to C(60). Therefore, quantitative data on long-term C(60) clearance from the lungs are required. To estimate the clearance rate and deposition fraction of C(60) from inhalation exposure, the C(60) burden in the lungs, liver, and brain of rats was determined after intratracheal instillation and inhalation. Male Wistar rats were intratracheally instilled with different concentrations of a C(60) suspension prepared with Tween 80 (geometric mean [GM] of particle diameter based on number, 18-29 nm; geometric standard deviation [GSD] of particle diameter, 1.5; and doses, 100, 200, and 1000 micrograms per body) or exposed to a C(60) aerosol prepared with nebulizer (GM of particle diameter based on number, 96 nm; GSD of particle diameter, 2.0; and exposure level, 120 µg/m(3)). C(60) burden in the lungs, liver, and brain was determined at various time points (1 h to 6 months) by a newly developed sensitive high-performance liquid chromatography-ultraviolet absorptiometry combined with extraction and concentration of C(60) from the organs. C(60) clearance was evaluated using a 2-compartment model: fast clearance after deposition on lung surface and slow clearance after retention in the epithelium. The detection limit of our analysis method was 8.9 ng/g tissue. Pulmonary C(60) burden decreased with time and depended on the C(60) concentration administered. The concentration of C(60) in the liver and brain was below the detection limit: 8.9 ng/g tissue. The half-life of intratracheally instilled C(60) was 15-28 days. The deposition mass fraction of inhaled C(60) was 0.14. Mode evaluation revealed that most instilled particles could be eliminated by the fast clearance pathway. This finding is consistent with the transmission electron microscopy finding that many particles were present in alveolar macrophages.

Identification of potential biomarkers from gene expression profiles in rat lungs intratracheally instilled with C(60) fullerenes.

The use of C(60) fullerenes is expected to increase in various industrial fields. Little is known about the potential toxicological mechanism of action of water-soluble C(60) fullerenes. In our previous research, gene expression profiling of the rat lung was performed after whole-body inhalation exposure to C(60) fullerenes to gain insights into the molecular events. These DNA microarray-based data closely matched the pathological findings that C(60) fullerenes caused no serious adverse pulmonary effects under the inhalation exposure condition. Taking advantage of this, we attempted to characterize time-dependent changes in the gene expression profiles after intratracheal instillation with C(60) fullerenes at different dosages and to identify the candidate expressed genes as potential biomarkers. The hierarchical cluster analysis revealed that the up- or downregulation of genes after intratracheal instillation with 1.0 mg C(60) fullerene particles in rat lung tissue was significantly over-represented in the "response to stimulus" and "response to chemical stimulus" categories of biological processes and in the "extracellular space" category of the cellular component. These results were remarkable for 1 week after the instillation with C(60) fullerenes. In the lung tissues instilled with 1.0 mg C(60) fullerene particles, many representative genes involved in "inflammatory response," such as the Cxcl2, Cxcl6, Orm1, and Spp1 genes, and in "matrix metalloproteinase activity," such as the Mmp7 and Mmp12 genes, were upregulated for over 6 months. The expression levels of 89 and 21 genes were positively correlated with the C(60) fullerene dose at 1 week and 6 months after the instillation, respectively. Most of them were involved in "inflammatory response", and the Ccl17, Ctsk, Cxcl2, Cxcl6, Lcn6, Orm1, Rnase9, Slc26a4, Spp1, Mmp7, and Mmp12 genes were overlapped. Meanwhile, the expression levels of 16 and 4 genes were negatively correlated with the C(60) fullerene dose at 1 week and 6 months after the instillation, respectively. Microarray-based gene expression profiling suggested that the expression of some genes is correlated with the dose of intratracheally instilled C(60) fullerenes. We propose that these genes are useful for identifying potential biomarkers in acute-phase or persistent responses to C(60) fullerenes in the lung tissue.