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

The health risks of inhalation exposure to engineered nanomaterials in the workplace are a major concern in recent years, and hazard assessments of these materials are being conducted. The pulmonary surfactant of lung alveoli is the first biological entity to have contact with airborne nanomaterials in inhaled air. In this study, we retrospectively evaluated the pulmonary surfactant components of rat lungs after a 4-week inhalation exposure to three different nanomaterials: fullerenes, nickel oxide (NiO) nanoparticles and multi-walled carbon nanotubes (MWCNT), with similar levels of average aerosol concentration (0.13-0.37 mg/m(3)). Bronchoalveolar lavage fluid (BALF) of the rat lungs stored after previous inhalation studies was analyzed, focusing on total protein and the surfactant components, such as phospholipids and surfactant-specific SP-D (surfactant protein D) and the BALF surface tension, which is affected by SP-B and SP-C. Compared with a control group, significant changes in the BALF surface tension and the concentrations of phospholipids, total protein and SP-D were observed in rats exposed to NiO nanoparticles, but not in those exposed to fullerenes. Surface tension and the levels of surfactant phospholipids and proteins were also significantly different in rats exposed to MWCNTs. The concentrations of phospholipids, total protein and SP-D and BALF surface tension were correlated significantly with the polymorphonuclear neutrophil counts in the BALF. These results suggest that pulmonary surfactant components can be used as measures of lung inflammation.

Expression of cytokine-induced neutrophil chemoattractant in rat lungs following an intratracheal instillation of micron-sized nickel oxide nanoparticle agglomerates.

OBJECTIVE: In our previous study, we reported that the micron-sized nickel oxide nanoparticle agglomerates induced neutrophil infiltration and the gene expression of the cytokine-induced neutrophil chemoattractant (CINC)-2αß in a rat lung. In this study, we examined the expression of the CINCs family in the lung using the same rat model exposed to micron-sized nickel oxide nanoparticle agglomerates. METHODS: The count median diameter of nickel oxide nanoparticle agglomerates suspended in saline was 1.34 µm (primary diameter: 8.41 nm). Male Wistar rats received an intratracheal instillation of 1 mg (3.3 mg/kg) of nickel oxide nanoparticles and were dissected at 3 days, 1 week, 1 month, 3 months, and 6 months after the instillation. The negative control group received an instillation of saline. The concentration of CINC-1 in the lung and the bronchoalveolar lavage fluid (BALF), CINC-2αß in the BALF, and CINC-3 in the lung and the BALF was examined. RESULTS: The concentration of CINC-1 was elevated at 3 days, 3 months, and 6 months in the lung tissue and from 3 days to 6 months in the BALF. The concentration of CINC-2αß was elevated from 3 days to 3 months in the BALF. The concentration of CINC-3 was also elevated at 3 days, 1 week, 3 months, and 6 months in the lung tissue. Infiltration of neutrophil and alveolar macrophage was observed mainly in the alveoli during the observed time period. CONCLUSION: These results suggest that CINC-1 to -3 were totally involved in the lung injury caused by micron-sized nickel oxide nanoparticle agglomerates.

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

Multi-walled carbon nanotubes (MWCNTs) are interesting new materials, but there is some concern about their harmfulness due to their fibrous nature. To determine the difference in the biological effects of MWCMTs by fiber length, we prepared two MWCNT samples from one bulk sample. One consisted of cut up short fibers (Short; average length=0.94 µm) and the other was just dispersed (Long; average length=3.4 µm). The samples were administered to male Wistar rats by intratracheal instillation at doses of 0.2 mg and 1 mg/animal (Short) and 0.2 mg and 0.6 mg/animal (Long). The animals were sacrificed at time points from 3 d to 12 months after administration. Bronchoalveolar lavage fluid (BALF) was taken from the lungs and pathological specimens were prepared. The concentrations of phospholipids, total protein and surfactant protein D (SP-D) in the pulmonary surfactant of the BALF were determined, the surface tension of BALF was measured, and the inflammation score was determined by the point-counting method to assess pulmonary tissue inflammation. The present study suggests that inflammatory response in the lung was slightly higher for long MWCNTs than for short MWCNTs when compared at the same mass dose. The correlation between pulmonary surfactant components and BALF surface tension was also evaluated. The Spearman's rank correlation coefficients obtained for the phospholipid, total protein and SP-D concentrations were -0.068 (p=0.605), -0.360 (p=0.005) and -0.673 (p=0.000), respectively. Surface tension, measured by a simple method, should be reflected in the change of a surfactant protein, such as SP-D.

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

Inhalation studies and intratracheal instillation studies using laboratory animals are commonly conducted for pulmonary toxicity tests of nanomaterials. In our study, male Wister rats were exposed to nickel oxide (NiO) particles including a nano-scale, even for aerosols and suspensions, in a 4-week inhalation and intratracheal instillation. Using polymorphonuclear neutrophils (PMNs) in bronchoalveolar lavage fluid as a biomarker of inflammation, we attempted to quantify the relationship between responses to inhalation and intratracheal instillation of the nanoparticles, based on surface area doses. Four kinds of NiO suspension samples with different specific surface areas were singly injected via the tracheas of the rats. The relationship between the instilled doses and PMN production was examined 3 days and 1 month after the instillation. In parallel, 4-week inhalation studies, using two of the suspensions, were conducted for aerosols generated by a pressurized nebulizer. NiO samples induced PMN responses 3 days after instillation according to the surface area doses, but not the mass doses, as has been reported in many studies. When the same NiO samples were tested in a 4-week inhalation and intratracheal instillation, the amount of pulmonary deposition of the sample after the 4-week inhalation, and an intratracheally instilled dose about ten-times higher, induced similar PMN responses 3 days after termination of inhalation and instillation. Using the relationship between these responses to 4-week inhalation and intratracheal instillation, it may be possible to estimate what aerosol concentrations of other nanomaterials might cause the same responses of PMN production as intratracheal instillation tests.

Analysis of bronchoalveolar lavage fluid adhering to lung surfactant. Experiment on intratracheal instillation of nickel oxide with different diameters.

Nickel oxide with two different particle sizes, micron size (NiO) and submicron size (nNiOm), as well as crystalline silica as a positive control and titanium dioxide as a negative control, were intratracheally instilled in rats and the phospholipid concentration and the protein concentration and surface tension of bronchoalveolar lavage fluid (BALF), which are used in surfactant assessment, were measured to see if they could be effective biomarkers in toxicity assessment. The results showed that the NiO instilled group showed no significant difference compared to the control group throughout the observation period. In contrast, a significant difference was found in the nNiOm instilled group compared to the control group throughout the observation period. Moreover, a significant difference was found in the crystalline silica instilled group for each measurement compared to the control group while for the titanium dioxide group, almost no significant difference was found. These results indicate that submicronsized particles of nickel oxide with smaller median diameters potentially have a stronger biological effect than micron size particles. They also indicate that screening can be done by measuring the phospholipid concentration and the protein concentration and surface tension of BALF.

Pulmonary toxicity of well-dispersed single-wall carbon nanotubes after inhalation.

Single-wall carbon nanotubes (SWCNTs) were well-dispersed by ultrasonication to conduct an inhalation study. SWCNTs were generated using a pressurised nebuliser with liquid suspension of SWCNTs. Wistar rats were exposed to the well-dispersed SWCNT (diameter of bundle: 0.2 µm; length of bundle: 0.7 µm) for 4 weeks. The low and high mass concentrations of SWCNTs were 0.03 ± 0.003 and 0.13 ± 0.03 mg/m(3), respectively. The rats were sacrificed at 3 days, 1 month, and 3 months after the end of exposure. There were no increases of total cell or neutrophil counts in the bronchoalveolar lavage fluid (BALF), or the concentration of cytokine-induced neutrophil chemoattractant in the lungs or BALF in both the high and low concentration-exposed groups. Pulmonary infiltration of neutrophils was not observed in either exposed group throughout the observation period. Well-dispersed SWCNT did not induce neutrophil inflammation in the lung under the conditions in the present study.

Pulmonary toxicity of well-dispersed multi-wall carbon nanotubes following inhalation and intratracheal instillation.

Multi-walled carbon nanotubes (MWCNTs), dispersed in suspensions consisting mainly of individual tubes, were used for intratracheal instillation and inhalation studies. Rats intratracheally received a dose of 0.2 mg, or 1 mg of MWCNTs and were sacrificed from 3 days to 6 months. MWCNTs induced a pulmonary inflammation, as evidenced by a transient neutrophil response in the low-dose groups, and presence of small granulomatous lesion and persistent neutrophil infiltration in the high-dose groups. In the inhalation study, rats were exposed to 0.37 mg/m(3) aerosols of well-dispersed MWCNTs (>70% of MWCNTs were individual fibers) for 4 weeks, and were sacrificed at 3 days, 1 month, and 3 months after the end of exposure. The inhalation exposures delivered less amounts of MWCNTs into the lungs, and therefore less pulmonary inflammation responses was observed, as compared to intratracheal instillation. The results of our study show that well-dispersed MWCNT can produce pulmonary lesions, including inflammation.

Biopersistence of inhaled MWCNT in rat lungs in a 4-week well-characterized exposure.

It is important to conduct a risk assessment that includes hazard assessment and exposure assessment for the safe production and handling of newly developed nanomaterials. We conducted an inhalation study of a multi-wall carbon nanotube (MWCNT) as a hazard assessment. Male Wistar rats were exposed to well-dispersed MWCNT for 4 weeks by whole body inhalation. The exposure concentration in the chamber was 0.37 ± 0.18 mg/m³. About 70% of the MWCNTs in the chamber were single fiber. The geometric mean diameter (geometric standard deviation, GSD) and geometric mean length (GSD) of the aerosolized MWCNTs in the chamber were 63 nm (1.5) and 1.1 µm (2.7), respectively. The amounts of MWCNT deposited in the rat lungs were determined by the X-ray diffraction method and elemental carbon analysis. The average deposited amounts at 3 days after the inhalation were 68 µg/lung by the X-ray diffraction method and 76 µg/lung by elemental carbon analysis. The calculated deposition fractions were 18% and 20% in each analysis. The amount of retained MWCNT in the lungs until 3 months after the inhalation decreased exponentially and the calculated biological half times of MWCNT were 51 days and 54 days, respectively. The clearance was not delayed, but a slight increase in lung weight at 3 days after the inhalation was observed.

Expression of heme oxygenase-1 in the lungs of rats exposed to potassium octatitanate whiskers.

OBJECTIVES: Oxidative stress is thought to be the pathogenesis of pulmonary fibrosis induced by asbestos, and heme oxygenase-1 (HO-1) protects lung tissue against oxidative stress. We hypothesized that HO-1 is also associated with oxidative lung injury caused by exposure to potassium octatitanate whiskers (PT1), which is one of the asbestos substitutes. METHODS: Male Wistar rats were administered 1 mg or 2 mg PT1 suspended in saline by a single intratracheal instillation and were sacrificed after recovery for 3 days, 1 wk, 1 mo, 3 mo or 6 mo. Gene expression of HO-1 protein and mRNA and immunostaining were investigated in rat lungs. RESULTS: HO-1 protein expression was increased from 3 days to 1 mo and at 6 mo in the 1 or 2 mg PT1-exposed groups, and the gene expression of HO-1 mRNA was also increased at 3 days and from 1 mo to 6 mo. HO-1-positive cells were mainly found in the alveolar macrophages and the bronchial epithelial cells in immunostaining. CONCLUSIONS: These findings suggest that HO-1 is involved in lung damage caused by PT1.

Pathological features of rat lung following inhalation and intratracheal instillation of C(60) fullerene.

We evaluated the pulmonary pathological features of rats that received a single intratracheal instillation and a 4-week inhalation of a fullerene. We used fullerene C(60) (nanom purple; Frontier Carbon Co. Ltd, Japan) in this study. Male Wistar rats received intratracheal dose of 0.1, 0.2, or 1 mg of C(60), and were sacrificed at 3 days, 1 week, 1 month, 3 months, 6 months, and 12 months. In the inhalation study, Wistar rats received C(60) or nickel oxide by whole-body inhalation for 6 h/day, 5 days/week, 4 weeks, and were sacrificed at 3 days, 1 month, and 3 months after the end of exposure. During the observation period, no tumors or granulomas were observed in either study. Histopathological evaluation by the point counting method (PCM) showed that a high dose of C(60) (1 mg) instillation led to a significant increase of areas of inflammation in the early phase (until 1 week). In the inhalation study of the C(60)-exposed group, PCM evaluation showed significant changes in the C(60)-exposed group only at 3 days after exposure; after 1 month, no significant changes were observed. The present study demonstrated that the pulmonary inflammation pattern after exposure to well-characterized C(60) via both intratracheal and inhalation instillation was slight and transient. These results support our previous studies that showed C(60) has no significant adverse effects in intratracheal and inhalation instillation studies.

Pulmonary toxicity following an intratracheal instillation of nickel oxide nanoparticle agglomerates.

OBJECTIVE: We examined the pulmonary toxicity of nickel oxide nanoparticle agglomerates in the rat lung following an intratracheal instillation. METHODS: The weighted average surface primary diameter of nickel oxide nanoparticles was 8.41 nm, and the count median diameter of nickel oxide nanoparticle agglomerates suspended in saline was 1.34 µm. Male Wistar rats were exposed to 1 mg (3.3 mg/kg) of nickel oxide nanoparticles intratracheally. The control group received intratracheal instillation of saline. Rats were dissected 3 days, 1 wk, 1 mo, 3 mo, and 6 mo after the instillation. Cytokine-induced neutrophil chemoattractant (CINC)-2αß in the lung tissue was determined by quantitative measurement of protein by ELISA. RESULTS: The total cell count in bronchoalveolar lavage fluid (BALF) was increased persistently from 3 days to 6 mo. The neutrophil counts in BALF were also increased at 3 days, 1 wk, 3 mo, and 6 mo. In the lung tissue, infiltration of mainly neutrophils and alveolar macrophages was observed in alveoli from 3 days to 6 mo. The CINC-2αß concentration was elevated from 3 days to 6 mo in the lung tissue. CONCLUSIONS: These results showed that micron-sized nickel oxide nanoparticle agglomerates also induced a persistent inflammatory response.

Investigation of gene expression of MMP-2 and TIMP-2 mRNA in rat lung in inhaled nickel oxide and titanium dioxide nanoparticles.

In order to investigate whether or not dispersed nanoparticles have an effect of inflammation and fibrosis on animals, we developed a nanoparticle generation system and examined the gene expression of matrix metalloproteinase (MMP) and tissue inhibitor matrix proteinase (TIMP) in rat lung containing inhaled nickel oxide (NiO) or titanium dioxide (TiO(2)) nanoparticles. In both experiments, Wistar male rats were exposed to NiO or TiO(2) nanoparticles for 4 wk (6 h/day). The geometric mean diameters of NiO and TiO(2) in the chamber were 139 ± 12 nm and 51 ± 9 nm, respectively. The average concentration of the particle number of NiO and TiO(2) was 1.0E+05 /cm(3) and 2.8E+05 /cm(3), respectively. At 4 d, 1 and 3 months after the end of the inhalation, the rats exposed to these particles were sacrificed and the gene expressions of MMP-2, TIMP-2 and type I collagen were measured using RT-PCR. Pathological finding revealed that there was minimum inflammation with nickel oxide only at 4 d and no change with titanium oxide. However, there were no changes of the gene expression of MMP-2, TIMP-2, and type I collagen in either the NiO or TiO(2) exposure groups. In this study, inhalation of nickel oxide and titanium dioxide nanoparticles did not induce the gene expression of MMP-2 and TIMP-2 mRNA in rat lungs.

Biopersistence of potassium hexatitanate in inhalation and intratracheal instillation studies.

An inhalation study and an intratracheal instillation study were conducted to evaluate the biological effects of the new chemical, potassium hexatitanate (PH). For the inhalation study, Wistar male rats were exposed to PH for 6 h a day, 5 days a week for a period of 3 months. The mass median aerodynamic diameter of PH in the exposure chamber was 4.9 µm (1.8) and the mean concentration during the exposure was 2.3 ± 0.1 mg/m(3). After the 3-month inhalation period, rats were dissected at 3 days, 1 month, 3 months, 6 months, and 12 months. The initial PH burden was 0.17 ± 0.03 mg/lung, and this decreased exponentially up to 6 months after inhalation. After 6 months, the rate at which the burden decreased slowed. The biological halftime up to 6 months after exposure was 2.3 months. No difference was found in the dimension of PH fibers in the lung during the observation period and the histopathological examination found no remarkable inflammation or fibrosis. For the intratracheal instillation study, the rats were given a single 2-mg dose of PH suspended in a 0.4 ml saline solution. The geometric mean diameter was 4.3 µm (2.3). After instillation, the rats were dissected at 3 days to 12 months. The PH burden in the lungs decreased exponentially and the biological halftime was 3.1 months. The results of the dimension of PH and histopathological findings were the same as those for the inhalation study. These data suggest that the toxicity of PH in the lung is low in these doses.

Hazard assessments of manufactured nanomaterials.

BACKGROUND: It has been difficult to make reliable hazard assessments of manufactured nanomaterials, because the nanomaterials form large agglomerations in both in vitro and in vivo studies. OBJECTIVE: In the New Energy and Industrial Technology Development Organization (NEDO) Project of Japan, the physicochemical properties of many manufactured nanomaterials are being measured, and in vitro and in vivo studies are being performed to determine which endpoints are correspond to the hazards and risks of nanomaterials. Focusing on titanium dioxide, fullerenes and carbon nanotubes, we introduce findings made in inhalation and intratracheal installation studies overseas, and together with the findings made in the NEDO project, and also assess the hazards presented by manufactured nanomaterials. RESULTS AND CONCLUSION: A project by NEDO has succeeded in ensuring the stability of dispersion (nanoscale <100 nm) of manufactured nanomaterials, and is developing hazard assessments of manufactured nanomaterials. In these interim reports, the acceptable exposure concentration of titanium dioxide and fullerene was proposed to be 1.2 mg/m(3) and 0.8 mg/m(3) respirable dust in working environment, respectively.

Expression of inflammation-related cytokines following intratracheal instillation of nickel oxide nanoparticles.

The objective of this study was to examine what kinds of cytokines are related to lung disorder by well-dispersed nanoparticles. The mass median diameter of nickel oxide in distilled water was 26 nm. Rats intratracheally received 0.2 mg of nickel oxide suspended in distilled water, and were sacrificed from three days to six months. The concentrations of 21 cytokines including inflammation, fibrosis and allergy-related ones were measured in the lung. Infiltration of alveolar macrophages was observed persistently in the nickel oxide-exposed group. Expression of macrophage inflammatory protein-1alpha showed a continued increase in lung tissue and broncho-alveolar lavage fluid (BALF) while interleukin-1alpha (IL-1alpha), IL-1beta in lung tissue and monocyte chemotactic protein-1 in BALF showed transient increases. Taken together, it was suggested that nano-agglomerates of nickel oxide nanoparticles have a persistent inflammatory effect, and the transient increase in cytokine expression and persistent increases in CC chemokine were involved in the persistent pulmonary inflammation.

Hazard Assessments of Manufactured Nanomaterials.

Background: It has been difficult to make reliable hazard assessments of manufactured nanomaterials, because the nanomaterials form large agglomerations in both in vitro and in vivo studies. Objective: A project by the New Energy and Industrial Technology Development Organization (NEDO) of Japan has succeeded in ensuring the stability of dispersion (nanoscale <100 nm) of manufactured nanomaterials, and is developing hazard assessments of manufactured nanomaterials. Results and Conclusion: Focusing on titanium dioxide, fullerenes and carbon nanotubes, we introduce findings made in inhalation and intratracheal installation studies overseas, and together with the findings made in the NEDO project, and also assess the hazards presented by manufactured nanoparticles.

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.

Inflammogenic effect of well-characterized fullerenes in inhalation and intratracheal instillation studies.

BACKGROUND: We used fullerenes, whose dispersion at the nano-level was stabilized by grinding in nitrogen gas in an agitation mill, to conduct an intratracheal instillation study and an inhalation exposure study. Fullerenes were individually dispersed in distilled water including 0.1% Tween 80, and the diameter of the fullerenes was 33 nm. These suspensions were directly injected as a solution in the intratracheal instillation study. The reference material was nickel oxide in distilled water. Wistar male rats intratracheally received a dose of 0.1 mg, 0.2 mg, or 1 mg of fullerenes and were sacrificed after 3 days, 1 week, 1 month, 3 months, and 6 months. In the inhalation study, Wistar rats were exposed to fullerene agglomerates (diameter: 96 +/- 5 nm; 0.12 +/- 0.03 mg/m3; 6 hours/days for 5 days/week) for 4 weeks and were sacrificed at 3 days, 1 month, and 3 months after the end of exposure. The inflammatory responses and gene expression of cytokine-induced neutrophil chemoattractants (CINCs) were examined in rat lungs in both studies. RESULTS: In the intratracheal instillation study, both the 0.1 mg and 0.2 mg fullerene groups did not show a significant increase of the total cell and neutrophil count in BALF or in the expression of CINC-1,-2alphabeta and-3 in the lung, while the high-dose, 1 mg group only showed a transient significant increase of neutrophils and expression of CINC-1,-2alphabeta and -3. In the inhalation study, there were no increases of total cell and neutrophil count in BALF, CINC-1,-2alphabeta and-3 in the fullerene group. CONCLUSION: These data in intratracheal instillation and inhalation studies suggested that well-dispersed fullerenes do not have strong potential of neutrophil inflammation.

Expression of cytokine-induced neutrophil chemoattractant in rat lungs by intratracheal instillation of nickel oxide nanoparticles.

Since nanoparticles easily agglomerate to form larger particles, it is important to maintain the size of their agglomerates at the nano-level to evaluate the harmful effect of the nanoparticles. We prevented agglomeration of nickel oxide nanoparticles by ultrasound diffusion and filtration, established an acute exposure model using animals, and examined inflammation and chemokine expression. The mass median diameter of nickel oxide nanoparticle agglomerates suspended in distilled water for intratracheal instillation was 26 nm (8.41 nm weighted average surface primary diameter). Male Wistar rats received intratracheal instillation of nickel oxide nanoparticles at 0.1 mg (0.33 mg/kg) or 0.2 mg (0.66 mg/kg), and were dissected 3 days, 1 week, 1 month, 3 months, and 6 months after the instillation. The control group received intratracheal instillation of distilled water. Three chemokines (cytokine-induced neutrophil chemoattractant-1 (CINC-1), CINC-2alphabeta, and CINC-3) in the lung tissue and bronchoalveolar lavage fluid (BALF) were determined by quantitative measurement of protein by ELISA. Both CINC-1 and CINC-2alphabeta concentration was elevated from day 3 to 3 months in lung tissue and from day 3 to 6 months in BALF. On the other hand, CINC-3 was elevated on day 3 in both lung tissue and BALF, and then decreased. The total cell and neutrophil counts in BALF were increased from day 3 to 3 months. In lung tissue, infiltration of mainly neutrophils and alveolar macrophages was observed from day 3 to 6 months in alveoli. These results suggest that CINC was involved in lung injury by nickel oxide nanoparticles.