Inhaled Fine Particles Induce Alveolar Macrophage Death and Interleukin-1α Release to Promote Inducible Bronchus-Associated Lymphoid Tissue Formation.

Particulate pollution is thought to function as an adjuvant that can induce allergic responses. However, the exact cell types and immunological factors that initiate the lung-specific immune responses are unclear. We found that upon intratracheal instillation, particulates such as aluminum salts and silica killed alveolar macrophages (AMs), which then released interleukin-1α (IL-1α) and caused inducible bronchus-associated lymphoid tissue (iBALT) formation in the lung. IL-1α release continued for up to 2 weeks after particulate exposure, and type-2 allergic immune responses were induced by the inhalation of antigen during IL-1α release and iBALT formation, even long after particulate instillation. Recombinant IL-1α was sufficient to induce iBALTs, which coincided with subsequent immunoglobulin E responses, and IL-1-receptor-deficient mice failed to induce iBALT formation. Therefore, the AM-IL-1α-iBALT axis might be a therapeutic target for particulate-induced allergic inflammation.

Investigation of pulmonary inflammatory responses following intratracheal instillation of and inhalation exposure to polypropylene microplastics.

BACKGROUND: Microplastics have been detected in the atmosphere as well as in the ocean, and there is concern about their biological effects in the lungs. We conducted a short-term inhalation exposure and intratracheal instillation using rats to evaluate lung disorders related to microplastics. We conducted an inhalation exposure of polypropylene fine powder at a low concentration of 2 mg/m3 and a high concentration of 10 mg/m3 on 8-week-old male Fischer 344 rats for 6 h a day, 5 days a week for 4 weeks. We also conducted an intratracheal instillation of polypropylene at a low dose of 0.2 mg/rat and a high dose of 1.0 mg/rat on 12-week-old male Fischer 344 rats. Rats were dissected from 3 days to 6 months after both exposures, and bronchoalveolar lavage fluid (BALF) and lung tissue were collected to analyze lung inflammation and lung injury. RESULTS: Both exposures to polypropylene induced a persistent influx of inflammatory cells and expression of CINC-1, CINC-2, and MPO in BALF from 1 month after exposure. Genetic analysis showed a significant increase in inflammation-related factors for up to 6 months. The low concentration in the inhalation exposure of polypropylene also induced mild lung inflammation. CONCLUSION: These findings suggest that inhaled polypropylene, which is a microplastic, induces persistent lung inflammation and has the potential for lung disorder. Exposure to 2 mg/m3 induced inflammatory changes and was thought to be the Lowest Observed Adverse Effect Level (LOAEL) for acute effects of polypropylene. However, considering the concentration of microplastics in a real general environment, the risk of environmental hazards to humans may be low.

The Effects of Endoplasmic Reticulum Stress via Intratracheal Instillation of Water-Soluble Acrylic Acid Polymer on the Lungs of Rats.

Polyacrylic acid (PAA), an organic chemical, has been used as an intermediate in the manufacture of pharmaceuticals and cosmetics. It has been suggested recently that PAA has a high pulmonary inflammatory and fibrotic potential. Although endoplasmic reticulum stress is induced by various external and intracellular stimuli, there have been no reports examining the relationship between PAA-induced lung injury and endoplasmic reticulum stress. F344 rats were intratracheally instilled with dispersed PAA (molecular weight: 269,000) at low (0.5 mg/mL) and high (2.5 mg/mL) doses, and they were sacrificed at 3 days, 1 week, 1 month, 3 months and 6 months after exposure. PAA caused extensive inflammation and fibrotic changes in the lungs' histopathology over a month following instillation. Compared to the control group, the mRNA levels of endoplasmic reticulum stress markers Bip and Chop in BALF were significantly increased in the exposure group. In fluorescent immunostaining, both Bip and Chop exhibited co-localization with macrophages. Intratracheal instillation of PAA induced neutrophil inflammation and fibrosis in the rat lung, suggesting that PAA with molecular weight 269,000 may lead to pulmonary disorder. Furthermore, the presence of endoplasmic reticulum stress in macrophages was suggested to be involved in PAA-induced lung injury.

Serum Extracellular Vesicle-Derived hsa-miR-2277-3p and hsa-miR-6813-3p Are Potential Biomarkers for Major Depression: A Preliminary Study.

The aim of the present study was to examine the association between miRNA levels in extracellular vesicles (EVs) from serum and the severity of Major Depression (MD). Patient sera from 16 MD cases were collected at our university hospital. The miRNAs contained in EVs were extracted using a nanofiltration method, and their expression levels were analyzed using miRNA microarrays. Intergroup comparisons were performed to validate the diagnostic performance of miRNAs in EVs. Furthermore, candidate miRNAs in EVs were added to neural progenitor cells, astrocytes, and microglial cells in vitro, and the predicted target genes of the candidate miRNAs were extracted. The predicted target genes underwent enrichment analysis. The expression levels of hsa-miR-6813-3p and hsa-miR-2277-3p were significantly downregulated with increasing depression severity of MD. The pathway enrichment analysis suggests that hsa-miR-6813-3p may be involved in glucocorticoid receptor and gamma-aminobutyric acid receptor signaling. Additionally, hsa-miR-2277-3p was found to be involved in the dopaminergic neural pathway. The analysis of serum miRNAs in EVs suggests that hsa-miR-6813-3p and hsa-miR-2277-3p could serve as novel biomarkers for MD, reflecting its severity. Moreover, these miRNAs in EVs could help understand MD pathophysiology.

Inflammogenic effect of polyacrylic acid in rat lung following intratracheal instillation.

BACKGROUND: Some organic chemicals are known to cause allergic disorders such as bronchial asthma and hypersensitivity pneumonitis, and it has been considered that they do not cause irreversible pulmonary fibrosis. It has recently been reported, however, that cross-linked acrylic acid-based polymer, an organic chemical, might cause serious interstitial lung diseases, including pulmonary fibrosis. We investigated whether or not intratracheal instillation exposure to cross-linked polyacrylic acid (CL-PAA) can cause lung disorder in rats. METHODS: Male F344 rats were intratracheally instilled with dispersed CL-PAA at low (0.2 mg/rat) and high (1.0 mg/rat) doses, and were sacrificed at 3 days, 1 week, 1 month, 3 months and 6 months after exposure to examine inflammatory and fibrotic responses and related gene expressions in the lungs. Rat lungs exposed to crystalline silica, asbestos (chrysotile), and NiO and CeO2 nanoparticles were used as comparators. RESULTS: Persistent increases in total cell count, neutrophil count and neutrophil percentage, and in the concentration of the cytokine-induced neutrophil chemoattractant (CINC)-1, CINC-2 and C-X-C motif chemokine 5 (CXCL5), which correlated with lung tissue gene expression, were observed in bronchoalveolar lavage fluid (BALF) from 3 days until at least 1 month following CL-PAA intratracheal instillation. Persistent increases in heme oxygenase-1 (HO-1) in the lung tissue were also observed from 3 days to 6 months after exposure. Histopathological findings of the lungs demonstrated that extensive inflammation at 3 days was greater than that in exposure to silica, NiO nanoparticles and CeO2 nanoparticles, and equal to or greater than that in asbestos (chrysotile) exposure, and the inflammation continued until 1 month. Fibrotic changes also progressed after 1 month postexposure. CONCLUSION: Our results suggested that CL-PAA potentially causes strong neutrophil inflammation in the rat and human lung.

Effect of Different Molecular Weights of Polyacrylic Acid on Rat Lung Following Intratracheal Instillation.

BACKGROUND: We conducted intratracheal instillations of different molecular weights of polyacrylic acid (PAA) into rats in order to examine what kinds of physicochemical characteristics of acrylic acid-based polymer affect responses in the lung. METHODS: F344 rats were intratracheally exposed to a high molecular weight (HMW) of 598 thousand g/mol or a low molecular weight (LMW) of 30.9 thousand g/mol PAA at low and high doses. Rats were sacrificed at 3 days, 1 week, 1 month, 3 months and 6 months post exposure. RESULTS: HMW PAA caused persistent increases in neutrophil influx, cytokine-induced neutrophil chemoattractants (CINC) in the bronchoalveolar lavage fluid (BALF), and heme oxygenase-1 (HO-1) in the lung tissue from 3 days to 3 months and 6 months following instillation. On the other hand, LMW PAA caused only transient increases in neutrophil influx, CINC in BALF, and HO-1 in the lung tissue from 3 days to up to 1 week or 1 month following instillation. Histopathological findings of the lungs demonstrated that the extensive inflammation and fibrotic changes caused by the HMW PAA was greater than that in exposure to the LMW PAA during the observation period. CONCLUSION: HMW PAA induced persistence of lung disorder, suggesting that molecular weight is a physicochemical characteristic of PAA-induced lung disorder.

Crosslinked Structure of Polyacrylic Acid Affects Pulmonary Fibrogenicity in Rats.

We conducted intratracheal instillations of polyacrylic acid (PAA) with crosslinking and non-crosslinking into rats in order to examine what kinds of physicochemical characteristics of acrylic-acid-based polymers affect responses in the lung. F344 rats were intratracheally exposed to similar molecular weights of crosslinked PAA (CL-PAA) (degree of crosslinking: ~0.1%) and non-crosslinked PAA (Non-CL-PAA) at low and high doses. Rats were sacrificed at 3 days, 1 week, 1 month, 3 months, and 6 months post-exposure. Both PAAs caused increases in neutrophil influx, cytokine-induced neutrophil chemoattractants (CINC) in the bronchoalveolar lavage fluid (BALF), and heme oxygenase-1 (HO-1) in the lung tissue from 3 days to 6 months following instillation. The release of lactate dehydrogenase (LDH) activity in the BALF was higher in the CL-PAA-exposed groups. Histopathological findings of the lungs demonstrated that the extensive fibrotic changes caused by CL-PAA were also greater than those in exposure to the Non-CL- PAA during the observation period. CL-PAA has more fibrogenicity of the lung, suggesting that crosslinking may be one of the physicochemical characteristic factors of PAA-induced lung disorder.

Examination of Surfactant Protein D as a Biomarker for Evaluating Pulmonary Toxicity of Nanomaterials in Rat.

This work studies the relationship between lung inflammation caused by nanomaterials and surfactant protein D (SP-D) kinetics and investigates whether SP-D can be a biomarker of the pulmonary toxicity of nanomaterials. Nanomaterials of nickel oxide and cerium dioxide were classified as having high toxicity, nanomaterials of two types of titanium dioxides and zinc oxide were classified as having low toxicity, and rat biological samples obtained from 3 days to 6 months after intratracheal instillation of those nanomaterials and micron-particles of crystalline silica were used. There were different tendencies of increase between the high- and low-toxicity materials in the concentration of SP-D in bronchoalveolar-lavage fluid (BALF) and serum and in the expression of the SP-D gene in the lung tissue. An analysis of the receiver operating characteristics for the toxicity of the nanomaterials by SP-D in BALF and serum showed a high accuracy of discrimination from 1 week to 3 or 6 months after exposure. These data suggest that the differences in the expression of SP-D in BALF and serum depended on the level of lung inflammation caused by the nanomaterials and that SP-D can be biomarkers for evaluating the pulmonary toxicity of nanomaterials.

Silica crystals and aluminum salts regulate the production of prostaglandin in macrophages via NALP3 inflammasome-independent mechanisms.

Particulates such as silica crystal (silica) and aluminum salts (alum) activate the inflammasome and induce the secretion of proinflammatory cytokines in macrophages. These particulates also induce the production of immunoglobulin E via a T helper 2 (Th2) cell-associated mechanism. However, the mechanism involved in the induction of type 2 immunity has not been elucidated. Here, we showed that silica and alum induced lipopolysaccharide-primed macrophages to produce the lipid mediator prostaglandin E2 (PGE2) and interleukin-1ß (IL-1ß). Macrophages deficient in the inflammasome components caspase 1, NALP3, and ASC revealed that PGE2 production was independent of the NALP3 inflammasome. PGE2 expression was markedly reduced in PGE synthase-deficient (Ptges⁻/⁻) macrophages, and Ptges⁻/⁻ mice displayed reduced antigen-specific serum IgE concentrations after immunization with alum or silica. Our results indicate that silica and alum regulate the production of PGE2 and that the induction of PGE2 by particulates controls the immune response in vivo.

Induction of potent cell growth inhibition by schizophyllan/K-ras antisense complex in combination with gemcitabine.

Antisense oligonucleotides (AS-ODNs) specifically hybridize with target mRNAs, resulting in interference with the splicing mechanism or the regulation of protein translation. In our previous reports, we demonstrated that ß-glucan schizophyllan (SPG) can form a complex with AS-ODNs attached with oligo deoxyadenosine dA40 (AS-ODN-dA40/SPG), and that this complex can be recognized by ß-glucan receptor Dectin-1 on antigen presenting cells and lung cancer cells. In many types of cancer cell, activating K-ras mutations related to malignancy are frequently observed. In this study, we first designed 78 AS-ODNs for K-ras to optimize the sequence for highly efficient gene suppression. The selected AS-ODN (K-AS07) having dA40 made a complex with SPG. The resultant complex (K-AS07-dA40/SPG) showed an effect of silencing the ras gene in the cells (PC9: human adenocarcinoma differentiated from lung tissue) expressing Dectin-1, leading to the suppression of cell growth. Furthermore, the cytotoxic effect was enhanced when used in combination with the anticancer drug gemcitabine. Gemcitabine, a derivative of cytidine, was shown to interact with dA40 in a sequence-dependent manner. This interaction did not appear to be so strong, with the gemcitabine being released from the complex after internalization into the cells. SPG and the dA40 part of K-AS07-dA40 play roles in carriers for K-AS07 and gemcitabine, respectively, resulting in a strong cytotoxic effect. This combination effect is a novel feature of the AS-ODN-dA40/SPG complexes. These results could facilitate the clinical application of these complexes for cancer treatment.

Usefulness of myeloperoxidase as a biomarker for the ranking of pulmonary toxicity of nanomaterials.

BACKGROUND: In order to examine whether myeloperoxidase (MPO) can be a useful marker for evaluating the pulmonary toxicity of nanomaterials, we analyzed MPO protein in bronchoalveolar lavage fluid (BALF) samples obtained from previous examinations of a rat model. In those examinations we performed intratracheal instillation exposures (dose: 0.2-1.0 mg) and inhalation exposures (exposure concentration: 0.32-10.4 mg/m3) using 9 and 4 nanomaterials with different toxicities, respectively. Based on those previous studies, we set Nickel oxide nanoparticles (NiO), cerium dioxide nanoparticles (CeO2), multi wall carbon nanotubes with short or long length (MWCNT (S) and MWCNT (L)), and single wall carbon nanotube (SWCNT) as chemicals with high toxicity; and titanium dioxide nanoparticles (TiO2 (P90) and TiO2 (Rutile)), zinc oxide nanoparticles (ZnO), and toner with external additives including nanoparticles as chemicals with low toxicity. We measured the concentration of MPO in BALF samples from rats from 3 days to 6 months following a single intratracheal instillation, and from 3 days to 3 months after the end of inhalation exposure. RESULTS: Intratracheal instillation of high toxicity NiO, CeO2, MWCNT (S), MWCNT (L), and SWCNT persistently increased the concentration of MPO, and inhalation of NiO and CeO2 increased the MPO in BALF. By contrast, intratracheal instillation of low toxicity TiO2 (P90), TiO2 (Rutile), ZnO, and toner increased the concentration of MPO in BALF only transiently, and inhalation of TiO2 (Rutile) and ZnO induced almost no increase of the MPO. The concentration of MPO correlated with the number of total cells and neutrophils, the concentration of chemokines for neutrophils (cytokine-induced neutrophil chemoattractant (CINC)-1 and heme oxygenase (HO)-1), and the activity of released lactate dehydrogenase (LDH) in BALF. The results from the receiver operating characteristics (ROC) for the toxicity of chemicals by the concentration of MPO proteins in the intratracheal instillation and inhalation exposures showed that the largest areas under the curves (AUC) s in both examinations occurred at 1 month after exposure. CONCLUSION: These data suggest that MPO can be a useful biomarker for the ranking of the pulmonary toxicity of nanomaterials, especially at 1 month after exposure, in both intratracheal instillation and inhalation exposure.

Cancer Risks of Hexavalent Chromium in the Respiratory Tract.

Hexavalent chromium (Cr(VI)) compounds are recognized as carcinogens in the respiratory tract, giving rise to cancers of the lung, nose and nasal sinuses, especially in certain occupational environments. Inhalation exposure of Cr(VI)-containing particles, dusts and fumes commonly occurs in chromium-related occupational environments, such as chromium production, plating, welding of chromium-containing metals and alloys, electroplating, chromium-containing pigments and paints. Epidemiological surveys of chromium compounds have shown strong associations between exposure to Cr(VI) and mortality due to lung cancer, as well as positive associations with cancers of the nose and nasal cavity. Nasal symptoms, such as nasal irritation, ulceration and perforation of the nasal septum, nasal turbinate engorgement and hypertrophy, are important signs for the early diagnosis of lung cancer and cancers of the nose and nasal cavity in those with an occupational history of Cr(VI) exposure. Cr(VI) exposure in the workplace remains a serious problem as a cause of lung cancer and cancers of nose and nasal cavity, especially in relatively small enterprises that use chromium compounds. Appropriate protection for workers should be considered in occupations that involve exposure to chromium compounds.

Oxidative DNA damage in the rat lung induced by intratracheal instillation and inhalation of nanoparticles.

Nanoparticles are widely used as useful industrial materials. Therefore, their possible adverse health effects must be appraised. We assessed and compared the oxidative DNA damage caused by four different nanoparticles (TiO2, NiO, ZnO and CeO2). The effects of the administration methods, intratracheal instillation and inhalation, were also evaluated. Rats were subjected to intratracheal instillations or 4 weeks of inhalation exposure to the nanoparticles, and the 8-hydroxydeoxyguanosine (8-OHdG) levels in the lung were analyzed by an HPLC-EC detector method. The 8-OHdG levels were increased in a dose-dependent manner with the inhalation of NiO. ZnO also increased the 8-OHdG levels with inhalation. In comparison with the control, the 8-OHdG levels were significantly and persistently higher with the CeO2 nanoparticle administration, by both intratracheal instillation and inhalation. In contrast, there were no significant differences in the 8-OHdG levels between the control and TiO2 nanoparticle-treated groups, with either intratracheal instillation or inhalation during the observation period. These results indicated that NiO, ZnO and CeO2 nanoparticles generate significant amounts of free radicals, and oxidative stress may be responsible for the lung injury caused by these nanoparticles. In addition, both intratracheal instillation and inhalation exposure induced similar tendencies of oxidative DNA damage with these nanoparticles.

Evaluating the mechanistic evidence and key data gaps in assessing the potential carcinogenicity of carbon nanotubes and nanofibers in humans.

In an evaluation of carbon nanotubes (CNTs) for the IARC Monograph 111, the Mechanisms Subgroup was tasked with assessing the strength of evidence on the potential carcinogenicity of CNTs in humans. The mechanistic evidence was considered to be not strong enough to alter the evaluations based on the animal data. In this paper, we provide an extended, in-depth examination of the in vivo and in vitro experimental studies according to current hypotheses on the carcinogenicity of inhaled particles and fibers. We cite additional studies of CNTs that were not available at the time of the IARC meeting in October 2014, and extend our evaluation to include carbon nanofibers (CNFs). Finally, we identify key data gaps and suggest research needs to reduce uncertainty. The focus of this review is on the cancer risk to workers exposed to airborne CNT or CNF during the production and use of these materials. The findings of this review, in general, affirm those of the original evaluation on the inadequate or limited evidence of carcinogenicity for most types of CNTs and CNFs at this time, and possible carcinogenicity of one type of CNT (MWCNT-7). The key evidence gaps to be filled by research include: investigation of possible associations between in vitro and early-stage in vivo events that may be predictive of lung cancer or mesothelioma, and systematic analysis of dose-response relationships across materials, including evaluation of the influence of physico-chemical properties and experimental factors on the observation of nonmalignant and malignant endpoints.

The utility of electron microscopy in detecting asbestos fibers and particles in BALF in diffuse lung diseases.

BACKGROUND: In patients with diffuse lung diseases, differentiating occupational lung diseases from other diseases is clinically important. However, the value of assessing asbestos and particles in bronchoalveolar lavage fluid (BALF) in diffuse lung diseases by electron microscopy (EM) remains unclear. We evaluated the utility of EM in detecting asbestos fibers and particles in patients with diffuse lung diseases. METHODS: The BALF specimens of 107 patients with diffuse lung diseases were evaluated. First, detection of asbestos by EM and light microscopy (LM) were compared. Second, the detection of asbestos using surgically obtained lung tissues of 8 of 107 patients were compared with the results of EM and LM in BALF. Third, we compared the results of mineralogical components of particles in patients with (n = 48) and without (n = 59) a history of occupational exposure to inorganic dust. RESULTS: BALF asbestos were detected in 11 of 48 patients with a history of occupational exposure by EM; whereas asbestos as asbestos bodies (ABs) were detected in BALF in 4 of these 11 patients by LM. Eight of 107 patients in whom lung tissue samples were surgically obtained, EM detected BALF asbestos at a level of >1,000 fibers/ml in all three patients who had ABs in lung tissue samples by LM at a level of >1,000 fibers/g. The BALF asbestos concentration by EM and in lung tissue by LM were positively correlated. The particle fractions of iron and phosphorus were increased in patients with a history of occupational exposure and both correlated with a history of occupational exposure by a multiple regression analysis. CONCLUSIONS: EM using BALF seemed to be superior to LM using BALF and displayed a similar sensitivity to LM using surgically-obtained lung tissue samples in the detection of asbestos. Our results also suggest that detection of elements, such as iron and phosphorus in particles, is useful for evaluating occupational exposure. We conclude that the detection of asbestos and iron and phosphorus in particles in BALF by EM is very useful for the evaluation of occupational exposure.

Biopersistence of NiO and TiO2 Nanoparticles Following Intratracheal Instillation and Inhalation.

The hazards of various types of nanoparticles with high functionality have not been fully assessed. We investigated the usefulness of biopersistence as a hazard indicator of nanoparticles by performing inhalation and intratracheal instillation studies and comparing the biopersistence of two nanoparticles with different toxicities: NiO and TiO2 nanoparticles with high and low toxicity among nanoparticles, respectively. In the 4-week inhalation studies, the average exposure concentrations were 0.32 and 1.65 mg/m³ for NiO, and 0.50 and 1.84 mg/m³ for TiO2. In the instillation studies, 0.2 and 1.0 mg of NiO nanoparticles and 0.2, 0.36, and 1.0 mg of TiO2 were dispersed in 0.4 mL water and instilled to rats. After the exposure, the lung burden in each of five rats was determined by Inductively Coupled Plasma-Atomic Emission Spectrometer (ICP-AES) from 3 days to 3 months for inhalation studies and to 6 months for instillation studies. In both the inhalation and instillation studies, NiO nanoparticles persisted for longer in the lung compared with TiO2 nanoparticles, and the calculated biological half times (BHTs) of the NiO nanoparticles was longer than that of the TiO2 nanoparticles. Biopersistence also correlated with histopathological changes, inflammatory response, and other biomarkers in bronchoalveolar lavage fluid (BALF) after the exposure to nanoparticles. These results suggested that the biopersistence is a good indicator of the hazards of nanoparticles.

Significance of Intratracheal Instillation Tests for the Screening of Pulmonary Toxicity of Nanomaterials.

Inhalation tests are the gold standard test for the estimation of the pulmonary toxicity of respirable materials. Intratracheal instillation tests have been used widely, but they yield limited evidence of the harmful effects of respirable materials. We reviewed the effectiveness of intratracheal instillation tests for estimating the hazards of nanomaterials, mainly using research papers featuring intratracheal instillation and inhalation tests centered on a Japanese national project. Compared to inhalation tests, intratracheal instillation tests induced more acute inflammatory responses in the animal lung due to a bolus effect regardless of the toxicity of the nanomaterials. However, nanomaterials with high toxicity induced persistent inflammation in the chronic phase, and nanomaterials with low toxicity induced only transient inflammation. Therefore, in order to estimate the harmful effects of a nanomaterial, an observation period of 3 months or 6 months following intratracheal instillation is necessary. Among the endpoints of pulmonary toxicity, cell count and percentage of neutrophil, chemokines for neutrophils and macrophages, and oxidative stress markers are considered most important. These markers show persistent and transient responses in the lung from nanomaterials with high and low toxicity, respectively. If the evaluation of the pulmonary toxicity of nanomaterials is performed in not only the acute but also the chronic phase in order to avoid the bolus effect of intratracheal instillation and inflammatory-related factors that are used as endpoints of pulmonary toxicity, we speculate that intratracheal instillation tests can be useful for screening for the identification of the hazard of nanomaterials through pulmonary inflammation.

Long-term retention of pristine multi-walled carbon nanotubes in rat lungs after intratracheal instillation.

As a result of the growing potential industrial and medical applications of multi-walled carbon nanotubes (MWCNTs), people working in or residing near facilities that manufacture them may be exposed to airborne MWCNTs in the future. Because of concerns regarding their toxicity, quantitative data on the long-term clearance of pristine MWCNTs from the lungs are required. We administered pristine MWCNTs well dispersed in 0.5 mg ml(-1) Triton-X solution to rats at doses of 0.20 or 0.55 mg via intratracheal instillation and investigated clearance over a 12-month observation period. The pristine MWCNTs pulmonary burden was determined 1, 3, 7, 28, 91, 175 and 364 days after instillation using a method involving combustive oxidation and infrared analysis, combined with acid digestion and heat pretreatment. As 0.15- and 0.38-mg MWCNTs were detected 1 day after administration of 0.20 and 0.55 mg MWCNTs, respectively, approximately 30% of administrated MWCNTs may have been cleared by bronchial ciliary motion within 24 h of administration. After that, the pulmonary MWCNT burden did not decrease significantly over time for up to 364 days after instillation, suggesting that MWCNTs were not readily cleared from the lung. Transmission electron microscopy (TEM) showed that alveolar macrophages internalized the MWCNTs and retained in the lung for at least 364 days after instillation. MWCNTs were not detected in the liver or brain within the 364-day study period (<0.04 mg per liver, < 0.006 mg per brain).

Usefulness of Intratracheal Instillation Studies for Estimating Nanoparticle-Induced Pulmonary Toxicity.

Inhalation studies are the gold standard for the estimation of the harmful effects of respirable chemical substances, while there is limited evidence of the harmful effects of chemical substances by intratracheal instillation. We reviewed the effectiveness of intratracheal instillation studies for estimating the hazards of nanoparticles, mainly using papers in which both inhalation and intratracheal instillation studies were performed using the same nanoparticles. Compared to inhalation studies, there is a tendency in intratracheal instillation studies that pulmonary inflammation lasted longer in the lungs. A difference in pulmonary inflammation between high and low toxicity nanoparticles was observed in the intratracheal instillation studies, as in the inhalation studies. Among the endpoints of pulmonary toxicity, the kinetics of neutrophil counts, percentage of neutrophils, and chemokines for neutrophils and macrophages, heme oxygenase-1 (HO-1) in bronchoalveolar lavage fluid (BALF), reflected pulmonary inflammation, suggesting that these markers may be considered the predictive markers of pulmonary toxicity in both types of study. When comparing pulmonary inflammation between intratracheal instillation and inhalation studies under the same initial lung burden, there is a tendency that the inflammatory response following the intratracheal instillation of nanoparticles is greater than or equal to that following the inhalation of nanoparticles. If the difference in clearance in both studies is not large, the estimations of pulmonary toxicity are close. We suggest that intratracheal instillation studies can be useful for ranking the hazard of nanoparticles through pulmonary inflammation.

Evaluation of Pulmonary Toxicity of Zinc Oxide Nanoparticles Following Inhalation and Intratracheal Instillation.

We conducted inhalation and intratracheal instillation studies of zinc oxide (ZnO) nanoparticles in order to examine their pulmonary toxicity. F344 rats were received intratracheal instillation at 0.2 or 1 mg of ZnO nanoparticles with a primary diameter of 35 nm that were well-dispersed in distilled water. Cell analysis and chemokines in bronchoalveolar lavage fluid (BALF) were analyzed at three days, one week, one month, three months, and six months after the instillation. As the inhalation study, rats were exposed to a concentration of inhaled ZnO nanoparticles (2 and 10 mg/m³) for four weeks (6 h/day, 5 days/week). The same endpoints as in the intratracheal instillation study were analyzed at three days, one month, and three months after the end of the exposure. In the intratracheal instillation study, both the 0.2 and the 1.0 mg ZnO groups had a transient increase in the total cell and neutrophil count in the BALF and in the expression of cytokine-induced neutrophil chemoattractant (CINC)-1, CINC-2, chemokine for neutrophil, and heme oxygenase-1 (HO-1), an oxidative stress marker, in the BALF. In the inhalation study, transient increases in total cell and neutrophil count, CINC-1,-2 and HO-1 in the BALF were observed in the high concentration groups. Neither of the studies of ZnO nanoparticles showed persistent inflammation in the rat lung, suggesting that well-dispersed ZnO nanoparticles have low toxicity.