Contribution of toxicological pathology to occupational health: lung carcinogenicity of fibrous and particulate substances in rats.

In this review, we focus on the rat pulmonary carcinogenicity of two solid substances, fibrous multi-walled carbon nanotube (MWCNT) and particulate indium tin oxide (ITO). Inhalation exposure to MWNT-7, a type of MWCNTs, and ITO induced lung carcinogenicity in both male and female rats. Toxicity to the alveolar epithelium is induced by macrophages undergoing frustrated phagocytosis or frustrated degradation of engulfed particles (referred to as frustrated macrophages). Melted macrophage contents contribute significantly to development of hyperplasia of the alveolar epithelium, which eventually results in the induction of lung carcinoma. MWNT-7 and ITO induce secondary genotoxicity; consequently, a no-observed-adverse-effect level can be applied to these materials rather than benchmark doses that are used for non-threshold carcinogens. Thus, establishing occupational exposure limit values for MWNT-7 and ITO based on the existence of a carcinogenic threshold is reasonable.

Mechanisms of pulmonary disease in F344 rats after workplace-relevant inhalation exposure to cross-linked water-soluble acrylic acid polymers.

BACKGROUND: Recently in Japan, six workers at a chemical plant that manufactures resins developed interstitial lung diseases after being involved in loading and packing cross-linked water-soluble acrylic acid polymers (CWAAPs). The present study focused on assessing lung damage in rats caused by workplace-relevant inhalation exposure to CWAAP and investigated the molecular and cellular mechanisms involved in lung lesion development. METHODS: Using a whole-body inhalation exposure system, male F344 rats were exposed once to 40 or 100 mg/m3 of CWAAP-A for 4 h or to 15 or 40 mg/m3 of CWAAP-A for 4 h per day once per week for 2 months (9 exposures). In a separate set of experiments, male F344 rats were administered 1 mg/kg CWAAP-A or CWAAP-B by intratracheal instillation once every 2 weeks for 2 months (5 doses). Lung tissues, mediastinal lymph nodes, and bronchoalveolar lavage fluid were collected and subjected to biological and histopathological analyses. RESULTS: A single 4-h exposure to CWAAP-A caused alveolar injury, and repeated exposures resulted in regenerative changes in the alveolar epithelium with activation of TGFß signaling. During the recovery period after the last exposure, some alveolar lesions were partially healed, but other lesions developed into alveolitis with fibrous thickening of the alveolar septum. Rats administered CWAAP-A by intratracheal instillation developed qualitatively similar pulmonary pathology as rats exposed to CWAAP-A by inhalation. At 2 weeks after intratracheal instillation, rats administered CWAAP-B appeared to have a slightly higher degree of lung lesions compared to rats administered CWAAP-A, however, there was no difference in pulmonary lesions in the CWAAP-A and CWAAP-B exposed rats examined 18 weeks after administration of these materials. CONCLUSIONS: The present study reports our findings on the cellular and molecular mechanisms of pulmonary disease in rats after workplace-relevant inhalation exposure to CWAAP-A. This study also demonstrates that the lung pathogenesis of rats exposed to CWAAP-A by systemic inhalation was qualitatively similar to that of rats administered CWAAP-A by intratracheal instillation.

Pulmonary dust foci as rat pneumoconiosis lesion induced by titanium dioxide nanoparticles in 13-week inhalation study.

BACKGROUND: Most toxicological studies on titanium dioxide (TiO2) particles to date have concentrated on carcinogenicity and acute toxicity, with few studies focusing of pneumoconiosis, which is a variety of airspace and interstitial lung diseases caused by particle-laden macrophages. The present study examined rat pulmonary lesions associated with pneumoconiosis after inhalation exposure to TiO2 nanoparticles (NPs). METHODS: Male and female F344 rats were exposed to 6.3, 12.5, 25, or 50 mg/m3 anatase type TiO2 NPs for 6 h/day, 5 days/week for 13 weeks using a whole-body inhalation exposure system. After the last exposure the rats were euthanized and blood, bronchoalveolar lavage fluid, and all tissues including lungs and mediastinal lymph nodes were collected and subjected to biological and histopathological analyses. RESULTS: Numerous milky white spots were present in the lungs after exposure to 25 and 50 mg/m3 TiO2 NPs. Histopathological analysis revealed that the spots were alveolar lesions, characterized predominantly by the agglomeration of particle-laden macrophages and the presence of reactive alveolar epithelial type 2 cell (AEC2) hyperplasia. We defined this characteristic lesion as pulmonary dust foci (PDF). The PDF is an inflammatory niche, with decreased vascular endothelial cells in the interstitium, and proliferating AEC2 transformed into alveolar epithelial progenitor cells. In the present study, the AEC2 in the PDF had acquired DNA damage. Based on PDF induction, the lowest observed adverse effect concentration for pulmonary disorders in male and female rats was 12.5 mg/m3 and 6.3 mg/m3, respectively. The no observed adverse effect concentration for male rats was 6.3 mg/m3. There was a sex difference in lung lesion development, with females showing more pronounced lesion parameters than males. CONCLUSIONS: Inhalation exposure to TiO2 NPs caused PDF, an air-space lesion which is an alveolar inflammatory niche containing particle-laden macrophages and proliferating AEC2. These PDFs histopathologically resemble some pneumoconiosis lesions (pulmonary siderosis and hard metal pneumoconiosis) in workers and lung disease in smokers, suggesting that PDFs caused by exposure to TiO2 NPs in rats are an early pneumoconiosis lesion and may be a common alveolar reaction in mammals.

No evidence for carcinogenicity of titanium dioxide nanoparticles in 26-week inhalation study in rasH2 mouse model.

With the rapid development of alternative methods based on the spirit of animal welfare, the publications of animal studies evaluating endpoints such as cancer have been extremely reduced. We performed a 26-week inhalation exposure studies of titanium dioxide nanoparticles (TiO2 NPs) using CByB6F1-Tg(HRAS)2Jic (rasH2) mice model for detecting carcinogenicity. Male and female rasH2 mice were exposed to 2, 8 or 32 mg/m3 of TiO2 NPs for 6 h/day, 5 days/week for 26 weeks. All tissues and blood were collected and subjected to biological and histopathological analyses. TiO2 NPs exposure induced deposition of particles in lungs in a dose-dependent manner in each exposure group. Exposure to TiO2 NPs, as well as other organs, did not increase the incidence of lung tumors in any group, and pulmonary fibrosis and pre-neoplastic lesions were not observed in all groups. Finally, the cell proliferative activity of alveolar epithelial type 2 cells was examined, and it was not increased by exposure to TiO2 NPs. This is the first report showing the lack of pulmonary fibrogenicity and carcinogenicity (no evidence of carcinogenic activity) of TiO2 NPs in 26-week inhalation study in rasH2 mice exposed up to 32 mg/m3, which is considered to be a high concentration.

Dose-response relationship of pulmonary disorders by inhalation exposure to cross-linked water-soluble acrylic acid polymers in F344 rats.

BACKGROUND: In Japan, six workers handling cross-linked water-soluble acrylic acid polymer (CWAAP) at a chemical plant suffered from lung diseases, including fibrosis, interstitial pneumonia, emphysema, and pneumothorax. We recently demonstrated that inhalation of CWAAP-A, one type of CWAAP, causes pulmonary disorders in rats. It is important to investigate dose-response relationships and recoverability from exposure to CWAAPs for establishing occupational health guidelines, such as setting threshold limit value for CWAAPs in the workplace. METHODS: Male and female F344 rats were exposed to 0.3, 1, 3, or 10 mg/m3 CWAAP-A for 6 h/day, 5 days/week for 13 weeks using a whole-body inhalation exposure system. At 1 h, 4 weeks, and 13 weeks after the last exposure the rats were euthanized and blood, bronchoalveolar lavage fluid, and all tissues including lungs and mediastinal lymph nodes were collected and subjected to biological and histopathological analyses. In a second experiment, male rats were pre-treated with clodronate liposome or polymorphonuclear leukocyte-neutralizing antibody to deplete macrophages or neutrophils, respectively, and exposed to CWAAP-A for 6 h/day for 2 days. RESULTS: CWAAP-A exposure damaged only the alveoli. The lowest observed adverse effect concentration (LOAEC) was 1 mg/m3 and the no observed adverse effect concentration (NOAEC) was 0.3 mg/m3. Rats of both sexes were able to recover from the tissue damage caused by 13 weeks exposure to 1 mg/m3 CWAAP-A. In contrast, tissue damage caused by exposure to 3 and 10 mg/m3 was irreversible due to the development of interstitial lung lesions. There was a gender difference in the recovery from CWAAP-A induced pulmonary disorders, with females recovering less than males. Finally, acute lung effects caused by CWAAP-A were significantly reduced by depletion of alveolar macrophages. CONCLUSIONS: Pulmonary damage caused by inhalation exposure to CWAAP-A was dose-dependent, specific to the lung and lymph nodes, and acute lung damage was ameliorated by depleting macrophages in the lungs. CWAAP-A had both a LOAEC and a NOAEC, and tissue damage caused by exposure to 1 mg/m3 CWAAP-A was reversible: recovery in female rats was less than for males. These findings indicate that concentration limits for CWAAPs in the workplace can be determined.

MicroRNA induction by copy number gain is associated with poor outcome in squamous cell carcinoma of the lung.

Copy number gains in cancer genomes have been shown to induce oncogene expression and promote carcinogenesis; however, their role in regulating oncogenic microRNAs (onco-miRNAs) remains largely unknown. Our aim was to identify onco-miRNAs induced by copy number gains in human squamous cell carcinoma (Sq) of the lung. We performed a genome-wide screen of onco-miRNAs from 245 Sqs using data sets from RNA-sequencing, comparative genomic hybridization, and the corresponding clinical information from The Cancer Genome Atlas. Among 1001 miRNAs expressed in the samples, 231 were correlated with copy number alternations, with only 11 of these being highly expressed in Sq compared to adenocarcinoma and normal tissues. Notably, miR-296-5p, miR-324-3p, and miR-3928-3p expression was significantly associated with poor prognosis. Multivariate analysis using the Cox proportional hazards model showed that miRNA expression and smoking were independent prognostic factors and were associated with poor prognosis. Furthermore, the three onco-miRNAs inhibited FAM46C to induce MYC expression, promoting proliferation of Sq cells. We found that copy number gains in Sq of the lung induce onco-miRNA expression that is associated with poor prognosis.

Quantitative Analysis of the Accumulation of Marine-derived Tocopherol in the Tissue of Mice Fed with Salmon Roe Oil Using HPLC-fluorescence.

In this study, we measured the quantity of marine-derived tocopherol (MDT), a monounsaturated vitamin E (VE), stored in the body tissue of mice fed with a diet containing a VE-rich fraction extracted from salmon roe. We first prepared the calibration curves for the MDT concentration using an HPLC-fluorescence system. Ranging from 0.016 to 50 µg/mL, the slope was expressed as first-order equations, with R2 values = 0.99. The mice were fed with an AIN-93 based diet containing MDT in doses of 21.4 mg/kg for 4 weeks, and the storage in the heart, lung, liver, stomach, small intestine, large intestine, kidney, pancreas, spleen, testis, skeletal muscle, visceral white adipose tissue (WAT), subcutaneous WAT and brain was quantified. MDT was widely distributed in tissues throughout the whole body, with higher accumulations observed in the adipose tissue, liver and kidney. These results demonstrate means to estimating the MDT concentration in natural products and in the bodies of animals and contribute to the understanding of the physiological functions of MDT in relation to human health.

Comparison of single or multiple intratracheal administration for pulmonary toxic responses of nickel oxide nanoparticles in rats.

OBJECTIVES: In this study, we focused on the qualitative and quantitative differences of the lung lesions induced by single or multiple intratracheal administration of nickel oxide nanoparticles (NiO). METHODS: Male rats were randomized into groups receiving intratracheal administrations in a single dose or two to four divided doses of 2 mg/kg/bw. Bronchoalveolar lavage fluid (BALF) analyses were performed at 3 and 28 d post-dose. Histopathological analyses were performed at 28 and 91 d post-dose. RESULTS: BALF analyses revealed pulmonary injury, inflammation, and increases in the parameters indicating processing the foreign material in all the NiO-treated groups. Histopathological analyses showed the phagocytosis of NiO by alveolar macrophages, degeneration and necrosis of alveolar macrophages, and inflammatory responses. In the comparison between single and multiple administrations, the trend for stronger toxicity effects was observed after multiple application at 3 d post-dose, while the obvious toxicity effects were also seen in case of single administration. No particular differences of lung lesions depending on the frequency of administration at 28 and 91 d post-dose were evident. CONCLUSION: Intratracheal NiO administration induced strong toxic response thoroughly even by single administration. Therefore, single administration was concluded to be applicable to assess the inhalation toxicity of nanomaterials and can be used in the screening test.

Lung carcinogenicity of inhaled multi-walled carbon nanotube in rats.

BACKGROUND: Multi-walled carbon nanotubes (MWCNTs) constitute one of the most promising types of nanomaterials in industry today. With their increasing use, the potential toxicity and carcinogenicity of MWCNT needs to be evaluated in bioassay studies using rodents. Since humans are mainly exposed to MWCNT by inhalation, we performed a 104-week carcinogenicity study using whole-body inhalation exposure chambers with a fibrous straight type of MWCNT at concentrations of 0, 0.02, 0.2, and 2 mg/m3 using male and female F344 rats. RESULTS: Lung carcinomas, mainly bronchiolo-alveolar carcinoma, and combined carcinomas and adenomas were significantly increased in males exposed to 0.2 and 2 mg/m3 MWNT-7 and in females exposed to 2 mg/m3 MWNT-7 compared to the clean air control group. However, no development of pleural mesothelioma was observed. Concentration-dependent toxic effects in the lung such as epithelial hyperplasia, granulomatous change, localized fibrosis, and alteration in BALF parameters were found in MWNT-7 treatment groups of both sexes. There were no MWNT-7-specific macroscopic findings in the other organs, including the pleura and peritoneum. Absolute and relative lung weights were significantly elevated in male rats exposed to 0.2 and 2 mg/m3 MWNT-7 and in all exposed female groups. The lung burdens of MWNT-7 were clearly increased in a concentration-dependent as well as a duration-dependent manner. CONCLUSION: There is clear evidence that MWNT-7 is carcinogenic to the lungs of male and female F344 rats, however no plural mesothelioma was observed.

Thirteen-week study of toxicity of fiber-like multi-walled carbon nanotubes with whole-body inhalation exposure in rats.

Cancer development due to fiber-like straight type of multi-walled carbon nanotubes (MWCNTs) has raised concerns for human safety because of its shape similar to asbestos. To set concentrations of MWCNT for a rat carcinogenicity study, we conducted a 13-week whole body inhalation study. F344 male and female rats, 6-week-old at the commencement of the study, were exposed by whole-body inhalation to MWCNT at concentrations of 0, 0.2, 1 and 5 mg/m(3) with a generation and exposure system utilizing the cyclone sieve method. Measured concentrations in the exposure chambers were 0.20 ± 0.02, 1.01 ± 0.11 and 5.02 ± 0.25 mg/m(3) for 13 weeks. The MMAD (GSD) of MWCNT were 1.4-1.6 µm (2.3-3.0), and mean width and length were 94.1-98.0 nm and 5.53-6.19 µm, respectively, for each target concentration. Lung weights were increased 1.2-fold with 1 mg/m(3) and 1.3-fold with 5 mg/m(3) in both sexes compared to the controls. In the bronchoalveolar lavage fluid (BALF) analyses, inflammatory parameters were increased concentration-dependently in both sexes from 0.2 mg/m(3). Granulomatous changes in the lung were induced at 1 and 5 mg/m(3) in females and even at 0.2 mg/m(3) in males. Focal fibrosis of the alveolar wall was observed in both sexes at 1 mg/m(3) or higher. Inflammatory infiltration in the visceral pleural and subpleural areas was induced only at 5 mg/m(3). In conclusion, we determined 0.2 mg/m(3) as the low-observed-adverse-effect level (LOAEL) for respiratory tract toxicity in the present inhalation exposure study of rats.

Inhalation exposure to 1,2-dichloropropane: Distribution of blood and tissue concentrations of 1,2-dichloropropane in rats during and after exposure.

The present investigation was undertaken to determine the distribution and accumulation of 1,2-dichloropropane (DCP) in the blood, lung, liver, kidney, and abdominal fat of rats during and after inhalation exposure. Male rats were exposed to 80 or 500 ppm (v/v) DCP vapor for 360 min and the concentrations of DCP in the blood and tissues during the inhalation exposure period and after the end of the exposure period were measured. DCP accumulation in the abdominal fat was much greater than that in the blood and other tissues. Eighteen hours after the end of inhalation exposure, DCP could still be detected in the abdominal fat in the 80-ppm group, and in the blood, liver, kidney, and abdominal fat in the 500-ppm group. Our results are valuable data pertaining to the pharmacokinetics of DCP and to human health risk assessment of exposure to DCP vapor by inhalation.

Two-week Toxicity of Multi-walled Carbon Nanotubes by Whole-body Inhalation Exposure in Rats.

To evaluate pulmonary toxicity of multi-walled carbon nanotubes (MWCNTs), F344 rats of both sexes were exposed by inhalation to 0.2, 1 or 5 mg/m(3) MWCNT aerosol for 6 h/day, 5 days/week for 2 weeks using a whole-body exposure system. At the end of the 2-week exposure period, one-half of the rats were necropsied, and at the end of an additional 4-week postexposure period, the remaining rats were necropsied. MWCNTs were deposited in the lungs of all MWCNT-exposed groups and mostly remained in the lungs throughout the 4-week postexposure period. Granulomatous changes in the lung were found in the rats exposed to 5 mg/m(3) MWCNTs, and these changes were slightly aggravated at the end of the 4-week postexposure period. In the bronchoalveolar lavage fluid (BALF), the numbers of neutrophils, percentages of bi- and multinucleated alveolar macrophages, levels of ALP activity and concentrations of total protein and albumin were elevated in the rats exposed to 1 and 5 mg/m(3) MWCNTs. At the end of the 4-week postexposure period, the values of the BALF parameters tended to remain elevated. In addition, goblet cell hyperplasias in the nasal cavity and nasopharynx were observed in the rats exposed to 1 and 5 mg/m(3) MWCNTs, but these lesions had largely regressed by the end of the postexposure period. Based on the histopathological and inflammatory changes, the no-observed-adverse-effect level (NOAEL) for inhalation of MWCNTs for 2 weeks was 0.2 mg/m(3).

Failure to confirm an association between the PLXNA2 gene and schizophrenia in a Japanese population.

Plexins are receptors for multiple classes of semaphorins, either alone or in combination with neuropilins. Plexins participate in many cellular events that include axonal repulsion, axonal attraction, cell migration, axon pruning, and synaptic plasticity. PLXNA2 maps to chromosome 1q32. Several linkage studies reported schizophrenia susceptibility loci in the 1q22-42 region. A recent study reported that intronic single nucleotide polymorphisms (SNPs) of PLXNA2 were associated with schizophrenia in a European American population. We attempted to replicate this finding in a Japanese sample of 336 patients with schizophrenia and 304 controls. In addition, we examined 3 non-synonymous SNPs (Arg5Gln, GLn57Arg, and Ala267Thr) in PLXNA2. Genotyping was performed by the TaqMan allelic discrimination assay. There was no significant difference in genotype or allele distribution of either the 4 intronic SNPs or the 3 non-synonymous SNPs between patients and controls. Furthermore, haplotype-based analyses did not provide evidence for an association. These results suggest that PLXNA2 may not play a major role in the development of schizophrenia in our Japanese sample.

Association study of the vesicular monoamine transporter 1 (VMAT1) gene with schizophrenia in a Japanese population.

BACKGROUND: Vesicular monoamine transporters (VMATs) mediate accumulation of monoamines such as serotonin, dopamine, adrenaline, and noradrenaline from the cytoplasm into storage organelles. The VMAT1 (alternatively solute carrier family 18: SLC18A1) regulates such biogenic amines in neuroendocrine systems. The VMAT1 gene maps to chromosome 8p21.3, a locus with strong evidence of linkage with schizophrenia. A recent study reported that a non-synonymous single nucleotide polymorphism (SNP) of the gene (Pro4Thr) was associated with schizophrenia. METHODS: We attempted to replicate this finding in a Japanese sample of 354 schizophrenics and 365 controls. In addition, we examined 3 other non-synonymous SNPs (Thr98Ser, Thr136Ile, and Val392Leu). Genotyping was performed by the TaqMan allelic discrimination assay. RESULTS: There was no significant difference in genotype or allele distribution of the three SNPs of Pro4Thr, Thr136Ile, or Val392Leu between patients and controls. There was, however, a significant difference in genotype and allele distributions for the Thr98Ser polymorphism between the two groups (P = 0.01 for genotype and allele). When sexes were examined separately, significant differences were observed in females (P = 0.006 for genotype, P = 0.003 for allele), but not in males. The Thr98 allele was more common in female patients than in female controls (odds ratio 1.69, 95% CI 1.19-2.40, P = 0.003). Haplotype-based analyses also provided evidence for a significant association in females. CONCLUSION: We failed to replicate the previously reported association of Pro4Thr of the VMAT1 gene with schizophrenia. However, we obtained evidence for a possible role of the Thr98Ser in giving susceptibility to schizophrenia in women.