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.

IL-6-Mediated Upregulated miRNAs in Extracellular Vesicles Derived from Lund Human Mesencephalic (LUHMES) Cells: Effects on Astrocytes and Microglia.

Psychological stress plays a major role in depression, and interleukin-6 (IL-6) is elevated during depression and psychological stress. MicroRNAs (miRNAs) in extracellular vesicles (EVs), including exosomes and microvesicles, suppress mRNA expression in other cells when endocytosed. In this study, we analyzed the effect of IL-6 on EVs secreted by neural precursor cells. Cells from the human immortalized neural precursor cell line LUHMES were treated with IL-6. EVs were collected using a nanofiltration method. We then analyzed the uptake of LUHMES-derived EVs by astrocytes (ACs) and microglia (MG). Microarray analysis of miRNAs was performed using EV-incorporated RNA and intracellular RNA from ACs and MG to search for increased numbers of miRNAs. We applied the miRNAs to ACs and MG, and examined the cells for suppressed mRNAs. IL-6 increased several miRNAs in the EVs. Three of these miRNAs were originally low in ACs and MG (hsa-miR-135a-3p, hsa-miR-6790-3p, and hsa-miR-11399). In ACs and MG, hsa-miR-6790-3p and hsa-miR-11399 suppressed four mRNAs involved in nerve regeneration (NREP, KCTD12, LLPH, and CTNND1). IL-6 altered the types of miRNAs in EVs derived from neural precursor cells, by which mRNAs involved in nerve regeneration were decreased in ACs and MG. These findings provide new insights into the involvement of IL-6 in stress and depression.

Pulmonary toxicity of tungsten trioxide nanoparticles in an inhalation study and an intratracheal instillation study.

OBJECTIVES: We conducted inhalation and intratracheal instillation studies in order to examine the effects of tungsten trioxide (WO3 ) nanoparticles on the lung, and evaluated whether or not the nanoparticles would cause persistent lung inflammation. METHODS: In the inhalation study, male 10-week-old Fischer 334 rats were classified into 3 groups. The control, low-dose, and high-dose groups inhaled clean air, 2, and 10 mg/m3 WO3 nanoparticles, respectively, for 6 h each day for 4 weeks. The rats were dissected at 3 days, 1 month, and 3 months after the inhalation, and the bronchoalveolar lavage fluid (BALF) and lung tissue were examined. In the intratracheal instillation study, male 12-week-old Fischer 334 rats were divided into 3 subgroups. The control, low-dose, and high-dose groups were intratracheally instilled 0.4 ml distilled water, 0.2, and 1.0 mg WO3 nanoparticles, respectively, dissolved in 0.4 ml distilled water. The rats were sacrificed at 3 days, 1 week, and 1 month after the intratracheal instillation, and the BALF and lung tissue were analyzed as in the inhalation study. RESULTS: The inhalation and instillation of WO3 nanoparticles caused transient increases in the number and rate of neutrophils, cytokine-induced neutrophil chemoattractant (CINC)-1, and CINC-2 in BALF, but no histopathological changes or upregulation of heme oxygenase (HO)-1 in the lung tissue. CONCLUSION: Our results suggest that WO3 nanoparticles have low toxicity to the lung. According to the results of the inhalation study, we also propose that the no observed adverse effect level (NOAEL) of WO3 nanoparticles is 2 mg/m3 .

Binding assay of human Dectin-1 variants to DNA/ß-glucan complex for active-targeting delivery of antisense DNA.

The lectin Dectin-1 is a good target for ß-glucan-mediated drug delivery. Although many murine studies of Dectin-1 have been performed, its human analog has not been studied well in terms of being a drug delivery target. We thus analyzed human Dectin-1 cDNA obtained from chronic myelogenous leukemia-derived cells, CML-1, and confirmed the findings of previous studies that there are many isoforms of human Dectin-1 due to exon skipping, although murine Dectin-1 has only two forms. When we transfected the Dectin-1 gene into a non-Dectin-1-expressing cell line and examined cellular uptake of the antisense DNA/ß-glucan complex, we confirmed that expression of the target gene was effectively suppressed through ß-glucan/Dectin-1-mediated uptake. The present results suggest that the ß-glucan complex would be an effective tool to deliver antisense oligonucleotide (AS-ODN) to Dectin-1-expressing cells not only for mice but also for humans.

Selection of microRNAs in extracellular vesicles for diagnosis of malignant pleural mesothelioma by in vitro analysis.

Malignant pleural mesothelioma (MPM) is a malignant tumor which is a challenge for diagnosis and is associated with a poor patient prognosis. Thus, early diagnostic interventions will improve the quality of life and life expectancy of these patients. Recently, cellular microRNAs (miRNAs) have been found to be involved in maintaining homeostasis, and abnormal miRNA expression has often been observed in various diseases including cancer. Extracellular vesicles (EVs) released by many cells contain proteins and nucleic acids. miRNAs are secreted from all cells via EVs and circulate throughout the body. In this study, culture media were passed sequentially through membrane filters 220­50 nm in size, and EVs with diameters of 50 to 220 nm (EVcap50/220) were collected. miRNAs (EV50­miRNAs) in EVcap50/220 were purified, and microarray analysis was performed. EV50­miRNA expression profiles were compared between MPM cells and a normal pleural mesothelial cell line, and six EV50­miRNAs were selected for further investigation. Of these, hsa­miR­193a­5p and hsa­miR­551b­5p demonstrated higher expression in MPM­derived EVcap50/220. These miRNAs reduced the expression of several genes involved in cell­cell interactions and cell­matrix interactions in normal pleural mesothelial cells. Our data suggest that hsa­miR­193a­5p and hsa­miR­551b­5p in EVcap50/220 could be diagnostic markers for MPM.

Changes over time in pulmonary inflammatory response in rat lungs after intratracheal instillation of nickel oxide nanoparticles.

OBJECTIVE: Nickel oxide nanoparticles (NiONPs) are representative metal oxide NPs and are categorized as an insoluble nickel compound. Our previous studies suggested that NiONPs have more pulmonary toxicity than micron-sized NiO because they may dissolve slowly and produce many more Ni ions. We confirmed the hypothesis that the slow dissolution of NiONPs induces a change in inflammatory response over time. METHOD: We reanalyzed our previous data on intratracheally instilled NiONP to rats and focused on Ni retention in the lungs and the lung weight ratio for each rat to the mean of control rat lungs. We also measured the solubility of NiONPs and micron-sized NiO samples by means of an artificial lysosomal fluid (ALF, pH 4.5). RESULTS: The in vivo test of instilled NiONPs resulted in the biomarkers reaching their peak values at 1 week or 1 month, and not at 3 days, after instillation. We found that as the NiO mass in the lung increased, the lung weight ratios tended to increase. The relationships shifted to more toxic at 3 days to 1 month (P < .01). Compared to the dissolution of NiONPs in the ALF that took roughly 1 week, the dissolution of NiONPs in vivo was take about 1 month or more. CONCLUSION: When intratracheally instilled NiONPs dissolve slowly in the phagolysosomes of alveolar macrophages (AM), the resulting Ni ions cause the AM to transform into foamy cells at 1 month, and the inflammatory response persists even at 3 months after instillation.

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.

Long-term observation of pulmonary toxicity of toner with external additives following a single intratracheal instillation in rats.

OBJECTIVES: Along with technological innovations for improving the efficiency of printing, nanoparticles have been added to the surface of toners, and there is concern about the harmful effects of those components. We investigated, through a long-term observation following intratracheal instillation using rats, whether exposure to a toner with external additives can cause tumorigenesis. METHODS: Female Wistar rats were intratracheally instilled with dispersed toner at low (1 mg/rat) and high (2 mg/rat) doses, and the rats were sacrificed at 24 months after exposure, after which we examined pulmonary inflammation, histopathological changes, and DNA damage in the lung. Rats that had deceased before 24 months were dissected at that time as well, to compare tumor development. RESULTS: Although alveolar macrophages with pigment deposition in the alveoli were observed in the 1 and 2 mg exposure groups, no significant lung inflammation/fibrosis or tumor was observed. Since immunostaining with 8-OHdG or γ-H2AX did not show a remarkable positive reaction, it is thought that toner did not cause severe DNA damage to lung tissue. CONCLUSION: These results suggest that toner with external additives may have low toxicity in the lung.

UBE2L6 is Involved in Cisplatin Resistance by Regulating the Transcription of ABCB6.

BACKGROUND: Cisplatin is an important anticancer agent in cancer chemotherapy, but when resistant cells appear, treatment becomes difficult, and the prognosis is poor. OBJECTIVE: In this study, we investigated the gene expression profile in cisplatin sensitive and resistant cells, and identified the genes involved in cisplatin resistance. METHODS: Comparison of gene expression profiles revealed that UBE2L6 mRNA is highly expressed in resistant cells. To elucidate whether UBE2L6 is involved in the acquisition of cisplatin resistance, UBE2L6- overexpressing cells established from cisplatin-sensitive cells and UBE2L6-silenced cells developed from cisplatin- resistant cells were generated, and the sensitivity of cisplatin was examined. RESULTS: The sensitivity of the UBE2L6-overexpressing cells did not change compared with the control cells, but the UBE2L6-silenced cells were sensitized to cisplatin. To elucidate the mechanism of UBE2L6 in cisplatin resistance, we compared the gene expression profiles of UBE2L6-silenced cells and control cells and found that the level of ABCB6 mRNA involved in cisplatin resistance was decreased. Moreover, ABCB6 promoter activity was partially suppressed in UBE2L6-silenced cells. CONCLUSION: These results suggest that cisplatin-resistant cells have upregulated UBE2L6 expression and contribute to cisplatin resistance by regulating ABCB6 expression at the transcriptional level. UBE2L6 might be a molecular target that overcomes cisplatin resistance.

Update of occupational lung disease.

OBJECTIVE: Occupational Lung Disease is an oldest but still a biggest problem in occupational health. METHODS: Steering Committee members of the Japan Society for Occupational Health (JSOH) Occupational Lung Disease Study Group selected and summarized current topics on occupational lung diseases based on expert opinion, as informed by governmental regulation, public health concerns, and frequently discussed in related academic conferences. RESULTS: The topics included in this review are professional education in medical screening skills, 2014 update of Helsinki Criteria, respiratory diseases found in the earthquake and tsunami affected regions, newly recognized occupational lung diseases, and potential respiratory health hazards. DISCUSSIONS: Although occupational lung diseases seem to stay as one of the major concerns in occupational health, screening tools and control measures are standardized for the better prevention of the diseases. As this health problem usually occurs in where the most actively economically developing area is, the patients tend to increase in emerging economic powers with huge population.

Complex consisting of antisense DNA and ß-glucan promotes internalization into cell through Dectin-1 and hybridizes with target mRNA in cytosol.

Antisense oligonucleotides (AS-ODNs) hybridize with specific mRNAs, resulting in interference with the splicing mechanism or the regulation of protein translation. We previously demonstrated that the ß-glucan schizophyllan (SPG) can form a complex with AS-ODNs with attached dA40 (AS-ODNs/SPG), and this complex can be incorporated into cells, such as macrophages and dendritic cells, expressing the ß-glucan receptor Dectin-1. We have achieved efficient gene silencing in animal models, but the uptake mechanism and intracellular distribution are unclear. In this study, we prepared the complex consisting of SPG and AS-ODNs (AS014) for Y-box binding protein-1 (YB-1). After treatment with endocytosis inhibitor Pitstop 2 and small interfering RNA targeting Dectin-1, we found that AS014/SPG complexes are incorporated into cells by Dectin-1-mediated endocytosis and inhibit cell growth in a Dectin-1 expression level-dependent manner. After treatment with AS014/SPG complexes, we separated the cell lysate into endosomal and cytoplasmic components by ultracentrifugation and directly determined the distribution of AS014 by reverse transcription PCR using AS014 ODNs as a template or a reverse transcription primer. In the cytoplasm, AS014 clearly hybridized with YB-1 mRNAs. This is the first demonstration of the distinct distribution of the complex in cells. These results could facilitate the clinical application of the complex.

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.

Review of toxicity studies of carbon nanotubes.

OBJECTIVE: We reviewed studies on pulmonary, reproductive, and developmental toxicity caused by carbon nanotubes (CNTs). In paricular, we analyzed how CNT exposure affects the several processes of pulmonary toxicity, including inflammation, injury, fibrosis, and pulmonary tumors. METHODS: In pulmonary toxicity, there are various processes, including inflammation, injury, fibrosis, respiratory tumor in the lungs, and biopersistence of CNTs and genotoxicity as tumor-related factors, to develop the respiratory tumor. We evaluated the evidence for the carcinogenicity of CNTs in each process. In the fields of reproductive and developmental toxicity, studies of CNTs have been conducted mainly with mice. We summarized the findings of reproductive and developmental toxicity studies of CNTs. RESULTS: In animal studies, exposure to CNTs induced sustained inflammation, fibrosis, lung cancer following long-term inhalation, and gene damage in the lung. CNTs also showed high biopersistence in animal studies. Fetal malformations after intravenous and intraperitoneal injections and intratracheal instillation, fetal loss after intravenous injection, behavioral changes in offsprings after intraperitoneal injection, and a delay in the delivery of the first litter after intratracheal instillation were reported in mice-administered multi-walled carbon nanotubes (MWCNTs). Single-walled carbon nanotubes (SWCNTs) appeared to be embryolethal and teratogenic in mice when given by intravenous injection; moreover, the tubes induced death and growth retardation in chicken embryos. CONCLUSION: CNTs are considered to have carcinogenicity and can cause lung tumors. However, the carcinogenicity of CNTs may attenuate if the fiber length is shorter. The available data provide initial information on the potential reproductive and developmental toxicity of CNTs.

GalNAc-T6 in the relationship with invasion ability of endometrial carcinomas and prognostic significance.

O-glycosylation in the field of carcinogenesis has been a critical topic of concern for several decades. The abnormal function of enzymes catalyzing the first step of this process, named polypeptide N-acetylgalactosaminyltransferases (ppGalNAc-Ts) has been determined to play an important role in cancer development and metastasis. Accordingly, we investigated the expression of GalNAc-T6 in endometrial carcinoma and evaluated the relationship between invasion characteristics and the cellular level of GalNAc-T6. The results suggested that positive GalNAc-T6 expression is significantly associated with histological grade of tumors and myometrial invasion characteristic. In vitro experiments showed that the over expression of GalNAc-T6 had strong association with the decrease of endometrial cell invasiveness. Taken together, our data support the use of GalNAc-T6 as a potential indicator of good prognosis and noninvasive tumor in patients with endometrial carcinoma.

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.

Assessment of Pulmonary Toxicity Induced by Inhaled Toner with External Additives.

We investigated the harmful effects of exposure to a toner with external additives by a long-term inhalation study using rats, examining pulmonary inflammation, oxidative stress, and histopathological changes in the lung. Wistar rats were exposed to a well-dispersed toner (mean of MMAD: 2.1 µm) at three mass concentrations of 1, 4, and 16 mg/m3 for 22.5 months, and the rats were sacrificed after 6 months, 12 months, and 22.5 months of exposure. The low and medium concentrations did not induce statistically significant pulmonary inflammation, but the high concentration did, and, in addition, a histopathological examination showed fibrosis in the lung. Although lung tumor was observed in one sample of high exposure for 22.5 months, the cause was not statistically significant. On the other hand, a persistent increase in 8-OHdG was observed in the high exposure group, indicating that DNA damage by oxidative stress with persistent inflammation leads to the formation of tumorigenesis. The results of our studies show that toners with external additives lead to pulmonary inflammation, oxidative stress, and fibrosis only at lung burdens beyond overload. These data suggest that toners with external additives may have low toxicity in the lung.