Dietary Intervention with Quercetin Attenuates Diesel Exhaust Particle-Instilled Pulmonary Inflammation and Behavioral Abnormalities in Mice.

Exposure to diesel exhaust particles (DEPs) is inevitable and closely linked with increased health hazards, causing pulmonary abnormalities by increasing inflammation, hypoxia, and so on. Moreover, long-term exposure to DEPs may trigger whole-body toxicity with behavioral alterations. Therefore, nutritional intervention with natural components may be desirable to prevent and/or ameliorate DEP-inducible pathophysiology in mammals. Quercetin has been demonstrated to reduce metabolic complications by possessing antioxidative, anti-inflammatory, and antimutagenic effects. In this study, we investigated the effects of quercetin on pulmonary inflammation and behavioral alteration in male C57BL/6 mice against DEP instillation. The experimental mice were separated into four treatment groups (n = 8 per group), which include: vehicle control, DEP instillation, dietary intervention with a low dose of quercetin (20 mg/kg) for 14 days with DEP instillation for 7 days, or dietary intervention with a high dose of quercetin (100 mg/kg) for 14 days with DEP instillation for 7 days. Compared with the DEP-instilled group, dietary intervention with quercetin significantly attenuated eosinophils in the bronchoalveolar lavage fluid analysis, pulmonary cytokine, and hypoxic mRNA expressions regardless of quercetin concentrations. DEP instillation triggered hyperactivities in the experimental mice, while quercetin pretreatment successfully normalized DEP-inducible abnormalities regardless of the dosage. Therefore, dietary intervention with quercetin may be an applicable means to prevent DEP-triggered pulmonary and behavioral abnormalities.

Inhalation toxicity of polyhexamethylene guanidine-phosphate in rats: A 4-week inhalation exposure and 24-week recovery period study.

Humidifier disinfectant (HD) is a causative agent of atypical lung injury reported in 2011 in South Korea, and various diseases caused by HD after exposure cessation have been reported to date. However, there is limited research on most of the reported diseases in terms of their association with HD exposure, and information on the progression of diseases caused by HD exposure is also limited. Therefore, we investigated the effects of HD inhalation on the body in rats. Rats were exposed to 0.15, 0.50, and 1.60 mg/m3 polyhexamethylene guanidine-phosphate (PHMG-p), which is the major component of HDs and most closely related to HD-associated lung injury. We conducted necropsy four times during the recovery period (0, 4, 12, and 24 weeks) and evaluated general systemic toxicities. There were significant changes in the mortality rate, body weight, and food consumption in the PHMG-p-exposed groups. Hematology revealed changes in hemoglobin level, hematocrit, red blood cell, reticulocyte, and white blood cell counts until 12 weeks of the recovery period. PHMG-p induced a delay in prothrombin time until 12 weeks of the recovery period. The aspartate aminotransferase, alanine aminotransferase, total bilirubin, and triglyceride levels were higher in the PHMG-p-exposed groups than in the control group at week 4 of the recovery period, and these parameters normalized after 12 weeks of the recovery period. Histopathological examination in PHMG-p exposed groups revealed several changes in the lungs, including the presence of alveolar macrophages, chronic inflammation, squamous metaplasia, alveolar emphysema, and pulmonary fibrosis. The severity of these symptoms was maintained or exacerbated till 24 weeks. Overall, PHMG-p inhalation can induce irreversible histological changes in the lungs and cause various types of damage throughout the body, even after exposure ends.

Intratracheal exposure to polyhexamethylene guanidine phosphate disrupts coordinate regulation of FXR-SHP-mediated cholesterol and bile acid homeostasis in mouse liver.

A public health crisis in the form of a significant incidence of fatal pulmonary disease caused by repeated use of humidifier disinfectants containing polyhexamethylene guanidine phosphate (PHMG) recently arose in Korea. Although the mechanisms of pulmonary fibrosis following respiratory exposure to PHMG are well described, distant-organ effect has not been reported. In this study, we investigated whether intratracheal administration of PHMG affects liver pathophysiology and metabolism. Our PHMG mouse model showed a significant decrease in liver cholesterol level. An mRNA-seq analysis of liver samples revealed an alteration in the gene expression associated with cholesterol biosynthesis and metabolism to bile acids. The expression of genes involved in cholesterol synthesis was decreased in a real-time PCR analysis. To our surprise, we found that the coordinate regulation of cholesterol and bile acid homeostasis was completely disrupted. Despite the decreased cholesterol synthesis and low bile acid levels, the farnesoid X receptor/small heterodimer partner pathway, which controls negative feedback of bile acid synthesis, was activated in PHMG mice. As a consequence, gene expression of Cyp7a1 and Cyp7b1, the rate-limiting enzymes of the classical and alternative pathways of bile acid synthesis, was significantly downregulated. Notably, the changes in gene expression were corroborated by the hepatic concentrations of the bile acids. These results suggest that respiratory exposure to PHMG could cause cholestatic liver injury by disrupting the physiological regulation of hepatic cholesterol and bile acid homeostasis.

Pre-validation of a Calu-3 epithelium cytotoxicity assay for predicting acute inhalation toxicity of chemicals.

Although in vivo inhalation toxicity tests have been widely conducted, the testing of many chemicals is limited for economic and ethical reasons. Therefore, we previously developed an in vitro acute inhalation toxicity test method. The goal of the present pre-validation study was to evaluate the transferability, reproducibility, and predictive capacity of this method. After confirming the transferability of the Calu-3 epithelium cytotoxicity assay, reproducibility was evaluated using 20 test substances at three independent institutions. Cytotoxicity data were analyzed using statistical methods, including the intra-class correlation coefficient and Bland-Altman plots for within- and between-laboratory reproducibility. The assay for the 20 test substances showed excellent agreement within and between laboratories. To evaluate the predictive capacity, 77 test substances were analyzed for acute inhalation toxicity. Accuracy was measured using a cutoff of 40%, and the relevance was analyzed as a receiver-operating characteristic (ROC) curve. An accuracy of 72.73% was obtained, and the area under the ROC curve was 0.77, indicating moderate performance. In this study, we found that the in vitro acute inhalation toxicity test method demonstrated good reliability and relevance for predicting the acute toxicity of inhalable chemicals. Hence, this assay has potential as an alternative test for screening acutely toxic inhalants.

Formaldehyde exposure induces regulatory T cell-mediated immunosuppression via calcineurin-NFAT signalling pathway.

In this study, we investigated the effects of Formaldehyde (FA) exposure on splenic immune responses wherein helper T cells become activated and differentiate into effector T and regulatory T cells. BALB/c mice were exposed to two FA concentrations (1.38 mg/m3 and 5.36 mg/m3) for 4 h/day and 5 days/week for 2 weeks. FA-induced immune responses were examined by the production of cytokines, expression of mRNAs, and distributions of helper T cells and regulatory T cells. Moreover, expression of calcineurin and NFATs, regulatory T cell-related signalling proteins, were evaluated. FA exposure suppressed Th2-, Th1-, and Th17-related splenic cytokines in a dose-dependent manner. mRNA expression of splenic cytokines was also decreased by FA exposure, which correlated with decreased cytokine expression. In parallel, FA exposure promoted T cell differentiation into regulatory T cells in a dose-dependent manner supported by the expression of calcineurin and NFAT1. Taken together, our results indicated that FA exposure increases the number of regulatory T cells via calcineurin-NFAT signalling, thereby leading to effector T cell activity suppression with decreased T cell-related cytokine secretion and mRNA expression. These findings provide insight into the mechanisms underlying the adverse effects of FA and accordingly have general implications for human health, particularly in occupational settings.

Laminaria japonica Extract Enhances Intestinal Barrier Function by Altering Inflammatory Response and Tight Junction-Related Protein in Lipopolysaccharide-Stimulated Caco-2 Cells.

In the normal physiological state, intestinal epithelial cells act as a defensive frontline of host mucosal immunity to tolerate constant exposure to external stimuli. In this study, we investigated the potential anti-inflammatory and gut permeability protective effects of Laminaria japonica (LJ) water extract (LJE) and three types of fermented Laminaria japonica water extracts (LJE-F1, LJE-F2, and LJE-F3) in lipopolysaccharide (LPS)-stimulated Caco-2, human intestinal epithelial cells. All four extracts significantly decreased the production of nitric oxide and interleukin-6 induced by LPS stimulus. In addition, LJE and the three types of LJE-Fs also inhibited LPS-induced loss of monolayer permeability, as assessed by changes in transepithelial electrical resistance. All four LJ extracts significantly prevented the inhibition of the protein levels of occludin, whereas LJE, LJE-F1, and LJE-F3 significantly attenuated the reduction in phosphorylation of adenosine monophosphate-activated protein kinase compared with the LPS-treated group in Caco-2 cells. In conclusion, LJE and its fermented water extracts appear to have potential gut health-promoting effects by reducing inflammation and partially regulating the tight junction-related proteins in human intestinal epithelial cells. Thus, additional studies are warranted to evaluate Laminaria japonica as a therapeutic agent for inflammatory bowel diseases.

Inhalation of carbon black nanoparticles aggravates pulmonary inflammation in mice.

An increasing number of recent studies have focused on the impact of particulate matter on human health. As a model for atmospheric particulate inhalation, we investigated the effects of inhaled carbon black nanoparticles (CBNP) on mice with bleomycin-induced pulmonary fibrosis. The CNBPs were generated by a novel aerosolization process, and the mice were exposed to the aerosol for 4 hours. We found that CBNP inhalation exacerbated lung inflammation, as evidenced by histopathology analysis and by the expression levels of interleukin-6 protein, fibronectin, and interferon-γ mRNAs in lung tissues. Notably, fibronectin mRNA expression showed a statistically significant increase in expression after CBNP exposure. These data suggest that the concentration of CBNPs delivered (calculated to be 12.5 µg/m(3)) can aggravate lung inflammation in mice. Our results also suggest that the inhalation of ultrafine particles like PM 2.5 is an impactful environmental risk factor for humans, particularly in susceptible populations with predisposing lung conditions.

Polyhexamethyleneguanidine phosphate induces severe lung inflammation, fibrosis, and thymic atrophy.

Polyhexamethyleneguanidine phosphate (PHMG-P) has been widely used as a disinfectant because of its strong bactericidal activity and low toxicity. However, in 2011, the Korea Centers for Disease Control and Prevention and the Ministry of Health and Welfare reported that a suspicious outbreak of pulmonary disease might have originated from humidifier disinfectants. The purpose of this study was to assess the toxicity of PHMG-P following direct exposure to the lung. PHMG-P (0.3, 0.9, or 1.5 mg/kg) was instilled into the lungs of mice. The levels of proinflammatory markers and fibrotic markers were quantified in lung tissues and flow cytometry was used to evaluate T cell distribution in the thymus. Administration of PHMG-P induced proinflammatory cytokines elevation and infiltration of immune cells into the lungs. Histopathological analysis revealed a dose-dependent exacerbation of both inflammation and pulmonary fibrosis on day 14. PHMG-P also decreased the total cell number and the CD4(+)/CD8(+) cell ratio in the thymus, with the histopathological examination indicating severe reduction of cortex and medulla. The mRNA levels of biomarkers associated with T cell development also decreased markedly. These findings suggest that exposure of lung tissue to PHMG-P leads to pulmonary inflammation and fibrosis as well as thymic atrophy.

Nasal and pulmonary toxicity of titanium dioxide nanoparticles in rats.

In recent decades, titanium dioxide (TiO2) nanoparticles have been used in various applications, including paints, coatings, and food. However, data are lacking on the toxicological aspects associated with their use. The aim of this study was to assess the inhalation toxicity of TiO2 nanoparticles in rats by using inhalation exposure. Male Wistar rats were exposed to TiO2 nanoparticles for 2 weeks (6 hr/day, 5 days/week) at a mean mass concentration of 11.39 ± 0.31 mg/m(3). We performed time-course necropsies at 1, 7, and 15 days after exposure. Lung inflammation and injury were assessed on the basis of the total and individual cell counts in bronchoalveolar lavage fluid (BALF), and by biochemical assays, including an assay for lactate dehydrogenase (LDH). Furthermore, histopathological examination was performed to investigate the lungs and nasal cavity of rats. There were no statistically significant changes in the number of BALF cells, results of biochemical assays of BALF and serum, and results of cytokine analysis. However, we did observe histopathological changes in the nasal cavity tissue. Lesions were observed at post-exposure days 1 and 7, which resolved at post-exposure day 15. We also calculated the actual amounts of TiO2 nanoparticles inhaled by the rats. The results showed that the degree of toxicity induced by TiO2 nanoparticles correlated with the delivered quantities. In particular, exposure to small particles with a size of approximately 20 nm resulted in toxicity, even if the total particle number was relatively low.

Standardization of bronchoalveolar lavage method based on suction frequency number and lavage fraction number using rats.

Bronchoalveolar lavage (BAL) is a useful tool in researches and in clinical medicine of lung diseases because the BAL fluid contains biochemical and cytological indicators of the cellular response to infection, drugs, or toxicants. However, the variability among laboratories regarding the technique and the processing of the BAL material limits clinical research. The aim of this study was to determine the suction frequency and lavage fraction number necessary to reduce the variability in lavage using male Sprague-Dawley rats. We compared the total cell number and protein level of each lavage fraction and concluded that more cells and protein can be obtained by repetitive lavage with a suction frequency of 2 or 3 than by lavage with a single suction. On the basis of total cell recovery, approximately 70% of cells were obtained from fractions 1~3. The first lavage fraction should be used for evaluation of protein concentration because fractions 2~5 of lavage fluid were diluted in manifolds. These observations were confirmed in bleomycin-induced inflamed lungs of rats. We further compared the BAL data from the whole lobes with data from the right lobes and concluded that BAL data of the right lobes represented data of the whole lobes. However, this conclusion can only be applied to general lung diseases. At the end, this study provides an insight into the technical or analytical problems of lavage study in vivo.

Dose-response Effects of Bleomycin on Inflammation and Pulmonary Fibrosis in Mice.

Many studies have reported that bleomycin, anti-cancer drug, induces pulmonary fibrosis as a side effect. However, few investigations have focused on the dose-response effects of bleomycin on pulmonary fibrosis. Therefore, in the present study, we investigated the effects of different doses of bleomycin in male mice. ICR mice were given 3 consecutive doses of bleomycin: 1, 2, or 4 mg/kg in bleomycin-treated (BT) groups and saline only in vehicle control (VC) groups. The animals were sacrificed at 7 and 24 days postinstillation. The severity of pulmonary fibrosis was evaluated according to inflammatory cell count and lactate dehydrogenase (LDH) activity in the broncho alveolar lavage fluid (BALF) , and lung tissues were histologically evaluated after hematoxylin and eosin (H&E) , and Masson's trichrome staining. BT groups exhibited changed cellular profiles in BAL fluid compared to the VC group, which had an increased number of total cells, neutrophils, and lymphocytes and a modest increase in the number of macrophages at 7 days post-bleomycin instillation. Moreover, BT groups showed a dose-dependent increase in LDH levels and inflammatory cell counts. However, at 24 days after treatment, collagen deposition, interstitial thickening, and granulomatous lesions were observed in the alveolar spaces in addition to a decrease in inflammatory cells. These results indicate that pulmonary fibrosis induced by 4 mg/kg bleomycin was more severe than that induced by 1 or 2 mg/kg. These data will be utilized in experimental animal models and as basic data to evaluate therapeutic candidates through non-invasive monitoring using the pulmonary fibrosis mouse model established in this study.