Non-contact microfluidic analysis of the stiffness of single large extracellular vesicles from IDH1-mutated glioblastoma cells.

In preparation for leveraging extracellular vesicles (EVs) for disease diagnostics and therapeutics, fundamental research is being done to understand EV biological, chemical, and physical properties. Most published studies have investigated nanoscale EVs and focused on EV biochemical content. There is much less understanding of large microscale EV characteristics and EV mechanical properties. We recently introduced a non-contact microfluidic technique that measures the stiffness of large EVs (>1 µm diameter). This pilot study probes the robustness of the microfluidic technique to distinguish between EV populations by comparing stiffness distributions of large EVs derived from glioblastoma cell lines. EVs derived from cells expressing the IDH1 mutation, a common glioblastoma mutation known to disrupt lipid metabolism, were stiffer than those expressed from wild-type cells in a statistical comparison of sample medians. A supporting lipidomics analysis showed that the IDH1 mutation increased the amount of saturated lipids in EVs. Taken together, these data encourage further investigation into the potential of high-throughput microfluidics to distinguish between large EV populations that differ in biomolecular composition. These findings contribute to the understanding of EV biomechanics, in particular for the less studied microscale EVs.

Electrokinetically enhanced label-free plasmonic sensing for rapid detection of tumor-derived extracellular vesicles.

ANALYSIS: of rare circulating extracellular vesicles (EV) from early cancers or different types of host cells requires extremely sensitive EV sensing technologies. Nanoplasmonic EV sensing technologies have demonstrated good analytical performances, but their sensitivity is often limited by EVs' diffusion to the active sensor surface for specific target EV capture. Here, we developed an advanced plasmonic EV platform with electrokinetically enhanced yields (KeyPLEX). The KeyPLEX system effectively overcomes diffusion-limited reactions with applied electroosmosis and dielectrophoresis forces. These forces bring EVs toward the sensor surface and concentrate them in specific areas. Using the keyPLEX, we showed significant improvements in detection sensitivity by ∼100-fold, leading to the sensitive detection of rare cancer EVs from human plasma samples in 10 min. The keyPLEX system could become a valuable tool for point-of-care rapid EV analysis.

Plasmon-Enhanced Characterization of Single Extracellular Vesicles.

Extracellular vesicles (EVs) represent heterogeneous populations of membrane-bound vesicles shed from almost all kinds of cells. Although superior to conventional methods, most newly developed EV sensing platforms still require a certain number of EVs, measuring bulk signals from a group of vesicles. A new analytical approach that enables single EV analysis could be extremely valuable for understanding EVs' subtypes, heterogeneity, and production dynamics during disease development and progression. Here, we describe a new nanoplasmonic sensing platform for sensitive single EV analysis. Termed nPLEX-FL (nano-plasmonic EV analysis with enhanced fluorescence detection), the system amplifies EVs' fluorescence signals using periodic gold nanohole structures, enabling sensitive, multiplexed analysis of single EVs.

Fibrinogen on extracellular vesicles derived from polyhexamethylene guanidine phosphate-exposed mice induces inflammatory effects via integrin ß.

Polyhexamethylene guanidine phosphate (PHMG-p), used as a humidifier disinfectant, causes interstitial lung disease, obliterative bronchiolitis, and lung fibrosis; however, little is known about its effect on intercellular interactions. Extracellular vesicles (EVs), which carry diverse compounds including proteins, RNA, and DNA to mediate cell-to-cell communication through their paracrine effects, have been highlighted as novel factors in lung fibrogenesis. This study aimed to identify the effect of proteins on small EVs (sEVs) from bronchoalveolar lavage fluid (BALF) of the recipient cells after PHMG-p exposure. A week after intratracheal administration of PHMG-p, sEVs were isolated from BALF of tissue showing overexpressed inflammatory and fibrosis markers. To investigate the role of sEVs in inflammation, naïve macrophages were cultured with sEVs, which induced their activation. To identify sEV proteins that are associated with these responses, proteomics analysis was performed. In the gene ontology analysis, coagulation, fibrinolysis, and hemostasis were associated with the upregulated proteins in sEVs. The highest increase was observed in fibrinogen levels, which was also related to those gene ontologies. We validated role of exosomal fibrinogen in inflammation using recombinant fibrinogen and an inhibitor of the integrin, which is the binding receptor for fibrinogen. Overall, we elucidated that increased fibrinogen levels in the early sEVs-PHMG activated inflammatory response during early fibrosis. These results suggest that sEVs from the BALF of PHMG-p-exposed mice could aggravate fibrogenesis by activating naïve macrophages via various proteins in the sEVs, Furthermore, this finding will be broadening the spectrum of communicating mediators.

Plasmon-Enhanced Single Extracellular Vesicle Analysis for Cholangiocarcinoma Diagnosis.

Cholangiocarcinoma (CCA) is a fatal disease often detected late in unresectable stages. Currently, there are no effective diagnostic methods or biomarkers to detect CCA early with high confidence. Analysis of tumor-derived extracellular vesicles (tEVs) harvested from liquid biopsies can provide a new opportunity to achieve this goal. Here, an advanced nanoplasmonic sensing technology is reported, termed FLEX (fluorescence-amplified extracellular vesicle sensing technology), for sensitive and robust single EV analysis. In the FLEX assay, EVs are captured on a plasmonic gold nanowell surface and immunolabeled for cancer-associated biomarkers to identify tEVs. The underlying plasmonic gold nanowell structures then amplify EVs' fluorescence signals, an effective amplification process at the single EV level. The FLEX EV analysis revealed a wide heterogeneity of tEVs and their marker levels. FLEX also detected small tEVs not detected by conventional EV fluorescence imaging due to weak signals. Tumor markers (MUC1, EGFR, and EPCAM) are identified in CCA, and this marker combination is applied to detect tEVs in clinical bile samples. The FLEX assay detected CCA with an area under the curve of 0.93, significantly better than current clinical markers. The sensitive and accurate nanoplasmonic EV sensing technology can aid in early CCA diagnosis.

Polystyrene microplastic particles induce autophagic cell death in BEAS-2B human bronchial epithelial cells.

The detection of high levels of microplastics in indoor and outdoor air has increased concerns regarding its toxic effects on the respiratory system. They are not easily degradable and can be deposited deep in the lungs. Although several studies have reported inhalation toxicities of microplastics, they are still controversial due to a lack of evidence. Herein, we evaluated the inhalation toxicities of three differently charged polystyrene microplastics (PS-MPs), the most abundant microplastics in the air. Cytotoxicity and ROS generation were evaluated using WST-1 and DCF-DA assays, respectively. To evaluate the toxic effects on the lung, inflammatory responses were analyzed after repeated exposure to the PS-MPs through intratracheal instillation. To explore the mechanism of toxicity, autophagy and ER stress-associated proteins were analyzed. Only the positively charged PS-MPs (NH2 -PS-MPs) showed cytotoxicity and increased ROS generation in BEAS-2B cells. Similarly, only NH2 -PS-MPs significantly increased the expression and secretion of the pro-inflammatory cytokine IL-ß in the animal experiments. The expression of ER stress proteins indicated that NH2 -PS-MPs increased ER stress via PERK-EIF2α and ATF4-CHOP pathways. Moreover, accumulation of NH2 -PS-MPs in lysosomes and deformity of the nucleus were observed in BEAS-2B cells with autophagy induction. Taken together, our results demonstrated that NH2 -PS-MPs induced autophagic cell death in bronchial epithelial cells, leading to inflammatory responses in the lungs. These results suggest that repeated inhalation of microplastics can result in inflammatory responses in the lung through cellular damage of lung epithelial cells, and that inhalation microplastics should be monitored to reduce inhalation health risks.

Recent Advances in Molecular Diagnosis of Pulmonary Fibrosis for Precision Medicine.

Pulmonary fibrosis is a serious, progressive lung disease characterized by scarring and stiffening lung tissues, affecting the respiratory system and leading to organ failure. It is a complex disease consisting of alveolar damage, chronic inflammation, and a varying degree of lung fibrosis. Significant challenges with pulmonary fibrosis include the lack of effective means to diagnose the disease at early stages, identify patients at higher risks of progress, and assess disease progression and treatment response. Precision medicine powered by accurate molecular profiling and phenotyping could significantly improve our understanding of the disease's heterogeneity, potential biomarkers for diagnosis and prognosis, and molecular targets for treatment development. This Review discusses various translational model systems, including organoids and lung-on-a-chip systems, biomarkers in single cells and extracellular vesicles, and functional pharmacodynamic markers. We also highlight emerging sensing technologies for molecular characterization of pulmonary fibrosis and biomarker detection.

Physisorption of Affinity Ligands Facilitates Extracellular Vesicle Detection with Low Non-Specific Binding to Plasmonic Gold Substrates.

Plasmonic biosensors are increasingly being used for the analysis of extracellular vesicles (EVs) originating from disease areas. However, the high non-specific binding of EVs to a gold-sensing surface has been a critical problem and hindered the true translational potential. Here, we report that direct antibody immobilization on the plasmonic gold surface via physisorption shows excellent capture of cancer-derived EVs with ultralow non-specific binding even at very high concentrations. Contrary to commonly used methods that involve thiol-based linker attachment and an EDC/sulfo-NHS reaction, we show a higher specific capture rate and >50-fold lower non-specific on citrate-capped plain and nanopatterned gold surfaces. The method provides a simple, fast, and reproducible means to functionalize plasmonic gold surfaces with antibodies for robust EV biosensing.

Comparison of 3D airway models for the assessment of fibrogenic chemicals.

Lung epithelial cells and fibroblasts play key roles in pulmonary fibrosis and are involved in fibrotic signaling and production of the extracellular matrix (ECM), respectively. Recently, 3D airway models consisting of both cell types have been developed to evaluate the fibrotic responses while facilitating cell-cell crosstalk. This study aimed to evaluate the fibrotic responses in these models using different fibrogenic agents, which are known as key events in adverse outcome pathways of pulmonary fibrosis. We quantified cell injury and several sequential steps in fibrogenesis, including inflammation, the epithelial-mesenchymal transition (EMT), fibroblast activation, and ECM accumulation, using two different 3D airway models, the EpiAirway™-full thickness (Epi/FT) and MucilAir™-human fibroblast (Mucil/HF) models. In the Epi/FT model, fibrogenic agents induced the expression of inflammation and EMT-associated markers, while in the Mucil/HF model, they induced fibroblast activation and ECM accumulation. Using this information, we conducted gene ontology term network analysis. In the Epi/FT model, the terms associated with cell migration and response to stimulus made up a large part of the network. In the Mucil/HF model, the terms associated with ECM organization and cell differentiation and proliferation constituted a great part of the network. Collectively, our data suggest that polyhexamethyleneguanidine phosphate and bleomycin induce different responses in the two 3D airway models. While Epi/FT was associated with inflammatory/EMT-associated responses, Mucil/HF was associated with fibroblast-associated responses. This study will provide an important basis for selecting proper 3D airway models and fibrogenic agents to further research or screen chemicals causing inhalation toxicity.

Reprogrammed lung epithelial cells by decrease of miR-451a in extracellular vesicles contribute to aggravation of pulmonary fibrosis.

Extracellular vesicles (EVs) play novel roles in homeostasis through cell-to-cell communication in human airways via transferring miRNAs. However, the contribution of EV miRNAs to pulmonary phenotypic homeostasis is not clearly understood. Hence, the aim of this study was to elucidate the functional role of miRNAs obtained from epithelium-derived EVs in lung fibrogenesis. Pulmonary fibrosis was induced by exposure of polyhexamethylene guanidine phosphate (PHMG-p)-instilled mice. In histopathological changes, a clear phenotypic change was observed in bronchial epithelium. For figuring out the role of EVs derived from conditioned media of untreated cells (EV-Con) and PHMG-p-treated BEAS-2B (EV-PHMG), significant increase in EVs released from PHMG-p-treated BEAS-2B was detected. Functional analysis with targets of differentially expressed miRNAs in EVs was annotated to epithelial-mesenchymal transition (EMT). Especially, the most abundant miRNA, miR-451a, was downregulated in EV of PHMG-p-treated BEAS-2B cells. We found that odd-skipped related 1 (OSR1) was a putative target for miR-451a, which had been known as a transcription factor of several fibrosis-associated genes. Transfer of decreased miR-451a via EV-PHMG upregulated OSR1 and induced EMT compared to Con-EV-treated cells. In pulmonary fibrosis mice, miR-451a levels were significantly reduced in EV derived from bronchoalveolar lavage fluid and OSR1 expression was increased in lung tissues of mice with PHMG-p exposure. MiR-451a-transfected EVs markedly alleviated fibrogenesis in the PHMG-p-exposed lungs. Low level of miR-451a in EVs modulated EMT and fibrogenesis in recipient cells by increasing OSR1 levels in vitro and in vivo. Our results suggest that transferring EV miR-451a induces anti-fibrotic autocrine effect by downregulating its target, OSR1 maintaining pulmonary homeostasis disrupted by PHMG-p exposure, which can be a potential therapeutic target.

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.

Polyhexamethylene Guanidine Phosphate Induces Apoptosis through Endoplasmic Reticulum Stress in Lung Epithelial Cells.

Airway epithelial cell death contributes to the pathogenesis of lung fibrosis. Polyhexamethylene guanidine phosphate (PHMG-p), commonly used as a disinfectant, has been shown to be strongly associated with lung fibrosis in epidemiological and toxicological studies. However, the molecular mechanism underlying PHMG-p-induced epithelial cell death is currently unclear. We synthesized a PHMG-p-fluorescein isothiocyanate (FITC) conjugate and assessed its uptake into lung epithelial A549 cells. To examine intracellular localization, the cells were treated with PHMG-p-FITC; then, the cytoplasmic organelles were counterstained and observed with confocal microscopy. Additionally, the organelle-specific cell death pathway was investigated in cells treated with PHMG-p. PHMG-p-FITC co-localized with the endoplasmic reticulum (ER), and PHMG-p induced ER stress in A549 cells and mice. The ER stress inhibitor tauroursodeoxycholic acid (TUDCA) was used as a pre-treatment to verify the role of ER stress in PHMG-p-induced cytotoxicity. The cells treated with PHMG-p showed apoptosis, which was inhibited by TUDCA. Our results indicate that PHMG-p is rapidly located in the ER and causes ER-stress-mediated apoptosis, which is an initial step in PHMG-p-induced lung fibrosis.

Dendropanoxide, a Triterpenoid from Dendropanax morbifera, Ameliorates Hepatic Fibrosis by Inhibiting Activation of Hepatic Stellate Cells through Autophagy Inhibition.

Hepatic fibrosis results from chronic liver damage and is characterized by excessive accumulation of extracellular matrix (ECM). In this study, we showed that dendropanoxide (DPX), isolated from Dendropanax morbifera, had anti-fibrotic effects on hepatic fibrosis by inhibiting hepatic stellate cell (HSC) activation. DPX suppressed mRNA and protein expression of α-SMA, fibronectin, and collagen in activated HSCs. Moreover, DPX (40 mg/kg) treatment significantly lowered levels of liver injury markers (aspartate aminotransferase and alanine transaminase), expression of fibrotic markers, and deposition of ECM in a carbon tetrachloride-induced mouse model. Anti-fibrotic effects of DPX were comparable to those of silymarin in a hepatic fibrosis mouse model. As a possible mechanism of anti-fibrotic effects, we showed that DPX inhibited autophagosome formation (LC3B-II) and degradation of p62, which have important roles in HSC activation. These findings suggest that DPX inhibits HSC activation by inhibiting autophagy and can be utilized in hepatic fibrosis therapy.

(-)-Catechin-7-O-ß-d-Apiofuranoside Inhibits Hepatic Stellate Cell Activation by Suppressing the STAT3 Signaling Pathway.

Hepatic fibrosis is characterized by the abnormal deposition of extracellular matrix (ECM) proteins. During hepatic fibrogenesis, hepatic stellate cell (HSC) activation followed by chronic injuries is considered a key event in fibrogenesis, and activated HSCs are known to comprise approximately 90% of ECM-producing myofibroblasts. Here, we demonstrated that (-)-catechin-7-O-ß-d-apiofuranoside (C7A) significantly inhibited HSC activation via blocking the signal transducer and activator of transcription 3 (STAT3) signaling pathway. This is the first study to show the hepatic protective effects of C7A with possible mechanisms in vitro and in vivo. In our bioactivity screening, we figured out that the EtOH extract of Ulmusdavidiana var. japonica root barks, which have been used as a Korean traditional medicine, inhibited collagen synthesis in HSCs. Four catechins isolated from the EtOAc fraction of the EtOH extract were compared with each other in terms of reduction in collagen, which is considered as a marker of hepatic protective effects, and C7A showed the strongest inhibitory effects on HSC activation in protein and qPCR analyses. As a possible mechanism, we investigated the effects of C7A on the STAT3 signaling pathway, which is known to activate HSCs. We found that C7A inhibited phosphorylation of STAT3 and translocation of STAT3 to nucleus. C7A also inhibited expressions of MMP-2 and MMP-9, which are downstream genes of STAT3 signaling. Anti-fibrotic effects of C7A were evaluated in a thioacetamide (TAA)-induced liver fibrosis model, which indicated that C7A significantly inhibited ECM deposition through inhibiting STAT3 signaling. C7A decreased serum levels of aspartate amino transferase and alanine transaminase, which were markedly increased by TAA injection. Moreover, ECM-associated proteins and mRNA expression were strongly suppressed by C7A. Our study provides the experimental evidence that C7A has inhibitory effects on HSC activation after live injury and has preventive and therapeutic potentials for the management of hepatic fibrosis.

Effects of ß-Sitosterol from Corn Silk on TGF-ß1-Induced Epithelial-Mesenchymal Transition in Lung Alveolar Epithelial Cells.

Pulmonary fibrosis is a chronic lung disease characterized by abnormal accumulation of the extracellular matrix (ECM). Chronic damage of the alveolar epithelium leads to a process called "epithelial-mesenchymal transition" (EMT) and increases synthesis and deposition of ECM proteins. Therefore, inhibition of EMT might be a promising therapeutic approach for the treatment of pulmonary fibrosis. ß-Sitosterol is one of the most abundant phytosterols in the plant kingdom and the major constituent in corn silk, which is derived from the stigma and style of maize (Zea mays). In this study, we elucidated that ß-sitosterol inhibited transforming growth factor-ß1 (TGF-ß1)-induced EMT and consequently had an antifibrotic effect. ß-Sitosterol (1-10 µg/mL) significantly downregulated the TGF-ß1-induced fibrotic proteins, such as collagen, fibronectin, and α-smooth muscle actin in human alveolar epithelial cells (p < 0.01). After 24 h, relative wound density (RWD) was increased in TGF-ß1 treated group (82.16 ± 5.70) compare to the control group (64.63 ± 2.21), but RWD was decreased in ß-sitosterol cotreated group (10 µg/mL: 71.54 ± 7.39; 20 µg/mL: 65.69 ± 6.42). In addition, the changes of the TGF-ß1-induced morphological shape and protein expression of EMT markers, N-cadherin, vimentin, and E-cadherin, were significantly blocked by ß-sitosterol treatment (p < 0.01). The effects of ß-sitosterol on EMT were found to be associated with the TGF-ß1/Snail pathway, which is regulated by Smad and non-Smad signaling pathways. Taken together, these findings suggest that ß-sitosterol can be used to attenuate pulmonary fibrosis through suppression of EMT by inhibiting the TGF-ß1/Snail pathway.

ß-Peltoboykinolic Acid from Astilbe rubra Attenuates TGF-ß1-Induced Epithelial-to-Mesenchymal Transitions in Lung Alveolar Epithelial Cells.

Epithelial-to-mesenchymal transition (EMT) is increasingly recognized as contributing to the pathogenesis of idiopathic pulmonary fibrosis. Therefore, novel plant-based natural, active compounds have been sought for the treatment of fibrotic EMT. The aim of the present study was to investigate the inhibitory effects of Astilbe rubra on TGF-ß1-induced EMT in lung alveolar epithelial cells (A549). A. rubra was subjected to extraction using 70% ethanol (ARE), and ethanol extracts of the aerial part and that of the rhizome were further partitioned using various solvents. Protein expression and cell motility were investigated to evaluate the inhibitory effects of ARE on EMT. EMT occurred in A549 cells treated with TGF-ß1, but was prevented by co-treatment with ARE. The dichloromethane fractions showed the strongest inhibitory effect on TGF-ß1-induced EMT. ß-Peltoboykinolic acid was isolated from the dichloromethane fractions of A. rubra by activity-oriented isolation. ß-Peltoboykinolic acid not only attenuated TGF-ß1-induced EMT, but also the overproduction of extracellular matrix components including type I collagen and fibronectin. The Smad pathway activated by TGF-ß1 was inhibited by co-treatment with ß-peltoboykinolic acid. Taken together, these results indicate that ß-peltoboykinolic acid from A. rubra and dichloromethane fractions shows potential as an antifibrotic agent in A549 cells treated with TGF-ß1.

Akt and Notch pathways mediate polyhexamethylene guanidine phosphate-induced epithelial-mesenchymal transition via ZEB2.

Polyhexamethylene guanidine phosphate (PHMG-p), an antimicrobial additive, was used as a humidifier disinfectant in Korea and caused severe lung injuries, including lung fibrosis, in hundreds of victims. As PHMG-p-induced lung fibrosis is different from that induced by known fibrogenic agents such as bleomycin, it is important to understand the molecular mechanisms underlying this effect. A recent study showed that epithelial-mesenchymal transition (EMT) could play key roles in PHMG-p-induced pulmonary fibrosis. Therefore, we aimed to characterize the molecular mechanisms associated with PHMG-p-induced EMT. We observed EMT, macrophage infiltration, and fibrosis in mouse lung tissues after intratracheal instillation of PHMG-p. Furthermore, PHMG-p-induced EMT was observed in A549 cells by the evaluation of cell morphology and quantitation of mRNA and protein expression. The use of EMT inhibitors revealed that PHMG-p induced EMT through the activation of Akt and Notch signaling. Moreover, the transcription factor ZEB2 was observed in PHMG-p-treated A549 cells and mouse lungs. The results indicated that upstream regulators, including Akt and Notch 1, acted as intracellular effectors that triggered ZEB2 expression after exposure to PHMG-p. Attenuation of PHMG-p-induced EMT following inhibition or silencing of Akt and Notch signaling or ZEB2 implied that PHMG-p-induced EMT was a result of Akt, Notch, and ZEB2 activation. Our findings showed that PHMG-p induced EMT through Akt/Notch signaling pathways and that ZEB2 played an important role in PHMG-p-induced lung toxicity. This study will help to understand the mechanisms of action of PHMG-p associated with lung fibrogenesis.

Guanidine-based disinfectants, polyhexamethylene guanidine-phosphate (PHMG-P), polyhexamethylene biguanide (PHMB), and oligo(2-(2-ethoxy)ethoxyethyl guanidinium chloride (PGH) induced epithelial-mesenchymal transition in A549 alveolar epithelial cells.

Abstracts Objective: The major active ingredient of humidifier disinfectant, polyhexamethylene guanidine-phosphate (PHMG-P), caused hundreds of deaths with pulmonary fibrosis. However, structurally similar guanidine-based disinfectants are still in use in various fields. Moreover, as they are precursors of excellent antimicrobial compounds, new chemicals with guanidine-based structures have been synthesized and introduced. In this study, we evaluated pulmonary fibrotic responses induced by PHMG-P, polyhexamethylene biguanide (PHMB), and oligo(2-(2-ethoxy)ethoxyethyl guanidinium chloride (PGH) and their toxicity mechanisms in type II alveolar epithelial A549 cells. Materials and methods: Cellular damage was compared by using the cytotoxicity test (WST-1 assay) and plasma membrane toxicity tests (Lactate dehydrogenase leakage detection assay and plasma membrane staining). As a measure of fibrotic response, induction of the epithelial-mesenchymal transition (EMT) was evaluated by measuring E-cadherin and α-smooth muscle actin (α-SMA) protein expression (epithelial and mesenchymal marker, respectively). Results: All tested compounds showed membrane damage; PHMG-P and PGH induced the highest and lowest damage, respectively. Moreover, they induced EMT when the test chemicals were treated with similar cytotoxic concentrations. Conclusions: Our study indicates that three guanidine-based disinfectants are potential fibrosis-inducing chemicals that induce EMT through cellular damage. Therefore, use of guanidine-based polymers should be strictly regulated by considering their potential adverse effects on the lungs.

In vitro model for predicting acute inhalation toxicity by using a Calu-3 epithelium cytotoxicity assay.

INTRODUCTION: As the current methods to predict the inhalation toxicity of chemicals using animal models are limited, alternative methods are required. We present a new in vitro prediction method for acute inhalation toxicity using the Calu-3 epithelial cytotoxicity assay applicable for water-soluble inhalable chemicals. METHOD: To confirm the characteristics of the optimal Calu-3 epithelium, tight-junction formation, morphology, and mucus secretion were verified using scanning electron microscopy, transepithelial electrical resistance analysis, and immunofluorescence after growth in an air-liquid interface (ALI). Sixty chemicals, including 38 positive and 22 negative for acute inhalation toxicity, were selected from the European Chemical Agency chemical database. The cell viability of the exposed cells was assessed using an MTT assay to predict the acute inhalation toxicity by calculating the area under the receiver operating characteristic (ROC) curve and accuracy. RESULTS: When cultivated in an ALI, the epithelium was thicker and secreted more mucin than that under submerged cultivation, characteristic of the in vivo respiratory epithelium. The areas under the ROC curve were 0.75 and 0.78 when exposed to chemicals at concentrations of 2.5 and 5%, respectively. The highest accuracy of the methods was 68 and 78% at cut-off values of 85 and 40% cell viability, respectively. DISCUSSION: The in vitro model was moderately accurate with good prediction. It is replicable because of its advantages, i.e., the use of cultured cells and the simplicity of the method. Overall, the Calu-3 epithelial cytotoxicity assay may be a useful and simple approach to identify substances that cause acute inhalation toxicity.

Polyhexamethylene guanidine phosphate-induced upregulation of MUC5AC via activation of the TLR-p38 MAPK and JNK axis.

Epidemiological and toxicological studies indicate that polyhexamethylene guanidine phosphate (PHMG-p) is a guanidine-based cationic disinfectant strongly associated with interstitial lung diseases. As individuals exposed to aerosolized PHMG-p complain of respiratory problems (asthma and rhinitis), whether PHMG-p can cause respiratory diseases other than interstitial fibrosis should be investigated. MUC5AC, the predominant mucin gene expressed in airways, is associated with obstructive respiratory disease pathogenesis. Therefore, in this study, we elucidated the relationship between PHMG-p and MUC5AC overexpression. First, in immunofluorescence studies, the bronchial epithelia of mice intratracheally administrated PHMG-p appeared to be sloughing and tethered by MUC5AC. Second, Calu-3 cells exposed to PHMG-p showed concentration-dependent increases in MUC5AC mRNA and protein expression. c-Jun N-terminal kinase (JNK), p38, and c-jun were phosphorylated in cells exposed to PHMG-p. SP600125 and SB203580, JNK and p38 inhibitors, respectively, reduced the upregulation of MUC5AC by PHMG-p in Calu-3 cells. When toll-like receptor (TLR)2, 4, and 6 were silenced, PHMG-p-induced JNK and p38 phosphorylation decreased. Furthermore, TLR2-, 4-, and 6-silenced cells showed reduced levels of MUC5AC mRNA and protein induced by PHMG-p, with TLR6 knockdown showing the greatest effect. In conclusion, PHMG-p induced MUC5AC overexpression via activation of the TLR-p38 MAPK and JNK axis.