Cytotoxicity In Lung Epithelial Cells Research Articles

Forced expression of microRNA-221-3p exerts protective effects against manganese-induced cytotoxicity in human lung epithelial cells

Manganese (Mn)-induced pulmonary toxicity and the underlying molecular mechanisms remain largely enigmatic. Further, in recent years, microRNAs (miRNAs) have emerged as regulators of several pollutants-mediated toxicity. In this context, our study aimed at elucidating whether miRNAs are involved in manganese (II) chloride (MnCl2) (Mn2+)-induced cytotoxicity in lung epithelial cells. Growth inhibition of Mn2+ towards normal human bronchial epithelial (BEAS-2B) and adenocarcinomic human alveolar basal epithelial (A549) cells was analyzed by MTT assay following 24 or 48 h treatment. Reactive oxygen species (ROS) generation, mitochondrial membrane potential (ΔΨm), cell cycle arrest, and apoptosis were evaluated by flow cytometry. RT-qPCR and Western blot were performed to analyze the expression of cyclins, anti-oxidant genes, and miRNAs. We used small RNA sequencing to investigate Mn2+-induced changes in miRNA expression patterns. In both cell lines, Mn2+ treatment inhibited growth in a dose-dependent manner. Further, compared with vehicle-treated cells, Mn2+ (250 μM) treatment induced ROS generation, cell cycle arrest, apoptosis, and decreased ΔΨm as well as altered the expression of cyclins and anti-oxidant genes. Sequencing data revealed that totally 296 miRNAs were differentially expressed in Mn2+-treated cells. Among them, miR-221-3p was one of the topmost down-regulated miRNAs in Mn2+-treated cells. We further confirmed this association in A549 cells. In addition, transient transfection was performed to study gain-of-function experiments. Forced expression of miR-221-3p significantly improved cell viability and reduced Mn2+-induced cell cycle arrest and apoptosis in BEAS-2B cells. In conclusion, miR-221-3p may be the most likely target that accounts for the cytotoxicity of Mn2+−exposed lung epithelial cells.

Ambient Particulate Matter in Valdivia, Chile: Temporal Analysis and Compared Cytotoxicity in Lung Epithelial Cells

Ambient Particulate Matter in Valdivia, Chile: Temporal Analysis and Compared Cytotoxicity in Lung Epithelial Cells

Ethyl maltol enhances copper mediated cytotoxicity in lung epithelial cells

Ethyl maltol (EM) is a flavoring agent commonly used in foods that falls under the generally recognized as safe category. It is added to many commercial e-cigarette vaping fluids and has been detected in the aerosols. Considering that EM facilitates heavy metal transport across plasma membranes, and that heavy metals have been detected in aerosols generated from e-cigarettes, this study examines whether EM enhances heavy metal mediated toxicity. A decrease in viability was observed in the Calu-6 and A549 lung epithelial cell lines co-exposed to EM and copper (Cu) but no decrease was observed after co-exposure to EM with iron (Fe). Interestingly, co-exposure to EM and Fe decreased viability of the HEK293 and IMR-90 fibroblast cell lines but co-exposure to EM and Cu did not. Increases in the apoptotic markers Annexin V staining and fragmented nuclei were observed in Calu-6 cells co-exposed to EM and Cu. Co-exposure to EM and Cu in Calu-6 cells resulted in DNA damage as indicated by activation of ATM and expression of γH2A.x foci. Finally, co-exposure to EM and Cu caused oxidative stress as indicated by increases in the generation of reactive oxygen species and the expression of ferritin light chain mRNA and hemeoxygenase-1 mRNA and protein. These data show that co-exposure to EM and Cu, at concentrations that are not toxic for either chemical individually, induce apoptosis and evoke oxidative stress and DNA damage in lung epithelial cells. We suggest that there is a greater risk of lung damage in users of c-cigarette who vape with vaping fluid containing EM.

Concentration‐ and time‐dependent effects of benzalkonium chloride in human lung epithelial cells; fate of necrosis, apoptosis, or epithelial mesenchymal transition

Benzalkonium chloride (BAC), an antimicrobial agent in inhalable medications and household sprays, has been reported to be toxic to pulmonary organs. Although cell membrane damage has been considered as the main cytotoxic action of BAC, its concentration‐ and time‐dependent cellular effects on lung epithelium has been not fully understood. In the present study, human lung epithelial (H358) cells were exposed to 0.2–40 μg/mL of BAC for 30 min to 21 days. Cell membranes were rapidly disrupted by 30 min exposure, but 24 h incubation of BAC (4–40 μg/mL) dominantly caused apoptosis rather than necrosis. BAC (2–4 μg/mL) induced mitochondrial depolarization which may be related to the increased pro‐apoptotic proteins (caspase‐3, PARP, Bax, p53, and p21), and the decreased anti‐apoptotic Bcl‐2. IRE1α, BiP, CHOP, and p‐JNK proteins were also elevated by BAC (2–4 μg/mL) suggesting the possible involvement of ER stress in the apoptosis. Long‐term (7–21 days) incubations of BAC (0.2–0.6 μg/mL) did not affect the cell viabilities but led to epithelial mesenchymal transition (EMT) as shown as the decrease of E‐cadherin, and the increases of N‐cadherin, fibronectin, and vimentin by up‐regulating the EMT transcription factors, Snail, Slug, Twist1, Zeb1, and Zeb2. Therefore, we conclude that apoptosis can be an important mechanism of acute BAC cytotoxicity in lung epithelial cells, and chronic exposure to BAC even at sub‐toxic doses can promote pulmonary EMT.Support or Funding InformationThis study was funded by Nano‐Material Technology Development Program through the National Research Foundation of Korea (NRF) funded by Ministry of Science and ICT (NRF‐ 2018M3A7B4071233).

Concentration- and Time-Dependent Effects of Benzalkonium Chloride in Human Lung Epithelial Cells: Necrosis, Apoptosis, or Epithelial Mesenchymal Transition.

Benzalkonium chloride (BAC), an antimicrobial agent in inhalable medications and household sprays, has been reported to be toxic to pulmonary organs. Although cell membrane damage has been considered as the main cytotoxic mechanism of BAC, its concentration- and time-dependent cellular effects on lung epithelium have not been fully understood. In the present study, human lung epithelial (H358) cells were exposed to 0.2–40 μg/mL of BAC for 30 min or 21 days. Cell membranes were rapidly disrupted by 30 min exposure, but 24 h incubation of BAC (4–40 μg/mL) predominantly caused apoptosis rather than necrosis. BAC (2–4 μg/mL) induced mitochondrial depolarization, which may be associated with increased expression of pro-apoptotic proteins (caspase-3, PARP, Bax, p53, and p21), and decreased levels of the anti-apoptotic protein Bcl-2. The protein expression levels of IRE1α, BiP, CHOP, and p-JNK were also elevated by BAC (2–4 μg/mL) suggesting the possible involvement of endoplasmic reticulum stress in inducing apoptosis. Long-term (7–21 days) incubation with BAC (0.2–0.6 μg/mL) did not affect cell viability but led to epithelial-mesenchymal transition (EMT) as shown by the decrease of E-cadherin and the increase of N-cadherin, fibronectin, and vimentin, caused by the upregulation of EMT transcription factors, such as Snail, Slug, Twist1, Zeb1, and Zeb2. Therefore, we conclude that apoptosis could be an important mechanism of acute BAC cytotoxicity in lung epithelial cells, and chronic exposure to BAC even at sub-lethal doses can promote pulmonary EMT.

(2S)-5,6,7,3′,4′-pentamethoxyflavanone, a citrus polymethoxyflavone ameliorates arsenic- and cigarette smoke extract-induced cytotoxicity via activating Nrf2-mediated defense system

Exogenous toxicants (e.g. arsenic, cigarette smoke), trigger oxidative stress, and initiate the pathogenesis of many human diseases. Induction of nuclear factor E2-related factor 2 (Nrf2) counteracts exogenous toxicant-induced oxidative insults. Using a high-throughput screen, we firstly identified a citrus polymethoxyflavone, (2S)-5,6,7,3′,4′-pentamethoxyflavanone (PMF), to be an Nrf2 activator, and an investigation targeting Nrf2 pathway was performed. Our study indicated that: (i) PMF activated Nrf2 and its downstream genes, NQO1 and γ-GCS, and enhanced the stabilization of Nrf2 through inhibiting Nrf2 ubiquitination. (ii) Inhibition of PI3K, p38 MAPK, PKC and PERK by specific kinase inhibitors suppressed PMF-induced activation of Nrf2. (iii) PMF conferred Nrf2-dependent protection against sodium arsenite- and cigarette smoke extract- induced cytotoxicity in lung epithelial cells. Collectively, our data demonstrate that PMF is a novel Nrf2 activator with potential prevention against oxidative insults. The diverse pharmacological functions of citrus polymethoxyflavone might be related to their ability of activating Nrf2 pathway.

Differential expression of heat shock proteins and activation of mitogen‐activated protein kinases in A549 alveolar epithelial cells exposed to cigarette smoke extract

What is the central question of this study? What is the effect of cigarette smoke on cell death, oxidative damage, expression of heat shock proteins (HSPs) and activation of mitogen-activated protein kinases (MAPKs) in A549 alveolar epithelial cells? What is the main finding and its importance? Cigarette smoke induces cytotoxicity and oxidative damage to A549 cells, increases expression of different HSPs and activates MAPK signalling pathways. This could be related to inflammatory response and apoptosis observed in lungs of patients with smoking-related diseases. Cigarette smoking is one of the main risk factors for development of chronic obstructive pulmonary disease (COPD). We previously reported that cigarette smoke (CS) induces damage to proteins and their ineffective degradation. Here, we hypothesize that CS could induce oxidative stress and cytotoxicity in lung epithelial cells through alterations of heat shock protein (HSP) expression and mitogen-activated protein kinase (MAPK) signalling pathways. We exposed A549 alveolar epithelial cells to various concentrations of cigarette smoke extract (CSE). Higher concentrations of CSE caused apoptosis of A549 cells after 4h, while after 24h cell viability was decreased, and lactate dehydrogenase in cell culture medium was increased as well as the number of necrotic cells. Concentrations of malondialdehyde (MDA) were elevated, while total thiol groups were decreased. Changes in the expression of HSPs (HSP70, HSP32 and HSP27) were time-dependent. After 6h, CSE caused an increase in the expression of HSP70 and HSP32, while after 8h all examined HSPs were up-regulated and remained increased up to 48h. Treatment of A549 cells with CSE stimulated phosphorylation of extracellular signal-regulated kinase and p38 in a dose-dependent manner, while c-Jun N-terminal kinase activation was not detected. By using specific inhibitors, we demonstrated that MAPKs and HSPs interplay in CSE effects. In conclusion, our results show that MAPKs and HSPs are involved in the mechanism underlying CSE-induced cytotoxicity and oxidative damage to A549 alveolar epithelial cells. These processes could be related to inflammatory response and apoptosis observed in lungs of patients with smoking-related diseases, such as COPD.

The use of a 0.20 μm particulate matter filter decreases cytotoxicity in lung epithelial cells following air-liquid interface exposure to motorcycle exhaust

This study was designed to investigate whether the use of a 0.20 μm particulate matter (PM) filter reduced the cytotoxicity induced by motorcycle exhaust (ME), a mixture of gases and particles, in lung epithelial cells cultured in air-liquid interface (ALI) inserts. The concentrations of PM, carbon monoxide, carbon dioxide, total hydrocarbons (THC), total volatile organic compounds, and nitrogen oxides in both filtered ME (fME) by a 0.20 μm filter and non-filtered ME (non-fME) were measured. Lung epithelial cells were exposed to clean air, fME, or non-fME in the ALI chamber. Cell relative viabilities (CRV) and the reactive oxygen species (ROS) generation were determined. Our results revealed that PM2.5 was the main compound of PM in ME. After filtration, PM and THC levels were significantly reduced, as compared with non-fME. When compared with the clean air exposed group, the CRV in both fME and non-fME-exposed group was significantly reduced (p < 0.001), while their ROS generation were markedly increased (p < 0.001). When compared with non-fME-exposed group, the CRV and ROS generation were significantly improved following fME exposure (p < 0.05). As a result, of PM and THC concentrations were decreased approximately 90% and 22.71%, respectively, the CRV was improved from 40.4% (non-fME) to 55.7% (fME), and the increased ROS generation by non-fME was decreased about 51.6%. When BEAS-2B cells were exposed to fME, a time-dependent reduction in CRV was observed. In conclusion, our findings suggest that ME-exposure in the ALI system induces cytotoxicity and oxidative stress responses. The addition of a 0.20 μm PM filter significantly modifies the particulate composition in PM and the concentration of THC, and shows protective effects by improving the survival of exposed lung epithelial cells and reducing the ROS generation. Therefore, emission factors such as different size of PM and THC from motorcycles may play a role in ME-induced toxicity.

Identification of a bacteriolysis-associated virulence factor against lung epithelial cells in Pseudomonas aeruginosa PAO-1 cell lysate

The precise identities of the virulence factors of Pseudomonas aeruginosa after bacteriolysis are still unknown. In the present study, we identified PA0423 protein, which was isolated from the Pseudomonas PAO-1 strain, as the factor responsible for cytotoxicity in lung epithelial cells. Whole bacterial cell lysate of P. aeruginosa PAO-1 caused cytotoxicity in A549 lung epithelial cells. This cytotoxic factor could be partially purified via gel-filtration and anion-exchange column chromatography, and its activity was attenuated by proteinase K treatment. The cytotoxic fraction increased caspase-3/7 activity in A549 cells, suggesting the induction of apoptosis. This fraction was then subjected to amino-acid sequence analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, resulting in the identification of 7 matches, 4 of which were with known proteins (PA0122, PA2687, PA3406, and PA0423). Deletion mutant analysis of these 7 candidates revealed that only the PA0423 mutation led to reduced cytotoxicity, indicating that this protein is the virulence factor. Furthermore, PA0423 recombinant protein was constructed, purified, and refolded. Transduction of recombinant PA0423, but not PA0122, into A549 cells engendered a dose-dependent cytotoxic effect. These results show the first evidence that specific bacteriolysis-induced virulence factor PA0423 from Pseudomonas is toxic to lung epithelial cells.

Human 8-oxoguanine DNA glycosylase increases resistance to hyperoxic cytotoxicity in lung epithelial cells and involvement with altered MAPK activity

It is unknown whether base excision DNA repair (BER) proteins interact with mitogen-activated protein kinases (MAPK) under oxidation. Here, we explored roles of BER proteins in signaling transduction involving MAPK during hyperoxia. We demonstrated that ERK1/2 phosphorylation in A549 cells was increased in 95% O2. p38 activity in A549 cells was also increased by exposure to 95% O2. To evaluate regulatory roles of MAPK, we have transduced A549 cells and primary alveolar epithelial type II cells (AECII) to overexpress 8-oxoguanine DNA glycosylase (hOgg1). Overexpression of hOgg1 reduced hyperoxic toxicity in A549 and AECII cells. Furthermore, protection by BER against hyperoxia appeared to involve an upregulation of ERK1/2 and downregulation of p38. These observations demonstrate, for the first time, that reduction of hyperoxic toxicity by BER proteins may be involved with MAPK activity, thereby impacting cell survival. Furthermore, our studies suggest that modulation of MAPK may be used in combination with BER proteins to counteract hyperoxic toxicity.