Alleviating effects of reduced graphene oxide against lead-induced cytotoxicity and oxidative stress in human alveolar epithelial (A549) cells

Broad application of reduced graphene oxide (rGO) and ubiquitous lead (Pb) pollution may increase the possibility of combined exposure of humans. Information on the combined effects of rGO and Pb in human cells is scarce. This work was designed to explore the potential effects of rGO on Pb-induced toxicity in human alveolar epithelial (A549) cells. Prepared rGO was polycrystalline in nature. The formation of a few layers of visible creases and silky morphology due to high aspect ratio was confirmed. Low level (25 μg/mL) of rGO was not toxic to A549 cells. However, Pb exposure (25 μg/mL) induced cell viability reduction, lactate dehydrogenase enzyme leakage with rounded morphology in A549 cells. Remarkably, Pb-induced cytotoxicity was significantly mitigated by rGO co-exposure. Pb-induced mitochondrial membrane potential loss, cell cycle arrest and higher activity of caspase-3 and -9 enzymes were also alleviated by rGO co-exposure. Moreover, we observed that Pb exposure causes generation of pro-oxidants (e.g., reactive oxygen species, hydrogen peroxide and lipid peroxidation) and antioxidant depletion (e.g., glutathione and antioxidant enzymes). In addition, the effects of Pb on pro-oxidant and antioxidant markers were significantly reverted by GO co-exposure. Inductively coupled plasma-mass spectrometry suggested that due to the adsorption of Pb on rGO sheets, accessibility of Pb ions for A549 cells was limited. Hence, rGO reduced the toxicity of Pb in A549 cells. This research warrants further study to work on detailed underlying mechanisms of the mitigating effects of rGO against Pb-induced toxicity on a molecular level.     

PM2.5-induced oxidative stress triggers autophagy in human lung epithelial A549 cells

Exposure to higher levels of air pollution particulate matter (PM) with an aerodynamic diameter of less than 2.5 μm (PM_2.5) links with an increased risk of cardiovascular and respiratory deaths and hospital admission as well as lung cancer. Although the mechanism underlying the correlation between PM_2.5 exposure and adverse effects has not fully elucidated, PM_2.5-induced oxidative stress has been considered as an important molecular mechanism of PM_2.5-mediated toxicity. In this work, human lung epithelial A549 cells were used to further investigate the biological effects of PM_2.5 on autophagy. The cell viability showed both time- and concentration-dependent decrease when exposure to PM_2.5, which can be attributed to increase of the levels of extracellular lactate dehydrogenase (LDH) release and intracellular reactive oxygen species (ROS) generation in A549 cells. Moreover, PM_2.5-induced oxidative damage in A549 cells was observed through the alteration of superoxide dismutase (SOD) and catalase (CAT) activities compared to the unexposed control cells. PM_2.5-induced autophagy was indicated by an increase in microtubule-associated protein light chain-3 (LC3) puncta, and accumulation of LC3 in both time- and concentration-dependent manner. PM_2.5-induced mRNA expression of autophagy-related protein Atg5 and Beclin1 was also observed compared with those of the unexposed control cells. These results suggest the possibility that PM_2.5-induced oxidative stress probably plays a key role in autophagy in A549 cells, which may contribute to PM_2.5-induced impairment of pulmonary function.     

Synergistic neurotoxicity of oxygen-glucose deprivation and tetrabromobisphenol A in vitro: role of oxidative stress

BackgroundTetrabromobisphenol A (TBBPA) is a toxic brominated flame retardant. Previous studies have demonstrated that exposure of primary cultures of rat cerebellar granule cells (CGC) to ≥ 10 μM TBBPA induces toxicity and excitotoxicity, and the underlying mechanism may involve calcium imbalance and oxidative stress. Here we examined whether the application of TBBPA at subtoxic concentrations may exacerbate acute damage of CGC challenged with oxygen-glucose deprivation (OGD), and evaluated with fluorescent indicators the involvement of calcium imbalance, mitochondrial depolarization and oxidative stress.MethodsSurvival of CGC was assessed 24 h after OGD/TBBPA using fluorescent dyes. An OGD challenge lasting for 45, 60 or 75 min induced a duration-dependent injury to the neurons.ResultsApplication of 2.5, 5 or 7.5 μM TBBPA for 45 min to normoxic and glucose-containing incubation medium did not reduce the viability of cultured CGC, but this compound exacerbated the toxic effects of OGD in a concentration-dependent way. Moreover, TBBPA had a slight effect on calcium homeostasis and mitochondrial membrane potential, but significantly activated the production of reactive oxygen species in CGC. The application of H₂O₂ at 5, 10 and 25 μM mimicked the effects of TBBPA on OGD toxicity, while 0.1 mM ascorbic acid or 1 mM glutathione ameliorated this toxicity.ConclusionThese results suggest the involvement of oxidative stress in the synergistic neurotoxic effects of TBBPA and OGD.     

Biotransformation and cytotoxicity of a brominated flame retardant, tetrabromobisphenol A, and its analogues in rat hepatocytes

The metabolism and cytotoxic effects of tetrabromobisphenol A (TBBPA), a phenolic flame retardant, and its analogues were studied in freshly isolated rat hepatocytes and isolated hepatic mitochondria, respectively. The exposure of hepatocytes to TBBPA caused not only concentration (0.25-1.0 mM)- and time- (0-3 h) dependent cell death accompanied by the loss of cellular ATP, adenine nucleotide pools, reduced glutathione, and protein thiols, but also the accumulation of oxidized glutathione and malondialdehyde, indicating lipid peroxidation. TBBPA at a weakly toxic level (0.25 mM) was metabolized to monoglucuronide and monosulfate conjugates: the amounts of glucuronide rather than sulfate conjugate predominantly increased, accompanied by a loss of the parent compound, with time. In comparative effects based on cell viability, mitochondrial membrane potential and some toxic parameters, bisphenol A (BPA) was less toxic than TBBPA and tetrachlorobisphenol A (TCBPA), which are not significant differences in these parameters. In mitochondria isolated from rat liver, TBBPA and TCBPA caused an increase in the rate of State 4 oxygen consumption in the presence of succinate, indicating an uncoupling effect and a decrease in the rate of State 3 oxygen consumption in a concentration-dependent manner (5-25 microM). Taken collectively, our results indicate that (i) mitochondria are target organelles for TBBPA, which elicits cytotoxicity through mitochondrial dysfunction related to oxidative phosphorylation at an early stage and subsequently lipid peroxidation at a later stage; and (ii) the toxicity of TBBPA and TCBPA is greater than that of BPA, suggesting the participation of halogen atoms such as bromine and chlorine in the toxicity.     

苯乙双胍通过影响线粒体功能促进A549/DDP细胞凋亡

目的 探究苯乙双胍抑制 A549/DDP细胞增殖并促进其凋亡的可能机制。方法 MTS法检测顺铂（顺铂 组）和苯乙双胍（苯乙双胍组）对 A549/DDP 细胞和 A549 细胞的细胞活力（24 h 和 48 h）的影响。苯乙双胍组以 2.5 mmol/L苯乙双胍处理,对照组采用PBS处理,MTS法检测苯乙双胍对A549/DDP细胞增殖的影响,克隆形成实验检测 苯乙双胍对 A549/DDP细胞克隆形成能力的影响,流式细胞术检测苯乙双胍对 A549/DDP细胞凋亡和活性氧（ROS） 胞内线粒体质量（Mitotracker）、钙离子（Rhod-2）的影响,实时荧光定量PCR检测线粒体DNA（mtDNA）变化,三磷酸腺 苷（ATP）试剂盒检测 ATP的变化,Western blot检测苯乙双胍对线粒体氧化磷酸化复合物表达的影响。结果 与顺 铂组比较,苯乙双胍组A549/DDP细胞在24 h和48 h的细胞活力降低（P＜0.05）,而2组间A549细胞24 h和48 h活力 差异无统计学意义。顺铂处理48 h时A549/DDP细胞活力均高于A549细胞（P＜0.05）,而24 h间差异无统计学意义; 苯乙双胍处理的A549/DDP细胞活力低于A549细胞（P＜0.05）。与PBS组相比,苯乙双胍组A549/DDP细胞的24 h和 48 h 增殖率、集落形成数减低,而凋亡率升高,ROS 水平增高,Mitotracker、Rhod-2、mtDNA 及 ATP 水平降低（P＜ 0.05）,线粒体氧化磷酸化复合物Ⅰ中的NDUFB8蛋白、复合物Ⅱ中的SDHB蛋白和复合物Ⅳ中的MTCO1蛋白表达明 显降低（P＜0.05）。结论 （1）A549/DDP细胞较 A549细胞对苯乙双胍更为敏感,但对顺铂耐受性较 A549细胞强。 （2）苯乙双胍通过增加细胞内 ROS、减少线粒体质量、钙离子和 mtDNA、抑制线粒体氧化磷酸化复合物表达,减少 ATP生成,从而抑制A549/DDP细胞增殖并促进其凋亡。     

Perfluorooctane sulfonate induces apoptosis in lung cancer A549 cells through reactive oxygen species‐mediated mitochondrion‐dependent pathway

Perfluorooctane sulfonate (PFOS) is a widespread environmental contaminant that is detected in the lung of mammals. The mechanisms underlying PFOS‐induced lung cytotoxicity remain unclear. The main purpose of this study was to evaluate the cytotoxic effects of PFOS on human lung cancer A549 cells and its possible molecular mechanism. A549 cells were treated with PFOS (0, 25, 50, 100 and 200 μm ) and the cellular apoptosis, mitochondrial membrane potential as well as intracellular reactive oxygen species were determined. In this study, PFOS induced a dose‐dependent increase in A549 cell toxicity via an apoptosis pathway as characterized by increased percentage of sub‐G1, activation of caspase‐3 and ?9, and increased ratio of Bax/bcl‐2 mRNA expression. In addition, there was obvious oxidative stress, represented by decreased glutathione level, increased malondialdehyde level and superoxide dismutase activity. N‐Acetylcysteine, as an antioxidant that is a direct reactive oxygen species scavenger, can effectively block PFOS‐induced reactive oxygen species generation, mitochondrial membrane potential loss and cell apoptosis. These data indicate that PFOS induces apoptosis in A549 cells through a reactive oxygen species‐mediated mitochondrial dysfunction pathway mechanism. Copyright © 2012 John Wiley & Sons, Ltd.     

The surfactant dipalmitoylphophatidylcholine modifies acute responses in alveolar carcinoma cells in response to low‐dose silver nanoparticle exposure

Nanotechnology is a rapidly growing field with silver nanoparticles (AgNP) in particular utilized in a wide variety of consumer products. This has presented a number of concerns relating to exposure and the associated toxicity to humans and the environment. As inhalation is the most common exposure route, this study investigates the potential toxicity of AgNP to A549 alveolar epithelial carcinoma cells and the influence of a major component of lung surfactant dipalmitoylphosphatidylcholine (DPPC) on toxicity. It was illustrated that exposure to AgNP generated low levels of oxidative stress and a reduction in cell viability. While DPPC produced no significant effect on viability studies its presence resulted in increased reactive oxygen species formation. DPPC also significantly modified the inflammatory response generated by AgNP exposure. These findings suggest a possible interaction between AgNP and DPPC causing particles to become more reactive, thus increasing oxidative insult and inflammatory response within A549 cells. Copyright © 2015 John Wiley & Sons, Ltd.     

Silver nanoparticles induce toxicity in A549 cells via ROS-dependent and ROS-independent pathways

Silver nanoparticles (AgNPs) are incorporated into a large number of consumer and medical products. Several experiments have demonstrated that AgNPs can be toxic to the vital organs of humans and especially to the lung. The present study evaluated the in vitro mechanisms of AgNP (<100 nm) toxicity in relationship to the generation of reactive oxygen species (ROS) in A549 cells. AgNPs caused ROS formation in the cells, a reduction in their cell viability and mitochondrial membrane potential (MMP), an increase in the proportion of cells in the sub-G1 (apoptosis) population, S phase arrest and down-regulation of the cell cycle associated proliferating cell nuclear antigen (PCNA) protein, in a concentration- and time-dependent manner. Pretreatment of the A549 cells with N-acetyl-cysteine (NAC), an antioxidant, decreased the effects of AgNPs on the reduced cell viability, change in the MMP and proportion of cells in the sub-G1population, but had no effect on the AgNP-mediated S phase arrest or down-regulation of PCNA. These observations allow us to propose that the in vitro toxic effects of AgNPs on A549 cells are mediated via both ROS-dependent (cytotoxicity) and ROS-independent (cell cycle arrest) pathways.     

Evaluation of cytotoxic,genotoxic and inflammatory response in human alveolar and bronchial epithelial cells exposed to titanium dioxide nanoparticles

The toxicity of titanium dioxide nanoparticles (TiO₂‐NPs), used in several applications, seems to be influenced by their specific physicochemical characteristics. Cyto‐genotoxic and inflammatory effects induced by a mixture of 79% anatase/21% rutile TiO₂‐NPs were investigated in human alveolar (A549) and bronchial (BEAS‐2B) cells exposed to 1–40 µg ml^–1 30 min, 2 and 24 h to assess potential pulmonary toxicity. The specific physicochemical properties such as crystallinity, NP size and shape, agglomerate size, surface charge and specific surface area (SSA) were analysed. Cytotoxic effects were studied by evaluating cell viability using the WST1 assay and membrane damage using LDH analysis. Direct/oxidative DNA damage was assessed by the Fpg‐comet assay and the inflammatory potential was evaluated as interleukin (IL)‐6, IL‐8 and tumour necrosis factor (TNF)‐α release by enzyme‐linked immunosorbant assay (ELISA). In A549 cells no significant viability reduction and moderate membrane damage, only at the highest concentration, were detected, whereas BEAS‐2B cells showed a significant viability reduction and early membrane damage starting from 10 µg ml^–1. Direct/oxidative DNA damage at 40 µg ml^–1 and increased IL‐6 release at 5 µg ml^–1 were found only in A549 cells after 2 h. The secretion of pro‐inflammatory cytokine IL‐6, involved in the early acute inflammatory response, and oxidative DNA damage indicate the promotion of early and transient oxidative‐inflammatory effects of tested TiO₂‐NPs on human alveolar cells. The findings show a higher susceptibility of normal bronchial cells to cytotoxic effects and higher responsiveness of transformed alveolar cells to genotoxic, oxidative and early inflammatory effects induced by tested TiO₂‐NPs. This different cell behaviour after TiO₂‐NPs exposure suggests the use of both cell lines and multiple end‐points to elucidate NP toxicity on the respiratory system. Copyright © 2014 John Wiley & Sons, Ltd.     

Tephrosin-induced autophagic cell death in A549 non-small cell lung cancer cells

Anticancer effect of tephrosin (1) has been documented; however, the molecular mechanisms underlying the cytotoxicity of tephrosin in cancer cells remain unclear. In the present paper, the proliferation inhibition rate of several cancer cells was tested using the MTT assay; cell cycle, reactive oxygen species (ROS), and mitochondrial membrane potential (MMP) were determined by flow cytometry; poly(ADP-ribose) polymerase (PARP) cleavage and heat shock protein 90 (Hsp90) expression were evaluated by Western blotting; autophagy was examined by confocal microscopy and light chain 3 (LC3) conversion assay. The results showed that exposure of the cells to tephrosin induced significant proliferation inhibition in a dose-dependent manner, especially on A549 with G₂/M being arrested. Tephrosin was not found to induce cell apoptosis as PARP cleavage was not detected after 24 h treatment, but the formation of acidic vesicular organelle of autophagy character was found, and autophagy was further confirmed by the increase in the ratio of LC3-II to LC3-I. It was observed that tephrosin induced ROS generation and Hsp90 expression inhibition. These results indicate that tephrosin induces A549 cancer cell death via the autophagy pathway, and the roles of ROS generation and Hsp90 expression inhibition in this process need further study in the future.     

Tephrosin-induced autophagic cell death in A549 non-small cell lung cancer cells

Anticancer effect of tephrosin (1) has been documented; however, the molecular mechanisms underlying the cytotoxicity of tephrosin in cancer cells remain unclear. In the present paper, the proliferation inhibition rate of several cancer cells was tested using the MTT assay; cell cycle, reactive oxygen species (ROS), and mitochondrial membrane potential (MMP) were determined by flow cytometry; poly(ADP-ribose) polymerase (PARP) cleavage and heat shock protein 90 (Hsp90) expression were evaluated by Western blotting; autophagy was examined by confocal microscopy and light chain 3 (LC3) conversion assay. The results showed that exposure of the cells to tephrosin induced significant proliferation inhibition in a dose-dependent manner, especially on A549 with G(2)/M being arrested. Tephrosin was not found to induce cell apoptosis as PARP cleavage was not detected after 24?h treatment, but the formation of acidic vesicular organelle of autophagy character was found, and autophagy was further confirmed by the increase in the ratio of LC3-II to LC3-I. It was observed that tephrosin induced ROS generation and Hsp90 expression inhibition. These results indicate that tephrosin induces A549 cancer cell death via the autophagy pathway, and the roles of ROS generation and Hsp90 expression inhibition in this process need further study in the future.     

Mechanisms of crystalline silica-induced pulmonary toxicity revealed by global gene expression profiling

A proper understanding of the mechanisms underlying crystalline silica-induced pulmonary toxicity has implications in the management and potential prevention of the adverse health effects associated with silica exposure including silicosis, cancer and several auto-immune diseases. Human lung type II epithelial cells and rat lungs exposed to crystalline silica were employed as experimental models to determine global gene expression changes in order to understand the molecular mechanisms underlying silica-induced pulmonary toxicity. The differential gene expression profile induced by silica correlated with its toxicity in the A549 cells. The biological processes perturbed by silica exposure in the A549 cells and rat lungs, as identified by the bioinformatics analysis of the differentially expressed genes, demonstrated significant similarity. Functional categorization of the differentially expressed genes identified cancer, cellular movement, cellular growth and proliferation, cell death, inflammatory response, cell cycle, cellular development, and genetic disorder as top ranking biological functions perturbed by silica exposure in A549 cells and rat lungs. Results of our study, in addition to confirming several previously identified molecular targets and mechanisms involved in silica toxicity, identified novel molecular targets and mechanisms potentially involved in silica-induced pulmonary toxicity. Further investigations, including those focused on the novel molecular targets and mechanisms identified in the current study may result in better management and, possibly, reduction and/or prevention of the potential adverse health effects associated with crystalline silica exposure.     

Mechanisms of crystalline silica-induced pulmonary toxicity revealed by global gene expression profiling

A proper understanding of the mechanisms underlying crystalline silica-induced pulmonary toxicity has implications in the management and potential prevention of the adverse health effects associated with silica exposure including silicosis, cancer and several auto-immune diseases. Human lung type II epithelial cells and rat lungs exposed to crystalline silica were employed as experimental models to determine global gene expression changes in order to understand the molecular mechanisms underlying silica-induced pulmonary toxicity. The differential gene expression profile induced by silica correlated with its toxicity in the A549 cells. The biological processes perturbed by silica exposure in the A549 cells and rat lungs, as identified by the bioinformatics analysis of the differentially expressed genes, demonstrated significant similarity. Functional categorization of the differentially expressed genes identified cancer, cellular movement, cellular growth and proliferation, cell death, inflammatory response, cell cycle, cellular development, and genetic disorder as top ranking biological functions perturbed by silica exposure in A549 cells and rat lungs. Results of our study, in addition to confirming several previously identified molecular targets and mechanisms involved in silica toxicity, identified novel molecular targets and mechanisms potentially involved in silica-induced pulmonary toxicity. Further investigations, including those focused on the novel molecular targets and mechanisms identified in the current study may result in better management and, possibly, reduction and/or prevention of the potential adverse health effects associated with crystalline silica exposure.     

Differential cytotoxicity of copper ferrite nanoparticles in different human cells

Copper ferrite nanoparticles (NPs) have the potential to be applied in biomedical fields such as cell labeling and hyperthermia. However, there is a lack of information concerning the toxicity of copper ferrite NPs. We explored the cytotoxic potential of copper ferrite NPs in human lung (A549) and liver (HepG2) cells. Copper ferrite NPs were crystalline and almost spherically shaped with an average diameter of 35 nm. Copper ferrite NPs induced dose‐dependent cytotoxicity in both types of cells, evident by 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazoliumbromide and neutral red uptake assays. However, we observed a quite different susceptibility in the two kinds of cells regarding toxicity of copper ferrite NPs. Particularly, A549 cells showed higher susceptibility against copper ferrite NP exposure than those of HepG2 cells. Loss of mitochondrial membrane potential due to copper ferrite NP exposure was observed. The mRNA level as well as activity of caspase‐3 enzyme was higher in cells exposed to copper ferrite NPs. Cellular redox status was disturbed as indicated by induction of reactive oxygen species (oxidant) generation and depletion of the glutathione (antioxidant) level. Moreover, cytotoxicity induced by copper ferrite NPs was efficiently prevented by N‐acetylcysteine treatment, which suggests that reactive oxygen species generation might be one of the possible mechanisms of cytotoxicity caused by copper ferrite NPs. To the best of our knowledge, this is the first report showing the cytotoxic potential of copper ferrite NPs in human cells. This study warrants further investigation to explore the mechanisms of differential toxicity of copper ferrite NPs in different types of cells. Copyright © 2016 John Wiley & Sons, Ltd.     

Chloropicrin-induced toxic responses in human lung epithelial cells

Chloropicrin is a slowly evaporating toxic irritant that is known to cause damage in the respiratory system. Here we used a lung epithelial cell line (A549) to study the molecular responses underlying chloropicrin toxicity. Glutathione (GSH), synthetic peptide and 2′-deoxyguanosine were used as in vitro trapping agents to identify early markers of chloropicrin toxicity. Microscopy of the cells revealed massive vacuolization by chloropicrin exposure (80–100 μM). The number of apoptotic cells increased with the chloropicrin concentration as assessed by flow cytometry. Immunoblotting analysis revealed increases in the amount of four proteins (p53, p21, p27 and phospho-Erk1/2) that are involved in DNA-damage, cell cycle regulation and apoptosis. Chloropicrin evoked a dose-dependent increase in levels of reactive oxygen species within one hour of exposure. The treatment triggered also the formation of disulphide bonds between the model thiol-containing peptides as analysed by LC/MS. Chloropicrin did not form stable adducts with the model peptides or 2′-deoxyguanosine. N-acetyl-cysteine (1 mM NAC) fully prevented the vacuoles and chloropicrin-induced cytotoxicity. The results suggest that an oxidative insult, particularly modification of free sulfhydryl groups in proteins is involved in the acute toxicity evoked by chloropicrin in airway epithelial cells. The protective effect of NAC as a potential antidote in chloropicrin intoxication will require further investigation.     

Neurobehavioral effects of tetrabromobisphenol A,a brominated flame retardant,in mice

Tetrabromobisphenol A (TBBPA) is widely used as a flame retardant and is suspected to be stable in the environment with possible widespread human exposures. In the present study, we investigated the behavioral effects of TBBPA and measured the levels of TBBPA in the brain after oral administration in mice. Acute treatment with TBBPA (5 mg/kg body weight) 3 h before the open-field test induced an increase in the horizontal movement activities. In contextual fear conditioning paradigm, mice treated with TBBPA (0.1 mg/kg or 5 mg/kg body weight) showed more freezing behavior than vehicle-treated mice. In addition, TBBPA (0.1 mg/kg body weight) significantly increased the spontaneous alternation behavior in the Y-maze test. The levels of TBBPA in the brain following TBBPA treatment were determined by using LC/ESI-MS/MS system. In the brain regions examined, high amounts of TBBPA were detected in the striatum after treatment with 0.1 mg/kg or 5 mg/kg body weight TBBPA, whereas non-specific accumulation of TBBPA in the brain was found after treatment with 250 mg/kg body weight TBBPA. These results suggest that TBBPA accumulates in brain regions including the striatum and induces the behavioral alterations. Together, the possibility of widespread human exposure to TBBPA warrants further studies to characterize its neurotoxicity.     

Exposure to dibenzofuran triggers autophagy in lung cells

Environmental pollutants, such as dioxins and furans, are extremely toxic and related with pulmonary disease development. Exposure of A549 human lung cells to dibenzofuran showed both time- and concentration-dependent decreases in cell proliferation and MTT reduction, but no alterations in cell viability. No differences were observed in the number of apoptotic nuclei, which can be due to the energetic failure caused by dibenzofuran-induced ATP depletion. Moreover, cells in culture exposed to the pollutant showed an increase in the conversion of LC3, a protein involved in the autophagic process. Incubation of A549 lung cells with dibenzofuran caused an increase in Lysotracker Red staining, indicating an increase in lysosomal vacuoles content. These results suggest that exposure to dibenzofuran affects lung mitochondrial phosphorylative function, causing an increase in the population of dysfunctional mitochondria and an impairment in the energetic status maintenance, therefore stimulating autophagy as a possible rescue mechanism in this cell line.     

Lipopolysaccharide induces apoptotic insults to human alveolar epithelial A549 cells through reactive oxygen species-mediated activation of an intrinsic mitochondrion-dependent pathway

Alveolar type II epithelial cells can regulate immune responses to sepsis-induced acute lung injury. Lipopolysaccharide (LPS), an outer membrane component of Gram-negative bacteria, can cause septic shock. This study was designed to evaluate the cytotoxic effects of LPS on human alveolar epithelial A549 cells and its possible molecular mechanisms. Exposure of A549 cells to LPS decreased cell viability in concentration- and time-dependent manners. In parallel, LPS concentration- and time-dependently induced apoptosis of A549 cells. Meanwhile, LPS only at a high concentration of 10 μg/ml caused mildly necrotic insults to A549 cells. In terms of the mechanism, exposure of A549 cells to LPS increased the levels of cellular nitric oxide and reactive oxygen species (ROS). Pretreatment with N-acetylcysteine (NAC), an antioxidant, significantly lowered LPS-caused enhancement of intracellular ROS in A549 cells and simultaneously attenuated the apoptotic insults. Sequentially, treatment of A549 cells with LPS caused significant decreases in the mitochondrial membrane potential and biosynthesis of adenosine triphosphate. In succession, LPS triggered the release of cytochrome c from the mitochondria to the cytoplasm. Activities of caspase-9 and caspase-6 were subsequently augmented following LPS administration. Consequently, exposure of A549 cells induced DNA fragmentation in a time-dependent manner. Pretreatment of A549 cells with NAC significantly ameliorated LPS-caused alterations in caspase-9 activation and DNA damage. Therefore, this study shows that LPS specifically induces apoptotic insults to human alveolar epithelial cells through ROS-mediated activation of the intrinsic mitochondrion–cytochrome c-caspase protease mechanism.     

Evaluation of pulmonary toxicity of benzalkonium chloride and triethylene glycol mixtures using in vitro and in vivo systems

Benzalkonium chloride (BAC) is a widely used disinfectant/preservative, and respiratory exposure to this compound has been reported to be highly toxic. Spray‐form household products have been known to contain BAC together with triethylene glycol (TEG) in their solutions. The purpose of this study was to estimate the toxicity of BAC and TEG mixtures to pulmonary organs using in vitro and in vivo experiments. Human alveolar epithelial (A549) cells incubated with BAC (1‐10 μg/mL) for 24 hours showed significant cytotoxicity, while TEG (up to 1000 μg/mL) did not affect cell viability. However, TEG in combination with BAC aggravated cell damage and inhibited colony formation as compared to BAC alone. TEG also exacerbated BAC‐promoted production of reactive oxygen species (ROS) and reduction of glutathione (GSH) level in A549 cells. However, pretreatment of the cells with N‐acetylcysteine (NAC) alleviated the cytotoxicity, indicating oxidative stress could be a mechanism of the toxicity. Quantification of intracellular BAC by LC/MS/MS showed that cellular distribution/absorption of BAC was enhanced in A549 cells when it was exposed together with TEG. Intratracheal instillation of BAC (400 μg/kg) in rats was toxic to the pulmonary tissues while that of TEG (up to 1000 μg/kg) did not show any harmful effect. A combination of nontoxic doses of BAC (200 μg/kg) and TEG (1000 μg/kg) promoted significant lung injury in rats, as shown by increased protein content and lactate dehydrogenase (LDH) activity in bronchoalveolar lavage fluids (BALF). Moreover, BAC/TEG mixture recruited inflammatory cells, polymorphonuclear leukocytes (PMNs), in terminal bronchioles and elevated cytokine levels, tumor necrosis factor α (TNF‐α), and interleukin 6 (IL‐6) in BALF. These results suggest that TEG can potentiate BAC‐induced pulmonary toxicity and inflammation, and thus respiratory exposure to the air mist from spray‐form products containing this chemical combination is potentially harmful to humans.