Evaluation of cytotoxic,oxidative stress,proinflammatory and genotoxic effect of silver nanoparticles in human lung epithelial cells

Silver nanoparticles are increasingly used in various products, due to their antibacterial properties. Despite its wide spread use, only little information on possible adverse health effects exists. Therefore, the aim of this study was to assess the toxic potential of silver nanoparticles (<100 nm) in human lung epithelial (A549) cells and the underlying mechanism of its cellular toxicity. Silver nanoparticles induced dose and time‐dependent cytotoxicity in A549 cells demonstrated by MTT and LDH assays. Silver nanoparticles were also found to induce oxidative stress in dose and time‐dependent manner indicated by depletion of GSH and induction of ROS, LPO, SOD, and catalase. Further, the activities of caspases and the level of proinflammatory cytokines, namely interleukin‐1β (IL‐1β) and interleukin‐6 (IL‐6) were significantly higher in treated cells. DNA damage, as measured by single cell gel electrophoresis, was also dose and time‐dependent signicants in A549 cells. This study investigating the effects of silver nanoparticles in human lung epithelial cells has provided valuable insights into the mechanism of potential toxicity induced by silver nanoparticles and warrants more careful assessment of silver nanoparticles before their industrial applications. © 2013 Wiley Periodicals, Inc. Environ Toxicol 30: 149–160, 2015.     

Indium oxide nanoparticles induce lung intercellular toxicity between bronchial epithelial cells and macrophages

Concerns have been raised over the safety and health of industrial workers exposed to indium oxide nanoparticles (IO-NPs) when working. IO-NPs were previously shown in vitro and in vivo to be cytotoxic, but the mechanism of pathogenesis was unclear. In this study, the effects of IO-NPs on lung cells associated with respiratory and immune barriers and the toxic effects of intercellular cascades were studied. Here IO-NPs had acute toxicity to Wistar rats over a time course (5 days post-intratracheal instillation). Following treatment epithelial cells (16HBE) or macrophages (RAW264.7) with IO-NPs or IO fine particles (IO-FPs), the damage of 16HBE cells caused by IO-NPs was serious, mainly in the mitochondrial and rough endoplasmic reticulum. The lactate dehydrogenase level also showed that cytotoxicity in vitro was more serious for IO-NPs compared with IO-FPs. The level of In³⁺ (examined by inductively coupled plasma mass spectrometry) in 16HBE cells was 10 times higher than that in RAW cells. In³⁺, releasing from IO-NPs absorbed by 16HBE cells, could not only significantly inhibit the phagocytosis and migration of macrophages (P < .0001), but also stimulate RAW cells to secrete high levels of inflammatory cytokines. IO-NPs can directly damage pulmonary epithelial cells. The In³⁺ released by epithelial cells affect the phagocytosis and migration of macrophages, which may be a new point for the decrease in the clearance of alveolar surfactants and the development of IO-related pulmonary alveolar proteinosis.     

Comparison of manganese oxide nanoparticles and manganese sulfate with regard to oxidative stress, uptake and apoptosis in alveolar epithelial cells

Due to their physicochemical characteristics, metal oxide nanoparticles (NPs) interact differently with cells compared to larger particles or soluble metals. Oxidative stress and cellular metal uptake were quantified in rat type II alveolar epithelial cells in culture exposed to three different NPs: manganese(II,III) oxide nanoparticles (Mn₃O₄-NPs), the soluble manganese sulfate (Mn-salt) at corresponding equivalent doses, titanium dioxide (TiO₂-NPs) and cerium dioxide nanoparticles (CeO₂-NPs). In the presence of reactive oxygen species an increased apoptosis rate was hypothesized. Oxidative stress was assessed by detection of fluorescently labeled reactive oxygen species and by measuring intracellular oxidized glutathione. Catalytic activity was determined by measuring catalyst-dependent oxidation of thiols (DTT-assay) in a cell free environment. Inductively coupled plasma mass spectrometry was used to quantify cellular metal uptake. Apoptosis rate was determined assessing the activity of caspase-3 and by fluorescence microscopic quantification of apoptotic nuclei. Reactive oxygen species were mainly generated in cells treated with Mn₃O₄-NPs. Only Mn₃O₄-NPs oxidized intracellular glutathione. Catalytic activity could be exclusively shown for Mn₃O₄-NPs. Cellular metal uptake was similar for all particles, whereas Mn-salt could hardly be detected within the cell. Apoptosis was induced by both, Mn₃O₄-NPs and Mn-salt. The combination of catalytic activity and capability of passing the cell membrane contributes to the toxicity of Mn₃O₄-NPs. Apoptosis of samples treated with Mn-salt is triggered by different, potentially extracellular mechanisms.     

Toxic response of nickel nanoparticles in human lung epithelial A549 cells

Nickel nanoparticle (Ni NP) is increasingly used in modern industries such as catalysts, sensors and electronic applications. Due to wide-spread industrial applications the inhalation is the primary source of exposure to Ni NPs. However, data demonstrating the effect of Ni NPs on the pulmonary system remain scarce. The present study was designed to examine the toxic effect of human lung epithelial A549 cells treated with well characterized Ni NPs at the concentrations of 0, 1, 2, 5, 10 and 25 μg/ml for 24 and 48 h. Mitochondrial function (MTT assay), membrane leakage of lactate dehydrogenase (LDH assay), reduced glutathione (GSH), reactive oxygen species (ROS), membrane lipid peroxidation (LPO) and caspase-3 activity were assessed as toxicity end points. Results showed that Ni NPs reduced mitochondrial function and induced the leakage of LDH in dose and time-dependent manner. Ni NPs were also found to induce oxidative stress in dose and time-dependent manner indicated by depletion of GSH and induction of ROS and LPO. Further, activity of caspase-3 enzyme, marker of apoptosis was significantly higher in treated cells with time and Ni NPs dosage. The results exhibited significant toxicity of Ni NPs in human lung epithelial A549 cells which is likely to be mediated through oxidative stress. This study warrants more careful assessment of Ni NPs before their industrial applications.     

Copper oxide nanoparticles induce oxidative stress and cytotoxicity in airway epithelial cells

Metal oxide nanoparticles are often used as industrial catalysts and elevated levels of these particles have been clearly demonstrated at sites surrounding factories. To date, limited toxicity data on metal oxide nanoparticles are available. To understand the impact of these airborne pollutants on the respiratory system, airway epithelial (HEp-2) cells were exposed to increasing doses of silicon oxide (SiO₂), ferric oxide (Fe₂O₃) and copper oxide (CuO) nanoparticles, the leading metal oxides found in ambient air surrounding factories. CuO induced the greatest amount of cytotoxicity in a dose-dependent manner; while even high doses (400 μg/cm²) of SiO₂ and Fe₂O₃ were non-toxic to HEp-2 cells. Although all metal oxide nanoparticles were able to generate ROS in HEp-2 cells, CuO was better able to overwhelm antioxidant defenses (e.g. catalase and glutathione reductase). A significant increase in the level of 8-isoprostanes and in the ratio of GSSG to total glutathione in cells exposed to CuO suggested that ROS generated by CuO induced oxidative stress in HEp-2 cells. Co-treatment of cells with CuO and the antioxidant resveratrol increased cell viability suggesting that oxidative stress may be the cause of the cytotoxic effect of CuO. These studies demonstrated that there is a high degree of variability in the cytotoxic effects of metal oxides, that this variability is not due to the solubility of the transition metal, and that this variability appears to involve sustained oxidative stress possibly due to redox cycling.     

Concentration‐dependent cytokine responses of silica nanoparticles and role of ROS in human lung epithelial cells

Reactive oxygen species (ROS) is regarded as a critical denominator in nanoparticle toxicology and inflammation. Previously, we have shown that silica nanoparticles sized 50 nm (Si50) induce release of CXCL8 and IL‐6 from BEAS‐2B cells, via mechanisms involving NFκB, p38 MAP kinase and TGF‐α‐activated EGF receptor. In the present study, the role of ROS‐mediated mechanisms in the concentration‐dependent Si50 induction of CXCL8 and IL‐6 responses was examined. Si50 (200 µg/mL) induced a time‐dependent ROS formation and a postponed increase in expression of haem oxygenase (HO‐1) mRNA and protein. Pre‐treatment with the ROS inhibitors N‐acetyl cysteine (NAC) and diphenyleneiodonium (DPI) partially attenuated CXCL8 and IL‐6 responses to 200 µg/mL, but not to 100 µg/mL Si50. The release of TGF‐α induced by Si50 (200 µg/mL) was significantly reduced by NAC, but not by DPI nor siRNA against NADPH oxidase DUOX‐1 (siDUOX‐1). Furthermore, siDUOX‐1 reduced Si50‐induced CXCL8, but not IL‐6. Both p38 and p65 phosphorylations were inhibited by siDUOX‐1, but for NAC only p65 phosphorylation reached a significant reduction. Neither NAC nor DPI reduced Si50‐induced CXCL8 and IL‐6 gene expressions. In conclusion, Si50‐induced CXCL8 and IL‐6 involved both ROS‐dependent and ROS‐independent mechanisms. Notably, the role of ROS seemed restricted to effects of higher concentrations of Si50 and not mediated via the gene expression.     

The effect of Fe2O3 and ZnO nanoparticles on cytotoxicity and glucose metabolism in lung epithelial cells

Metallic nanoparticles (NPs) have potential applications in industry and medicine, but they also have the potential to cause many chronic pulmonary diseases. Mechanisms for their cytotoxicity, glucose and energy metabolism responses need to be fully explained in lung epithelial cells after treatment with metallic nanoparticles. In our study, two different metallic nanoparticles (Fe₂O₃ and ZnO) and two cell‐based assays (BEAS‐2B and A549 cell lines) were used. Our findings demonstrate that ZnO nanoparticles, but not Fe₂O₃ nanoparticles, induce cell cycle arrest, cell apoptosis, reactive oxygen species (ROS) production, mitochondrial dysfunction and glucose metabolism perturbation, which are responsible for cytotoxicity. These results also suggest that the glucose metabolism and bioenergetics had a great potential in evaluating the cytotoxicity and thus were very helpful in understanding their underlying molecular mechanisms. Copyright © 2015 John Wiley & Sons, Ltd.     

氧化亚铜纳米粒对B16细胞上皮间质转化的影响

目的 制备氧化亚铜纳米粒（cuprous oxide nanoparticles,CONPs）并研究对小鼠黑色素瘤B16细胞上皮间质转化的影响。方法 采用水热法制备氧化亚铜纳米粒。将B16细胞分为正常培养组和CONPs（5、25、50 μg/ml）刺激培养组,于倒置相差显微镜下观察细胞形态学的变化,通过细胞划痕实验和Transwell检测CONPs对B16细胞迁移能力的影响,采用免疫荧光染色法和蛋白质印迹法检测B16细胞表型的相关分子标志物表达的变化。结果 合成的氧化亚铜纳米粒分布均匀,粒径约40 nm;体外细胞实验发现CONPs明显地抑制了B16细胞的迁移和侵袭能力;并刺激B16细胞上皮细胞表型的E-cadherin、Cytokeratin、Desmoplakin的表达明显升高,而间质细胞表型的N-cadherin、Vimentin的表达降低。结论 CONPs能明显地抑制黑色素瘤细胞的侵袭转移和上皮-间质转化（EMT）过程。     

Thiolated polyacrylic acid-modified iron oxide nanoparticles for in vitro labeling and MRI of stem cells

Purpose: The purpose of this study was to develop and characterize new surface-modified iron oxide nanoparticles demonstrating the efficiency to be internalized by human endothelial progenitor cells (EPCs) from umbilical cord blood.

Methods: Iron oxide nanoparticles were coated with polyacrylic acid-cysteine (PAA-Cys) by either in situ precipitation or postsynthesis. The nanoparticles were characterized by X-ray powder diffraction. EPCs were labeled with PAA-Cys-modified iron oxide nanoparticles or with uncoated nanoparticles. The relaxivity of uncoated and coated iron oxide nanoparticles as well as EPCs labeled with PAA-Cys-modified iron oxide were determined.

Results: Addition of PAA-Cys increased the particle size from 10.4 to 144 and 197?nm, respectively. The X-ray powder diffraction pattern revealed that the particles consist of Fe₃O₄ with a spinal structure. Postsynthesis coated particles showed a cellular uptake of 85% and 15.26 pg iron/cell. For both types of particles the relaxivity ratio was at least 2-fold higher than that of the gold standard Resovist^®.

Conclusion: The PAA-Cys coated iron oxide nanoparticles are a promising tool for labeling living cells such as stem cells for diagnostic and therapeutic application in cell-based therapies due to their high relaxivities and their easy uptake by cells.     

No evidence of the genotoxic potential of gold,silver, zinc oxide and titanium dioxide nanoparticles in the SOS chromotest

Gold nanoparticles (Au NPs), silver nanoparticles (Ag NPs), zinc oxide nanoparticles (ZnO NPs) and titanium dioxide nanoparticles (TiO₂ NPs) are widely used in cosmetic products such as preservatives, colorants and sunscreens. This study investigated the genotoxicity of Au NPs, Ag NPs, ZnO NPs and TiO₂ NPs using the SOS chromotest with Escherichia coli PQ37. The maximum exposure concentrations for each nanoparticle were 3.23 mg l^–1 for Au NPs, 32.3 mg l^–1 for Ag NPs and 100 mg l^–1 for ZnO NPs and TiO₂ NPs. Additionally, in order to compare the genotoxicity of nanoparticles and corresponding dissolved ions, the ions were assessed in the same way as nanoparticles. The genotoxicity of the titanium ion was not assessed because of the extremely low solubility of TiO₂ NPs. Au NPs, Ag NPs, ZnO NPs, TiO₂ NPs and ions of Au, Ag and Zn, in a range of tested concentrations, exerted no effects in the SOS chromotest, evidenced by maximum IF (IFmax) values of below 1.5 for all chemicals. Owing to the results, nanosized Au NPs, Ag NPs, ZnO NPs, TiO₂ NPs and ions of Au, Ag and Zn are classified as non‐genotoxic on the basis of the SOS chromotest used in this study. To the best of our knowledge, this is the first study to evaluate the genotoxicity of Au NPs, Ag NPs, ZnO NPs and TiO₂ NPs using the SOS chromotest. Copyright © 2012 John Wiley & Sons, Ltd.     

Comparative study of cyto‐ and genotoxic potential with mechanistic insights of tungsten oxide nano‐ and microparticles in lung carcinoma cells

The exigency of semiconductor and super capacitor tungsten oxide nanoparticles (WO₃ NPs) is increasing in various sectors. However, limited information on their toxicity and biological interactions are available. Hence, we explored the underlying mechanisms of toxicity induced by WO₃ NPs and their microparticles (MPs) using different concentrations (0–300 μg ml^–1) in human lung carcinoma (A549) cells. The mean size of WO₃ NPs and MPs by transmission electron microscopy was 53.84 nm and 3.88 μm, respectively. WO₃ NPs induced reduction in cell viability, membrane damage and the degree of induction was size‐ and dose‐dependent. There was a significant increase in the percentage tail DNA and micronuclei formation at 200 and 300 μg ml^–1 after 24 hours of exposure. The DNA damage induced by WO₃ NPs could be attributed to increased oxidative stress and inflammation through reactive oxygen species generation, which correlated with the depletion of reduced glutathione content, catalase and an increase in malondialdehyde levels. Cellular uptake studies unveiled that both the particles were attached/surrounded to the cell membrane according to their size. In addition, NP inhibited the progression of the cell cycle in the G₂/M phase. Other studies such as caspase‐9 and ‐3 and Annexin‐V‐fluorescein isothiocyanate revealed that NPs induced intrinsic apoptotic cell death at 200 and 300 μg ml^–1 concentrations. However, in comparison to NPs, WO₃ MPs did not incite any toxic effects at the tested concentrations. Under these experimental conditions, the no‐observed‐significant‐effect level of WO₃ NPs was determined to be ≤200 μg ml^–1 in A549 cells.     

Airway irritation,inflammation, and toxicity in mice following inhalation of metal oxide nanoparticles

Metal oxide nanoparticles are used in a broad range of industrial processes and workers may be exposed to aerosols of the particles both during production and handling. Despite the widespread use of these particles, relatively few studies have been performed to investigate the toxicological effects in the airways following inhalation. In the present study, the acute (24?h) and persistent (13 weeks) effects in the airways after a single exposure to metal oxide nanoparticles were studied using a murine inhalation model. Mice were exposed 60?min to aerosols of either ZnO, TiO₂, Al₂O₃ or CeO₂ and the deposited doses in the upper and lower respiratory tracts were calculated. Endpoints were acute airway irritation, pulmonary inflammation based on analyses of bronchoalveolar lavage (BAL) cell composition, DNA damage assessed by the comet assay and pulmonary toxicity assessed by protein level in BAL fluid and histology. All studied particles reduced the tidal volume in a concentration-dependent manner accompanied with an increase in the respiratory rate. In addition, ZnO and TiO₂ induced nasal irritation. BAL cell analyses revealed both neutrophilic and lymphocytic inflammation 24-h post-exposure to all particles except TiO₂. The ranking of potency regarding induction of acute lung inflammation was Al₂O₃ = TiO_2?<_?CeO₂ ? ZnO. Exposure to CeO₂ gave rise to a more persistent inflammation; both neutrophilic and lymphocytic inflammation was seen 13 weeks after exposure. As the only particles, ZnO caused a significant toxic effect in the airways while TiO₂ gave rise to DNA-strand break as shown by the comet assay.     

Zinc oxide nanoparticles inhibit Ca2+-ATPase expression in human lens epithelial cells under UVB irradiation

Epidemiological and experimental studies have revealed that lens epithelial cells exposed to ultraviolet B (UVB) light could be induced apoptosis, and lens epithelial cell apoptosis can initiate cataractogenesis. Posterior capsular opacification (PCO), the most frequent complication after cataract surgery, is induced by the proliferation, differentiation, migration of lens epithelial cells. Thus, inhibiting the proliferation of lens epithelial cells could reduce the occurrence of PCO. It is reported that zinc oxide (ZnO) nanoparticles have great potential for the application of biomedical field including cancer treatment. In the present study, we investigated the cytotoxic effect of ZnO nanoparticles on human lens epithelial cell (HLEC) viability. In addition, changes in cell nuclei, apoptosis, reactive oxygen species and intracellular calcium ion levels were also investigated after cells treated with ZnO nanoparticles in the presence and absence of UVB irradiation. Meanwhile, the expression of plasma membrane calcium ATPase 1 (PMCA1) was also determined at gene and protein levels. The results indicate that ZnO nanoparticles and UVB irradiation have synergistic inhibitory effect on HLEC proliferation in a concentration-dependent manner. ZnO nanoparticles can increase the intracellular calcium ion level, disrupt the intracellular calcium homeostasis, and decrease the expression level of PMCA1. UVB irradiation can strengthen the effect of reduced expression of PMCA1, suggesting that both UVB irradiation and ZnO nanoparticles could exert inhibitory effect on HLECs via calcium-mediated signaling pathway. ZnO nanoparticles have great potential for the treatment of PCO under UVB irradiation.     

Nickel compounds induce apoptosis in human bronchial epithelial Beas-2B cells by activation of c-Myc through ERK pathway

Nickel compounds are carcinogenic to humans and have been shown to alter epigenetic homeostasis. The c-Myc protein controls 15% of human genes and it has been shown that fluctuations of c-Myc protein alter global epigenetic marks. Therefore, the regulation of c-Myc by nickel ions in immortalized but not tumorigenic human bronchial epithelial Beas-2B cells was examined in this study. It was found that c-Myc protein expression was increased by nickel ions in non-tumorigenic Beas-2B and human keratinocyte HaCaT cells. The results also indicated that nickel ions induced apoptosis in Beas-2B cells. Knockout of c-Myc and its restoration in a rat cell system confirmed the essential role of c-Myc in nickel ion-induced apoptosis. Further studies in Beas-2B cells showed that nickel ion increased the c-Myc mRNA level and c-Myc promoter activity, but did not increase c-Myc mRNA and protein stability. Moreover, nickel ion upregulated c-Myc in Beas-2B cells through the MEK/ERK pathway. Collectively, the results demonstrate that c-Myc induction by nickel ions occurs via an ERK-dependent pathway and plays a crucial role in nickel-induced apoptosis in Beas-2B cells.     

In vitro cytotoxicity of superparamagnetic iron oxide nanoparticles on neuronal and glial cells. Evaluation of nanoparticle interference with viability tests

Superparamagnetic iron oxide nanoparticles (ION) have attracted great interest for use in several biomedical fields. In general, they are considered biocompatible, but little is known of their effects on the human nervous system. The main objective of this work was to evaluate the cytotoxicity of two ION (magnetite), coated with silica and oleic acid, previously determining the possible interference of the ION with the methodological procedures to assure the reliability of the results obtained. Human neuroblastoma SHSY5Y and glioblastoma A172 cells were exposed to different concentrations of ION (5–300 µg ml^–1), prepared in complete and serum‐free cell culture medium for three exposure times (3, 6 and 24 h). Cytotoxicity was evaluated by means of the MTT, neutral red uptake and alamar blue assays. Characterization of the main physical–chemical properties of the ION tested was also performed. Results demonstrated that both ION could significantly alter absorbance readings. To reduce these interferences, protocols were modified by introducing additional washing steps and cell‐free systems. Significant decreases in cell viability were observed for both cell lines in specific conditions by all assays. In general, oleic acid‐coated ION were less cytotoxic than silica‐coated ION; besides, a serum‐protective effect was observed for both ION studied and cell lines. These results contribute to increase the knowledge of the potential harmful effects of ION on the human nervous system. Understanding these effects is essential to establish satisfactory regulatory policies on the safe use of magnetite nanoparticles in biomedical applications. Copyright © 2015 John Wiley & Sons, Ltd.     

Silver nanoparticles induce anti-proliferative effects on airway smooth muscle cells. Role of nitric oxide and muscarinic receptor signaling pathway

Silver nanoparticles (AgNPs) are used to manufacture materials with new properties and functions. However, little is known about their toxic or beneficial effects on human health, especially in the respiratory system, where its smooth muscle (ASM) regulates the airway contractility by different mediators, such as acetylcholine (ACh) and nitric oxide (NO). The aim of this study was to evaluate the effects of AgNPs on ASM cells. Exposure to AgNPs induced ACh-independent expression of the inducible nitric oxide synthase (iNOS) at 100 μg/mL, associated with excessive production of NO. AgNPs induced the muscarinic receptor activation, since its blockage with atropine and blockage of its downstream signaling pathway inhibited the NO production. AgNPs at 10 and 100 μg/mL induced ACh-independent prolonged cytotoxicity and decreased cellular proliferation mediated by the muscarinic receptor-iNOS pathway. However, the concentration of 100 μg/mL of AgNPs induced muscarinic receptor-independent apoptosis, suggesting the activation of multiple pathways. These data indicate that AgNPs induce prolonged cytotoxic and anti-proliferative effects on ASM cells, suggesting an activation of the muscarinic receptor-iNOS pathway. Further investigation is required to understand the full mechanisms of action of AgNPs on ASM under specific biological conditions.     

Isolation and characterization of metabolically competent pulmonary epithelial cells from pig lung tissue

Administration of drugs by inhalation opens new possibilities for entry into the systemic circulation and cultures of porcine pulmonary epithelial cells (PECs) may prove to be valuable in the prediction of pulmonary metabolism of drugs in humans. This paper, therefore, reports a method for the routine isolation and cultivation of PECs from slaughterhouse animals. On average 1.5?×?10⁶ cells g^?1 tissue were isolated by discontinuous density-gradient centrifugation. Cells were subsequently cultivated on collagen-coated plates and characterized by staining for alkaline phosphatase, by tannic acid staining of lamellar bodies and by surfactant protein (SP) expression at days 0, 3 and 6 in culture. Over 70% of purified cells were positive for SP-C and tannic acid staining and thus defined as epithelial cells of alveolar origin (AECs). The AEC phenotype was also confirmed by specific binding of marker lectins (Maclura pomifera and Helix pomatia) and by studying gene expression and activity of cytochrome P450 monooxygenases. Testosterone, ethoxyresorufin, benzyloxyresorufin and verapamil were used as substrates for cytochrome P450-catalysed oxidations and cultured cells were found to be differentiated as well as metabolically competent during cultivation. Therefore, this culture system enables in depth pulmonary biotransformation and toxicity studies.