Cellular responses induced by cerium oxide nanoparticles: induction of intracellular calcium level and oxidative stress on culture cells

Cerium oxide (CeO(2)) is an important metal oxide used for industrial products. Many investigations about the cellular influence of CeO(2) nanoparticles have been done, but results are contradictory. It has been reported that CeO(2) nanoparticles have an anti-oxidative effect in cells, but it has also been reported that CeO(2) nanoparticles induce oxidative stress. We investigated the potential influence on cells and the mechanisms induced by CeO(2) nanoparticles in vitro. We prepared a stable CeO(2) culture medium dispersion. Cellular responses in CeO(2) medium-exposed cells were examined. Cellular uptake of CeO(2) nanoparticles was observed. After 24-h exposure, a high concentration of CeO(2) nanoparticles (～200 mg/ml) induced an increase in the intracellular level of reactive oxygen species (ROS); a low concentration of CeO(2) nanoparticles induced a decrease in the intracellular ROS level. On the other hand, exposure of CeO(2) nanoparticle for 24 h had little influence on the cell viability. Exposure of CeO(2) nanoparticles increased the intracellular Ca(2+) concentration and also Calpain was activated. These results suggest that CeO(2) nanoparticles have a potential to induce intracellular oxidative stress and increase the intracellular Ca(2+) level, but these influences are small.     

Association of zinc ion release and oxidative stress induced by intratracheal instillation of ZnO nanoparticles to rat lung

Zinc oxide (ZnO) nanoparticles are one of the important industrial nanoparticles. The production of ZnO nanoparticles is increasing every year. On the other hand, it is known that ZnO nanoparticles have strong cytotoxicity. In vitro studies using culture cells revealed that ZnO nanoparticles induce severe oxidative stress. However, the in vivo influence of ZnO nanoparticles is still unclear. In the present study, rat lung was exposed to ZnO nanoparticles by intratracheal instillation, and the influences of ZnO nanoparticles to the lung in the acute phase, particularly oxidative stress, were examined. Additionally, in vitro cellular influences of ZnO nanoparticles were examined using lung carcinoma A549 cells and compared to in vivo examinations. The ZnO nanoparticles used in this study released zinc ion in both dispersions. In the in vivo examinations, ZnO dispersion induced strong oxidative stress in the lung in the acute phase. The oxidative stress induced by the ZnO nanoparticles was stronger than that of a ZnCl(2) solution. Intratracheal instillation of ZnO nanoparticles induced an increase of lipid peroxide, HO-1 and alpha-tocopherol in the lung. The ZnO nanoparticles also induced strong oxidative stress and cell death in culture cells. Intracellular zinc level and reactive oxygen species were increased. These results suggest that ZnO nanoparticles induce oxidative stress in the lung in the acute phase. Intracellular ROS level had a high correlation with intracellular Zn(2+) level. ZnO nanoparticles will stay in the lung and continually release zinc ion, and thus stronger oxidative stress is induced.     

Iron Oxide Nanoparticles Induce Oxidative Stress,DNA Damage,and Caspase Activation in the Human Breast Cancer Cell Line

Broad applications of iron oxide nanoparticles require an improved understanding of their potential effects on human health. In the present study, we explored the underlying mechanism through which iron oxide nanoparticles induce toxicity in human breast cancer cells (MCF-7). MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) and lactate dehydrogenase assays were used to examine mechanisms of cytotoxicity. Concentration- and time-dependent cytotoxicity was observed in MCF-7 cells. Iron oxide nanoparticles were found to induce oxidative stress evidenced by the elevation of reactive oxygen species generation, lipid peroxidation, and depletion of superoxide dismutase, glutathione, and catalase activities in MCF-7 cells. Nuclear staining was performed using 4′, 6-diamidino-2-phenylindole (DAPI), and cells were analyzed with a fluorescence microscope. Iron oxide nanoparticles (60 μg/ml) induced substantial apoptosis that was identified by morphology, condensation, and fragmentation of the nuclei of the MCF-7 cells. It was also observed that the iron oxide NPs induced caspase-3 activity. DNA strand breakage was detected by comet assay, and it occurred in a concentration- and time-dependent manner. Thus, the data indicate that iron oxide nanoparticles induced cytotoxicity and genotoxicity in MCF-7 cells via oxidative stress. This study warrants more careful assessment of iron oxide nanoparticles before their industrial applications.     

Protective Effects of Flavonoids Against Oxidative Stress Induced by Simulated Microgravity in SH-SY5Y Cells

Many lines of evidence suggest that microgravity results in increased oxidative stress in the nervous system. In order to protect neuronal cells from oxidative damage induced by microgravity, we selected some flavonoids that might prevent oxidative stress because of their antioxidant activities. Among the 20 flavonoids we examined, we found that isorhamnetin and luteolin had the best protective effects against H₂O₂ or SIN-1-induced cytotoxicity in SH-SY5Y cells. Using a clinostat to simulate microgravity, we found that isorhamnetin and luteolin treatment protected SH-SY5Y cells by preventing microgravity-induced increases in reactive oxygen species (ROS), nitric oxide (NO) and 3-nitrotyrosine (3-NT) levels, and a decrease in antioxidant power (AP). Moreover, isorhamnetin and luteolin treatment downregulated the expression of inducible nitric oxide synthase (iNOS), and oxidative stress was significantly inhibited by an iNOS inhibitor in SH-SY5Y cells exposed to simulated microgravity (SMG). These results indicate that isorhamnetin and luteolin could protect against microgravity-induced oxidative stress in neuroblastoma SH-SY5Y cells by inhibiting the ROS-NO pathway. These two flavonoids may have potential for preventing oxidative stress induced by space flight or microgravity.     

The comparative study of the effects of Fe2O3 and TiO2 micro- and nanoparticles on oxidative states of lung and bone marrow tissues and colony stimulating factor secretion

Nowadays, increased use of nanomaterials in industry and biomedicine poses potential risks to human health and the environment. Studying their possible toxicological effects is therefore of great significance. The present investigation was designed to examine the status of oxidative stress induced by nanoparticles (NPs) of ferric oxide (Fe₂O ₃) and titanium oxide (TiO ₂) with their micro-sized counterpart on mouse lung and bone marrow–derived normal tissue cells. We assessed the induction of oxidative stress by measuring its indicators such as antioxidant scavenging activity of superoxide dismutase and catalase as well as malondialdehyde concentration. Moreover, colony formation of bone marrow cells was assayed following induction with colony stimulating factor (CSF) from lung cells. NPs had a more potent stimulatory effect on the oxidative stress status than their micron-sized counterparts. In addition, the highest level of oxidative stress derived from TiO ₂ NPs was observed in both tissue types. Cotreatment with NPs and the antioxidant α-tocopherol reduced antioxidant activities and membrane lipid peroxidation (LPO) in the lung cells, but increased CSF-induced colony formation activity of bone marrow cells, suggesting that oxidative stress may be the cause of the cytotoxic effects of NPs. It is concluded that free radicals generated following exposure to NPs resulted in significant oxidative stress in mouse cells, indicated by increased LPO and antioxidant enzyme activity and decreased colony formation.     

Specificity of resistance to oxidative stress

Two clonal nerve-like cell lines derived from HT22 and PC12 have been selected for resistance to glutamate toxicity and amyloid toxicity, respectively. In the following experiments it was asked if these cell lines show cross-resistance toward amyloid beta peptide (Abeta) and glutamate as well as toward a variety of additional neurotoxins. Conversely, it was determined if inhibitors of oxytosis, a well-defined oxidative stress pathway, also protect cells from the neurotoxins. It is shown that both glutamate and amyloid resistant cells are cross resistant to most of the other toxins or toxic conditions, while inhibitors of oxytosis protect from glutathione and cystine depletion and H2O2 toxicity, but not from the toxic effects of nitric oxide, rotenone, arsenite or cisplatin. It is concluded that while there is a great deal of cross-resistance to neurotoxins, the components of the cell death pathway which has been defined for oxytosis are not used by many of the neurotoxins.     

Protein nitration increased by simulated weightlessness and decreased by melatonin and quercetin in PC12 cells.

A variety of experiments suggest that space flight is associated with an increase in oxidative stress in organism. To explore the effects of oxidative stress on neuronal cells during microgravity, we used rat pheochromocytoma (PC12) cells as a neuronal cell model, cultured in a clinostat, which could simulate microgravity, to investigate the effects of reactive nitrogen species on protein nitration in PC12 cells during clinorotation. The effects of melatonin and quercetin on protein nitration in PC12 cells were also assayed to evaluate the possible protective role of melatonin or quercetin as an antioxidant. The results of immunological staining showed that after the 3 days' clinorotation the protein expressions of neuronal nitric oxide synthase and inducible nitric oxide synthesis were up-regulated. Our data also reflected that the concentrations of nitric oxide and nitrotyrosine were significantly increased after clinorotation, and they were reduced markedly in cells that were treated with 50 micromol/L melatonin or 0.5 micromol/L quercetin during simulated microgravity, when compared to those of control cells. These results suggest that clinorotation-induced weightlessness increases oxidative stress responses in PC12 cells, and melatonin or quercetin was shown to protect PC12 cells from oxidative damage during simulated weightlessness.     

Prion protein reduces both oxidative and non-oxidative copper toxicity

The prion protein is a membrane tethered glycoprotein that binds copper. Conversion to an abnormal isoform is associated with neurodegenerative diseases known as prion diseases. Expression of the prion protein has been suggested to prevent cell death caused by oxidative stress. Using cell based models we investigated the potential of the prion protein to protect against copper toxicity. Although prion protein expression effectively protected neurones from copper toxicity, this protection was not necessarily associated with reduction in oxidative damage. We also showed that glycine and the prion protein could both protect neuronal cells from oxidative stress. Only the prion protein could protect these cells from the toxicity of copper. In contrast glycine increased copper toxicity without any apparent oxidative stress or lipid peroxidation. Mutational analysis showed that protection by the prion protein was dependent upon the copper binding octameric repeat region. Our findings demonstrate that copper toxicity can be independent of measured oxidative stress and that prion protein expression primarily protects against copper toxicity independently of the mechanism of cell death.     

Possible use of quercetin, an antioxidant, for protection of cells suffering from overload of intracellular Ca2+: a model experiment

Quercetin is known to protect the cells suffering from oxidative stress. The oxidative stress elevates intracellular Ca(2+) concentration, one of the phenomena responsible for cell death. Therefore, we hypothesized that quercetin would protect the cells suffering from overload of intracellular Ca(2+). To test the hypothesis, the effects of quercetin on the cells suffering from oxidative stress and intracellular Ca(2+) overload were examined by using a flow cytometer with appropriate fluorescence probes (propidium iodide, fluo-3-AM, and annexin V-FITC) and rat thymocytes. The concentrations (1-30 microM) of quercetin to protect the cells suffering from intracellular Ca(2+) overload by A23187, a calcium ionophore, were similar to those for the cells suffering from oxidative stress by H(2)O(2). The cell death respectively induced by H(2)O(2) and A23187 was significantly suppressed by removal of external Ca(2+). Furthermore, quercetin greatly delayed the process of Ca(2+)-dependent cell death although it did not significantly affect the elevation of intracellular Ca(2+) concentration by H(2)O(2) and A23187, respectively. It is concluded that quercetin can protect the cells from oxidative injury in spite of increased concentration of intracellular Ca(2+). Results suggest that quercetin is also used for protection of cells suffering from overload of intracellular Ca(2+).     

Stress induction and mitochondrial localization of Oxr1 proteins in yeast and humans

Reactive oxygen species (ROS) are critical molecules produced as a consequence of aerobic respiration. It is essential for cells to control the production and activity of such molecules in order to protect the genome and regulate cellular processes such as stress response and apoptosis. Mitochondria are the major source of ROS within the cell, and as a result, numerous proteins have evolved to prevent or repair oxidative damage in this organelle. The recently discovered OXR1 gene family represents a set of conserved eukaryotic genes. Previous studies of the yeast OXR1 gene indicate that it functions to protect cells from oxidative damage. In this report, we show that human and yeast OXR1 genes are induced by heat and oxidative stress and that their proteins localize to the mitochondria and function to protect against oxidative damage. We also demonstrate that mitochondrial localization is required for Oxr1 protein to prevent oxidative damage.     

Reflectance structured illumination imaging of internalized cerium oxide nanoparticles modulating dose-dependent reactive oxygen species in breast cancer cells

Cerium oxide nanoparticles have been shown to sensitize cancer cells to radiation damage. Their unique redox properties confer excellent therapeutic potential by augmenting radiation dose with reactive oxygen species mediating bystander effects. Owing to its metallic properties, cerium oxide nanoparticles can be visualized inside cells using reflected light and optical sectioning. This can be advantageous in settings requiring none or minimal sample preparation and modification. We investigated the use of reflectance imaging for the detection of unmodified nanoceria in MDA MB231 breast cancer cells along with differential interference contrast imaging and fluorescent nuclear labeling. We also performed studies to evaluate the uptake capability, cellular toxicity and redox properties of nanocaria in these cells. Our results demonstrate that reflectance structured illumination imaging can effectively localize cerium oxide nanoparticles in breast cancer cells, and when combining with differential interference contrast and fluorescent cell label imaging, effective compartmental localization of the nanoparticles can be achieved. The total number of cells taking up the nanoparticles and the amount of nanoparticle uptake increased significantly in proportion to the dose, with no adverse effects on cell survival. Moreover, significant reduction in reactive oxygen species was also observed in proportion to increasing nanoceria concentrations attesting to its ability to modulate oxidative stress. In conclusion, this work serves as a pre-clinical scientific evaluation of the effective use of reflectance structured illumination imaging of cerium oxide nanoparticles in breast cancer cells and the safe use of these nanoparticles in MDA MB231 cells for further therapeutic applications.     

Toxicological effects of inorganic nanoparticles on human lung cancer A549 cells

Many researches have shown that anionic clays can be used as delivery carriers for drug or gene molecules due to their efficient cellular uptake in vitro, and enhanced permeability and retention effect in vivo. It is, therefore, highly required to establish a guideline on their potential toxicity for practical applications. The toxicity of anionic clay, layered metal hydroxide nanoparticle, was evaluated in two human lung epithelial cells, carcinoma A549 cells and normal L-132 cells, and compared with that in other human cancer cell lines such as cervical adenocarcinoma cells (HeLa) and osteosarcoma cells (HOS). The present nanoparticles showed little cytotoxic effects on the proliferation and viability of four cell lines tested at the concentrations used (<250 μg/ml) within 48 h. However, exposing cancer cells to high concentrations (250-500 μg/ml) for 72 h resulted in an inflammatory response with oxidative stress and membrane damage, which varied with the cell type (A549 > HOS > HeLa). On the other hand, the toxicity mechanism seems to be different from that of other inorganic nanoparticles frequently studied for biological and medicinal applications such as iron oxide, silica, and single walled carbon nanotubes. Iron oxide caused cell death associated with membrane damage, while single walled carbon nanotube induced oxidative stress followed by apoptosis. Silica triggered an inflammation response without causing considerable cell death for both cancer cells and normal cells, whereas layered metal hydroxide nanoparticle did not show any cytotoxic effects on normal L-132 cells in terms of inflammation response, oxidative stress, and membrane damage at the concentration of less than 250 μg/ml. It is , therefore, highly expected that the present nanoparticle can be used as a efficient vehicle for drug delivery and cancer cell targeting as well.     

Superparamagnetic iron oxide nanoparticles as radiosensitizer via enhanced reactive oxygen species formation

Internalization of citrate-coated and uncoated superparamagnetic iron oxide nanoparticles by human breast cancer (MCF-7) cells was verified by transmission electron microscopy imaging. Cytotoxicity studies employing metabolic and trypan blue assays manifested their excellent biocompatibility. The production of reactive oxygen species in iron oxide nanoparticle loaded MCF-7 cells was explained to originate from both, the release of iron ions and their catalytically active surfaces. Both initiate the Fenton and Haber-Weiss reaction. Additional oxidative stress caused by X-ray irradiation of MCF-7 cells was attributed to the increase of catalytically active iron oxide nanoparticle surfaces.     

Titanium dioxide particles phosphorylate histone H2AX independent of ROS production

Zinc oxide (ZnO) nanoparticles are finding applications in a wide range of products including cosmetics, food packaging, imaging, etc. This increases the likelihood of human exposure to these nanoparticles through dermal, inhalation and oral routes. Presently, the majority of the studies concerning ZnO nanoparticle toxicity have been conducted using in vitro systems which lack the complex cell-cell, cell-matrix interactions and hormonal effects found in the in vivo scenario. The present in vivo study in mice was aimed at investigating the oral toxicity of ZnO nanoparticles. Our results showed a significant accumulation of nanoparticles in the liver leading to cellular injury after sub-acute oral exposure of ZnO nanoparticles (300 mg/kg) for 14 consecutive days. This was evident by the elevated alanine aminotransferase (ALT) and alkaline phosphatase (ALP) serum levels and pathological lesions in the liver. ZnO nanoparticles were also found to induce oxidative stress indicated by an increase in lipid peroxidation. The DNA damage in the liver and kidney cells of mice was evaluated by the Fpg-modified Comet assay which revealed a significant (p<0.05) increase in the Fpg-specific DNA lesions in liver indicating oxidative stress as the cause of DNA damage. The TUNEL assay revealed an induction of apoptosis in the liver of mice exposed to ZnO nanoparticles compared to the control. Our results conclusively demonstrate that sub-acute oral exposure to ZnO nanoparticles in mice leads to an accumulation of nanoparticles in the liver causing oxidative stress mediated DNA damage and apoptosis. These results also suggest the need for a complete risk assessment of any new engineered nanoparticle before its arrival into the consumer market.     

Involvement of the Leishmania donovani virulence factor A2 in protection against heat and oxidative stress

Leishmania is an obligate intracellular protozoan parasite that infects cells of the reticulo-endothelial system. Host defences against Leishmania include fever and oxidant production, and the parasite has developed a number of defence mechanisms to neutralize the host response. The Leishmania donovani A2 family of proteins has been shown to be essential for survival in mammalian visceral organs. Here we provide evidence that A2 proteins protect the parasite against host defences, namely heat stress (fever) and oxidative stress. A2 is however unable to protect the cells from endoplasmic reticulum stress induced by dithiothreitol. To downregulate A2 protein expression, L. donovani was transfected with an A2 antisense RNA expressing-vector, resulting in significant reduction of A2 levels. The resulting A2-deficient cells were more sensitive to heat shock and this was associated with increased production of internal oxidants during heat shock. Moreover, axenic amastigotes with downregulated A2 expression had increased internal oxidants and decreased viability following treatment with hydrogen peroxide or a nitric oxide donor when compared to control cells. Overall, these results suggest that A2 protects L. donovani from a variety of stresses, thereby allowing it to survive in the internal organs of the mammalian host and to cause visceral disease.     

Evaluation of cellular influences of platinum nanoparticles by stable medium dispersion

Platinum nanoparticles have industrial application, for example in catalysis, and are used in consumer products such as cosmetics and supplements. Therefore, among the many nanoparticles, platinum is one of the more accessible nanoparticles for consumers. Most platinum nanoparticles that are used in cosmetics and supplements which have an anti-oxidant activity are modified particles. However, the cellular influences of pristine platinum nanoparticles are still unclear, although it has been reported that platinum nanoparticles induce oxidative stress. In this study, we investigated the cellular influences induced by pure pristine platinum nanoparticles. Platinum nanoparticles of 100% purity were dispersed in a cell culture medium and stable medium dispersion was obtained. The platinum nanoparticle medium dispersion was applied to two kinds of cultured cells, A549 and HaCaT cells, and the cellular influences were examined. Cell viability (MTT assay), cell proliferation (clonogenic assay), apoptosis induction (caspase-3 activity), intracellular ROS level (DCFH assay), and lipid peroxidation level (DPPP assay) were measured as markers of cellular influences. Transmission electron microscope observation showed cellular uptake of platinum nanoparticles. However, the platinum nanoparticles did not drive any markers. It is known that some metal oxide nanoparticles such as NiO and CuO show severe cytotoxicity via metal ion release. Compared with these toxic nanoparticles, the platinum nanoparticles used in this study did not release platinum ions into the culture media. These results suggest that the physically and chemically inactive cellular influences of platinum nanoparticles are small.     

Metallothionein protects bone marrow stromal cells against hydrogen peroxide-induced inhibition of osteoblastic differentiation

Metallothionein (MT), a cysteine-rich, metal-binding protein, is involved in homeostatic regulation of essential metals and protection of cells against oxidative injury. It has been shown that oxidative stress is associated with pathogenesis of osteoporosis and is capable of inhibiting osteoblastic differentiation of bone cells by nuclear factor-kappaB (NF-kappaB). In this study, the effect of MT on oxidative stress-induced inhibition of osteoblast differentiation was examined. 50-200 microM hydrogen peroxide-induced oxidative stress suppressed the osteoblastic differentiation process of primary mouse bone marrow stromal cells (BMSCs), manifested by a reduction in the differentiation marker alkaline phosphatase (ALP). The presence of exogenous MT (20-500 microM) or induction of endogenous MT by ZnCl2 (50-200 microM) could protect BMSCs against H2O2-induced inhibition of osteoblastic differentiation, manifested by a resumption of H2O2-inhibited ALP activity and ALP positive cells. Furthermore, adding exogenous MT or inducing endogenous MT expression impaired H2O2-stimulated NF-kappaB signaling. These data indicate the ability of MT to protect BMSCs against oxidative stress-induced inhibition of osteoblastic differentiation.     

Bursopentin (BP5) Protects Dendritic Cells from Lipopolysaccharide-Induced Oxidative Stress for Immunosuppression

Dendritic cells (DCs) play a vital role in the regulation of immune-mediated inflammatory diseases. Thus, DCs have been regarded as a major target for the development of immunomodulators. However, oxidative stress could disturb inflammatory regulation in DCs. Here, we examined the effect of bursopentine (BP5), a novel pentapeptide isolated from chicken bursa of fabricius, on the protection of DCs against oxidative stress for immunosuppression. BP5 showed potent protective effects against the lipopolysaccharide (LPS)-induced oxidative stress in DCs, including nitric oxide, reactive oxygen species and lipid peroxidation. Furthermore, BP5 elevated the level of cellular reductive status through increasing the reduced glutathione (GSH) and the GSH/GSSG ratio. Concomitant with these, the activities of several antioxidative redox enzymes, including glutathione peroxidase (GPx), catalase (CAT) and superoxide dismutase (SOD), were obviously enhanced. BP5 also suppressed submucosal DC maturation in the LPS-stimulated intestinal epithelial cells (ECs)/DCs coculture system. Finally, we found that heme oxygenase 1 (HO-1) was remarkably upregulated by BP5 in the LPS-induced DCs, and played an important role in the suppression of oxidative stress and DC maturation. These results suggested that BP5 could protect the LPS-activated DCs against oxidative stress and have potential applications in DC-related inflammatory responses.     

IRE1-dependent activation of AMPK in response to nitric oxide

While there can be detrimental consequences of nitric oxide production at pathological concentrations, eukaryotic cells have evolved protective mechanisms to defend themselves against this damage. The unfolded-protein response (UPR), activated by misfolded proteins and oxidative stress, is one adaptive mechanism that is employed to protect cells from stress. Nitric oxide is a potent activator of AMP-activated protein kinase (AMPK), and AMPK participates in the cellular defense against nitric oxide-mediated damage in pancreatic β-cells. In this study, the mechanism of AMPK activation by nitric oxide was explored. The known AMPK kinases LKB1, CaMKK, and TAK1 are not required for the activation of AMPK by nitric oxide. Instead, this activation is dependent on the endoplasmic reticulum (ER) stress-activated protein IRE1. Nitric oxide-induced AMPK phosphorylation and subsequent signaling to AMPK substrates, including Raptor, acetyl coenzyme A carboxylase, and PGC-1α, is attenuated in IRE1α-deficient cells. The endoribonuclease activity of IRE1 appears to be required for AMPK activation in response to nitric oxide. In addition to nitric oxide, stimulation of IRE1 endoribonuclease activity with the flavonol quercetin leads to IRE1-dependent AMPK activation. These findings indicate that the RNase activity of IRE1 participates in AMPK activation and subsequent signaling through multiple AMPK-dependent pathways in response to nitrosative stress.