Potential neurological lesion after nasal instillation of TiO(2) nanoparticles in the anatase and rutile crystal phases

Nanoscale titanium dioxide (TiO(2)) is massively produced and widely used in living environment, which hence make the potential risk to human health. Central nervous system (CNS) is the potential susceptible target of inhaled nanoparticles, but the studies on this aspect are limited so far. We report the accumulation and toxicity results in vivo of two crystalline phases of TiO(2) nanoparticles (80nm, rutile and 155nm, anatase; purity >99%). The female mice were intranasally instilled with 500microg of TiO(2) nanoparticles suspension every other day for 30 days. Synchrotron radiation X-ray fluorescence analysis (SRXRF) and inductively coupled plasma mass spectrometry (ICP-MS) were used to determine the contents of titanium in murine brain. Then, the pathological examination of brain tissue, oxidative stress-mediated responses, and levels of neurochemicals in the brain of exposed mice were also analyzed. The obvious morphological changes of hippocampal neurons and increased GFAP-positive astrocytes in the CA4 region were observed, which were in good agreements with higher Ti contents in the hippocampus region. Oxidative stress occurred obviously in whole brain of exposed mice such as lipid peroxidation, protein oxidation and increased activities of catalase, as well as the excessive release of glutamic acid and nitric oxide. These findings indicate anatase TiO(2) nanoparticles exhibited higher concern on some tested biological effects. To summarize, results provided the preliminary evidence that nasal instilled TiO(2) nanoparticles could be translocated into the central nervous system and cause potential lesion of brain, and the hippocampus would be the main target within brain.     

Lectin-mediated transport of nanoparticles across Caco-2 and OK cells

Recent experiments by a number of workers have suggested that it may be possible to use various targeting molecules, which bind to the intestinal epithelium, to promote the uptake and transport of nanoparticles from the intestine to the circulation. We have used commercial nanoparticles to examine the effect of size, density and inhibitors on uptake of lectin-coated nanoparticles by epithelial cells. The degree of uptake was most influenced by the density of lectin on the particle, with size and type of lectin being less important. Uptake could be inhibited by the presence of specific sugars or free lectin. These studies should provide a good basis for the design of targetable biodegradable drug-loadable particles suitable for oral delivery.     

Cytotoxicity of monodispersed chitosan nanoparticles against the Caco-2 cells

Published toxicology data on chitosan nanoparticles (NP) often lack direct correlation to the in situ size and surface characteristics of the nanoparticles, and the repeated NP assaults as experienced in chronic use. The aim of this paper was to breach these gaps. Chitosan nanoparticles synthesized by spinning disc processing were characterised for size and zeta potential in HBSS and EMEM at pHs 6.0 and 7.4. Cytotoxicity against the Caco-2 cells was evaluated by measuring the changes in intracellular mitochondrial dehydrogenase activity, TEER and sodium fluorescein transport data and cell morphology. Cellular uptake of NP was observed under the confocal microscope. Contrary to established norms, the collective data suggest that the in vitro cytotoxicity of NP against the Caco-2 cells was less influenced by positive surface charges than by the particle size. Particle size was in turn determined by the pH of the medium in which the NP was dispersed, with the mean size ranging from 25 to 333 nm. At exposure concentration of 0.1%, NP of 25 ± 7 nm (zeta potential 5.3 ± 2.8 mV) was internalised by the Caco-2 cells, and the particles were observed to inflict extensive damage to the intracellular organelles. Concurrently, the transport of materials along the paracellular pathway was significantly facilitated. The Caco-2 cells were, however, capable of recovering from such assaults 5 days following NP removal, although a repeat NP exposure was observed to produce similar effects to the 1st exposure, with the cells exhibiting comparable resiliency to the 2nd assault.     

Doxorubicin decreases paraquat accumulation and toxicity in Caco-2 cells

P-glycoprotein (P-gp) is an efflux pump belonging to the ATP-binding cassette transporter superfamily expressed in several organs. Considering its potential protective effects, the induction of de novo synthesis of P-gp could be used therapeutically in the treatment of intoxications by its substrates. The herbicide paraquat (PQ) is a P-gp substrate responsible for thousands of fatal intoxications worldwide that still lacks an effective antidote.     

Effects of red wine on ochratoxin A toxicity in intestinal Caco-2/TC7 cells.

Ochratoxin A (OTA) is found in a variety of foods and beverages, including red wine. OTA was reported to be nephrotoxic, immunotoxic, hepatotoxic and a potential carcinogen, with yet uncharacterized mechanisms. Consumption of contaminated wines might contribute up to 13% of OTA daily human intake. Potentially chronic exposure has therefore raised public health concern. OTA toxicity in the presence of de-alcoholated red wine was investigated in human intestinal Caco-2/TC7 cells, differentiated on filter supports, by measuring tight junction (TJ) permeability, morphological alterations of TJ proteins and occurrence of apoptosis. Cells were treated with OTA, in the presence of de-alcoholated red wine, for 48h and the ability to recover from the effects of OTA was evaluated after 24h in complete medium. OTA treatment increased TJ permeability and caused intracellular redistribution of claudin-4. However, cells were able to restore permeability and correct localization of claudin-4 following 24h recovery. Conversely, in the presence of red wine, OTA produced faster and irreversible increase in TJ permeability, intracellular delocalization of claudin-4 and extensive apoptosis. Our results point at a possible synergy between OTA and some red wine components, such as polyphenols, in the induction of apoptotic cell death.     

Uptake of different crystal structures of TiO2 nanoparticles by Caco-2 intestinal cells

The gastrointestinal uptake of different crystal structures of TiO₂ was investigated using Caco-2 intestinal cells. Caco-2 monolayers exhibited time-dependent, saturable uptake of Ti from TiO₂ exposures of 1 mg l⁻¹ over 24 h, which was influenced by crystal type. Initial uptake rates were 5.3, 3.73, 3.58 and 4.48 nmol mg⁻¹ protein h⁻¹ for bulk, P25, anatase and rutile forms respectively. All exposures caused elevations of Ti in the cells relative to the control (ANOVA P < 0.05). Electron micrographs of the Caco-2 monolayer showed the presence of particles inside the cells, and energy dispersive spectroscopy (EDS) confirmed the composition as TiO₂. Incubating the cells with 120 IU nystatin (putative endocytosis inhibitor) or 100 μmol l⁻¹ vanadate (ATPase inhibitor) caused large increases in Ti accumulation for all crystal types relative to controls (ANOVA P < 0.05), except for the rutile form with vanadate. Incubating the cells with 90 μmol l⁻¹ genistein (tyrosine kinase inhibitor) or 27 μmol l⁻¹ chloropromazine (clathrin-mediated endocytosis inhibitor) caused a large decrease in Ti accumulation relative to the controls (ANOVA P < 0.05). Cell viability measures were generally good (low LDH leak, normal cell morphology), but there were some changes in the electrolyte composition (K⁺, Na⁺, Ca²⁺, Mg²⁺) of exposed cells relative to controls. A rise in total Ca²⁺ concentration in the cells was observed for all TiO₂ crystal type exposures. Overall, the data shows that Ti accumulation for TiO₂ NP exposure in Caco-2 cells is crystal structure-dependent, and that the mechanism(s) involves endocytosis of intact particles.     

Acute respiratory and systemic toxicity of pulmonary exposure to rutile Fe-doped TiO(2) nanorods

Nanomaterials are extensively used in medicines, industry and daily life, but little is known about their possible health effects. Titanium dioxide (TiO₂) nonmaterial-based photocatalysis is useful in the complete mineralization of organic pollutants in waste water and air. While the Fe-doping of TiO₂ enhances their photocatalytic activity, their potential pathophysiologic effects are unknown. Here, rutile Fe-doped (9%) pure titanium dioxide (TiO₂) nanorods were prepared and characterized. Subsequently, we assessed the acute (24 h) pulmonary and extrapulmonary effects of intratracheal (i.t.) instillation of these nanorods (1 and 5 mg/kg) in Wistar rats. In the bronchoalveolar lavage, the treatment induced a significant and dose-dependent increase of neutrophils, an increase of interleukin-6 (IL-6, at 5 mg/kg), and caused a dose-dependent-decrease of superoxide dismutase (SOD) activity. The lung sections of rats exposed to rutile Fe–TiO₂ nanorods showed infiltration of inflammatory cells in dose-dependent manner. Similarly, the heart rate, systolic blood pressure, plasma IL-6, and leukocyte and platelet numbers were increased at 5 mg/kg. The plasma SOD and reduced glutathaione activities were dose-dependently decreased after exposure to the nanorods. Histopathologically, the liver showed mild inflammatory cells infiltration of few portal tracts, but the kidneys and heart were unaffected. In plasma, the levels of lactate dehydrogenase and hepatic enzymes, i.e., alanine aminotranferease and aspartate aminotransferase were increased significantly. The in vitro exposure of human lung cancer cells NCI-H460-Luc2 and human hepatoma cells HepG2 to FeTiO₂ (6.25–100 μg/ml) dose-dependently reduced cellular viability. Also, the In vitro direct addition of these nanorods (0.1–1 μg/ml) to untreated rat blood, significantly and dose-dependently induced platelet aggregation. In conclusion, exposure to rutile Fe–TiO₂ promotes pulmonary and systemic inflammation and oxidative stress. It affects the liver, enhances thrombotic potential, heart rate and systolic blood pressure. Moreover, the rutile Fe–TiO₂ elicited direct toxicity on NCI-H460-Luc2 and HepG2 cells.     

Vitamin B12-mediated transport of nanoparticles across Caco-2 cells

Several studies have shown that Caco-2 cells have the capability to transport peptides and proteins from their apical to basal surfaces when these molecules are linked to vitamin B12 (VB12). In this study we have extended these studies and have shown that Caco-2 cells are also able to internalize and transport VB12-modifed nanoparticles from their apical to basal surfaces. Uptake and transport of nanoparticles was found to occur in both a VB12-dependent intrinsic factor (IF)-independent manner as well as in a VB12-dependent IF-dependent manner. Both IF-independent and IF-dependent VB12-mediated uptake and transport were dependent upon the surface density of VB12 as a reduction in surface modification of the nanoparticles with VB12 resulted in a reduced level of both VB12-mediated and IF-mediated uptake. At lower levels of VB12 modification there was no apparent non-IF-mediated uptake; however, VB12-IF-mediated uptake was still measurable. These studies show that Caco-2 cell cultures are a suitable model for the study of VB12-mediated uptake and transport of nanoparticles, and suggest that for effective oral uptake of VB12-coated nanoparticles high surface densities of VB12 are required.Copyright     

Alternariol induce toxicity via cell death and mitochondrial damage on Caco-2 cells

Alternariol (AOH), a mycotoxin produced by Alternaria sp, appears as food contaminant in fruit, vegetables and cereal products. Its toxicity has been demonstrated, but the mechanisms involved have not been elucidated yet. In this study, the pathways triggered by AOH and degradation products generated on Caco-2 cells were evaluated. Cells were exposed to AOH sub-cytotoxic concentrations of 15, 30 and 60 μM. Cell cycle disruption, the induction of apoptosis/necrosis and changes in mitochondrial membrane potential (Δψm) after 24 and 48 h was asses by flow cytometry. Also, AOH and its degradation products were evaluated after 24 and 48 h by high-performance liquid chromatography with tandem mass spectrometric (LC-MS/MS) to detect and quantify its levels. Cell cycle was significantly decreased at G1 phase and increased at S and G2/M phase at the time of exposure. AOH induced necrosis, apoptosis/necrosis and loss of Δψm in a dose and time-dependent manner. The concentrations of AOH quantified in the culture media exposed to AOH decreased as the exposure time was increased. In conclusion, AOH caused cytotoxic effects supported by blocking cell cycle, decreasing cell proliferation and increasing apoptosis/necrosis cells.     

Contact-dependent transfer of TiO2 nanoparticles between mammalian cells

Cellular organelles have been shown to shuttle between cells in co-culture. We hereby show that titanium dioxide (TiO₂) nanoparticles (NPs) can be transferred in such a manner, between cells in direct contact, along with endosomes and lysosomes. A co-culture system was employed for this purpose and the NP transfer was observed in mammalian cells including normal rat kidney (NRK) and HeLa cells. We found that the small GTPase Arf6 facilitates the intercellular transfer of smaller NPs and agglomerates. Spherical, anatase nano-TiO₂ with sizes of 5 (Ti5) and 40?nm (Ti40) were used in this study. Humans are increasingly exposed to TiO₂ NPs from external sources such as constituents of foods, cosmetics, and pharmaceuticals, or from internal sources represented by Ti-based implants, which release NPs upon abrasion. Exposure to 5?mg/l of Ti5 and Ti40 for 24?h did not affect cellular viability but modified their ability to communicate with surrounding cells. Altogether, our results have important implications for the design of nanomedicines, drug delivery and toxicity.     

Assessment of cellular toxicity of TiO2 nanoparticles for cardiac tissue engineering applications

Abstract

Because of the increased use of titanium dioxide (TiO₂) nanoparticles (NPs) in tissue engineering (TE), and in new constructs for cardiac TE, their effect was studied on three relevant cell types: Adult rat ventricular cardiomyocytes, human embryonic stem cell-derived cardiomyocytes (hESC-CM) and fibroblasts. For adult rat myocytes, 10 μg/mL TiO₂ NPs showed no significant effect on myocyte survival over 24 h or acute myocyte contractility. Increasing the concentration to 100 μg/mL was seen to reduce contraction amplitude (p < 0.05). For hESC-CM, 10 μg/mL TiO₂ reduced the beating rate significantly by 24 h. No arrhythmias or cessation of beating were observed in either cell type. Culturing fibroblasts in 5–150 μg/mL TiO₂ significantly reduced cell proliferation at day 4 and increased cell death. We conclude that there may be modest but potentially adverse effects of TiO₂ NPs if used in fast degrading polymers for myocardial tissue engineering (MTE) applications.     

Iron oxide/hydroxide nanoparticles with negatively charged shells show increased uptake in Caco-2 cells

The absorption of commonly used ferrous iron salts from intestinal segments at neutral to slightly alkaline pH is low, mainly because soluble ferrous iron is easily oxidized to poorly soluble ferric iron and because ferrous iron, but not ferric iron, is carried by the divalent metal transporter DMT-1. Moreover, ferrous iron frequently causes gastrointestinal side effects. Iron hydroxide nanoparticles with neutral and hydrophilic carbohydrate shells are alternatively used to ferrous salts. In these formulations gastrointestinal side effects are rare because hundreds of ferric iron atoms are safely packed in nanoscaled cores surrounded by the solubilizing shell; nevertheless, iron bioavailability is even worse compared to ferrous salts. In this study the cell uptake of iron hydroxide and iron oxide nanoparticles (FeONP) with negatively charged shells of different chemical types and sizes was compared to the uptake of those with neutral hydrophilic shells, ferrous sulfate and ferric chloride. The nanoparticle uptake was measured in Caco-2 cells with the iron detecting ferrozine method and visualized by transmission electron microscopy. The toxicity was evaluated using the MTT assay. For nanoparticles with a negatively charged shell the iron uptake was about 40 times higher compared to those with neutral hydrophilic carbohydrate shell or ferric chloride and in the same range as ferrous sulfate. However, in contrast to ferrous sulfate, nanoparticles with negatively charged shells showed no toxicity. Two different uptake mechanisms were proposed: diffusion for hydroxide nanoparticles with neutral hydrophilic shell and adsorptive endocytosis for nanoparticles with negatively charged shells. It needs to be determined whether iron hydroxide nanoparticles with negatively charged shells also show improved bioavailability in iron-deficient patients compared to iron hydroxide nanoparticles with a neutral hydrophilic shell, which exist in the market today.     

The effects of baicalein or baicalin on the colloidal stability of ZnO nanoparticles (NPs) and toxicity of NPs to Caco-2 cells

Recent study suggested that the presence of phytochemicals in food could interact with nanoparticles (NPs) and consequently reduce the toxicity of NPs, which has been attributed to the antioxidant properties of phytochemicals. In this study, we investigated the interactions between ZnO NPs and two flavonoids baicalein (Ba) or baicalin (Bn) as well as the influence of the interactions on the toxicity of ZnO NPs to Caco-2 cells. The antioxidant properties of Ba and Bn were confirmed by 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) assays, with Ba being stronger. However, the presence of Ba or Bn did not significantly affect cytotoxicity, intracellular superoxide or release of inflammatory cytokines of Caco-2 cells after ZnO NP exposure. When Ba was present, the cellular viability of Caco-2 cells after exposure to ZnO NPs was slightly increased, associated with a modest decrease of intracellular Zn ions, but these effects were not statistically different. Ba was more effective than Bn at changing the hydrodynamic sizes, Zeta potential and UV–Vis spectra of ZnO NPs, which indicated that Ba might increase the colloidal stability of NPs. Taken together, the results of the present study indicated that the anti-oxidative phytochemical Ba might only modestly protected Caco-2 cells from the exposure to ZnO NPs associated with an insignificant reduction of the accumulation of intracellular Zn ions. These results also indicated that when assessing the combined effects of NPs and phytochemicals to cells lining gastrointestinal tract, it might be necessary to evaluate the changes of colloidal stability of NPs altered by phytochemicals.     

The effects of endoplasmic reticulum stress inducer thapsigargin on the toxicity of ZnO or TiO2 nanoparticles to human endothelial cells

It was recently shown that ZnO nanoparticles (NPs) could induce endoplasmic reticulum (ER) stress in human umbilical vein endothelial cells (HUVECs). If ER stress is associated the toxicity of ZnO NPs, the presence of ER stress inducer thapsigargin (TG) should alter the response of HUVECs to ZnO NP exposure. In this study, we addressed this issue by assessing cytotoxicity, oxidative stress and inflammatory responses in ZnO NP exposed HUVECs with or without the presence of TG. Moreover, TiO₂ NPs were used to compare the effects. Exposure to 32?μg/mL ZnO NPs (p?2 NPs (p?>?0.05), significantly induced cytotoxicity as assessed by WST-1 and neutral red uptake assay, as well as intracellular ROS. ZnO NPs dose-dependently increased the accumulation of intracellular Zn ions, and ZnSO₄ induced similar cytotoxic effects as ZnO NPs, which indicated a role of Zn ions. The release of inflammatory proteins tumor necrosis factor α (TNFα) and interleukin-6 (IL-6) or the adhesion of THP-1 monocytes to HUVECs was not significantly affected by ZnO or TiO₂ NP exposure (p?>?0.05). The presence of 250?nM TG significantly induced cytotoxicity, release of IL-6 and THP-1 monocyte adhesion (p?p?>?0.05). ANOVA analysis indicated no interaction between exposure to ZnO NPs and the presence of TG on almost all the endpoints (p?>?0.05) except neutral red uptake assay (p?相似文献   

Nanoparticle size and combined toxicity of TiO2 and DSLS (surfactant) contribute to lysosomal responses in digestive cells of mussels exposed to TiO2 nanoparticles

The aim of this investigation was to understand the bioaccumulation, cell and tissue distribution and biological effects of disodium laureth sulfosuccinate (DSLS)-stabilised TiO₂ nanoparticles (NPs) in marine mussels, Mytilus galloprovincialis. Mussels were exposed in vivo to 0.1, 1 and 10?mg Ti/L either as TiO₂ NPs (60 and 180?nm) or bulk TiO₂, as well as to DSLS alone. A significant Ti accumulation was observed in mussels exposed to TiO₂ NPs, which were localised in endosomes, lysosomes and residual bodies of digestive cells, and in the lumen of digestive tubules, as demonstrated by ultrastructural observations and electron probe X-ray microanalysis. TiO₂ NPs of 60?nm were internalised within digestive cell lysosomes to a higher extent than TiO₂ NPs of 180?nm, as confirmed by the quantification of black silver deposits after autometallography. The latter were localised mainly forming large aggregates in the lumen of the gut. Consequently, lysosomal membrane stability (LMS) was significantly reduced upon exposure to both TiO₂ NPs although more markedly after exposure to TiO₂-60 NPs. Exposure to bulk TiO₂ and to DSLS also affected the stability of the lysosomal membrane. Thus, effects on the lysosomal membrane depended on the nanoparticle size and on the combined biological effects of TiO₂ and DSLS.     

Differential toxicity and gene expression in Caco-2 cells exposed to arsenic species

Inorganic arsenic [As(V) + As(III)] and its metabolites, especially the trivalent forms [monomethylarsonous acid, MMA(III), and dimethylarsinous acid, DMA(III)], are considered the forms of arsenic with the highest degree of toxicity, linked to certain types of cancer and other pathologies. The gastrointestinal mucosa is exposed to these forms of arsenic, but it is not known what toxic effect these species may have on it. The aim of the present work was to evaluate the toxicity and some mechanisms of action of inorganic arsenic and its metabolites [monomethylarsonic acid, MMA(V), dimethylarsinic acid, DMA(V), MMA(III) and DMA(III)] in intestinal epithelial cells, using the Caco-2 human cell line as a model.     

Cytotoxicity and oxidative DNA damage by nanoparticles in human intestinal Caco-2 cells

Abstract

The use of engineered nanoparticles in the food sector is anticipated to increase dramatically, whereas their potential hazards for the gastrointestinal tract are still largely unknown. We investigated the cytotoxic and DNA-damaging effects of several types of nanoparticles and fine particles relevant as food additives (TiO₂ and SiO₂) or for food packaging (ZnO and MgO) as well as carbon black on human intestinal Caco-2 cells. All particles, except for MgO, were cytotoxic (LDH and WST-1 assay). ZnO, and to lesser extent SiO₂, induced significant DNA damage (Fpg-comet), while SiO₂ and carbon black were the most potent in causing glutathione depletion. DNA damage by TiO₂ was found to depend on sample processing conditions. Interestingly, application of different TiO₂ and ZnO particles revealed no relation between particle surface area and DNA damage. Our results indicate a potential hazard of several food-related nanoparticles which necessitate investigations on the actual exposure in humans.     

Permeability and toxicity characteristics of L-cysteine and 2-methyl-thiazolidine-4-carboxylic acid in Caco-2 cells

Acetaldehyde is a known mutagenic substance and has been classified as a group-one carcinogen by the WHO. It is possible to bind acetaldehyde locally in the gastrointestinal (GI) tract with the semi-essential amino acid l-cysteine, which reacts covalently with acetaldehyde and forms compound 2-methyl-thiozolidine-4-carboxylic acid (MTCA). The Caco-2 cell line was used to determine the permeation of l-cysteine and MTCA, as well as the possible cell toxicity of both substances. Neither of the substances permeated through the Caco-2 cells at the concentrations used in this study, and only the highest concentration of MTCA affected the viability of the cells in the MTT (3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium bromide) test. These results showed that when l-cysteine is administered in formulations releasing it locally in the lower parts of GI tract, it is not absorbed but can react with acetaldehyde, and that neither l-cysteine nor MTCA is harmful to the cells when present locally in the upper parts of GI tract. This study also shows that MTCA is sensitive at a lower pH of 5.5. Since stable MTCA is desired in different parts of the GI tract, this observation raises concern over the influence of lower pH on l-cysteine-containing product ability to bind and eliminate carcinogenic acetaldehyde.     

Lecithin in mixed micelles attenuates the cytotoxicity of bile salts in Caco-2 cells

This study was designed to investigate the cytotoxicity of bile salt–lecithin mixed micelles on the Caco-2 cell model. Cell viability and proliferation after mixed micelles treatments were evaluated with the MTT assay, and the integrity of Caco-2 cell monolayer was determined by quantitating the transepithelial electrical resistance and the flux of tracer, FITC-dextran 4400. The apoptosis induced by mixed micelles treatments was investigated with the annexin V/PI protocol. The particle size of mixed micelles was all smaller than 100 nm. The mixed micelles with lower than 0.2 mM sodium deoxycholate (SDC) had no significant effects on cell viability and proliferation. When the level of SDC was higher than 0.4 mM and the lecithin/SDC ratio was lower than 2:1, the mixed micelles caused significant changes in cell viability and proliferation. Furthermore, the mixed micelles affected tight junctions in a composition-dependent manner. Specifically, the tight junctions were transiently opened rather than damaged by the mixed micelles with SDC of between 0.2 and 0.6 mM. The mixed micelles with more lecithin also induced less apoptosis. These results demonstrate that relatively higher concentrations of mixed micelles are toxic to Caco-2 cells, while phospholipids can attenuate the toxicity of the bile salts.