Article pubs.acs.org/crt
TiO2 Nanoparticles Induce Dysfunction and Activation of Human Endothelial Cells Angélica Montiel-Dávalos,†,‡,§ José Luis Ventura-Gallegos,∥ Ernesto Alfaro-Moreno,‡ Elizabeth Soria-Castro,⊥ Ethel García-Latorre,§ José Gerardo Cabañas-Moreno,# María del Pilar Ramos-Godinez,∇ and Rebeca López-Marure*,† †
Departamento de Biología Celular, Instituto Nacional de Cardiología “Ignacio Chávez”, México Subdirección de Investigación Básica, Instituto Nacional de Cancerología, México § Posgrado en CQB, Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, México ∥ Departamento de Bioquímica, Instituto Nacional de Ciencias Médicas y Nutrición “Salvador Zubirán”, and Departamento de Medicina Genómica y Toxicología Ambiental IIB, UNAM, México ⊥ Departamento de Patología, Instituto Nacional de Cardiología “Ignacio Chávez”, México # Centro de Nanociencias y Micro y Nanotecnologías, Instituto Politécnico Nacional, México ∇ Departamento de Microscopía Electrónica, Instituto Nacional de Cancerología, México ‡
ABSTRACT: Nanoparticles can reach the blood and cause inflammation, suggesting that nanoparticles−endothelial cells interactions may be pathogenically relevant. We evaluated the effect of titanium dioxide nanoparticles (TiO2) on proliferation, death, and responses related with inflammatory processes such as monocytic adhesion and expression of adhesion molecules (E- and P-selectins, ICAM-1, VCAM-1, and PECAM-1) and with inflammatory molecules (tissue factor, angiotensin-II, VEGF, and oxidized LDL receptor-1) on human umbilical vein endothelial cells (HUVEC). We also evaluated the production of reactive oxygen species, nitric oxide production, and NF-κB pathway activation. Aggregates of TiO2 of 300 nm or smaller and individual nanoparticles internalized into HUVEC inhibited proliferation strongly and induced apoptotic and necrotic death starting at 5 μg/cm2. Besides, TiO2 induced activation of HUVEC through an increase in adhesion and in expression of adhesion molecules and other molecules involved with the inflammatory process. These effects were associated with oxidative stress and NF-κB pathway activation. In conclusion, TiO2 induced HUVEC activation, inhibition of cell proliferation with increased cell death, and oxidative stress.
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INTRODUCTION Nanoparticles of titanium dioxide (TiO2), products of nanotechnology, are nanomaterials that have diverse applications, including photocatalysts, cosmetics, and pharmaceuticals, because of their high stability, anticorrosiveness, and photocatalytic properties.1−4 The National Institute for Occupational Safety and Health (NIOSH) has reviewed relevant animal and human data for assessing the carcinogenicity of TiO2 and has indicated that occupational exposures (mainly via inhalation and dermal contact) to low concentrations of TiO2 produce a negligible risk of lung cancer in workers; therefore, it recommends exposure limits of 1.5 mg/m3 for fine TiO2 and 0.1 mg/m3 for ultrafine TiO2, as time-weighted average concentrations (TWA) for up to 10 h/day during a 40 h work week.5 As mentioned above, TiO2 is used in a variety of products to which consumers can be exposed. A major consumption seems to occur as part of solid matrixes; therefore, nanocoated materials should undergo further emission evaluations. When TiO2 are used in “liquid products”, this is © 2012 American Chemical Society
likely to be a major route of exposure, whereas inhalation may be significant if is used as spray.6 One study showed that in sun cream exposures, applied as spray-on formulations, a possible concentration of 35 mg/m3 could occur during their use, a value that represents an acute/short-term exposure event; however, consumer products used for longer periods could be more dangerous.7 Experimental studies provide evidence that inhaled particles have the ability to cross the lung−circulation barrier, suggesting that they may translocate to the bloodstream, target tissues distant from the port of entry, and cause an inflammatory response.8,9 They can reach the vascular system, activating vessel endothelial cells or even distant organs; therefore, endothelial cells could be playing an important role in response to TiO2 due to their involvement in pro-inflammatory events. Received: December 20, 2011 Published: February 21, 2012 920
dx.doi.org/10.1021/tx200551u | Chem. Res. Toxicol. 2012, 25, 920−930
Chemical Research in Toxicology
Article
diffraction to determine composition (Bruker D8 Advance with Cu Kα radiation and a Lynxeye Bruker detector Bruker, Karlsruhe, Germany). TEM was used to determine the uptake and internalization of TiO2 by HUVEC. The BET surface area of nanoparticles was determined in an ASAP 2050 Xtended Pressure Sorption Analyzer (Micromeritics Instrument Corporation, Norcross, GA). In experimental assays, nanoparticles were not sonicated because we did not observe differences of sonicated or nonsonicated nanoparticles on the biological effect. Cell Culture. HUVEC were obtained from primary human endothelial cells by proteolytic dissociation of umbilical cord veins from normal deliveries, treated with collagenase type II (0.2 mg/mL), and cultured on gelatin-coated culture dishes in M199 supplemented with 10% FBS, glutamine (2 mM), heparin (1 mg/mL), and endothelial mitogen (20 μg/mL), as previously described.17 Cells were used for all experiments on their second passage. The phenotype of HUVEC cultures was confirmed by Von Willebrand antigen staining. Cultures exposed to human recombinant TNF-α (10 ng/mL) were used as a positive control of endothelial activation. On the basis of previous studies, we chose to expose HUVEC at 5, 10, 20, and 40 μg/cm2 of TiO2. Human leukemia pro-monocytic U937 cells were cultured with RPMI-1640 medium supplemented with 10% FBS and Lglutamine (2 mM). Cell Proliferation. The cell number was evaluated by crystal violet staining. HUVEC were plated on 96-multiwell plates and cultured without and with TiO2 for 24 and 72 h. At the end of these treatments, cells were fixed with 100 μL of ice cold glutaraldehyde (1.1% in PBS) for 15 min at 4 °C. Plates were washed three times by submersion in deionized water, air-dried, and stained for 20 min with 100 μL of a 0.1% crystal violet solution (in 200 mM phosphoric acid buffer at pH 6). After careful aspiration of the crystal violet solution, the plates were extensively washed with deionized water and air-dried prior to the solubilization of the bound dye with 100 μL of a 10% acetic acid solution incubated during 30 min. The optical density of the plates was measured at 595 nm in a multiplate spectrophotometer. Cell Death Determination. Phosphatidylserine translocation was used to determine apoptosis. Cells were treated with TiO2 for 24 h, then washed with PBS, and centrifuged at 200 rpm for 5 min. The cell pellet was resuspended in 100 μL of labeling solution (20 μL annexinV-fluos labeling reagent in 1 mL of Hepes buffer [10 mM Hepes/ NaOH, pH 7.4, 140 mM NaCl, and 5 mM CaCl2]) and 1 μg/mL propidium iodide and incubated for 15 min. The volume was increased to 500 μL with Hepes buffer, and the samples were analyzed on the flow cytometer at 488 (excitation) and 515 nm bandpass filter for fluorescein detection and a filter >560 nm for propidium iodide detection. Cells stained with annexin-V-fluos alone were considered apoptotic, whereas double-stained cells (annexin-V-fluos + propidium iodide) were considered late apoptotic or necrotic and analyzed with the cytometer using the Cell Quest Software Program. Adhesion of U937 Cells to Endothelial Cells. Adhesion was evaluated using U937 cells that were labeled with [3H]-thymidine; 1 × 105 HUVEC were seeded in 24-well tissue culture plates with 1 mL of supplemented M199 medium and treated with TNF-α and different concentrations of TiO2 for 3 h, whereas 6 × 106 U937 cells were incubated with 30 μCi of [3H]-thymidine for 48 h. Pretreated HUVEC were cocultivated for 3 h with 5 × 105 U937 cells/well. Each well was washed to eliminate U937 cells not attached to HUVEC. After this, cells were fixed with 95% methanol and lyzed with NaOH (200 mM) for 12 h, and radioactivity was determined in a scintillation counter (Packard model 2200CA). Counts per minute (cpm) were considered directly proportional to the number of U937 cells adhered to the endothelial cells. Evaluation of the Adhesion Molecule Expression by Flow Cytometry. Two million HUVEC were seeded each in a 100 mm diameter Petri dish and treated with TiO2 for 3 or 24 h to determine the expression of early and late adhesion molecules, respectively. After this time, cells were detached with collagenase (0.4 mg/mL) and centrifuged at 1200 rpm for 3 min. Cells were incubated with the different FITC-labeled human adhesion molecules monoclonal antibodies diluted 1:20. After 1 h of incubation, cells were washed
One of the initiating events of inflammation is endothelial activation, and the initiating dysfunction event is followed by smooth muscle proliferation and architectural disruption.10 Monocytes-macrophages are then recruited by the endothelium and invade the subintimal space through a complex mechanism characterized by the enhanced expression of chemoattractant interleukins, adhesion molecule proteins, and various cellmembrane cytokine receptors. In this process, oxidized lowdensity lipoproteins (LDL) induce adhesion molecules expression (ICAM-1, VCAM-1, PECAM-1, and E- and Pselectins) on endothelial cells. Oxidized LDL also activates the signal transduction pathways that lead to enhanced gene expression of the transcription factor nuclear transcription factor κB (NF-κB) and IκB protein degradation. These intracellular signals increase reactive oxygen species (ROS) and NO production and cause an increase in the expression of factors such as vascular endothelial growth factor (VEGF), angiotensin II (AII), and tissue factor (TF).11−13 Recent studies have demonstrated TiO2-induced oxidative stress and pro-inflammatory responses on a bronchial epithelial cell line.14,15 Other investigations showed that TiO2 have the ability to cause cell death, mitochondrial damage, and oxidative stress of the human lung adenocarcinoma epithelial cell line A549.16 In this work, we determined the effect of TiO2 on human umbilical vein endothelial cells (HUVEC) activation. TiO2 induced an increase in monocytic cells adhesion to HUVEC, an increase of early (E- and P-selectin) and late (ICAM-1, VCAM-1, and PECAM-1) adhesion molecules expression, an increase in ROS and NO production, NF-κB translocation, and development of apoptotic death. In addition, TiO2 induced an increase in AII, TF, VEGF, and oxidized LDL receptor-1 (LOX-1) expression.
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EXPERIMENTAL PROCEDURES
Materials. RPMI 1640, M199 medium, and trypsin were purchased from GIBCO/BRL (Grand Island, NY) and fetal bovine serum (FBS) was from HyClone (Logan, UT). Annexin V was purchased from Roche (Mannheim, Germany). Sterile plastic material for tissue culture was from NUNC and COSTAR. Flow cytometry reagents were purchased from Becton Dickinson, Immunocytometry Systems (San José, CA). Tumor necrosis factor α (TNF-α) was purchased from R&D Systems (Minneapolis, MN). Peroxidase-labeled monoclonal antibody against Von Willebrand factor was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). 2,7-Dichlorodihydrofluorescein diacetate (H2DCFDA) was purchased from Molecular Probes. [γ-32P]ATP was purchased from New England Nuclear (Boston, MA). T4 polynucleotide kinase was purchased from New England Biolabs (Beverly, MA). TiO2 were purchased from Paris Drugstore (Mexico City, Mexico). All other chemicals were purchased from Sigma Aldrich (St. Louis, MO). Titanium Dioxide Nanoparticles (TiO2). TiO2 were handled under light-free conditions at all of the times. TiO2 were sterilized by autoclave (1.5 atm, 20 min). A stock solution of TiO2 was prepared by resuspending 1 mg of sterile particles in 1 mL of phosphate buffer solution (150 mM NaCl, 4.4 mM KCl, 10.9 mM Hepes, and 12.2 mM glucose, pH 7.4), immediately before treatment of cells. During the incubation periods, the exposed cells remained in the light-free incubator. A suspension of TiO2 at 20 μg/cm2 in M199 medium supplemented with 10% FBS was used to characterize the nanoparticles by means of transmission electron microscopy (TEM) (Jeol 1010 with an AMT camera system at 60 Kv) and scanning electron microscopy (SEM) (Zeiss DSM 950 at 15 Kv). The size of the particles and the ζ-potential were analyzed in a Zetasizer Nano series model ZS. To this measuring, nanoparticles were suspended in M199 medium plus 10% FBS and sonicated at 33 W for 15 min to their better dispersion. Nanoparticles were also analyzed by X-ray 921
dx.doi.org/10.1021/tx200551u | Chem. Res. Toxicol. 2012, 25, 920−930
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Figure 1. Morphologial evaluation and uptake of TiO2. (A) Aggregates of TiO2 dispersed in culture medium supplemented with FBS and dispersed in only PBS solution (inset box); direct magnification, 100 000×. (B) TiO2 dispersed in PBS observed by SEM at magnification of 40 000×. (C and D). Cells were treated with TiO2 at a concentration of 5 μg/cm2. After 24 h of treatment, cells were washed three times with PBS and were analyzed by TEM at a direct magnification of 20 000× (C) and 60 000× (D). cultures were used as negative controls. After treatment, 100 μL of conditioned medium was diluted 1:2 with 100 μL of Griess solution and incubated for 15 min at room temperature. Previously, a standard curve was obtained using known concentrations of NaNO2 (range of 0.4−100 μM). The optical density of the plates was measured at 540 nm (Microplate autoreader EL311, Bio-Tek Instruments). Concentrations of NaNO2 in control and exposed cultures were plotted against the standard. Nuclear Extract Preparation. Cells (3 × 106) were seeded in 100 mm diameter Petri dishes and treated with TiO2 at 5 and 20 μg/cm2 or TNF-α for 1 h. Afterward, the cells were scraped in PBS, collected, and centrifuged at 3000 rpm for 5 min. To recover the cellular nuclei, the pellet was frozen in liquid nitrogen for 15 s and resuspended gently in 100 μL of hypotonic solution (10 mM Hepes, pH 7.9, 10 mM KCl, 1.5 mM MgCl2, and 1 mM DTT). The integrity of nuclei was evaluated by staining with trypan blue and microscopic observation. Nuclear suspensions were centrifuged at 1000 rpm for 10 min at 4 °C. Nuclear pellets were resuspended in 15 μL of hypertonic solution (10 mM Hepes, pH 7.9, 0.4 mM NaCl, 1.5 mM MgCl2, 25% glycerol, 0.2 mM EDTA, 1 mM DTT, and 0.5 mM PMSF), incubated for 30 min with gentle mixing at 4 °C, and centrifuged at 18000 rpm for 20 min. Supernatants containing nuclear proteins were collected, diluted 1:2 with an HDKE buffer (20 mM Hepes, pH 7.9, 50 mM KCl, 25% glycerol, 0.2 mM EDTA, 1 mM DTT, and 0.5 mM PMSF), and stored at −70 °C. Nuclear protein concentrations were determined using the Bradford method. Electrophoretic Mobility Shift Assay (EMSA). Nuclear protein extracts (10 μg) were assayed for DNA interaction by EMSA. The [32P]-labeled double-stranded oligonucleotides used were (5′-AGTTGAGGGGACTTTCCCAGGC-3′) that contain the underlined NF-κB consensus sequence or the mutated sequence (5′-AGTTGAGGCGACTTTCCCAGGC-3′) (negative control). Specificity was evaluated by incubating with 80× excess of unlabeled oligonucleotide (cold competition). Binding reactions were carried out by incubating samples in a reaction buffer (50 mM KCl, 20% glycerol, 0.2 mM EDTA, 1 mM dithiothreitol, 0.5 mM phenymethylsulfonyl fluoride, 20 mM HEPES, pH 7.9, 1 μg/μL BSA, and 1 μg/μL poly dI-dC). DNA−
twice with PBS-albumin (8% albumin and 0.02% sodium azide), resuspended in 500 μL PBS, and immediately subjected to cytometric analysis performed with a Becton Dickinson Fascalibur Instrument. Results are expressed as percentage of expression compared to control cultures. To calculate the percentage of expression, we used the positive cell number to FITC (FL1-H) multiplied by the mean of the fluorescence units (FU). Results in FU for control cultures were considered as 100%. Evaluation of the AII, FT, VEGF, and LOX Expression by Flow Cytometry. Two million HUVEC were seeded in 100 mm Petri dishes and treated with TNF-α or TiO2 at 5 and 20 μg/cm2 for 24 h. After this time, cells were detached with collagenase (0.4 mg/mL) and centrifuged at 1200 rpm for 3 min. Cells were incubated with the different FITC-labeled human adhesion molecules monoclonal antibodies against TF, AII, VEGF, and LOX-1 diluted 1:20. After 1 h of incubation, cells were washed twice with PBS at 8% albumin and 0.02% sodium azide, resuspended in 500 μL of PBS, and immediately subjected to cytometric analysis performed with a Becton Dickinson Fascalibur Instrument. Results were recovered as relative fluorescence intensity units and converted to percent expression with respect to untreated controls. Measurement of ROS. Oxidation of H2DCFDA into 2,7dichlorofluorescein (DCF) was used to assess ROS generation in HUVEC. Cells were incubated with H2DCFDA (10 μM) for 30 min at 37 °C and washed twice with PBS. HUVEC were then cultured in the presence or absence of TiO2 at 5 and 20 μg/cm2 for 1 h. H2O2 (500 μM) was used as a positive control to induce oxidative stress. After an extensive wash, the fluorescence was evaluated by flow cytometry (Fascalibur, Becton Dickinson). The mean fluorescence intensity was calculated by multiplying the number of events (fluorescent cells) by the mean of the intensity presented by the Cell Quest software used for the analysis. Production of NO. Quantification of sodium nitrite (NaNO2) was used as an indirect method to determine NO production. Cells were seeded in 96 well plates (NUNC) at a density of 1 × 105 cells/well in M199 (phenol red free) and 5% FBS. Cells were exposed to TiO2 at 5 and 20 μg/cm2 or TNF-α for 24, 48, and 72 hours. Unexposed 922
dx.doi.org/10.1021/tx200551u | Chem. Res. Toxicol. 2012, 25, 920−930
Chemical Research in Toxicology
Article
protein complexes were resolved on 5% polyacrylamide gel at 100 V over 2 h. The gel was dried and exposed to a Storage Phosphor Screen (Molecular Dynamics, San Francisco, CA) that was read in a Storm 850 Phosphorimager (Molecular Dynamics) and analyzed with ImageQuant software (Molecular Dynamics). Western Blotting. Fifty micrograms of cytoplasmic protein was loaded per lane, resolved by SDS/PAGE on 7.5% polyacrylamide gels, and transferred to nitrocellulose membranes. Membranes were blocked with a suspension of 5% fat-free milk powder in 20 mM Tris, 137 mM NaCl, 3 mM KCl, and 0.1% Tween-20, pH 7.6 (TBST). After 2 h of incubation with monoclonal anti-IκB-α (diluted 1:500) or β-actin (1:5000), blots were washed for 5 min in TBS-T twice. A peroxidase goat antimouse antibody diluted 1:2500 was added for 1 h before washing blots three times with TBS-T. Peroxidase bound to blot was detected by enhanced chemiluminescence using the supersignal system (Pierce Rockford, IL) on X-ray film (Kodak, United States) and quantified by densitometry with the Molecular Imaging Kodak MI software (Eastman Kodak Company). Statistical Analysis. All experiments were performed in at least three independent trials. Results are expressed as means ± standard errors of the mean (SEMs). The results were analyzed by ANOVA test (INERStat v2.0), and differences were considered significant when P < 0.05. When a temporal curve was used to evaluate nitrite production, the exposed cultures were compared with controls at the respective time point.
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RESULTS TiO2 Nanoparticles Characterization and Cellular Uptake. TEM and SEM images show that TiO2 used in the present study formed aggregates of spheres of less than 50 nm (Figure 1A,B). The size distribution for aggregates used for the biological evaluations was between 105 and 1281 nm (data not shown), with a mean size of 421 nm (Table 1 and Figure 2A).
Figure 2. Characterization of TiO2. Size of TiO2 nanoparticles and ζpotentials were analyzed in a Zetasizer Nano series model ZS (A). The crystalline structure of TiO2 was determined by X-ray diffraction (B). For these analyses, a concentration of 20 μg/mL was evaluated.
Table 1. Physicochemical Properties of TiO2 NPsa mean size of particle mean size of aggregate ζ-potential purity anatase rutile BET surface area electrophoretic mobility
