Anatase Mixed Phase Nanoparticles

Article pubs.acs.org/crt

Distinctive Toxicity of TiO2 Rutile/Anatase Mixed Phase Nanoparticles on Caco-2 Cells Kirsten Gerloff,†,∥ Ivana Fenoglio,§ Emanuele Carella,§ Julia Kolling,† Catrin Albrecht,† Agnes W. Boots,† Irmgard Förster,‡ and Roel P. F. Schins*,† †

Particle Research and ‡Molecular Immunology, Leibniz Institut für Umweltmedizinische Forschung (IUF) at the Heinrich Heine University Düsseldorf, Germany § Dip. di Chimica Inorganica, Fisica e dei Materiali, Interdepartmental Centre “G. Scansetti” for Studies on Asbestos and Other Toxic Particulates and Interdepartmental Center for Nanostructured Interfaces and Surfaces, University of Torino, Italy ∥ Immunity, Infection and Inflammation Program, Mater Medical Research Institute and the University of Queensland, Mater Health Services, South Brisbane, QLD, Australia S Supporting Information *

ABSTRACT: Titanium dioxide has a long-standing use as a food additive. Micrometric powders are, e.g., applied as whiteners in confectionary or dairy products. Possible hazards of ingested nanometric TiO2 particles for humans and the potential influence of varying specific surface area (SSA) are currently under discussion. Five TiO2-samples were analyzed for purity, crystallinity, primary particle size, SSA, ζ potential, and aggregation/agglomeration. Their potential to induce cytotoxicity, oxidative stress, and DNA damage was evaluated in human intestinal Caco-2 cells. Only anatase-rutile containing samples, in contrast to the pure anatase samples, induced significant LDH leakage or mild DNA damage (Fpg-comet assay). Evaluation of the metabolic competence of the cells (WST-1 assay) revealed a highly significant correlation between the SSA of the anatase samples and cytotoxicity. The anatase/rutile samples showed higher toxicity per unit surface area than the pure anatase powders. However, none of the samples affected cellular markers of oxidative stress. Our findings suggest that both SSA and crystallinity are critical determinants of TiO2-toxicity toward intestinal cells.

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INTRODUCTION The use of particles as food additives has been well established throughout the last decades. TiO2 for example is well appreciated for its inert capacities and as such widely used as a white food coloring compound. Indeed, it can be found in many foods, for example, dairy products,1 and is accepted by the EU with the E-number 171. Currently, a lot of research is in progress to expand the application areas of this additive by using nanometric TiO2 particles, for example, as a coating in confectionary products to prevent melting or improve shelf life.2 Bulk TiO2 is regarded as a highly biocompatible material. However, TiO2 nanoparticles have been found to elicit toxic responses in various in vivo and in vitro systems.3−6 The observed adverse effects in these studies have been attributed to the small particle size, the high specific surface area (SSA), and the reactivity of nanometric TiO2 powders. On the level of the cell, the formation of reactive oxygen species (ROS) and possible resulting induction of oxidative stress have been considered as the underlying molecular mechanism implicated in the cytotoxic, inflammatory, and DNA damaging effects of nanoparticles, including TiO2. Consequently, investigations on possible adverse effects of ingested TiO2-nanoparticles are © 2012 American Chemical Society

necessary to rule out any health risks to humans consuming nanoparticle-containing products in the future. Previously, we have investigated the potential of a range of nanosized materials to induce cytotoxicity and DNA damage in Caco-2 cells.7 Low cytotoxic effects could be determined by using an anatase-rutile containing TiO2 particle with a SSA value of about 50 m2/g. In the absence of light exposure, this particular material appeared to have no significant DNA damaging potential after 4 h of treatment with a dose of 20 μg/ cm2. Because of the expected importance of nano-TiO2 for food products in the near future, we intended to further evaluate a possible influence of particle SSA on the cytotoxic and oxidative stress inducing potential of TiO2. In the present study, we have determined the SSA of five different TiO2 samples as well as their mean primary particle size and organization using N2 adsorption according to Brunauer, Emmett, and Teller (BET), transmission electron microscopy (TEM), dynamic light scattering (DLS), and Flow Particle Image Analyzer. We used two samples of high SSA values (>280 m2/g), two samples of medium SSA values (∼50 Received: August 9, 2011 Published: January 20, 2012 646

dx.doi.org/10.1021/tx200334k | Chem. Res. Toxicol. 2012, 25, 646−655

Chemical Research in Toxicology

Article

m2/g), and a micrometric TiO2 powder. The potential role of the surface area and crystalline phase of TiO2-particles on cytotoxicity and DNA damage was evaluated in human intestinal Caco-2 cells by the WST-1 and LDH assays and by the formamidopyrimidine glycosylase (Fpg)-modified comet assay, respectively. The role of oxidative stress was further investigated by analysis of the depletion of total cellular glutathione (GSH) and the mRNA expression of the oxidative stress marker genes heme oxygenase-1 (HO-1) and γ-glutamyl cysteine synthetase (γ-GCS).

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Instruments, Worcestershire, U.K.). In this technique, the velocity of particles in an oscillating electric field, which is proportional to their ζ potential, was measured by light scattering. TiO2 particles were suspended in ultrapure water and then sonicated for 2 min with a probe sonicator (100 W, 60 kHz, Sonoplus, Bandelin, Berlin, Germany). The ζ potential at pH 7.4 was obtained by interpolation of a curve obtained by measuring the ζ potential at different pH (2−9) by adding 0.1 M HCl or NaOH to the suspension (see Supporting Information). Morphological Characterization. The mean size of primary particles was evaluated by electron transmission microscopy TEM (JEOL 3010-UHR instrument operating at 300 kV, equipped with a 2k × 2k pixels Gatan US1000 CCD camera). The powders were suspended in water, sonicated to reduce the agglomeration of particles, deposited on a grid, and the solvent evaporated. Aggregation Degree. The evaporation of the solvent used to prepare the samples for TEM evaluations may lead to agglomeration of the particles. Therefore, the size of aggregates was confirmed by dynamic light scattering (DLS) (Zetasizer Nano-ZS, Malvern Instruments, Worcestershire, U.K., detection limits 1 nm−6 μm). This technique measures a hydrodynamic size correlated to the measurement of dynamic fluctuations of light-scattering intensity caused by the Brownian motion of the particles during movement in ultrapure water. The powders were suspended in water at pH 9 and sonicated for 2 min with a probe sonicator (100 W, 20 kHz, Sonoplus, Bandelin, Berlin, Germany). In these conditions, the high electrostatic repulsion between particles inhibits the formation of agglomerates. The presence of aggregates having a diameter larger than 6 μm was evaluated by optical microscopy analysis (DFC295, Leica, 100×). The experiments were performed on five different suspensions. Aggregation and Agglomeration Degree in Cellular Media. The size of aggregates/agglomerates in the serum free cellular media that was used for all experiments was evaluated by suspending the powders in the culture medium used for the in vitro experiments, according to the protocol described above. DLS analysis performed on the suspensions did not detect any particles in the nanometer range for all samples. Conversely, aggregates/agglomerates having a size larger than the detection limit of DLS analysis were observed by optical microscopy in all samples. Statistical analysis of the size of aggregates/ agglomerates was obtained by using a flow particle image analyzer (Sysmex FPIA-3000, Malvern Instrument Ltd., UK, detection limits 0.8−300 μm). This instrument measures the diameter of a circle having the same projected area as the particle image optically detected. The analysis of the particle size distributions has been performed on five different suspensions (see Supporting Information). Culture and Treatment of the Cells. The human colon adenocarcinoma cell line Caco-2 was obtained from DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Germany) and grown in MEM with Earle's salts and nonessential amino acids, supplemented with 20% FCS, 1% L-glutamine, and 30 IU/mL penicillin−streptomycin. For cytotoxicity assays, DNA damage detection, and measurement of oxidative stress, cells were prepared and treated in serum free cellular media as described earlier.7 Cytotoxicity. Cytotoxicity was determined by the lactate dehydrogenase (LDH) assay as a marker of cell membrane integrity as well as the water-soluble tetrazolium salt (WST-1) assay as a measure of the metabolic activity of the cells. For both the LDH and the WST-1 assay, 96-well tissue culture plates were used, and cells were treated at the indicated concentrations and time intervals with the particles as described above. LDH was determined using a commercial diagnostic kit. The cleavage of the tetrazolium salt WST-1 to formazan dye via mitochondrial dehydrogenases was measured using a commercial WST-1 diagnostic kit. Cells were incubated for additional 2 h with WST-1 and analyzed directly. Detection of DNA Strand Breakage and Oxidative DNA Damage by Fpg-Modified Comet Assay. The Fpg-modified comet assay was used to measure DNA strand breaks and specifically oxidative DNA damage in the cells, based on the method described by Speit et al.8 with some minor modifications described earlier.7 To maintain absence of cytotoxicity and prevent DNA damage repair,

MATERIALS AND METHODS

Materials. Trypsin, Dulbecco’s Ca2+/Mg2+-free phosphate buffered saline (PBS), agarose, low melting point (LMP) agarose, Triton X100, dimethyl sulfoxide (DMSO), ethidium bromide, glutathionereductase, reduced L-glutathione, fetal calf serum (FCS), βnicotinamide adenine dinucleotide phosphate (β-NADPH), 5,5′dithio-bis-2-nitrobenzoic acid (DTNB), and BCA-assay kit (bicinchoninic acid- and copper(II) sulfate solution) were all purchased from Sigma (Germany). Lactate dehydrogenase (LDH) Cytotoxicity Detection Kit and Cell Proliferation Reagent WST-1 were both obtained from Hoffmann-LaRoche (Switzerland). Minimum essential medium (MEM) with Earle's salts and nonessential amino acids (catalog number 10370), L-glutamine, and penicillin−streptomycin were purchased from invitrogen (Germany). iScript cDNA Synthesis Kit was purchased from BioRad Laboratories (USA). RNeasy Mini kit and QuantiFast SYBR Green PCR Kit were obtained from Qiagen (Germany). [R]-1-[(10-Chloro-4-oxo-3-phenyl-4H-benzo[α]quinolizin-1-yl)-carbonyl]-2-pyrrolidinemethanol (Ro19-8022) was a kind gift from Hoffmann-LaRoche (Switzerland). Formamidopyrimidine-glycosylase (Fpg)-enzyme was kindly provided by Dr. Andrew Collins, Institute for Nutrition Research, University of Oslo, Norway. All other chemicals were from Merck (Germany). TiO2 Samples. A set of five different TiO2 samples was used: a pyrogenic nanometric anatase/rutile powder (Aeroxide P25) purchased from Degussa-Evonik (Germany) (TUFA/RI); a nanometric anatase/rutile powder purchased from Sigma (Germany) (TUFA/RII); a nanometric anatase powder (JRC12), supplied by the Japan Catalysis Society (TUFAI); and a nanometric anatase powder (Hombikat UV100) obtained from Sachtleben (Germany) (TUFAII). Finally, a fine (micrometric) anatase powder purchased from Aldrich (Germany) (TFA) was used as negative control; fine and nanometric TiO2 powders, including those which we selected for the present investigations, have been used by several investigators as model particles to address the role of particle surface area in in vitro and in vivo toxicity.3−7 For the incubation of Caco-2 cells with the TiO2 powders, the samples were suspended in serum free cellular media, sonicated for 10 min in a water bath sonicator (Bandelin Sonorex RK 52, 120 W) and used directly. XRD Spectroscopy. XRD spectra were collected on a diffractometer (PW1830, Philips) using CoKα radiation, in the (20−90) 2θ range, with step width 2θ = 0.05, and diffraction peaks have been indexed according to the ICDD database (International Centre for Diffraction Data). The spectra have been elaborated (X’pert Highscore 1.0c, PANalytical B.V.) in order to assess the primary particle mean diameter of the different specimens. Surface Area Measurements. The surface area of the particles was measured by means of the Brunauer, Emmett, and Teller (BET) method based on N2 adsorption at 77 K (Micrometrics ASAP 2010). Elemental Analysis. The TiO2 samples were analyzed using an EDAX Eagle III energy dispersive micro-XRF spectrometer equipped with a Rh X-ray tube and a polycapillary exciting a circular area of nominally 30 μm diameter. Data collection occurred at each point for 200 s detector live time, with X-ray tube settings adjusted for 30% dead time. About 1 × 106 Cps were counted per scan. At least 4 points were collected for each sample. ζ Potential. The ζ potential was evaluated by means of electrophoretic light scattering (ELS) (Zetasizer Nano-ZS, Malvern 647



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Table 1. Characterization of the Investigated Materials sample

crystalline phasesa

purity (%)

ζ potential (water, pH 7.4)

particle specific surface area (m2/g)

primary particles mean diameter (nm)

TFA

100% anatase

>99b

−48.9

10

215 ± 2d

TUFA/RI

>99.6c

−21.5

52.6 ± 0.1

25.20 ± 0.20e

>99.7c

−23.5

52.8 ± 0.4

21.90 ± 0.30e

TUFAI

77% anatase, 23% rutile 90% anatase 10% rutile 100% anatase

>99.3c

−36.5

282.3 ± 1.9

6.7 ± 1.3d

TUFAII

100% anatase

>99.5b

−38.3

342.4 ± 1.3

3.94 ± 0.05e

TUFA/RII

Z-average hydrodynamic diameter (nm) water, pH 9f 374.0 ± 5.5 (PDI: 0.152 ± 0.03) 214.5 ± 2.6 (PDI: 0.208 ± 0.1) 327.5 ± 27.6 (PDI: 0.320 ± 0.03) 455.2 ± 74.4 (PDI: 0.385 ± 0.06) 291.1 ± 56.6 (PDI: 0.313 ± 0.06)

CE diameter (μm) in serum free cellular mediag 6.2 ± 5.3 11.8 ± 10.9 12.0 ± 10.4 6.5 ± 4.6 6.1 ± 4.4

a

Evaluated by XRD. bAs declared by the supplier. cEvaluated by XRF. dCalculated from the XRD broadening peak using Scherrer’s equation. Evaluated by TEM. fEvaluated by dynamic light scattering (DLS). The diameter is expressed as the Z-average hydrodynamic diameter ± standard deviation. gEvaluated by Flow Particle Image Analyzer Sysmex FPIA-3000. The diameter is expressed as circle equivalent (CE) diameter ± standard deviation.

e

Figure 1. Morphological characterization of titanium dioxide particles. TEM images of (A) TUFA/RII, 30kX; (B) TUFA/RI, 40kX; (C) TUFAII, 40kX; and (D) TUFAI 12kX. In the insets, the HRTEM images (200kX, 500kX, 300kX, and 300kX, respectively) are shown. which will occur upon too long incubation times, the cells were incubated with the lower particle concentration (20 μg/cm2) and harvested after 4 h for analysis of DNA strand breakage and oxidative

DNA damage. As a positive control, the photosensitizer Ro-19 8022 was used, which induces specific oxidative lesions after 2 min of light exposure.9 Data are presented as % comet tail values in the absence of 648



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Fpg. Moreover, results are depicted as the calculated group means and standard deviations (SD) of the differences in % tail DNA as measured in the presence or absence of the Fpg enzyme: ΔFpg = [% tail DNA+Fpg] − [% tail DNA−Fpg]. Total Glutathione Content. Total glutathione content of the Caco-2 cells was determined as a marker for oxidative stress. Therefore, cells were treated for 4 h, rinsed twice with ice cold PBS, and processed as described earlier.7 The values were adjusted to the total protein content as determined by the BCA-assay. RNA Isolation and Quantitative Real-Time Reverse-Transcription Polymerase Chain Reaction (qRT-PCR). Cells were treated with 20 μg/cm2 particles for 4 h as described above. Total RNA was isolated from the cells using a commercial RNeasy kit according to the manufacturer’s instructions, and 0.5 μg per sample was transcribed into cDNA using iScript cDNA Synthesis Kit according to the protocol. Total amount of mRNA was analyzed in the Rotor-Gene Q (Qiagen) in 47 cycles, using 7.5 μL of QuantiFast SYBR Green PCR Kit, 25 ng of cDNA, and 0.5 μM primers. Human βActin was used as the internal reference gene. Primer sequences for HO-1 were 5′-AAC TTT CAG AAG GGC CAG GT-3′ (forward) and 5′-CCT CCA GGG CCA CAT AGA T-3′ (reverse), for γ-GCS (light chain) were 5′-GAC AAA ACA CAG TTG GAA CAG C-3′ (forward) and 5′-CAG TCA AAT CTG GTG GCA TC-3′ (reverse), and for βActin 5′-CCC CAG GCA CCA GGG CGT GAT-3′ (forward) and 5′GGT CAT CTT CTC GCG GTT GGC CTT GGG GT-3′ (reverse). Statistical Analysis. All treatment related effects were evaluated using one-way analysis of variance (ANOVA) with Dunnett's posthoc comparison. Data shown in the graphs represent mean and standard deviations (SD), with level of significance indicated by the number of asterisks, i.e., *p < 0.05, **p < 0.01, and ***p < 0.001. Linear regression analysis was used to determine the association between the toxicity of the samples and their SSA. Therefore, the dose was expressed as particle surface per unit cell culture dish surface area (cm2/cm2). Statistical analyses were performed using SPSS 18.

Figure 2. Crystalline phases in titanium dioxide particles. XRD patterns of (a) TFA; (b) TUFA/RI; (c) TUFA/RII; (d) TUFAII; and (e) TUFAI in the 20−80 = 2θ range. The diffraction peaks of anatase (*) and rutile (○) are indicated above the patterns.

were detected in both TEM and DLS analysis. The broadening of the diffraction peaks corresponding to the anatase phase in the XRD pattern (Figure 2) reveals a highly disordered structure which is a direct consequence of the small size of primary particles. The presence of amorphous TiO2 is confirmed by the low abundance of crystallographic planes in the HRTEM images (see insert of Figure 1C). A similar structure is likely for the other high surface area sample TUFAI: the broad diffraction peaks in the XRD patterns are indicative of a disordered anatase structure. As shown in Figure 1D, the size of the primary particle was difficult to evaluate by TEM. From the XRD diffraction pattern according to Scherrer’s equation, the estimated primary particle size of this sample was 6.7 nm (Table 1). TEM analysis of both samples revealed the presence of both nanometric and micrometric aggregates, thus confirming the high value of the polydispersity index (PDI) found in the DLS analysis (Table 1). TFA is a micrometric powder as suggested by the low SSA and by the crystallite size estimated from the XRD diffraction pattern according to Scherrer’s method.10 The powder is mainly composed by aggregates as suggested by the DLS measurement (Table 1). As expected, when the TiO2 powders were dispersed in the FCS-free cellular media, the formation of large agglomerates with sizes ranging from hundreds of nanometers to micrometers was observed for all samples as a consequence of the high ionic strength and of the neutral pH of the solutions. However, TUFA/RI and TUFA/RII exhibited mean diameters larger than those of the anatase samples (Table 1). In these conditions, no monodispersed particles or aggregates in nanometric size were detected for any sample by DLS analysis. The different crystalline phases of samples reflect the surface charge of particles measured as ζ potential at pH 7.4 (Table 1 and Supporting Information, Figure S1). As expected, the anatase samples (TFA, TUFAI, and TUFAII) exhibited a negative ζ potential as a consequence of the presence of slightly acidic hydroxyls at the surface. A less intense ζ potential was found for the mixed phases samples (TUFARI and TUFARII), suggesting a lower intensity of surface charge. This may be a consequence of both a minor abundance of acidic

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RESULTS The samples used in this study were all characterized by a high degree of purity (>99%) but differed to a great extent in terms of their crystalline phases, SSA values, and nanostructure (Table 1). Two samples, i.e., TUFA/RI and TUF A/RII appeared composed of anatase and rutile phases in different proportions, while the other three samples were pure anatase. TUFA/RI and TUFA/RII exhibited similar SSA values. The TEM images of these two samples show the presence of primary particles having a mean diameter of approximately 20 nm, mainly organized in nanometric and micrometric agglomerates (Figure 1A and B). However, single nanoparticles were also detected. A statistical analysis of the size of aggregates has been obtained by DLS analysis in water at pH 9. At this pH, TiO2 particles exhibit a highly negatively charged surface (see Supporting Information, Figure S1) and therefore, the formation of agglomerates is minimized because of the electrostatic repulsions among particles. Both samples exhibited aggregates having a wide range of diameters as suggested by the high polydispersity index (PDI) values. Well-defined crystallographic planes are visible in the HRTEM images (see inserts in Figure 1A and B), suggesting a high degree of crystallinity of the materials. The XRD patterns (Figure 2) reveal the presence of very sharp peaks corresponding to anatase and rutile phases, which further confirms the crystallinity of both samples. The high surface area sample TUFAII exhibits a very different structure: in this case, nanometric primary particles are strongly bonded together to form nanometric and micrometric aggregates (Figure 1C). No single monodisperse nanoparticles 649



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To further evaluate the cytotoxicity of the samples using an independent test, the metabolic activity of the Caco-2 cells was determined via the WST-1 assay after incubation for 4 and 24 h with 20 and 80 μg/cm2 particles (Figure 4A and B). After 4 h of

functionalities at the surface or a lower acidity of hydroxyl groups. The higher points of zero charge (PZC) observed for the mixed phases samples (Supporting Information, Figure S1) confirm the prevalence of the latter effect. To determine whether the observed differences in primary particle size, surface area, or crystalline phase of the TiO2 samples result in an altered cytotoxic potential to Caco-2 cells, all five particle types were simultaneously tested in the LDH and the WST-1 assay. Cell membrane integrity was measured after incubation for 4 and 24 h with 20 and 80 μg/cm2 particles, respectively, as the degree of LDH leakage in comparison to a positive control (100%), provided by the assay kit, and negative control (0%) (Figure 3A and B). No significant cytotoxic effect

Figure 4. Effects of titanium dioxide particles on the metabolic competence of Caco-2 cells measured by the WST-1 assay. Cells were incubated with 20 and 80 μg/cm2 particles for 4 (A) and 24 (B) h. SSA is indicated in m2/g. Mitochondrial enzyme activity was measured via the conversion of WST-1 as a marker of cell viability. Values are expressed as the mean and standard deviation, n = 3. * p < 0.05, ** p < 0.01, and *** p < 0.001 vs control.

Figure 3. Effects of titanium dioxide particles on Caco-2 cell membrane damage measured by the LDH assay. Cells were incubated with 20 and 80 μg/cm2 of the different TiO2 samples for 4 (A) and 24 (B) h. SSA is indicated in m2/g. Membrane integrity was measured as a marker of cell toxicity using the LDH assay. Values are expressed as the mean and standard deviation, n = 3. * p < 0.05, ** p < 0.01, and *** p < 0.001 vs control.

incubation, statistically significant reduction in metabolic activity could be observed for three out of the five samples, i.e., TFA, TUFA/RII, and TUFAI. Among these findings, however, only the effect of the TUFA/RII can be considered of biological significance (>10% reduction). The effects found after treatment with TFA and TUFAI were rather low and therefore regarded negligible (Figure 4A). In contrast, after 24 h of incubation, with the exception of the fine sample (TFA), all TiO2 samples induced decreases in cell viability (Figure 4B). In order to evaluate whether the observed effects were associated with the SSA of the samples, for all samples the dose was also expressed as particle surface area per unit cell culture dish surface area for the 24 h time point (Figure 5A). The reduction in metabolic activity as measured by the WST-1 assay was found to be significantly associated with surface area dose (Pearson’s r = −0.708, p < 0.05, n = 10). Interestingly, when the two anatase/rutile mixture samples were excluded from the analysis this association improved remarkably (Pearson’s r = −0.7986, p < 0.001, n = 6). Thus, the WST-1 effect appeared to

at all was found after incubation with the fine TiO2 sample (TFA). Both samples with the highest SSA and the lowest primary particle size (TUFAI and TUFAII) also did not induce any significant LDH leakage after 4 or 24 h. In contrast, TUFA/RI as well as TUFA/RII, both representing anatase and rutile-containing particle types, induced significant cytotoxicity after incubation with 80 μg/cm2 at both treatment time intervals. LDH levels in supernatants of lysed Caco-2 cells, incubated for 24 h with the highest particle concentrations, were identical to the LDH levels of the lysates that were incubated in the absence of particles (Supporting Information, Figure S3). As such, an underestimation of toxicity could be excluded. 650



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and hence can be also considered as an indicator of the induction of oxidative stress. The formation of DNA strand breaks and oxidative damage are shown in Figure 6A and B,

Figure 6. DNA damage in Caco-2 cells upon exposure to different titanium dioxide particles. DNA strand breakage was determined in Caco-2 cells using the alkaline comet assay following 4 h of treatment with 20 μg/cm2 of TiO2 samples varying in crystallinity and SSA (A). The amount of oxidative DNA damage was determined by applying the Fpg enzyme to the assay and is depicted as ΔFpg, calculated by the difference between +Fpg and −Fpg (B). SSA is indicated in m2/g. ctr = control. Values are expressed as the mean and standard deviation, n = 3. * p < 0.05 and *** p < 0.001 vs control.

Figure 5. Effects of titanium dioxide particles on metabolic competence as a function of particle surface dose. The metabolic competence of the Caco-2 cells is shown as the average WST-1 formation in % of control as a function of the treatment dose expressed as particle surface area (SSA) per culture dish surface area (cm2/cm2) after 24 h of incubation. The pure anatase samples are shown as solid symbols and the anatase/rutile samples as open symbols. Graph A represents the data for all five TiO2 samples as derived from panel B, tested at two concentrations (i.e., 20 and 80 μg/ cm2). The line in graph A represents the linear curve of fit for the anatase samples only (n = 6). Graph B shows the results after 24 h of incubation with TUFAII and TUFA/RI, each tested at five different concentrations (10, 20, 40, 80, and 160 μg/cm2). The values in graph B are expressed as the mean and standard error, n = 4. *** p < 0.001 vs control.

respectively. In the comet assay, DNA damage becomes visible as a “tail” of DNA fragments behind the cell core after electrophoresis and is scored by determination of the percentage of DNA in tail. The Fpg-enzyme is used to specifically identify oxidative DNA damage by cleavage of oxidative DNA lesions, particularly 8-OHdG sites. As a positive control, the photosensitizer Ro-19 8022, known to induce oxidative DNA damage by the production of singlet oxygen,11 was applied to verify the activity of the Fpg (see Figure 6). Among the tested TiO2 powders, a significant increase in DNA strand breakage could be observed only with TUFA/RI. Remarkably, both particles of the highest surface area and the lowest primary particle size (TUFAI and TUFAII) did not induce any significant DNA strand breakage (Figure 6A). None of the powders caused a significant induction of oxidative DNA damage (Figure 6B), suggesting the absence of oxidative stress. The role of oxidative stress in the above effects was additionally determined by analysis of the total intracellular GSH content and mRNA regulation of HO-1 and γ-GCS after 4 h of particle treatment with 20 μg/cm2 (Figures 7 and 8A and B). Interestingly, regardless of their SSA and crystallinity, neither of the particles tested in our study diminished the total

depend both on the SSA and the crystallinity of the material. As can be seen in Figure 5A, both anatase/rutile samples showed higher toxicity per unit surface area than the pure anatase powders. For further verification of this observation, additional experiments were performed at a wider concentration range, using TUFAII as a representative nanosize anatase sample and TUFA/RI as representative anatase/rutile sample. Results are shown in Figure 5B. The results of these experiments (Figure 5) confirm the marked dependence of surface area associated toxicity on the crystallinity. The Fpg-modified comet assay was used to determine the potential DNA damaging properties of the samples. Apart from measuring DNA strand breaks and alkali labile sites, this assay allows for the determination of specific oxidative DNA damage 651



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agglomerates are defined as undispersed clusters of aggregates during their production or generation and in suspension.12,13 The fine TiO2 powder (TFA) with a SSA of 10 m2/g was specifically included in this study as a negative control. Two of the TiO2 powders used (TUFAI and TUFAII) were found to have a very high SSA (around 300 m2/g), a composition of 100% anatase, and similar negative ζ potential values at pH 7.4 (Table 1). The high SSA of the two samples corresponded to a very low primary particle size (4 and 7 nm, respectively). The TUFA/RI as well as the TUFA/RII sample both exhibited ζ potential values at pH 7.4 of lower amplitude than those found for the anatase samples, an intermediate SSA (approximately 50 m2/g), and consisted of both anatase and rutile. Primary particles appeared organized in nano- and microsized aggregates in all samples. The cytotoxic potential of the materials was tested upon suspension in FCS free culture medium. In biological systems, the absorption of proteins to the surface of particles is known to play an important role in stabilizing a small primary particle size.14 However, it also results in an altered, often reduced particle reactivity, depending on the type and amount of adsorbed protein, and may influence particle toxicity either directly, by masking the surface, or indirectly by binding and depletion of proteins that are of advantage for cell growth and thus available only for nonparticle treated cells.15−18 Therefore, we decided to minimize the presence of interfering proteins in the cell treatment by using serum free cellular medium, to be able to put the observed effects down to the particle surface properties only. Nevertheless, the avoidance of stabilizing proteins in the suspension led to a pronounced agglomeration of the particles, resulting in a distribution of the size of agglomerates, which is in the micrometric range for all samples. As such, on the one hand, this might lead to an underestimation of “nanosize” effects. On the other hand, however, the observed similarities in agglomeration conditions allow for a more controlled evaluation of the effects of SSA and crystallinity in the Caco-2 cells. Particle size specific contrasts in mechanisms such as intracellular uptake and potential subcellular translocation, e.g., into the nucleus, have typically been observed with the use of highly monodisperse (colloidal) model nanoparticles. In contrast, the commercial TiO2 powders which were used in our present study are reported to occur in substantially aggregated and agglomerated form in various applications. In the present study, we used two independent tests to determine the cytotoxic potential of the TiO2 samples under the exclusion of photoactivation. In the LDH assay, only the two anatase/rutile containing samples that also possess an intermediate specific surface area (i.e., TUFA/RI and TUFA/RII) induced low (less than 20%) but significant membrane damage at both time points but high concentrations only. In contrast, after 24 h, in the WST-1 assay all samples, except for the fine powder, were found to reduce the metabolic activity, and this appeared to be associated with the surface area dose (Figure 5). However, when considered at equal surface area, in both cytotoxicity assays the anatase/rutile containing samples were found to be more toxic to the cells than the pure anatase samples (Figure 5A and B). For the lung, it has been shown that crystallinity may influence the toxic potential of TiO2 particles, but usually, anatase had a greater tendency to induce adverse effects such as cyto- or genotoxicity (reviewed in ref 5). If in vitro tests are performed under UV irradiation or even under ambient

Figure 7. Effects of different titanium dioxide particles on total glutathione content of Caco-2 cells. Cells were incubated with 20 μg/ cm2 particles for 4 h. Total glutathione content of the Caco-2 cells is expressed in nM/mg protein. SSA is indicated in m2/g. Values are expressed as the mean and standard deviation, n = 3.

Figure 8. Effects of different titanium dioxide particles on the mRNA expression of oxidative stress markers in Caco-2 cells. Expression of HO-1 (A) or γ-GCS (B) mRNA after 4 h of incubation with 20 μg/ cm2 TiO2 particles was determined in Caco-2 cells via qRT-PCR. SSA is indicated in m2/g. Values are expressed as the mean and standard deviation, n = 3.

intracellular GSH content (Figure 7). In accordance with these findings, the incubation with all TiO2 samples tested did not induce a change in the expression of the mRNA of the oxidative stress markers HO-1 (Figure 8A) and γ-GCS (Figure 8B).

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DISCUSSION

In the present study, we have investigated the possible hazards of a range of TiO2 samples with respect to cytotoxicity, induction of oxidative stress, and DNA damage in human intestinal Caco-2 cells. All five samples tested were analyzed in detail with regard to their purity, specific surface area, crystallinity, particle size, ζ potential, and aggregation as well as their agglomeration behavior in culture medium. Generally, aggregates are formed by fusion of primary particles, whereas 652



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illumination,19,20 this may be related to the higher photocatalytic activity of the anatase phase,17 while no such effect should be expected in tests performed in the dark. However, a previous study by some of us reported that TiO2 anatase or anatase/rutile may cause direct oxidative damage to organic molecules in the dark through a reaction not involving oxygenated free radicals. Conversely, nanometric rutile was inactive in this reaction but generated hydroxyl radicals when in contact with hydrogen peroxide in the dark.21 Gurr and co-workers compared the DNA damaging effects of rutile and anatase TiO2 particles of 200 nm primary particle size on human bronchial epithelial cells, BEAS-2B. They demonstrated a significant increase in the release of hydrogen peroxide in cells treated with rutile TiO2 but not by anatase TiO2 if incubated in the dark. Additionally, using the comet assay, they found that a mixture of anatase and rutile particles showed a more pronounced DNA damaging effect in the absence of light than pure anatase or pure rutile TiO2.22 More recently, Grassian and colleagues studied the possible influence of primary particle size on the inflammatory potential of nanoTiO2 in mice. Two samples were considered, having different particle size and, consequently, different SSA. The sample having smaller primary particles consisted of pure anatase, while those having the larger particles contained both anatase and rutile crystalline phases. Stronger effects were expected after inhalation or instillation of the higher SSA TiO2. Interestingly, however, exactly the opposite was observed: after a 4 h low dose inhalation of the anatase/rutile-containing TiO2, a significantly increased number of total cells in bronchoalveolar lavage was observed, whereas anatase TiO2 could cause this effect only at high exposure concentrations. A similar effect was found after the instillation of mice with a medium and high concentration of anatase/rutile-containing TiO2 but not by pure anatase TiO2. Additionally, a significant release of LDH and IL-1β was induced by the highest and an increased release of TNF-α by the medium and high concentration of anatase/ rutile TiO2.23 Toxic effects toward keratinocytes have been reported for both rutile and anatase TiO2,24 while micrometric rutile appeared more active in inducing IL-1β release in macrophage-like THP-1 cells than anatase TiO2.25 In our current study, only TUFA/RI, an anatase/rutile containing sample, was found to induce mild but significant induction of DNA strand breakage. This supports our observations in the cytotoxicity assays on the intrinsically higher reactivity of anatase/rutile TiO2. However, the actual level of DNA damage induced by TUFA/RI was relatively close to the background, in contrast to our positive control (i.e., the photosensitizer Ro-19 8022), and is therefore considered low. A main contributor to nanoparticle toxicity is its ability to induce ROS, leading to oxidative stress.3,12 Surprisingly, however, our effects found on cell viability and DNA integrity could not be explained by a marked induction of oxidative stress in three independent assays. Next to lacking findings on Fpg-specific effects in the comet assay, which is known to be a sensitive marker for oxidative stress, we analyzed the intracellular GSH level that is reduced when oxidative stress occurs.7,26 Here, the intracellular GSH level was unchanged after treatment with all TiO2 samples tested, in contrast to our previous studies in the same cell line with other types of nanoparticles including SiO2 and carbon black.7 Furthermore, we determined a potential regulation of two enzymes that play an important role in ROS detoxification, HO-1 and γ-GCS, the latter being the rate-limiting enzyme for the formation of GSH.

Expression changes of those genes are considered to represent sensitive indicators of the induction of oxidative stress by particles in vitro as well as in vivo,27,28 but in our hands, no significant mRNA regulation changes were detected in Caco-2 cells. In contrast, we could demonstrate significant mRNA expression increases in Caco-2 cells upon treatment with CuO and NiO nanoparticles (Supporting Information, Figure S4) which both have been shown to induce oxidative stress in human epithelial cells.29,30 In subsequent investigations, we also failed to detect increased ROS levels in the Caco-2 cells upon incubation with the TUFA/RI sample, using either lucigeninenhanced chemiluminescence or electron paramagnetic resonance (EPR) spectroscopy (Supporting Information, Figure S5). Taken together, our data do not support a role for ROS in the observed cytotoxic and DNA damaging effects of the tested TiO2 samples. As TiO2 is a material of low solubility, its genotoxic potential would be expected to be mainly driven by ROS production and hence indirect genotoxic effects.31 However, as we did not find any signs of oxidative stress and ROS production, other mechanisms might have led to the toxic responses reported in the current study, such as direct mechanical interference with cellular components or release of low amounts of containing (trace)metals. Mechanical interference might lead to lipid peroxidation and subsequent membrane and mitochondrial damage, and the mode of action is moreover dependent on the cell cycle. For example, NP interference with microtubule or centrosomes during the interface can lead to a delay in cellular trafficking.32,33 However, further mechanistical determinations need to be performed in future studies. The observed toxicity of the anatase/rutile mixed phases samples may be related to a synergic effect due to the coexistence of anatase and rutile particles in contact to each other in the aggregates rather than a simple additional contribute of the rutile phase. A similar effect has been suggested to justify the high photocatalytic efficiency of this type of powder.34,35 In the present case, the different crystalline phase resulted in a different surface charge of the TiO2 samples. This may contribute to the observed higher ability to cause damage to cell membrane of the mixed phases samples. While bulk TiO2 has been in widespread use for several decades as a food colorant, to our knowledge, none of the tested nanometric samples is currently being used or considered as a food additive. Yet, our current data are of importance in the light of the steadily expanding “nano-sizing” initiatives in novel food applications (e.g., functional food and food packaging) as well as in various other consumer applications.2 Moreover, one should consider that polydisperse powders may contain a considerable “nanosize tail” and that, depending on the physicochemical properties of the material, this proportion may increase as a result of gastrointestinal digestion processes. Finally, nanometric TiO2 may also reach the gastrointestinal tract via hand-to-mouth contact or following (occupational) inhalation exposure via the mucociliary clearance pathway.36

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CONCLUSIONS The present data show that both the specific surface area and the crystallinity of TiO2 particles are relevant for their toxic potential in human intestinal Caco-2 cells. The mere absence of evidence for a role of ROS-mediated oxidative stress in TiO2 nanoparticle-exposed cells points out the need to explore alternative mechanisms of actions. 653



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ASSOCIATED CONTENT

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AUTHOR INFORMATION

Article

(5) Johnston, H. J., Hutchison, G. R., Christensen, F. M., Peters, S., Hankin, S., and Stone, V. (2009) Identification of the mechanisms that drive the toxicity of TiO(2) particulates: the contribution of physicochemical characteristics. Part Fibre Toxicol. 17, 6:33. (6) Trouiller, B., Reliene, R., Westbrook, A., Solaimani, P., and Schiestl, R. H. (2009) Titanium dioxide nanoparticles induce DNA damage and genetic instability in vivo in mice. Cancer Res. 69, 8784− 8789. (7) Gerloff, K., Albrecht, C., Boots, A. W., Förster, I., and Schins, R. P. F. (2009) Cytotoxicity and oxidative DNA damage by nanoparticles in human intestinal Caco-2 cells. Nanotoxicology 3 (4), 355−364. (8) Speit, G., Schütz, P., Bonzheim, I., Trenz, K., and Hoffmann, H. (2004) Sensitivity of the FPG protein towards alkylation damage in the comet assay. Toxicol. Lett. 146 (2), 151−158. (9) Angelis, K. J., Dusinská, M., and Collins, A. R. (1999) Single cell gel electrophoresis: detection of DNA damage at different levels of sensitivity. Electrophoresis 20 (10), 2133−2138. (10) Bangkedphol, S., Keenan, H. E., Davidson, C,M., Sakultantimetha, A., Sirisaksoontorn, W., and Songsasen, A. (2010) Enhancement of tributyltin degradation under natural light by Ndoped TiO2 photocatalyst. J Hazard Mater. 184, 533−537. (11) Will, O., Gocke, E., Eckert, I., Schulz, I., Pflaum, M., Mahler, H. C., and Epe, B. (1999) Oxidative DNA damage and mutations induced by a polar photosensitizer, Ro19-8022. Mutat. Res. 435 (1), 89−101. (12) Donaldson, K., Tran, L., Jimenez, L. A., Duffin, R., Newby, D. E., Mills, N., MacNee, W., and Stone, V. (2005) Combustion-derived nanoparticles: a review of their toxicology following inhalation exposure. Part Fibre Toxicol. 2, 10. (13) Zhang, Y., Chen, Y., Westerhoff, P., Hristovski, K., and Crittenden, J. C. (2008) Stability of commercial metal oxide nanoparticles in water. Water Res. 42 (8−9), 2204−2212. (14) Meissner, T., Potthoff, A., and Richter, V. (2009) Physicochemical characterization in the light of toxicological effects. Inhal. Toxicol. 21 (Suppl 1), 35−39. (15) Borm, P. J. A., Robbins, D., Haubold, S., Kuhlbusch, T., Fissan, H., Donaldson, K., Schins, R. P. F., Stone, V., Kreyling, W., Lademann, J., Krutmann, J., Warheit, D., and Oberdorster, E. (2006) The potential risks of nanomaterials: a review carried out for ECETOC. Part Fibre Toxicol. 3, 11. (16) Horie, M., Nishio, K., Fujita, K., Endoh, S., Miyauchi, A., Saito, Y., Iwahashi, H., Yamamoto, K., Murayama, H., Nakano, H., Nanashima, N., Niki, E., and Yoshida, Y. (2009) Protein adsorption of ultrafine metal oxide and its influence on cytotoxicity toward cultured cells. Chem. Res. Toxicol. 16;22 (3), 543−553. (17) Fubini, B., Ghiazza, M., and Fenoglio, I. (2010) Physicochemical features of engineered nanoparticles relevant to their toxicity. Nanotoxicology 4, 347−363. (18) Fenoglio, I., Fubini, B., Ghibaudi, E., and Turci, F. (2011) Multiple aspects of the interaction of biomacromolecules with inorganic surfaces. Adv. Drug Delivery Rev. 63, 1186−1209. (19) Serpone, N., Dondi, D., and Albini, A. (2007) Inorganic and organic UV filters: their role and efficacy in sunscreens and suncare products. Inorg. Chim. Acta 360, 794−802. (20) Sayes, C. M., Wahi, R., Kurian, P. A., Liu, Y., West, J. L., Ausman, K. D., Warheit, D. B., and Colvin, V. L. (2006) Correlating nanoscale titania structure with toxicity: a cytotoxicity and inflammatory response study with human dermal fibroblasts and human lung epithelial cells. Toxicol. Sci. 92 (1), 174−185. (21) Fenoglio, I., Greco, G., Livraghi, S., and Fubini, B. (2009) NonUV-induced radical reactions at the surface of TiO2 nanoparticles that may trigger toxic responses. Chem.Eur. J. 15 (18), 4614−4621. (22) Gurr, J. R., Wang, A. S., Chen, C. H., and Jan, K. Y. (2005) Ultrafine titanium dioxide particles in the absence of photoactivation can induce oxidative damage to human bronchial epithelial cells. Toxicology 213 (1−2), 66−73. (23) Grassian, V. H., Adamcakova-Dodd, A., Pettibone, J. M., O’shaughnessy, P. T., and Thorne, P. S. (2007) Inflammatory response of mice to manufactured titanium dioxide nanoparticles: comparison

S Supporting Information *

The ζ potential of the TiO2 samples and cumulative size distribution curves; proof of the absence of interference with the LDH cytotoxicity assay and the absence of ROS formation by TiO2 as evaluated by ESR and chemiluminescence; and HO1 and γ-GCS mRNA regulation changes in Caco-2 cells treated with CuO and NiO nanoparticles. This material is available free of charge via the Internet at http://pubs.acs.org. Corresponding Author

*Leibniz Institut für umweltmedizinische Forschung (IUF), Auf’m Hennekamp 50, D-40225 Düsseldorf, Germany. Tel: +49-211-3389-269. Fax: +49-211-3389-331. E-mail: roel. [email protected]. Funding

This study was financially supported by a grant from the German Research Council (Deutsche Forschungsgemeinschaft DFG), Graduate College GRK-1427, a DFG Research Fellowship GE-2328/1-1 awarded to K.G., and by Regione Piemonte (Progetti di Ricerca Sanitaria Finalizzata 2009). E.C. was a recipient of a doctoral fellowship from the Istituto Nazionale per l′Assicurazione contro gli infortuni sul lavoro (INAIL) Piemonte, Italia. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are grateful to Dr. Gianmario Martra, Dip. di Chimica IFM, University of Torino, for providing two of the TiO2 samples and Dr. Giovanni Agostini for his kind help with TEM analysis. We thank Dr. Andrew Collins, Institute for Nutrition Research, University of Oslo, Norway, for kindly providing the Fpgenzyme.

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ABBREVIATIONS BCA, bicinchoninic acid; BET, Brunauer, Emmett, and Teller; DLS, dynamic light scattering; DTNB, 5,5′-dithio-bis-2-nitrobenzoic acid; FCS, fetal calf serum; Fpg, formamidopyrimidine glycosylase; γ-GCS, γ-glutamyl cysteine synthetase; HO-1, heme oxygenase-1; IU, international unit; LDH, lactate dehydrogenase; LMP, low melting point; PDI, polydispersity index; PZC, points of zero charge; qRT-PCR, quantitative realtime reverse-transcription PCR; Ro19-8022, [R]-1-[(10-chloro4-oxo-3-phenyl-4H-benzo[α]quinolizin-1-yl)-carbonyl]-2-pyrrolidinemethanol; SSA, specific surface area; TEM, transmission electron microscopy; TiO2, titanium dioxide; WST-1, water-soluble tetrazolium salt; XRD, X-ray diffraction; XRF, Xray fluorescence.

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REFERENCES

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Anatase Mixed Phase Nanoparticles

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