Research Article Received: 5 March 2015,

Revised: 30 March 2015,

Accepted: 10 April 2015

Published online in Wiley Online Library: 23 June 2015

(wileyonlinelibrary.com) DOI 10.1002/jat.3171

Biological effect of food additive titanium dioxide nanoparticles on intestine: an in vitro study Zheng-Mei Songa, Ni Chena, Jia-Hui Liub, Huan Tangb, Xiaoyong Denga, Wen-Song Xia, Kai Hana, Aoneng Caoa, Yuanfang Liua,b and Haifang Wanga* ABSTRACT: Titanium dioxide nanoparticles (TiO2 NPs) are widely found in food-related consumer products. Understanding the effect of TiO2 NPs on the intestinal barrier and absorption is essential and vital for the safety assessment of orally administrated TiO2 NPs. In this study, the cytotoxicity and translocation of two native TiO2 NPs, and these two TiO2 NPs pretreated with the digestion simulation fluid or bovine serum albumin were investigated in undifferentiated Caco-2 cells, differentiated Caco-2 cells and Caco-2 monolayer. TiO2 NPs with a concentration less than 200 μg ml–1 did not induce any toxicity in differentiated cells and Caco-2 monolayer after 24 h exposure. However, TiO2 NPs pretreated with digestion simulation fluids at 200 μg ml–1 inhibited the growth of undifferentiated Caco-2 cells. Undifferentiated Caco-2 cells swallowed native TiO2 NPs easily, but not pretreated NPs, implying the protein coating on NPs impeded the cellular uptake. Compared with undifferentiated cells, differentiated ones possessed much lower uptake ability of these TiO2 NPs. Similarly, the traverse of TiO2 NPs through the Caco-2 monolayer was also negligible. Therefore, we infer the possibility of TiO2 NPs traversing through the intestine of animal or human after oral intake is quite low. This study provides valuable information for the risk assessment of TiO2 NPs in food. Copyright © 2015 John Wiley & Sons, Ltd. Additional supporting information may be found in the online version of this article at the publisher’s web-site. Keywords: titanium dioxide nanoparticles; digestion; cytotoxicity; intestinal cell; traverse

Introduction

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* Correspondence to: Haifang Wang, Institute of Nanochemistry and Nanobiology, Shanghai University, Shanghai 200444, China. E-mail: [email protected] a

Institute of Nanochemistry and Nanobiology, Shanghai University, Shanghai, China

b Beijing National Laboratory for Molecular Sciences, College of Chemistry and Molecular Engineering, Peking University, Beijing, China

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As an additive, titanium dioxide (TiO2) is widely used in various food products (Buettner and Valentine, 2011). In the UK, the dietary consumption of TiO2 is estimated as 5.4 mg person–1 day–1. Recent studies have demonstrated that TiO2 nanoparticles (NPs) do exist in various consumer products to improve the quality of food (Chaudhry et al., 2008; Weir et al., 2012; Xu et al., 2010). Our group has found that there are TiO2 NPs in chewing gums and candies (Chen et al., 2013). Weir et al. (2012) conclude that about 36% TiO2 in food products are nanosized. In addition, TiO2 NPs in cosmetics, particularly in lip balm (Oomen et al., 2011) and spray (Chen et al., 2010) probably enter the human body through oral uptake. Therefore, the oral exposure to TiO2 NPs seems inevitable for consumers. Considering the gastrointestinal tract is the first stop for TiO2 NPs after they enter the human body orally, investigating the effect of TiO2 NPs on the intestine is necessary and urgent for the safety assessment of TiO2 NPs. Several studies have been performed (Wang et al., 2013). Some studies accepted that TiO2 NPs could cross the intestine and redistribute in various organs (Skocaj et al., 2011); however, Cho et al. (2013) reported that almost no Ti from TiO2 NPs could enter the blood stream even after mice were exposed to a high dose, 1041.5 mg kg–1, for 13 weeks. At the cellular level, contradictory results were found when both toxic (Gerloff et al., 2009, 2012) and non-toxic effects (Barone et al., 2011; Abbott Chalew and Schwab, 2013; De Angelis et al., 2013; Koeneman et al., 2010; Krüger et al., 2014) were reported. In addition, some studies showed that TiO2 NPs could enter cells and transport through

the Caco-2 monolayer (Brun et al., 2014; De Angelis et al., 2013; Koeneman et al., 2010). Markedly, they contradict the reports that TiO2 NPs were unlikely or seldom penetrate the Caco-2 monolayer (Gitrowski et al., 2014; Janer et al., 2014; Jones et al. 2015). These contradictory in vitro results might come from the different cells and culture conditions, and tested TiO2 NPs with various physicochemical properties, such as size and surface property. These further indicate that more investigations on the toxicity of TiO2 NPs to the intestine are needed. TiO2 NPs in food are impossible to keep as single and pure in composition; the coating of NPs with various food components is indispensable. In addition, TiO2 NPs have to endure and pass through saliva, gastric and intestinal juices before reaching the intestine. Under these conditions, the physiological parameters of TiO2 NPs such as pH, ionic strength, as well as protein content and composition change greatly. Therefore, the surface property of NPs will eventually largely change, hence influencing their bioeffects and results substantially (Chen et al., 2009; Faunce et al., 2008; Pfaller et al., 2010).

Z.-M. Song et al. Therefore, the bioeffects study of TiO2 NPs in a real situation is necessary while evaluating their safety. To simulate the real situation and study the effect of environments, two kinds of TiO2 NPs were pretreated with the most popular model protein bovine serum albumin (BSA) and simulated gastrointestinal fluids. The native and pretreated TiO2 NPs were used for toxicity, cellular uptake and translocation studies on the Caco-2 cells and monolayer, which are the standard intestinal in vitro models widely accepted by pharmacological industries and regulatory authorities. The cytotoxicity of these NPs was determined using a viability assay, lactate dehydrogenase (LDH) release, live/dead staining and cellular reactive oxygen species (ROS) level. The cellular uptake of Caco-2 cells was detected by flow cytometric analysis and transmission electron microscopy (TEM). Translocation through the Caco-2 monolayer was evaluated by the inductively coupled plasma mass spectrometry. The results indicate that all TiO2 NPs tested show low toxicity and are difficult to pass through the intestinal epithelial cells after 24 h exposure.

Materials and Methods Titanium Dioxide Samples and Their Characterization Two kinds of TiO2 NPs were used in this study: food additive TiO2 NPs, denoted as T1, were purchased from Jianghu Industrial Co. (Shanghai, China), and TiO2 NPs, denoted as T2, were purchased from Aladdin Industrial Co. (Shanghai, China). The purity was analyzed by X-ray fluorescence (S4-Explorer; Karlsruhe, Bruker, Germany). The crystal structure was determined by an X-ray diffraction (Rigaku Co., Tokyo, Japan). The size and morphology were investigated with a JEM-200CX TEM ( JEOL, Tokyo, Japan). In addition, the corresponding size distribution was measured from at least 600 particles in TEM images by Image J software. The hydrodynamic size and zeta potential of TiO2 NPs in aqueous solutions were determined by a Nanosizer (Nano ZS90; Malvern, UK). Fourier transform infrared spectroscopy (Avatar 370; Thermo Nicolet, Madison, USA) was adopted to characterize the surface group of TiO2 NPs. To simulate the real situation in the gastrointestinal tract, TiO2 NPs were pretreated with the in vitro digestive fluids following the method and process reported by Peters et al. (2012) with slight modifications. Briefly, treatment was carried out at 37 °C in a shaker (Scientific incubated tabletop orbital shaker; Thermo, Boston, MA, USA) at 200 rpm. First, saliva juice (10 ml) was introduced to 10 mg TiO2 NPs and incubated for 30 min. Subsequently, gastric juice (20 ml) was added. The mixture was adjusted to pH 2.0 ± 0.5 and shaken for 2 h. Finally, duodenal juice (20 ml), bile juice (10 ml) and NaHCO3 solution (3 ml) were added into the mixture. The obtained mixture was adjusted to pH 8.0 ± 0.5 and shaken for another 2 h. After that, the mixture was centrifuged (8000 g × 10 min) to collect the TiO2 NPs (denoted as T1-G and T2-G). In addition, considering the complex situation of TiO2 NPs in food, food additive T1 particles were pretreated with BSA to simulate the absorption of proteins in food. Briefly, 10 mg TiO2 NPs were added into the 10 ml 6 mg ml–1 BSA solution and cultured at 37 °C for 4 h and then centrifuged (8000 g × 10 min) to collect the TiO2 NPs (T1-B). Cell Lines and Cell Culture

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The human colon colorectal adenocarcinoma cell line Caco-2 (ATCC, no. TCHu146) was obtained from the Shanghai Institute of Cell Bank (China), and used at passages 20–40. The Caco-2 cells were cultured as previously described (Yang et al., 2014).

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To prepare the Caco-2 monolayer, Caco-2 cells were seeded in 12-well transwell plates (1.12 cm2 effective membrane growth area; Corning, NY, USA) with a density of 3.5 × 105 cells per insert. The apical side chamber was refreshed with 0.5 ml culture media every day and the basolateral side chamber was refreshed with 1 ml media every other day until 21 days. The integrity of monolayer was determined by monitoring the transepithelial electric resistance (TEER) and by assessing the permeability of monolayer to Lucifer Yellow (LY, MW450; Sigma, Louis, MO, USA) and fluorescein isothiocyanate dextran (FITC-dextran, MW4000; Sigma). The TEER value reflects the tight junction of cells in the monolayer. The measurement was conducted in freshly added Dulbecco modified Eagle’s minimal essential medium (DMEM) using Millipore Millicell-ERS (Millipore, Boston, MA, USA). The monolayer with TEER > 300 Ω · cm2 before the experiment was used in this study. Measurements were taken pre- and postexposure to particles. The percentage change of TEER (△%) was calculated using [(TEERi – TEERp)/TEERi] × 100, where TEERi and TEERp represent the pre- and postexposure TEER, respectively. The paracellular permeability of the monolayer was evaluated using LY and FITC-dextran with and without pretreatment with ethylene glycol bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA; Sigma, Louis, MO, USA), a tight junction and adherence junction disruptor (Sergent et al., 2006). EGTA solution prepared in Hank’s balanced salt solution was added apically and basolaterally to chambers with a final concentration of 2.5 mM and incubated for 60 min. Then the inserts were washed with Hank’s balanced salt solution and the monolayers were exposed to LY and FITC-dextran apically for 90 min. Finally, the content of LY and FITC-dextran in basolateral chambers was measured by a microplate reader (Varioskan Flash; Thermo) at the excitation/emission wavelength of 485/530 nm. Cell Viability and Cell Membrane Integrity Assay The cell viability was evaluated by a WST-8 cell counting kit (CCK-8; Dojindo Molecular Technologies Inc., Kumamoto, Japan). The LDH test kit (CytoTox 96W Non-Radioactive Cytotoxicity Assay; Promega Corp., Madison, WI, USA) was used to assess the cell membrane integrity. Caco-2 cells (1.5 × 104 per well) were seeded into 96-well plates and grown overnight (undifferentiated cells) or for 15 days (differentiated cells). Then the cells were exposed to the fresh culture medium containing TiO2 NPs for 24 h. After that, the culture medium was collected for LDH measurement by centrifugation (4500 g × 10 min) and cells for the viability assay, following the procedures reported previously (Chang et al., 2011; Yang et al., 2014). Cell Proliferation Assay Undifferentiated Caco-2 cells were plated in 12-well plates (15 × 104 cells per well). One day later, fresh culture media containing TiO2 NPs were introduced to the cells. The cells without exposure to TiO2 NPs were taken as the control. After culture for 24 h, cells were collected and counted on a blood cell counting plate under an optical microscope. Each well was counted three times. Parallel triplet samples were tested. Live/Dead Cell Staining The cell viability was quantified using the live/dead kit (L-3224; Invitrogen, Carlsbad, CA, USA). A mix of calceinacetoxymethyl

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Bioeffects of food additive TiO2 nanoparticles to intestinal cells (calcein AM) and ethidium homodimer-1 was used to differentiate live cells ( green) from dead cells (red). Intracellular esterase in live cells converted non-fluorescent, cell-permeable calcein AM to the fluorescent calcein (excitation 495 nm/emission 515 nm). The damaged membrane of dead cells allowed ethidium homodimer-1 to enter the cells and become fluorescent (excitation 495 nm/emission 635 nm) after binding to nucleic acids. After the Caco-2 cells or Caco-2 monolayer have been exposed to 200 μg ml–1 TiO2 NPs for 24 h, the culture medium was removed and the dyes dissolved in phosphate-buffered saline buffer were introduced to cells. After being incubated for 15 min, the cells were fixed in 4% paraformaldehyde aqueous solution, washed and detected under a fluorescence microscope (DML3000; Leica, Solms, Germany).

plates. Medium in the basolateral chamber was collected and digested by 0.5 ml hydrofluoric acid and 7 ml nitric acid in a microwave digestion system (CEM; Mars, CEM Mars, New York, NY, USA). Ti content of the digested samples was analyzed by inductively coupled plasma mass spectrometry (ELAN DRC-e; PerkinElmer Co., Fremont, CA, USA). Statistical Analysis All means were calculated from at least three independent experiments, and are expressed as means ± SD. The analysis of statistical significance was done using the Student’s t-test. The results were considered significant if P < 0.05.

Results Intracellular Reactive Oxygen Species Generation Measurement The ROS level was detected using 2′,7′-dichlorofluorescin diacetate (DCFH-DA; Sigma, USA), which could enter cells and be hydrolyzed into a fluorescent DCFH probe. After being seeded into 96-well plates with a density of 1.5 × 104 cells per well and grown overnight (undifferentiated cells) or for 15 days (differentiated cells), Caco-2 cells was cultured in the fresh culture medium containing 200 μg ml–1 TiO2 NPs for predetermined periods. Then the ROS levels were determined following the protocol reported previously (Yang et al., 2014). The Caco-2 cell monolayers were investigated under the fluorescence microscope. Uptake of TiO2 Nanoparticles by Caco-2 Cells To investigate the cellular uptake of TiO2 NPs in undifferentiated and differentiated Caco-2 cells, thin sections of cells were prepared and investigated under TEM. The cells were plated in 25 cm2 culture flasks and incubated for 24 h. T1 and T1-G in a concentration of 200 μg ml–1 were introduced to undifferentiated Caco-2 cells, while 200 μg ml–1 T1 was introduced to differentiated Caco2 cells. After 24 h exposure, the cells were treated as described before (Yang et al., 2014). The thin sections were inspected with TEM (CM120; Philips, Amsterdam,The Netherlands). To confirm the uptake of TiO2 NPs by undifferentiated and differentiated Caco-2 cells, we also adopted flow cytometric light scatter analysis, which has proven an efficient method in studying the uptake of NPs (Suzuki et al., 2007; Wang et al., 2009). After cellular uptake, the cellular granularity increases and can be detected by flow cytometric analysis (BD Biosciences, Franklin Lakes, NJ) using the side scatter parameter. Using the granularity of the normal cells as the control, we counted the number of TiO2 NP-treated cells with a significantly higher granularity as an indicator of TiO2 uptake. The uptake was presented by the percentage of TiO2 NPs swallowed cells to entire cells. The cells in parallel experiments came from different donors. Translocation of TiO2 Nanoparticles through Caco-2 Monolayers

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The TiO2 NPs used in this study are two native NPs (T1 and T2), two TiO2 NPs pretreated with the in vitro digestive fluids (T1-G and T2-G) and T1 pretreated with BSA (T1-B). Figure 1(A,B) shows the morphology of T1 and T2. T1 has an average size of 99 ± 30 nm (Fig. 1C) and T2 26 ± 12 nm (Fig. 1D). The X-ray diffraction analysis (Fig. S1 in Supporting Information) confirms that both TiO2 NPs are of anatase phase. Their purity is over 99% according to the X-ray fluorescence analysis (Table 1). Fourier transform infrared spectra indicate that the surface of T1 and T2 is very clean, without any organic impurity adsorbed (Fig. S2 in Supporting Information). In aqueous solutions, TiO2 NPs aggregated severely. The hydrodynamic diameter of T2 (497 nm) is larger than T1 (233 nm), which may be due to smaller NPs having larger specific surface areas. Pretreatment with BSA and digestive fluids induced the coating of BSA and fluid components on to particles, making the hydrodynamic diameter increase to over 600 nm for BSA pretreatment and up to 2000 nm for the pretreatment with digestive fluids (Table 1). The aggregates of T1-B, T1-G and T2-G are confirmed by TEM investigation (Fig. S3 in Supporting Information). These are consistent with their zeta potentials (Table 1). In medium, T1 and T2 further aggregate, presumably because of the adsorption of proteins. However, the size of pretreated samples does not change markedly. One possible reason is that they have already adsorbed proteins and other components. The changes of zeta potential in medium support this conjecture. Viability of Undifferentiated and Differentiated Caco-2 Cells Following Exposure to TiO2 Nanoparticles Epithelial cells are the important components of gut. Therefore undifferentiated Caco-2 cells, human epithelial colorectal adenocarcinoma cells, are widely used to assess various gastrointestinal functions and physiologic responses of enterocytes to xenobiotics, including NPs. When undifferentiated Caco-2 cells are cultured under specific conditions, differentiated Caco-2 cells with longer microvilli are obtained; continual culture of differentiated Caco-2 cells induces the formation of a polarized epithelial cell monolayer with the characteristics of small intestine enterocytes, such as tight junctions, microvilli and membrane transporters. Caco-2 monolayers are widely used in the pharmaceutical industry as an in vitro model of the human small intestinal mucosa to evaluate the absorption of orally administered drugs. All these Caco-2 cells have been used in toxicity

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The translocation of the all types of TiO2 NPs in concentrations of 50 μg ml–1 and 200 μg ml–1 dispersed in fresh medium (DMEM) and medium containing 10% FBS (complete medium, cDMEM) was tested at 2 h, 4 h and 24 h postexposure. The medium (0.5 ml) containing TiO2 NPs was added to the apical chamber of the Caco-2 monolayer cultured in 12-well transwell

Characterization of TiO2 Nanoparticles

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Figure 1. Characterization of TiO2 nanoparticles. Transmission electron microscopy image of (A) T1 and (B) T2. (C) Size distribution of T1. (D) Size distribution of T2.

Table 1. Physicochemical properties of TiO2 nanoparticles

T1 T1-B T1-G T2 T2-G

Purity (%)

Size in water (nm)

Size in medium (nm)

zeta potential in water (mV)

zeta potential in medium (mV)

99.2 – – 99.2 –

233 ± 12 625 ± 27 2124 ± 285 497 ± 137 2762 ± 141

719 ± 56 727 ± 9 2694 ± 232 1785 ± 237 2915 ± 216

–47.2 ± 1.5 –20.6 ± 1.2 –15.7 ± 0.4 –32.2 ± 1.0 –12.8 ± 1.8

–7.3 ± 0.3 –8.1 ± 0.9 –4.4 ± 0.5 –8.4 ± 0.7 –4.4 ± 0.5

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studies. Thus, in this study undifferentiated Caco-2 cells, differentiated Caco-2 cells and Caco-2 monolayers were adopted to investigate the bioeffects of TiO2 NPs in food. To investigate whether TiO2 NPs influence the function of cells, the cell viability assay (CCK-8), which reflects the mitochondrial activity of cells, was carried out on both undifferentiated and differentiated Caco-2 cells. The results are shown in Fig. 2. After 24 h exposure, T1, T2 and T1-B, even at 200 μg ml–1, do not induce any clear loss of viability on both Caco-2 cells. T1-G and T2-G are non-toxic to differentiated Caco-2 cells. However, they induce a decrease in viability of the undifferentiated Caco-2 cells after 24 h exposure, although the viability remains higher than 86%. Clearly, differentiated Caco-2 cells were more tolerant to the TiO2 NP exposure compared with the undifferentiated Caco-2 cells. The membrane integrity of cells exposed to TiO2 NPs for 24 h was measured using the LDH kit. All TiO2 NPs tested, even at a high concentration of 200 μg ml–1, do not lead to the membrane damage of differentiated Caco-2 cells, indicating the low toxicity of TiO2 NPs (Fig. 3). In undifferentiated Caco-2

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cells, the increase in LDH level is seen for all TiO2 NPs, particularly at higher concentrations (Fig. 3). However, the increase in LDH level is several times lower than that of the positive control, indicating that membrane damage is limited. Clearly, all TiO2 NP samples are non-toxic to differentiated Caco-2 cells; however, it is difficult to conclude the toxicity towards undifferentiated Caco-2 cells. Therefore, more assays were performed on undifferentiated cells. First, the live/dead staining assay was conducted postexposure to 200 μg ml–1 TiO2 NPs for 24 h. The obtained images are summarized in Fig. 4, where green represents live cells and red represents dead cells. The difference between T1 and T2 is not apparent. Dead cells are speckled in the images, indicating the cell fatality rate is pretty low. However, we observe the decrease of cell density postexposure to T1-G and T2-G. We assume that exposure to TiO2 NPs pretreated with digestive fluids does not result in cell death, but inhibits the growth of undifferentiated Caco-2 cells. To confirm the results, the cell proliferation was determined (Fig. 5). The results are in accordance with those of the

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Bioeffects of food additive TiO2 nanoparticles to intestinal cells

Figure 2. The viability of undifferentiated (A) and differentiated (B) Caco-2 cells postexposed to TiO2 nanoparticles for 24 h. *P < 0.05 compared with the control.

Figure 3. The membrane integrity assay of undifferentiated (A) and differentiated (B) Caco-2 cells postexposed to TiO2 nanoparticles for 24 h. *P < 0.05 compared with the control. LDH, lactate dehydrogenase.

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Figure 4. Live/dead staining images of undifferentiated Caco-2 treated with different TiO2 nanoparticles at 200 μg ml for 24 h. Cont, control.

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not influence the differentiated Caco-2 cells, even at a higher concentration. This indicates that toxicity of TiO2 NPs is dependent on the concentration and status of cells, and the undifferentiated Caco-2 cells are more vulnerable to invasion.

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live/dead staining assay. The significant differences are found for T1-G and T2-G at 200 μg ml–1, compared with the control. Taken altogether, T1-G and T2-G were slightly toxic to undifferentiated cells at a higher concentration by inhibiting the cell growth. The native and all pretreated TiO2 NPs did

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Figure 5. The number of undifferentiated Caco-2 cells after 24 h exposure to different TiO2 nanoparticles. The number following the TiO2 samples in –1 the abscissa means the corresponding concentration with unit of μg ml . *P < 0.05 compared with the control. Cont, control.

Reactive Oxygen Species Generation in Caco-2 Cells after Exposure to TiO2 Nanoparticles ROS levels in Caco-2 cells postexposure to different TiO2 NPs were measured and the results are shown in Fig. 6. All TiO2 NP samples do not upregulate the ROS level in differentiated cells. In contrast, in undifferentiated Caco-2 cells, ROS generation is pretty significant, particularly for the samples at a higher concentration.

Effect of TiO2 Nanoparticles on Caco-2 Monolayer In this study, the Caco-2 monolayer was used to simulate the intestinal epithelium to reflect the situation of TiO2 NPs in intestine. First, the integrity of monolayer was checked by investigating the morphology with TEM, measuring the TEER value and the translocation of the paracellular transport markers (Fig. S4 in

Supporting Information). The orderly array of villi in the apical side and tight connection between cells can be observed under TEM. The TEER value of each insert reaches a plateau of about 500 Ω · cm2 at the end of the culture period (Table 2). The translocation of transport marker LY and FITC-dextran is less than 3%; however, the value increases significantly after addition of the junction disruptor EGTA (Sergent et al., 2006). All these data demonstrate that the Caco-2 monolayer has good integrity and is suitable for the experiments. The exposure to TiO2 NPs does not have any marked effect on the TEER values (Table 2). In addition, after exposure to TiO2 NPs for 24 h, the translocation of FITC-dextran is similar to that of the control monolayer (Fig. 7). This demonstrates that exposure to high concentrations of all TiO2 NP samples do not disrupt the tight junctions of the monolayer. Next, cell viability of the monolayer was tested postexposure to TiO2 NPs for 24 h. The monolayers were stained with the live/dead kit, fixed and analyzed with the fluorescence microscope (Fig. 8). Similar to Caco-2 cells, cell death is seldom observed for T1, T2 and T1-B samples. Comparatively speaking, the cell death is more distinct for T1-G and T2-G samples. Correspondingly, the intracellular ROS level of the Caco-2 monolayer was also assessed by the DCFH-DA assay. Similar to differentiated Caco-2 cells, no significant generation of ROS is observed (Fig. S5 in Supporting Information). In general, TiO2 NPs do not induce evident toxicity in the Caco-2 monolayer postexposure to TiO2 NPs for 24 h.

Uptake by Caco-2 Cells and Translocation through Caco-2 Monolayer We measured the uptake of TiO2 NPs in undifferentiated and differentiated Caco-2 cells using both flow cytometric light scatter analysis and TEM investigation. In flow cytometric light scatter analysis, the internalized TiO2 NPs in cells are identified by the

Figure 6. TiO2 nanoparticles induce reactive oxygen species generation in undifferentiated (A) and differentiated (B) Caco-2 cells postexposed to TiO2 nanoparticles for 24 h. *P < 0.05 compared with the control. DCF, 2′,7′-dichlorofluorescin.

Table 2. The TEER (Ω · CM2) values of Caco-2 monolayer before and after exposure to 200 μg ml–1 TiO2 nanoparticles for 24 h (n = 3)

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Sample

T1

T1-B

T1-G

T2

T2-G

Before After △%

540 ± 14 519 ± 34 3±3

554 ± 13 529 ± 40 4±4

562 ± 34 571 ± 21 7±2

527 ± 15 487 ± 13 8±1

602 ± 25 533 ± 51 12 ± 4

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Bioeffects of food additive TiO2 nanoparticles to intestinal cells When the model is changed to Caco-2 monolayers, a tiny proportion of TiO2 NPs traverses the monolayer (Table 3). No matter what the concentration, what the culture condition (culture time and medium), the highest translocation rate is less than 1%. This indicates that the translocation of TiO2 NPs is very small, even negligible. The change of surface property, concentration, culture time and culture medium does not enhance the translocation markedly. Moreover, no TiO2 NPs are found in the ultrasections of the Caco-2 monolayer after exposure (Fig. S6 in Supporting Information).

Discussion

Figure 7. The translocation of fluorescein isothiocyanate-dextran through –1 the Caco-2 monolayer postexposed to 200 μg ml TiO2 nanoparticles for 24 h or EGTA for 1 h. Cont, control.

higher side scatter compared with control cells (Fig. 9). Although it is difficult to obtain an accurate quantitative amount of TiO2 NPs internalized in cells, this method is rather simple and efficient to show the cellular uptake of NPs (Suzuki et al., 2007). Percentages of the TiO2 NP swallowed cells to entire cells are summarized in Fig. 9. These semiquantitative data show that the cellular uptake of T1 is higher than that of T2 in both Caco-2 cells. Although T2 has a smaller primary diameter (26 nm), T2 aggregates in culture medium are bigger than T1. It illustrates that the uptake of TiO2 NPs is greatly influenced by their aggregation. In general, the uptake ability of TiO2 NPs follows the order T1 > T1-B > T1-G in both Caco-2 cells, demonstrating that the surface coating decreases the cellular uptake. In addition, our results show that the uptake of TiO2 NPs is much higher in undifferentiated Caco-2 cells than differentiated Caco-2 cells (Fig. 9). The difference between two kinds of Caco-2 cells is clearly visible. Cellular uptake investigated by TEM (Fig. 10) supports the results shown above; uptake of T1-G is less than that of T1 in undifferentiated Caco-2 cells, and uptake of T1 is higher in undifferentiated Caco-2 cells than in differentiated ones.

Both undifferentiated and differentiated Caco-2 cells were used in this study. Compared with undifferentiated Caco-2 cells, differentiated cells are polarized cells retaining the integrity of the microvilli, which may impede the attachment of NPs on to cells and followed internalization, hence preventing cytotoxicity. Therefore, undifferentiated Caco-2 cells are more sensitive to TiO2 NPs. Our results are similar to those reported by Gerloff et al. (2013). ROS generation has been regarded as an important toxicity mechanism for many nanomaterials, including TiO2 NPs ( Jaeger et al., 2012; Jin et al., 2008; Yoo et al., 2012). In this study, TiO2 NPs could induce significant upregulation in undifferentiated cells, but not in differentiated cells. The ROS generation is concentrationdependent; higher concentration means higher ROS level. Although ROS generation was observed, the cytotoxicity of TiO2 NPs was low and was not concentration-dependent for T1, T2 and T1-B, indicating the ROS generation was not the main factor influencing the biosequence of TiO2 NPs in this study. These results are consistent with our previous work on TiO2 particles separated from chewing gums using GES-1 cells (Chen et al., 2013) and other reports (Bhattacharya et al., 2009; Shukla et al., 2011). Bhattacharya et al. (2009) demonstrated that, despite ROS production, no oxidative stress or DNA damage were observed when human bronchial epithelial cells were exposed to TiO2 NPs. The exposure to TiO2 NPs did not affect the function of the Caco-2 monolayer. TiO2 NPs, even T1-G and T2-G, did not disrupt the tight junction of the monolayer. This is consistent with the reports of Faust et al. (2014) and Janer et al. (2014). In addition,

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Figure 8. Live/dead assay for Caco-2 cell monolayer after incubated with TiO2 nanoparticles at 200 μg ml for 24 h. Cont, control.

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Figure 9. Scatter diagrams of undifferentiated (upper panel) and differentiated (lower panel) Caco-2 cells postexposed to 200 μg ml TiO2 nanoparticles for 24 h. Cont, control; D, differentiated; U, undifferentiated.

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Figure 10. Transmission electron microscopy images of undifferentiated and differentiated Caco-2 cells after exposure to 200 μg ml T1 or T1-G for 24 h. The arrows indicate TiO2 nanoparticles. D, differentiated; U, undifferentiated.

Table 3. Translocation of TiO2 nanoparticles through Caco-2 monolayer (n = 3)

T1 T1-B T1-G T2 T2-G

50 μg ml 1 (DMEM, 4 h)

200 μg ml 1 (DMEM, 2 h)

200 μg ml 1 (DMEM, 4 h)

200 μg ml 1 (cDMEM, 4 h)

200 μg ml 1 (cDMEM, 24 h)

0.89 ± 0.09 0.50 ± 0.23 0.46 ± 0.13 0.32 ± 0.01 0.89 ± 0.08

0.13 ± 0.04 0.18 ± 0.02 0.14 ± 0.07 0.13 ± 0.01 0.20 ± 0.07

0.26 ± 0.17 0.14 ± 0.08 0.21 ± 0.04 0.12 ± 0.06 0.23 ± 0.03

0.08 ± 0.02 0.07 ± 0.02 0.12 ± 0.07 0.08 ± 0.02 0.23 ± 0.01

0.17 ± 0.12 0.11 ± 0.13 0.20 ± 0.13 0.38 ± 0.21 0.21 ± 0.02

cDMEM, complete medium; DMEM, Dulbecco modified Eagle’s minimal essential medium.

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TiO2 NPs did not induce the ROS generation and cell viability decrease in the monolayer. The toxicity of TiO2 NPs to the monolayer is very similar to that of differentiated Caco-2 cells, inferring that differentiated Caco-2 cells are more suitable in vitro models for reflecting the cytotoxicity of NPs to intestine. Both flow cytometric light scatter analysis and the TEM investigation indicate the cell differentiation and surface coating decreases markedly the cellular uptake of TiO2 NPs. Studies have reported the uptake of macromolecules and even NPs by differentiated Caco-2 cells ( Jackman et al., 1994; Loo et al., 2012), but Janer et al. (2014) concluded that the microvilliated surface of the differentiated Caco-2 cells may preclude the endocytosis of larger NPs or NP aggregates. As surface coating decreases the cellular uptake of TiO2 NPs, similar results were reported previously that the uptake of NPs into human dermal fibroblasts via endocytosis was suppressed by the immobilization of BSA on the surface (Mikhaylova et al., 2004;

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Suzuki et al., 2007). A possible reason is that surface coating changes the particle aggregation and surface charge. Tiede et al. (2008) reported that the aggregation of particles is generally associated with a decrease in their uptake. In addition, the surface charge of particles changed the uptake of NPs into cells (Suzuki et al., 2007; Win and Feng, 2005). The cellular uptake of NPs did not affect their cytotoxicity in undifferentiated Caco-2 cells in this study. In undifferentiated cells, the native TiO2 NPs possessed the highest uptake, while digestive fluids pretreated TiO2 NPs show a much lower uptake rate. Nevertheless, it was the digestive fluids pretreated TiO2 NPs not the native TiO2 NPs that inhibited the cell growth. The difference between these TiO2 NPs is the surface property. When comparing T1 and T2, T1 shows a much higher cellular uptake, but both are non-toxic to the Caco-2 cells. Therefore, when we surmise the relationship between cell uptake and cytotoxicity,

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J. Appl. Toxicol. 2015; 35: 1169–1178

Bioeffects of food additive TiO2 nanoparticles to intestinal cells we also need to consider the surface property of NPs in biological systems. Koeneman et al. (2010) first reported that TiO2 NPs could pass through the Caco-2 monolayer. They detected that 14.4% of TiO2 NPs crossed the monolayer after exposure to 100 μg ml–1 TiO2 NPs. Others found that only a small amount of TiO2 NPs could pass through the monolayer (Gitrowski et al., 2014). This contradicts reports that TiO2 NPs were unlikely to penetrate the Caco-2 monolayer ( Janer et al., 2014; Jones et al., 2015). Brun et al. (2014) observed the TiO2 NPs in cells after the Caco-2 monolayer was exposed to 50 μg ml–1 TiO2 NPs; however, they did not provide the Ti content in the basolaterial chamber to confirm the transport of TiO2 NPs through the monolayer. In addition, we did not observe the marked traverse of TiO2 NPs through the monolayer. From the point of view of results, the contradiction comes from the different culture conditions and TiO2 NPs with various physicochemical properties, such as size and surface property. The non-traverse of TiO2 NPs through the intestine was also observed in animal experiments (Cho et al., 2013). Interestingly, a newly published experiment on humans also supports that it was very difficult for orally administrated TiO2 NPs to traverse the intestine ( Jones et al., 2015). Combined with our data, we surmise that it is very difficult for TiO2 NPs to traverse through the intestine and enter the blood circulation.

Conclusion Nowadays, TiO2 NPs are most widely used in various fields, including the agro-food industry. Therefore, oral exposure to TiO2 NPs is inevitable for humans. Understanding the behavior of TiO2 NPs in intestine is essential for the risk assessment of TiO2 NPs. To simulate the real situation in intestine, two native TiO2 NPs were pretreated with the digestion simulation fluids or protein BSA, and then their toxicity to undifferentiated Caco-2 cells, differentiated Caco-2 cells and the Caco-2 monolayer was studied systemically. Generally, two native TiO2 NPs and pretreated TiO2 NPs do not show marked toxicity to both Caco-2 cells and the Caco-2 monolayer, though they can be internalized by both cells. At a higher concentration, TiO2 NPs pretreated with digestive fluids inhibit cellular uptake and cell growth in undifferentiated Caco-2 cells. The ROS generation and particle uptake are not the mechanism of cytotoxicity of TiO2 NPs we studied. The traverse of TiO2 NPs through the Caco-2 monolayer was very difficult. These findings indicate that it is better to take differentiated Caco-2 cells as an in vitro model for toxicity evaluation of TiO2 NPs in intestine. We may infer from in vitro data that the possibility of TiO2 NPs traversing through the intestinal barrier and entering the blood circulation in animals or humans is low. Our work provides valuable information about the bioeffects of NPs in the intestinal tract after oral intake, which is important for the risk assessment of TiO2 NPs in food.

Acknowledgments We thank the National Basic Research Program of China (973 Program) (No. 2011CB933402) for financial supports.

Conflict of Interest

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The authors did not report any conflict of interest.

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J. Appl. Toxicol. 2015; 35: 1169–1178

Biological effect of food additive titanium dioxide nanoparticles on intestine: an in vitro study.

Titanium dioxide nanoparticles (TiO2 NPs) are widely found in food-related consumer products. Understanding the effect of TiO2 NPs on the intestinal b...
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