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undifferentiated human intestinal Caco-2 cells
Chemico-Biological Interactions 283 (2018) 38–46
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Effects of cerium oxide nanoparticles on differentiated/undifferentiated human intestinal Caco-2 cells
T
Laura Vilaa,1, Alba García-Rodrígueza,1, Constanza Cortésa, Antonia Velázqueza,b, Noel Xamenaa,b, Adriana Sampayo-Reyesc, Ricard Marcosa,b,∗, Alba Hernándeza,b,∗∗ a
Grup de Mutagènesi, Departament de Genètica i de Microbiologia, Facultat de Biociències, Universitat Autònoma de Barcelona, Bellaterra, Spain CIBER Epidemiología y Salud Pública, ISCIII, Spain c Universidad Autónoma de Nuevo León, Facultad de Biología, San Nicolás de los Garza, Nuevo León, Mexico b
A R T I C L E I N F O
A B S T R A C T
Keywords: Cerium-oxide nanoparticles Differentiated Caco-2 cells Monolayer-integrity Uptake Translocation Genotoxicity
Since ingestion constitute one of the main routes of nanoparticles (NPs) exposure, intestinal cells seems to be a suitable choice to evaluate their potential harmful effects. Caco-2 cells, derived from a human colon adenocarcinoma, have the ability to differentiate forming consistent cell monolayer structures. For these reasons Caco2 cells, both in their undifferentiated or differentiated state, are extendedly used. We have used well-structured monolayers of differentiated Caco-2 cells, as a model of intestinal barrier, to evaluate potential harmful effects associated to CeO2NPs exposure via ingestion. Different parameters such as cell toxicity, monolayer integrity and permeability, cell internalization, translocation through the monolayer, and induction of DNA damage were evaluated. No toxic effects of CeO2NPs were observed, independently of the differentiated state of the Caco2 cells. In the same way, no effects on the monolayer integrity/permeability were observed. Although important cell uptake was demonstrated in undifferentiated cells (by using confocal microscopy), CeO2NPs remained mostly attached to the apical membrane in the differentiated cells. In spite of this apparent lack of uptake in differentiated cells, translocation of CeO2NPs to the basolateral chamber was observed by using confocal microscopy. Finally no genotoxic effects were observed when the comet assay was used, although decreases in the levels of oxidized bases were observed, supporting the antioxidant role of CeO2NPs.
1. Introduction The exclusive and uncommon properties of nanomaterials (NMs) are expanding their use in many fields. Nevertheless, some doubts about their potential detrimental effects to human beings have raised. In this scenario, nanotoxicology is emerging as a new discipline aiming to study the toxicity of NMs and their potential human health risk [1,2]. An interesting model of nanomaterial is cerium dioxide nanoparticles (CeO2NP). This NM has multienzyme (i.e. superoxide oxidase, catalase, etc.) properties that make it a useful agent to be used in many biological fields, such as in bioanalysis, biomedicine and drug delivery, showing useful applications as antioxidant [3–5]. The documented human exposure to NMs (including CeO2NPs) via consumer products occurs mainly by inhalation (respiratory tract) and ingestion (GI, gastrointestinal tract) [2,6,7]. Once NMs have entered
into the body, the next step is to translocate through epithelial barriers to reach secondary targets like organs, tissues, blood vessels and vascular fluids. This has encouraged the research community to propose and describe a wide number of in vitro models/systems mimicking human epithelial barriers to study NMs translocation, as well as their toxicokinetics and toxicodynamics. This suggestion has led to use different in vitro models, depending on the NMs ways of entry into the human body. Some of the barrier models that have been set up are characterized by having good membrane integrity of tight junctions, forming epithelial monolayers [8]. Regarding the GI tract, Caco-2 cells (both as monoculture or forming part of co-cultures models) have been described and developed to study drug transport, from the intestinal lumen into the bloodstream. Caco2 cells have shown to express and synthesize brush border enzymes, involved in nutrient degradation and digestion, as well as those
∗ Corresponding author. Grup de Mutagènesi, Departament de Genètica i de Microbiologia, Universitat Autònoma de Barcelona, Edifici Cn, Campus de Bellaterra, 08193 Cerdanyola del Vallès, Barcelona, Spain. ∗∗ Corresponding author. Grup de Mutagènesi, Departament de Genètica i de Microbiologia, Universitat Autònoma de Barcelona, Edifici Cn, Campus de Bellaterra, 08193 Cerdanyola del Vallès, Barcelona, Spain. E-mail addresses: [email protected] (R. Marcos), [email protected] (A. Hernández). 1 Both persons contributed equally to the work.
https://doi.org/10.1016/j.cbi.2018.01.018 Received 9 November 2017; Received in revised form 6 January 2018; Accepted 22 January 2018 Available online 31 January 2018 0009-2797/ © 2018 Elsevier B.V. All rights reserved.
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serum (FBS), 1% non-essential amino acids (NEAA) (PAA Laboratories GmbH, Pashing, Austria) and 2.5 mg/mL Plasmocin (Invivo Gen, San Diego, CA). Cells were placed in humidified atmosphere of 5% CO2 and 95% air at 37 °C. Undifferentiated Caco-2 cells were seeded in 6-well culture plate and allowed to attach for 24 h. For differentiated Caco2 cells, cells were grown for 21 days in 12-well culture plates onto polyethylene terephthalate transwells with 1 μm mean pore size and a diameter of 1.12 cm (MCRP12H48, MerckMillipore). Cell culture medium was changed every 2 days. Both, undifferentiated and differentiated Caco-2 cells were treated with different concentrations of CeO2NPs for exposures lasting 24 h.
involved in the efflux and uptake of drug transporters [9,10]. Moreover, it has been demonstrated that permeability through a Caco-2 monolayer model correlates well with in vivo absorption in humans [11,12]. For this reason, the in vitro Caco-2 cell monolayer has been proposed as a suitable model to study the interaction of NMs with the intestinal barrier [13,14]. In this context, and taking into account the increasing use of CeO2NPs, more efforts are needed to understand their interaction across the different human exposure routes. Accordingly, we propose to simulate a putative interaction among this NPs and the GI tract by using the Caco-2 monolayer, as a suitable in vitro model. In addition to the toxic effects, structural and functional changes in the monolayer will also be evaluated. Potential translocation through the monolayer will be detected to determine whether CeO2NPs are able to cross the in vitro intestinal barrier. Finally potential genotoxic damage associated to CeO2NPs exposure will be determined. For these purposes, we evaluated the monolayer integrity after 24 h of CeO2NPs exposure by measuring the trans-epithelial electrical resistance (TEER) and by detecting the paracellular passage of Luciferyellow molecules. The potential induction of genotoxic and/or oxidative DNA damage was also evaluated through the comet assay. To study the CeO2NPs interaction with the Caco-2 monolayer, several qualitative and quantitative techniques have been carried out assessing its interaction. Mainly, microscopy techniques have been used to detect cell uptake and CeO2NPs translocation (i.e. transmission electron microscopy (TEM), fluorescent confocal microscopy, and TEM complemented with energy dispersive X-ray (EDX)). Although we assume that differentiated Caco-2 cells reflect better, at the morphological and functional level, the real physiologic environment of a human small intestine, most of the studies testing the toxicity of NMs used undifferentiated Caco-2 cells. For this reason, some effects of CeO2NPs over our barrier model of differentiated Caco-2 were compared with those induced in undifferentiated Caco-2 cells. In fact, differences in biological responses have already been related to the cell status, as reported for TiO2NPs exposures, where the uptake in undifferentiated cells was markedly higher than in differentiated cells [15].
2.3. CeO2NPs toxicity experiments To select the range of sub-toxic doses to be used in the different experiments, a preliminary viability study was carried out. Differentiated and undifferentiated Caco-2 cells were treated for 24 h with different doses of CeO2NPs, ranging from 1 to 200 μg/mL. After exposure, cells were washed three times with 0.5 mL of PBS (1%). Then, cells were incubated 3 min at 37 °C with 0.25 mL of trypsin (1.5%), to detach and separate them. Finally, cells were diluted in an ISOTON® solution (1/100) and analyzed with a ZTM Series coulter-counter (Beckman coulter Inc., CA) using a range of 7–21 μ. Viability values for each concentration were calculated from averaging three independent survival curves. This method has already been used to detect nanomaterials effect [18], and validated against other methods like the MTS cell proliferation assay. 2.4. Monolayer integrity and permeability evaluation To evaluate the putative and potential unfavorable effects of CeO2NPs on the integrity of the Caco-2 monolayer, trans-epithelial electrical resistance (TEER) measurements, and the Lucifer Yellow assay were carried out. The TEER value of each sampling time/concentration was determined with an epithelial voltmeter (Millicell-ERS volt-ohm meter). TEER values were analyzed at 7, 14 and 21 days after seeding, through the cell differentiation and monolayer formation. TEER measurements were done after 24 h of CeO2NPs treatment. Monolayers with TEER values over 350 Ω/cm2 were used for further assays. The paracellular passage of Lucifer-yellow (LY) through the monolayer was measured to determine its permeability. Following the 24 h of CeO2NPs treatments, Lucifer-yellow was added to the apical compartment at the final concentration of 0.4 mg/mL. Monolayers were placed into the incubator for 2 h and the amount of LY able to cross the monolayer was measured by using a fluorimeter plater reader at 405 nm excitation and 535 nm emission.
2. Materials and methods 2.1. Cerium dioxide nanoparticles characterization Cerium dioxide nanoparticles (CeO2NPs, NM212) were obtained from the EU Joint Research Center (Ispra, Italy), in the frame of the EU NANoREG project. It has been well characterized in the frame of different EU projects [16]. CeO2NPs were pre-wetted in 0.5% absolute ethanol and dispersed in 0.05% bovine serum albumin (BSA) using MilliQ water. After that, CeO2NPs were sonicated for 16 min in the dispersion medium, to obtain a stock dispersion of 2.56 mg/mL, according to the NanoGenotox protocol [17]. Transmission electron microscopy (TEM, JOEL-JEM-1400, Jeol LTD, Tokyo, Japan) was used to determine the nanoparticle size and its morphology. TEM images were processed using the Image-J software, to calculate the size diameter, by measuring over 100 particles in random fields of view. In addition, the hydrodynamic size and the z-potential were also evaluated by means of dynamic-light-scattering (DLS) and laser Doppler velocimetry (LDV) methodologies, in a Malvern ZetasizerNano-ZS zen3600 device (Malvern, UK).
2.5. Cell internalization of CeO2NPs Fluorescent confocal microscopy was used to seek and locate internalized CeO2NPs, in both differentiated and undifferentiated Caco2 cells. Previous to the confocal visualization, cells were treated with 100 μg/mL of CeO2NPs during 24 h and, after that, cells were stained with Hoechst 33342 and a red CellMask at 1/500 and 2/500, respectively, during 10 min CeO2NPs were visualized in green by its own reflective capability. The imaging of the stained samples was visualized by using a confocal laser-scanning microscope Leica TCS SP5. Confocal images were processed with Huygens essential 4.4.0p6 (Scientific Volume Imaging, Netherlands) and Imaris 7.2.1 (Bitplane, AG) software.
2.2. Caco-2 cell cultures and CeO2NPs exposure
2.6. Crossing and/or translocation of CeO2NPs through the differentiated Caco-2 monolayer
The human colon adenocarcinoma cell line Caco-2 was pleasantly provided by Dr. Isabella Angelis, from the Istituto Superiore di Sanità (ISS, Italia). Caco-2 cells were weekly maintained in Dulbecco's modified Eagle's High Glucose medium without pyruvate (DMEM w/o Pyruvate, Life Technologies NY) supplemented with 10% fetal bovine
To determine the passage of CeO2NPs from the apical to basolateral compartments, different methodologies and facilities were used. Thus, 39
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Fig. 1. (A) TEM images of CeO2NPs in dried form. (B) Size distribution of CeO2NPs over 100 randomly selected particles. (C) NPs average sizes (by TEM and DLS) by pre-wetting the nanomaterial with 0.5% absolute ethanol and steric stabilization using sterile-filtered 0.05% w/v BSA and posterior dilution in culture medium (100 and 10 μg/mL). Data represented as mean ± SD.
carried out. Then, 12 samples/drops were established for each concentration. Cells on GF were lysed in cold lysis buffer at 4 °C overnight and pH 10. After that, GF were washed twice (1 × 5 min, and 1 × 50 min) in enzyme buffer at 4 °C and pH 8.0, followed by an incubation of 30 min at 37 °C with enzyme buffer. One GF was incubated with enzyme buffer and FPG enzyme (1/10.000) and the other with a free-FPG enzyme buffer. The used FPG enzyme was pleasantly provided by Dr. A.R, Collins (Oslo University, Norway). GF were incubated with electrophoresis buffer (alkaline buffer) for 35 min followed by the electrophoresis step for 20 min at 20 V and 300 mA at 4 °C. Finally, GF were rinsed twice in cold PBS for 5 min, in distilled water for 1 min, fixed in absolute ethanol for at least 2 h, air-dried overnight at room temperature, and stained with SYBR Gold for 20 min. Each GF film was cut in two similar sized parts to fit into an acrylic slide (52.5 × 75 × 3 mm). A coverslip of 52.5 × 75 mm was placed on top of the drops effectively sealing the samples. Gels were observed using epifluorescence Olympus BX50 microscope, and damage was quantified measuring the percentage of DNA in tail, by using the Komet 5.5 Image analysis software. One hundred randomly selected comet images were analyzed per sample Treatments of 5 mM of potassium bromate (KBrO3) and 2.5 mM of methylmethanesulfonate (MMS) were used as positive controls of oxidative and genotoxic damage, respectively.
TEM with Energy-dispersive X-ray spectroscopy (TEM-EDX) (Hitachi H 7000), Inductively Coupled Plama-Mass Spectrometry (ICP-MS) 7500ce (Agilent Technologies) and Fluorescent Confocal Microscopy (LEICA TSC SP5) techniques were used to detect and quantify those CeO2NPs capable to cross the differentiated Caco-2 cells monolayer. Briefly, after 24 h of CeO2NPs treatment, total medium (1.5 mL) of each basal compartment was resuspended and collected in 1.5 mL microtubes. To eliminate the excess of inorganic material aggregation and crystallized proteins, samples were treated with proteinase K (100 μg/mL) and centrifuged in a speed vacuum at 37 °C for 2 h to concentrate the amount of CeO2NPs. This concentrate was used in the above-mentioned techniques. 2.7. Genotoxic and oxidative DNA damage evaluation The induction of genotoxic and oxidative DNA damage to differentiated Caco-2 cells was assessed by the alkaline comet assay after 24 h of CeO2NPs treatment. The addition of formamidopyrimidine DNA glycosylase (FPG enzyme) was used to measure oxidative DNA damage [19]. We applied the standard procedure followed by our group the use Gelbond films, as previously reported [5]. Briefly, once treated differentiated Caco-2 cells were washed twice with PBS, trypsinized (1% trypsin) and centrifuged at 1000 rpm for 8 min. The pellet was resuspended in PBS to obtain around of 17,500 cells/25 μL, and placed in ice at 4 °C to avoid DNA repair. Cells were mixed with 0.75% of LMP agarose at 37 °C and dropped (7 μL/drop and 3 drops/sample) onto Gelbond films (GF). Two replicates of each concentration were placed (3 drops + 2 drops) in each GF. Two replicates/experiments were
2.8. Statistical analysis All measurements were made by triplicate in at least three separate experiments. Results are expressed as mean ± standard error. Once demonstrated the normal distribution of the parameters tested, an 40
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unpaired Student's t-test, and a one-way ANOVA with Tukey's post-test were used to determine the differences among means, and between a group of means, respectively. Data was analyzed with GraphPad Prism version 5.00 for Windows (GraphPad Software, San Diego California USA, www.graphpad.com). In all cases, a two-sided P-value of P < 0.05 was considered statistically significant. 3. Results 3.1. Characterization of CeO2NPs Characteristics of CeO2NPs (NM212) were determined by using TEM and DLS methodologies. As indicated in Fig. 1, NM212 displayed a geometrical varied morphology including cubic structures, when evaluated by TEM (Fig. 1A). This methodology also permits to determine the mean size distribution, measuring over 100 particles in random fields of view. As shown in Fig. 1B, the obtained mean size was 70.33 ± 49.61 nm. When DLS was used to measure the average hydrodynamic radius in suspension, the observed value was 302.77 ± 7.64 (Fig. 1C). This value indicates some aggregation or agglomeration of CeO2NPs in water suspension. Interestingly, the hydrodynamic radius increased with the concentration. 3.2. Toxicity of CeO2NPs The toxic effects of CeO2NPs were evaluated in both, differentiated and undifferentiated Caco-2 cells. The use of two different states of differentiation was chosen to see whether such differentiation process affects the response to the exposure. As indicated in Fig. 2 no effects on cell viability were observed after exposures lasting for 24 h, independently of the state of differentiation. The obtained results indicate the lack of toxic effects associated to CeO2NPs exposure, since no changes in viability were observed in the range of doses used (up to 200 μg/mL). Interestingly, a tendency to increased viabilities was observed mainly in differentiated Caco-2 cells.
Fig. 3. TEER values evaluated during Caco-2 cells differentiation (A), and after 24 h of CeO2NPs treatment (B). Data represented as mean ± SEM. The columns over 21 d indicate the values of the samples before the respective exposures. Results were analyzed according to a one-way ANOVA with a Tukey post-test. **P < 0.01; ***P < 0.001.
TEER value can be considered as indicative of a well-structured barrier. Interestingly, no decreases in TEER values were observed after exposures to 10, 25 and 100 μg/mL, lasting for 24 h. Therefore, no alteration of the monolayer integrity was observed after exposure to CeO2NPs. To evaluate the permeability of the Caco-2 cells monolayer, the Lucifer yellow assay was carried out. Lucifer yellow is a paracellular transport compound that permits to measure tight-junction integrity, and the monolayer's permeability between cells. Fig. 4 shows that there were no significant differences in permeability to LY after exposure to CeO2NPs (10, 25 and 100 μg/mL), when comparing the observed values with those obtained in the untreated control. Taking all together, our results indicate that exposure to CeO2NPs does not alter the integrity/permeability of the monolayer of Caco2 cells, after treatments lasting for 24 h.
3.3. Assessment of the monolayer integrity and permeability NPs can be able to damage the established monolayer, regardless of its ability to internalize or to cross the barrier. To determine potential effects of CeO2NPs on the integrity of this differentiated Caco-2 monolayer, TEER and LY assays were carried out. As indicated in Fig. 3A, TEER values were measured during differentiation, and 24 h after the treatment with CeO2NPs (Fig. 3B). The obtained results indicate that TEER values progressively increase during differentiation of Caco-2 cells, reaching a value of 475.28 ± 45.97 Ω/cm2 at day 21. This
3.4. Cellular uptake of CeO2NPs Confocal microscopy offers a good alternative for the qualitative observation of metallic NPs internalization. This must be considered as a good complement to the information obtained by TEM, although it does not allow us to distinguish between individual NPs and NPs aggregates. This methodology was used for both differentiated and undifferentiated cells; that is, cells growing in transwells or in culture flasks. Since the aim was not quantifying the uptake, the highest concentration was used (100 μg/mL) to facilitate the observation of CeO2NPs, if they uptake. As shown in Fig. 5, the cell membrane was stained in red, nucleus in blue, and the CeO2NPs, although reflecting the polarized light, were
Fig. 2. Cell viability of undifferentiated (continuous line) and differentiated (discontinuous line) Caco-2 cells treated with CeO2NPs for 24 h in a range from 0 to 200 μg/ mL. Data are presented as a percentage of viable cells with respect to controls. Results were analyzed with an unpaired Student's t-test Data represented as mean ± SEM.
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practically non-existent. 3.5. Translocation of CeO2NPs through Caco-2 cells monolayer To determine if CeO2NPs were able to cross the barrier, different techniques including TEM + EDX, ICP-MS, and confocal microscopy were used to determine their presence in the basolateral medium. The obtained results are shown in Fig. 6. When the basolateral medium was analyzed by TEM-EDX a large amount of organic matter and mineral salts was observed. This, potentially, mask NPs avoiding their observation (Fig. 6A). Nevertheless, no cerium was observed in the basolateral medium after using EDX analysis (Fig. 6B). In this case, only the highest concentration (100 μg/ mL) was tested. In addition, potential translocation of CeO2NPs was chemically analyzed by ICP-MS on the apical and basolateral medium. It must be remembered that ICP-MS technique does not distinguish between ionic and NPs form, this method only detects the element studied. Our results indicate that no significant increases in Ce were observed in the basolateral medium (Fig. 6C), since Ce values were below the detection limit of the technique (0.01 μg). To test the methodology, the apical medium was also evaluated. In this case, the absolute amount (μg) of Ce in the apical chamber was elevated observing a concentration-related effect (Fig. 6D). In this case, the effects of three concentrations (10, 25 and 100 μg/mL) were evaluated. Finally, taking advantage of the ability of metal NPs to reflect polarized light, the basolateral medium was also analyzed by confocal microscopy. As observed, despite the previous reported negative results, confocal microscopy showed green spots corresponding to CeO2NPs in the basolateral medium after exposure (Fig. 6F). No signal was detected in the basolateral medium of the untreated cells (Fig. 6E).
Fig. 4. Percentage of LY in the basolateral (BL) chamber after treatment with CeO2NPs during 24 h. Data represented as mean ± SEM. Results were analyzed according to a oneway ANOVA with a Tukey post-test.
observed in green. This technique, by using a specific software, allows covering nuclei and NPs with a mask that allows locating structures in a three-dimensional space (Fig. 5 B,D). The results show that the undifferentiated cells present a much lower thickness, because they are not polarized, and do not express microvilli in the apical part. In these cells, CeO2NPs were able to internalize within the cell, reaching the nucleus (Fig. 5A and B). Nevertheless, in the differentiated state, practically all CeO2NPs were found attached to the apical membrane (Fig. 5C and D), and uptake was
Fig. 5. Confocal images from undifferentiated (A, B) and differentiated (C, D) Caco-2 cells, treated with 100 μg/mL of CeO2NPs. Cellular uptake was measured after 24 h of treatment. Nucleuses are stained in blue, cell membranes in red, and CeO2NPs in green. Arrows indicate the localization of CeO2NPs in cells. In A and B lateral and bottom boxes correspond to cut views. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
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Fig. 6. Translocation studies of CeO2NPs through Caco-2 monolayer. The basolateral extract was analyzed after 24 h of CeO2NPs exposure by using TEM: A (image) and B (TEM + EDX); by using ICP-MS: C (μg of Ce in the basolateral chamber) and D (μg of Ce in apical chamber); and by using confocal microscopy: E (control), and F (CeO2NPs). Results are represented as mean ± SEM and were analyzed according to a one-way ANOVA with a Tukey post-test.
observed in Fig. 7 no increases in DNA breaks were detected after our exposure conditions (A). Nevertheless, high and significant increases in the percentage of DNA in tail were observed when MMS (0.5 mM) was used as a positive control, supporting the suitability of the assay. In addition, no oxidative DNA damage was observed when FPG enzyme was used (Fig. 7B). It should be remembered that FPG enzyme detects oxidized bases and induces single-strand breaks after their excision. This means that no significant induction of oxidized bases was produced after 24 h of exposure (Fig. 7B), but the contrary since lower levels of oxidative damage were detected, suggesting an antioxidant effect. High and significant effects were observed after potassium bromate treatment, used as a positive pro-oxidant agent.
This would confirm the sensitivity of the technique showing that, in spite of the low uptake, some CeO2NPs were able to cross the monolayer. 3.6. Genotoxic and oxidative DNA damage induction Among the different potential harmful effects induced by NPs exposure, DNA damage is of crucial relevance. To assess general genotoxic damage resulting from DNA breaks, and to evaluate the oxidative damage induced on DNA bases, the alkaline comet assay, supplemented with FPG, was carried out. The effects of three concentrations (10, 25 and 100 μg/mL) were assayed in exposures lasting for 24 h. The percentage of DNA in tail was used as the biomarker of genotoxic effect. As 43
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Fig. 7. Analysis of genotoxic (A) and oxidative DNA damage (B) after 24 h of CeO2NPs exposure using the comet assay. Three drops per replicate were used per concentration. Results were analyzed according to a one-way ANOVA with a Tukey post-test. Data represented as mean ± SEM. *P < 0.05; ***P < 0.001.
4. Discussion
junctions, studies on the stability of this barrier are needed. In our Caco2 model, TEER values for the stabilised barrier were 475.28 ± 45.97 Ω/cm2 at the end of 21 days of differentiation. This value is similar to the TEER values reported by different authors [24] what means that our model of barrier was well constituted. Furthermore, permeability of the Caco-2 monolayer was evaluated by measuring the passive crossing of small hydrophilic molecules, as LY, through the paracellular space [33]. Its passage is easily detectable and provides a sensitive and reliable technique to confirm the integrity of the barrier [34]. In our study, 24 h of incubation with CeO2NPs did not alter the integrity or the permeability of the intestinal epithelium, regardless of the concentration. These negative findings agree with those obtained by other members of the NANoREG consortium [35]; this means that CeO2NPs exposure (under our experimental conditions) do not disrupt our model of Caco-2 monolayer. In addition to the studies on the potential alteration of the integrity of the barrier, it is also important to investigate whether NMs are able to internalize into Caco-2 monolayer or, even, if they are able to cross it. Confocal microscopy is a technique that allows the detection of metallic NMs such as cerium, because of their ability to reflect polarized light, although it does not distinguish between aggregates and individual NMs [36]. According to that, by using confocal microscopy as a new approach, we have been able to demonstrate that CeO2NPs strongly internalize in undifferentiated Caco-2 cells but few in differentiated Caco-2 cells. It is important to emphasize that this methodology permits to distinguish if NMs are located on the surface of the epithelia or if they are internalized, either in the cytoplasm or in the nucleus, due to differential staining between different cellular components (nucleus and plasmatic membrane) [37,38]. In our study, CeO2NPs were observed in both, inside cytoplasm and nucleus of undifferentiated Caco-2 cells. Nevertheless, they were mainly found outside cells, attached to plasmatic membrane, in differentiated Caco2 cells. This difference of internalization could be due to small intestinelike structure reached by differentiated cells. A more complex cell structure and morphology could complicate the penetration of NPs inside cells. Low, but relevant internalization of CeO2NPs inside differentiated Caco-2 cells were observed by confocal microscopy after 24 h of exposure [39], contrasting with our poor observed uptake. This discrepancy can be explained by methodological differences between studies, as Gaiser et al. [39] worked with 14 days-old Caco-2 monolayers and 14.9 ± 7.4 nm CeO2NPs, while we used 21 days-old Caco-2 monolayers and our NPs size were approximately 70.33 nm. Thus, a low
It is largely known that Caco-2 cells have the capability to differentiate spontaneously to enterocytes, when they arrive at confluence. In parallel, they acquire several characteristics associated with the physical and metabolic barrier of the intestinal epithelium such as cell polarity, formation of microvilli, presence of tight-junctions, expression of digestive enzymes in the brush border, secretion, and assembly of a matrix [20]. The European Center for the Validation of Alternative Methods (ECVAM) pre-validated the Caco-2 monolayer as a useful model to determine the effects of acute toxicity, and for the assessment of active transport of drugs and chemicals [21,22]; but not for evaluating the passive crossing of NPs. In spite of that, all the indicated characteristics make this model suitable to be used to determine potential translocation through intestinal barrier of ingested nanomaterials [23]. In our study no toxic effects of CeO2NPs in Caco-2 cells were observed, in neither undifferentiated nor differentiated cells. These results are in agreement with those obtained by Fisichella et al. [24] who found that CeO2NPs, did not induce cytotoxic effects on undifferentiated Caco-2 cells after 24 h or 72 h exposure, even at high concentrations (170 μg/mL). Although not statistically significant, an increase on cell viability was observed after CeO2NPs exposure to both cell states. This result would agree with those describing in vitro stimulation of cell proliferation by exposing primary mouse bone marrow stromal cells (BMSCs) [25], and primary mouse embryonic fibroblast [26] to cerium oxide nanoparticles. Despite the stimulation mechanisms are not totally clears, CeO2NPs could stimulate cell proliferation through reduction of intracellular levels of ROS by modulating the expression level of the major antioxidant enzymes [26]. Nevertheless, toxicity effects of CeO2NPs are controversial due to its reported prooxidant [27,28] or anti-oxidant role [29,30]. In general, it is assumed that the observed differences in toxicity of NPs depend on multiple factors, including: the manufacturer, the composition of the NPs, their shape and size, their ability to form aggregates, the method of preparation, and the dispersion protocol used [31,32]. In addition, the cells used in the study or even their clones, as in the case of the Caco-2 cell line [19], can also modulate the response to toxic agents. Epithelia are formed by cells that are attached to each other by tight-junctions. These junctions guarantee the integrity of the epithelium avoiding the crossing of undesirable compounds. Since the presence of NPs could alter the integrity of the barrier, by acting on tight44
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not suppose an important risk for human beings. Additionally, the resistance to NPs uptake showed by differentiated cells must be taken into consideration in the risk assessment of NPs exposure. In this way, data obtained in undifferentiated cells may overestimate the real risk of such exposures.
differentiated status and a more little NP diameter could enhance and trigger cell uptake. Nevertheless, and in spite of the great uptake of CeO2NPs in undifferentiated cells, no toxic effects were observed. Higher uptake in undifferentiated cells, with regard to differentiated cells, has also been found for TiO2NPs exposures [40]. Regarding these differences in cell uptake, and in order to avoid inconsistent results, we strongly suggest working with more physiologically realistic in vitro models as Caco-2 monolayers represent. Despite the apparent low internalization of CeO2NPs inside differentiated Caco-2 cells, NPs translocation was observed through the monolayer. Different techniques were used (EDX coupled to TEM, confocal microscopy, and ICP–MS) in order to detect NPs in the basolateral medium. Negative results were obtained by TEM-EDX and ICPMS, but confocal microscopy showed green spots in basolateral medium corresponding to CeO2NPs. These spots were not detected in the control (non-treated Caco-2 monolayer). This finding lead us to hypothesize that the main crossing route of CeO2NPs is intracellularly and that such NPs are readily uptake by the cells, remaining less than 24 h into the cytoplasm, as it was barely impossible to detect them into the cells. This result is surprisingly, due to the accepted high sensitivity of the ICP-MS methodology. Possibly the reflection of CeO2NPs made that, in spite of its low amount present in the basolateral chamber, the confocal microscopy is able to visualize them. Altogether, these results indicate the usefulness of confocal microscopy to detect metallic NPs like CeO2NPs. Moreover, more effort need to be done to further and exactly clarify the crossing mechanisms that CeO2NPs use to translocate through epithelial barriers. For instance, it could be interesting to use several time points enough close among them to specifically locate the CeO2NPs. To check changes at the transcriptional and translational level of tight-junction markers could be a useful tool to further demonstrate that the barrier integrity is not altered. At this point it should be indicated that TiO2NPs exposures up regulated the gene expression of impaired cell junction in others in vitro intestinal models, as a response to actively repair adverse effects in the barrier integrity [41]. The induction of oxidative stress is one of the common mechanisms of toxicity mediated by NMs, through reactive oxygen species (ROS) formation [42–44]. Nevertheless, the chemical composition of NMs, rather than size, seems to be the most determinant factor influencing ROS formation in exposed cells [45], which can damage DNA. In the present study, neither genotoxic nor oxidative DNA damage were produced by CeO2NPs in differentiated Caco-2. These negative results agree with other studies carried out by our group in BEAS-2B cells, where no increases in the levels of ROS were found after CeO2NPs exposure [46]. Such results would manifest the unique antioxidant properties of CeO2NPs, since its autoregenerative redox cycle can protect cells and tissues from oxidative stress induced damage [5,47]. Antioxidant properties of PEGylated CeO2NPs in HaCaT cells were reported showing that CeO2NPs were able to protect the cells from damage caused by the ROS resulting from the loss of cellular GSH production. Furthermore, exposure to PEGylated CeO2NPs did not induce any DNA damage; in fact, such pre-treated cells exhibited protection against nuclear fragmentation and micronucleus formation [48]. All this data would reinforce the view that CeO2NPs are not genotoxic, at least when the comet assay is used. Summing up, we can conclude that CeO2NPs are not toxic in Caco2 cells, independently of their state of differentiation. Furthermore, CeO2NPs exposure is not able to alter monolayer's integrity neither tight-junctions and, finally, CeO2NPs are not able to produce neither genotoxic nor oxidative DNA damage, as demonstrated by using the comet assay complemented with FPG enzyme. Although undifferentiated cells are able to uptake important amounts of CeO2NPs, differentiated cells present very low NP's internalization remaining mostly attached in the apical plasmatic membrane. In spite of this apparent low uptake in differentiated Caco-2 cells, some CeO2NPs are able to cross the monolayer, at least as visualized by confocal microscopy. All this together would reinforce the view that CeO2NPs exposure does
Conflicts of interest The authors report no conflict of interest. The authors alone are responsible for the content and writing of the paper. Acknowledgements This investigation has been partially supported by the Ministry of Economy and Competition (SAF2015-63519-R), and the EC-FP7NANoREG (Grant Agreement NMP4-LA-2013-310584). A. GarciaRodriguez and L. Vila were funded by postgraduate fellowships from the Universitat Autònoma de Barcelona and the Generalitat de Catalunya, respectively. Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.cbi.2018.01.018. References [1] G. Oberdörster, E. Oberdörster, J. Oberdörster, Nanotoxicology: an emerging discipline evolving from studies of ultrafine particles, Environ. Health Perspect. 113 (2005) 823–839. [2] H. Johnston, G. Pojana, S. Zuin, N.R. Jacobsen, P. Møller, S. Loft, M. SemmlerBehnke, C. McGuiness, D. Balharry, A. Marcomini, H. Wallin, W. Kreyling, K. Donaldson, L. Tran, V. Stone, Engineered nanomaterial risk. Lessons learnt from completed nanotoxicology studies: potential solutions to current and future challenges, Crit. Rev. Toxicol. 43 (2013) 1–20. [3] S. Das, J.M. Dowding, K.E. Klump, J.F. McGinnis, W. Self, S. Seal, Cerium oxide nanoparticles: applications and prospects in nanomedicine, Nanomedicine 8 (2013) 1483–1508. [4] C. Xu, X. Qu, Cerium oxide nanoparticle: a remarkably versatile rare earth nanomaterial for biological applications, NPG Asia Mater. 6 (2014) 1–16. [5] L. Rubio, B. Annangi, L. Vila, A. Hernández, R. Marcos, Antioxidant and anti-genotoxic properties of cerium oxide nanoparticles in a pulmonary-like cell system, Arch. Toxicol. 90 (2016) 269–278. [6] P.H. Hoet, I. Brüske-Hohlfeld, O.V. Salata, Nanoparticles -known and unknown health risks, J. Nanobiotechnol. 2 (2004) 12. [7] A. Kermanizadeh, D. Balharry, H. Wallin, S. Loft, P. Møller, Nanomaterial translocation–the biokinetics, tissue accumulation, toxicity and fate of materials in secondary organs–a review, Crit. Rev. Toxicol. 45 (2015) 837–872. [8] B. Srinivasan, A.R. Kolli, M.B. Esch, H.E. Abaci, M.L. Shuler, J.J. Hickman, TEER measurement techniques for in vitro barrier model systems, J. Lab. Autom. 20 (2015) 107–126. [9] C. Hilgendorf, G. Ahlin, A. Seithel, P. Artursson, A.L. Ungell, J. Karlsson, Expression of thirty-six drug transporter genes in human intestine, liver, kidney, and organotypic cell lines, Drug Metab. Dispos. 35 (2007) 1333–1340. [10] D.A. Volpe, Drug-permeability and transporter assays in Caco-2 and MDCK cell lines, Future Med. Chem. 3 (2011) 2063–2077. [11] S. Yamashita, T. Furubayashi, M. Kataoka, T. Sakane, H. Sezaki, H. Tokuda, Optimized conditions for prediction of intestinal drug permeability using Caco-2 cells, Eur. J. Pharm. Sci. 10 (2000) 195–204. [12] P. Artursson, K. Palm, K. Luthman, Caco-2 monolayers in experimental and theoretical predictions of drug transport, Adv. Drug Deliv. Rev. 46 (2001) 27–43. [13] A. Thubagere, B.M. Reinhard, Nanoparticle-induced apoptosis propagates through hydrogen-peroxide-mediated bystander killing: insights from a human intestinal epithelium in vitro model, ACS Nano 4 (2010) 3611–3622. [14] P. Ilina, S. Partti, J. Niklander, M. Ruponen, Y.R. Lou, M. Yliperttula, Effect of differentiation on endocytic profiles of endothelial and epithelial cell culture models, Exp. Cell Res. 332 (2015) 89–101. [15] Z.M. Song, N. Chen, J.H. Liu, H. Tang, X. Deng, W.S. Xi, K. Han, A. Cao, Y. Liu, H. Wang, Biological effect of food additive titanium dioxide nanoparticles on intestine: an in vitro study, J. Appl. Toxicol. 35 (2015) 1169–1178. [16] C. Singh, S. Friedrichs, G. Ceccone, N. Gibson, K.A. Jensen, M. Levin, H.G. Infante, D. Carlander, K. Rasmussen, Cerium Dioxide, NM-211, NM-212, NM-213. Characterization and Test Item Preparation, (2014) http://publications.jrc.ec. europa.eu/repository/bitstream/JRC89825/lbna26649enn.pdf. [17] Nanogenotox, (2011) http://www.nanogenotox.eu/files/PDF/Deliverables/ nanogeno tox%20deliverable%203_wp4_%20dispersion%20protocol.pdf. [18] B. Annangi, L. Rubio, M. Alaraby, J. Bach, R. Marcos, A. Hernández, Acute and
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Chemico-Biological Interactions journal homepage: www.elsevier.com/locate/chembioint
Effects of cerium oxide nanoparticles on differentiated/undifferentiated human intestinal Caco-2 cells
T
Laura Vilaa,1, Alba García-Rodrígueza,1, Constanza Cortésa, Antonia Velázqueza,b, Noel Xamenaa,b, Adriana Sampayo-Reyesc, Ricard Marcosa,b,∗, Alba Hernándeza,b,∗∗ a
Grup de Mutagènesi, Departament de Genètica i de Microbiologia, Facultat de Biociències, Universitat Autònoma de Barcelona, Bellaterra, Spain CIBER Epidemiología y Salud Pública, ISCIII, Spain c Universidad Autónoma de Nuevo León, Facultad de Biología, San Nicolás de los Garza, Nuevo León, Mexico b
A R T I C L E I N F O
A B S T R A C T
Keywords: Cerium-oxide nanoparticles Differentiated Caco-2 cells Monolayer-integrity Uptake Translocation Genotoxicity
Since ingestion constitute one of the main routes of nanoparticles (NPs) exposure, intestinal cells seems to be a suitable choice to evaluate their potential harmful effects. Caco-2 cells, derived from a human colon adenocarcinoma, have the ability to differentiate forming consistent cell monolayer structures. For these reasons Caco2 cells, both in their undifferentiated or differentiated state, are extendedly used. We have used well-structured monolayers of differentiated Caco-2 cells, as a model of intestinal barrier, to evaluate potential harmful effects associated to CeO2NPs exposure via ingestion. Different parameters such as cell toxicity, monolayer integrity and permeability, cell internalization, translocation through the monolayer, and induction of DNA damage were evaluated. No toxic effects of CeO2NPs were observed, independently of the differentiated state of the Caco2 cells. In the same way, no effects on the monolayer integrity/permeability were observed. Although important cell uptake was demonstrated in undifferentiated cells (by using confocal microscopy), CeO2NPs remained mostly attached to the apical membrane in the differentiated cells. In spite of this apparent lack of uptake in differentiated cells, translocation of CeO2NPs to the basolateral chamber was observed by using confocal microscopy. Finally no genotoxic effects were observed when the comet assay was used, although decreases in the levels of oxidized bases were observed, supporting the antioxidant role of CeO2NPs.
1. Introduction The exclusive and uncommon properties of nanomaterials (NMs) are expanding their use in many fields. Nevertheless, some doubts about their potential detrimental effects to human beings have raised. In this scenario, nanotoxicology is emerging as a new discipline aiming to study the toxicity of NMs and their potential human health risk [1,2]. An interesting model of nanomaterial is cerium dioxide nanoparticles (CeO2NP). This NM has multienzyme (i.e. superoxide oxidase, catalase, etc.) properties that make it a useful agent to be used in many biological fields, such as in bioanalysis, biomedicine and drug delivery, showing useful applications as antioxidant [3–5]. The documented human exposure to NMs (including CeO2NPs) via consumer products occurs mainly by inhalation (respiratory tract) and ingestion (GI, gastrointestinal tract) [2,6,7]. Once NMs have entered
into the body, the next step is to translocate through epithelial barriers to reach secondary targets like organs, tissues, blood vessels and vascular fluids. This has encouraged the research community to propose and describe a wide number of in vitro models/systems mimicking human epithelial barriers to study NMs translocation, as well as their toxicokinetics and toxicodynamics. This suggestion has led to use different in vitro models, depending on the NMs ways of entry into the human body. Some of the barrier models that have been set up are characterized by having good membrane integrity of tight junctions, forming epithelial monolayers [8]. Regarding the GI tract, Caco-2 cells (both as monoculture or forming part of co-cultures models) have been described and developed to study drug transport, from the intestinal lumen into the bloodstream. Caco2 cells have shown to express and synthesize brush border enzymes, involved in nutrient degradation and digestion, as well as those
∗ Corresponding author. Grup de Mutagènesi, Departament de Genètica i de Microbiologia, Universitat Autònoma de Barcelona, Edifici Cn, Campus de Bellaterra, 08193 Cerdanyola del Vallès, Barcelona, Spain. ∗∗ Corresponding author. Grup de Mutagènesi, Departament de Genètica i de Microbiologia, Universitat Autònoma de Barcelona, Edifici Cn, Campus de Bellaterra, 08193 Cerdanyola del Vallès, Barcelona, Spain. E-mail addresses: [email protected] (R. Marcos), [email protected] (A. Hernández). 1 Both persons contributed equally to the work.
https://doi.org/10.1016/j.cbi.2018.01.018 Received 9 November 2017; Received in revised form 6 January 2018; Accepted 22 January 2018 Available online 31 January 2018 0009-2797/ © 2018 Elsevier B.V. All rights reserved.
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serum (FBS), 1% non-essential amino acids (NEAA) (PAA Laboratories GmbH, Pashing, Austria) and 2.5 mg/mL Plasmocin (Invivo Gen, San Diego, CA). Cells were placed in humidified atmosphere of 5% CO2 and 95% air at 37 °C. Undifferentiated Caco-2 cells were seeded in 6-well culture plate and allowed to attach for 24 h. For differentiated Caco2 cells, cells were grown for 21 days in 12-well culture plates onto polyethylene terephthalate transwells with 1 μm mean pore size and a diameter of 1.12 cm (MCRP12H48, MerckMillipore). Cell culture medium was changed every 2 days. Both, undifferentiated and differentiated Caco-2 cells were treated with different concentrations of CeO2NPs for exposures lasting 24 h.
involved in the efflux and uptake of drug transporters [9,10]. Moreover, it has been demonstrated that permeability through a Caco-2 monolayer model correlates well with in vivo absorption in humans [11,12]. For this reason, the in vitro Caco-2 cell monolayer has been proposed as a suitable model to study the interaction of NMs with the intestinal barrier [13,14]. In this context, and taking into account the increasing use of CeO2NPs, more efforts are needed to understand their interaction across the different human exposure routes. Accordingly, we propose to simulate a putative interaction among this NPs and the GI tract by using the Caco-2 monolayer, as a suitable in vitro model. In addition to the toxic effects, structural and functional changes in the monolayer will also be evaluated. Potential translocation through the monolayer will be detected to determine whether CeO2NPs are able to cross the in vitro intestinal barrier. Finally potential genotoxic damage associated to CeO2NPs exposure will be determined. For these purposes, we evaluated the monolayer integrity after 24 h of CeO2NPs exposure by measuring the trans-epithelial electrical resistance (TEER) and by detecting the paracellular passage of Luciferyellow molecules. The potential induction of genotoxic and/or oxidative DNA damage was also evaluated through the comet assay. To study the CeO2NPs interaction with the Caco-2 monolayer, several qualitative and quantitative techniques have been carried out assessing its interaction. Mainly, microscopy techniques have been used to detect cell uptake and CeO2NPs translocation (i.e. transmission electron microscopy (TEM), fluorescent confocal microscopy, and TEM complemented with energy dispersive X-ray (EDX)). Although we assume that differentiated Caco-2 cells reflect better, at the morphological and functional level, the real physiologic environment of a human small intestine, most of the studies testing the toxicity of NMs used undifferentiated Caco-2 cells. For this reason, some effects of CeO2NPs over our barrier model of differentiated Caco-2 were compared with those induced in undifferentiated Caco-2 cells. In fact, differences in biological responses have already been related to the cell status, as reported for TiO2NPs exposures, where the uptake in undifferentiated cells was markedly higher than in differentiated cells [15].
2.3. CeO2NPs toxicity experiments To select the range of sub-toxic doses to be used in the different experiments, a preliminary viability study was carried out. Differentiated and undifferentiated Caco-2 cells were treated for 24 h with different doses of CeO2NPs, ranging from 1 to 200 μg/mL. After exposure, cells were washed three times with 0.5 mL of PBS (1%). Then, cells were incubated 3 min at 37 °C with 0.25 mL of trypsin (1.5%), to detach and separate them. Finally, cells were diluted in an ISOTON® solution (1/100) and analyzed with a ZTM Series coulter-counter (Beckman coulter Inc., CA) using a range of 7–21 μ. Viability values for each concentration were calculated from averaging three independent survival curves. This method has already been used to detect nanomaterials effect [18], and validated against other methods like the MTS cell proliferation assay. 2.4. Monolayer integrity and permeability evaluation To evaluate the putative and potential unfavorable effects of CeO2NPs on the integrity of the Caco-2 monolayer, trans-epithelial electrical resistance (TEER) measurements, and the Lucifer Yellow assay were carried out. The TEER value of each sampling time/concentration was determined with an epithelial voltmeter (Millicell-ERS volt-ohm meter). TEER values were analyzed at 7, 14 and 21 days after seeding, through the cell differentiation and monolayer formation. TEER measurements were done after 24 h of CeO2NPs treatment. Monolayers with TEER values over 350 Ω/cm2 were used for further assays. The paracellular passage of Lucifer-yellow (LY) through the monolayer was measured to determine its permeability. Following the 24 h of CeO2NPs treatments, Lucifer-yellow was added to the apical compartment at the final concentration of 0.4 mg/mL. Monolayers were placed into the incubator for 2 h and the amount of LY able to cross the monolayer was measured by using a fluorimeter plater reader at 405 nm excitation and 535 nm emission.
2. Materials and methods 2.1. Cerium dioxide nanoparticles characterization Cerium dioxide nanoparticles (CeO2NPs, NM212) were obtained from the EU Joint Research Center (Ispra, Italy), in the frame of the EU NANoREG project. It has been well characterized in the frame of different EU projects [16]. CeO2NPs were pre-wetted in 0.5% absolute ethanol and dispersed in 0.05% bovine serum albumin (BSA) using MilliQ water. After that, CeO2NPs were sonicated for 16 min in the dispersion medium, to obtain a stock dispersion of 2.56 mg/mL, according to the NanoGenotox protocol [17]. Transmission electron microscopy (TEM, JOEL-JEM-1400, Jeol LTD, Tokyo, Japan) was used to determine the nanoparticle size and its morphology. TEM images were processed using the Image-J software, to calculate the size diameter, by measuring over 100 particles in random fields of view. In addition, the hydrodynamic size and the z-potential were also evaluated by means of dynamic-light-scattering (DLS) and laser Doppler velocimetry (LDV) methodologies, in a Malvern ZetasizerNano-ZS zen3600 device (Malvern, UK).
2.5. Cell internalization of CeO2NPs Fluorescent confocal microscopy was used to seek and locate internalized CeO2NPs, in both differentiated and undifferentiated Caco2 cells. Previous to the confocal visualization, cells were treated with 100 μg/mL of CeO2NPs during 24 h and, after that, cells were stained with Hoechst 33342 and a red CellMask at 1/500 and 2/500, respectively, during 10 min CeO2NPs were visualized in green by its own reflective capability. The imaging of the stained samples was visualized by using a confocal laser-scanning microscope Leica TCS SP5. Confocal images were processed with Huygens essential 4.4.0p6 (Scientific Volume Imaging, Netherlands) and Imaris 7.2.1 (Bitplane, AG) software.
2.2. Caco-2 cell cultures and CeO2NPs exposure
2.6. Crossing and/or translocation of CeO2NPs through the differentiated Caco-2 monolayer
The human colon adenocarcinoma cell line Caco-2 was pleasantly provided by Dr. Isabella Angelis, from the Istituto Superiore di Sanità (ISS, Italia). Caco-2 cells were weekly maintained in Dulbecco's modified Eagle's High Glucose medium without pyruvate (DMEM w/o Pyruvate, Life Technologies NY) supplemented with 10% fetal bovine
To determine the passage of CeO2NPs from the apical to basolateral compartments, different methodologies and facilities were used. Thus, 39
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Fig. 1. (A) TEM images of CeO2NPs in dried form. (B) Size distribution of CeO2NPs over 100 randomly selected particles. (C) NPs average sizes (by TEM and DLS) by pre-wetting the nanomaterial with 0.5% absolute ethanol and steric stabilization using sterile-filtered 0.05% w/v BSA and posterior dilution in culture medium (100 and 10 μg/mL). Data represented as mean ± SD.
carried out. Then, 12 samples/drops were established for each concentration. Cells on GF were lysed in cold lysis buffer at 4 °C overnight and pH 10. After that, GF were washed twice (1 × 5 min, and 1 × 50 min) in enzyme buffer at 4 °C and pH 8.0, followed by an incubation of 30 min at 37 °C with enzyme buffer. One GF was incubated with enzyme buffer and FPG enzyme (1/10.000) and the other with a free-FPG enzyme buffer. The used FPG enzyme was pleasantly provided by Dr. A.R, Collins (Oslo University, Norway). GF were incubated with electrophoresis buffer (alkaline buffer) for 35 min followed by the electrophoresis step for 20 min at 20 V and 300 mA at 4 °C. Finally, GF were rinsed twice in cold PBS for 5 min, in distilled water for 1 min, fixed in absolute ethanol for at least 2 h, air-dried overnight at room temperature, and stained with SYBR Gold for 20 min. Each GF film was cut in two similar sized parts to fit into an acrylic slide (52.5 × 75 × 3 mm). A coverslip of 52.5 × 75 mm was placed on top of the drops effectively sealing the samples. Gels were observed using epifluorescence Olympus BX50 microscope, and damage was quantified measuring the percentage of DNA in tail, by using the Komet 5.5 Image analysis software. One hundred randomly selected comet images were analyzed per sample Treatments of 5 mM of potassium bromate (KBrO3) and 2.5 mM of methylmethanesulfonate (MMS) were used as positive controls of oxidative and genotoxic damage, respectively.
TEM with Energy-dispersive X-ray spectroscopy (TEM-EDX) (Hitachi H 7000), Inductively Coupled Plama-Mass Spectrometry (ICP-MS) 7500ce (Agilent Technologies) and Fluorescent Confocal Microscopy (LEICA TSC SP5) techniques were used to detect and quantify those CeO2NPs capable to cross the differentiated Caco-2 cells monolayer. Briefly, after 24 h of CeO2NPs treatment, total medium (1.5 mL) of each basal compartment was resuspended and collected in 1.5 mL microtubes. To eliminate the excess of inorganic material aggregation and crystallized proteins, samples were treated with proteinase K (100 μg/mL) and centrifuged in a speed vacuum at 37 °C for 2 h to concentrate the amount of CeO2NPs. This concentrate was used in the above-mentioned techniques. 2.7. Genotoxic and oxidative DNA damage evaluation The induction of genotoxic and oxidative DNA damage to differentiated Caco-2 cells was assessed by the alkaline comet assay after 24 h of CeO2NPs treatment. The addition of formamidopyrimidine DNA glycosylase (FPG enzyme) was used to measure oxidative DNA damage [19]. We applied the standard procedure followed by our group the use Gelbond films, as previously reported [5]. Briefly, once treated differentiated Caco-2 cells were washed twice with PBS, trypsinized (1% trypsin) and centrifuged at 1000 rpm for 8 min. The pellet was resuspended in PBS to obtain around of 17,500 cells/25 μL, and placed in ice at 4 °C to avoid DNA repair. Cells were mixed with 0.75% of LMP agarose at 37 °C and dropped (7 μL/drop and 3 drops/sample) onto Gelbond films (GF). Two replicates of each concentration were placed (3 drops + 2 drops) in each GF. Two replicates/experiments were
2.8. Statistical analysis All measurements were made by triplicate in at least three separate experiments. Results are expressed as mean ± standard error. Once demonstrated the normal distribution of the parameters tested, an 40
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unpaired Student's t-test, and a one-way ANOVA with Tukey's post-test were used to determine the differences among means, and between a group of means, respectively. Data was analyzed with GraphPad Prism version 5.00 for Windows (GraphPad Software, San Diego California USA, www.graphpad.com). In all cases, a two-sided P-value of P < 0.05 was considered statistically significant. 3. Results 3.1. Characterization of CeO2NPs Characteristics of CeO2NPs (NM212) were determined by using TEM and DLS methodologies. As indicated in Fig. 1, NM212 displayed a geometrical varied morphology including cubic structures, when evaluated by TEM (Fig. 1A). This methodology also permits to determine the mean size distribution, measuring over 100 particles in random fields of view. As shown in Fig. 1B, the obtained mean size was 70.33 ± 49.61 nm. When DLS was used to measure the average hydrodynamic radius in suspension, the observed value was 302.77 ± 7.64 (Fig. 1C). This value indicates some aggregation or agglomeration of CeO2NPs in water suspension. Interestingly, the hydrodynamic radius increased with the concentration. 3.2. Toxicity of CeO2NPs The toxic effects of CeO2NPs were evaluated in both, differentiated and undifferentiated Caco-2 cells. The use of two different states of differentiation was chosen to see whether such differentiation process affects the response to the exposure. As indicated in Fig. 2 no effects on cell viability were observed after exposures lasting for 24 h, independently of the state of differentiation. The obtained results indicate the lack of toxic effects associated to CeO2NPs exposure, since no changes in viability were observed in the range of doses used (up to 200 μg/mL). Interestingly, a tendency to increased viabilities was observed mainly in differentiated Caco-2 cells.
Fig. 3. TEER values evaluated during Caco-2 cells differentiation (A), and after 24 h of CeO2NPs treatment (B). Data represented as mean ± SEM. The columns over 21 d indicate the values of the samples before the respective exposures. Results were analyzed according to a one-way ANOVA with a Tukey post-test. **P < 0.01; ***P < 0.001.
TEER value can be considered as indicative of a well-structured barrier. Interestingly, no decreases in TEER values were observed after exposures to 10, 25 and 100 μg/mL, lasting for 24 h. Therefore, no alteration of the monolayer integrity was observed after exposure to CeO2NPs. To evaluate the permeability of the Caco-2 cells monolayer, the Lucifer yellow assay was carried out. Lucifer yellow is a paracellular transport compound that permits to measure tight-junction integrity, and the monolayer's permeability between cells. Fig. 4 shows that there were no significant differences in permeability to LY after exposure to CeO2NPs (10, 25 and 100 μg/mL), when comparing the observed values with those obtained in the untreated control. Taking all together, our results indicate that exposure to CeO2NPs does not alter the integrity/permeability of the monolayer of Caco2 cells, after treatments lasting for 24 h.
3.3. Assessment of the monolayer integrity and permeability NPs can be able to damage the established monolayer, regardless of its ability to internalize or to cross the barrier. To determine potential effects of CeO2NPs on the integrity of this differentiated Caco-2 monolayer, TEER and LY assays were carried out. As indicated in Fig. 3A, TEER values were measured during differentiation, and 24 h after the treatment with CeO2NPs (Fig. 3B). The obtained results indicate that TEER values progressively increase during differentiation of Caco-2 cells, reaching a value of 475.28 ± 45.97 Ω/cm2 at day 21. This
3.4. Cellular uptake of CeO2NPs Confocal microscopy offers a good alternative for the qualitative observation of metallic NPs internalization. This must be considered as a good complement to the information obtained by TEM, although it does not allow us to distinguish between individual NPs and NPs aggregates. This methodology was used for both differentiated and undifferentiated cells; that is, cells growing in transwells or in culture flasks. Since the aim was not quantifying the uptake, the highest concentration was used (100 μg/mL) to facilitate the observation of CeO2NPs, if they uptake. As shown in Fig. 5, the cell membrane was stained in red, nucleus in blue, and the CeO2NPs, although reflecting the polarized light, were
Fig. 2. Cell viability of undifferentiated (continuous line) and differentiated (discontinuous line) Caco-2 cells treated with CeO2NPs for 24 h in a range from 0 to 200 μg/ mL. Data are presented as a percentage of viable cells with respect to controls. Results were analyzed with an unpaired Student's t-test Data represented as mean ± SEM.
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practically non-existent. 3.5. Translocation of CeO2NPs through Caco-2 cells monolayer To determine if CeO2NPs were able to cross the barrier, different techniques including TEM + EDX, ICP-MS, and confocal microscopy were used to determine their presence in the basolateral medium. The obtained results are shown in Fig. 6. When the basolateral medium was analyzed by TEM-EDX a large amount of organic matter and mineral salts was observed. This, potentially, mask NPs avoiding their observation (Fig. 6A). Nevertheless, no cerium was observed in the basolateral medium after using EDX analysis (Fig. 6B). In this case, only the highest concentration (100 μg/ mL) was tested. In addition, potential translocation of CeO2NPs was chemically analyzed by ICP-MS on the apical and basolateral medium. It must be remembered that ICP-MS technique does not distinguish between ionic and NPs form, this method only detects the element studied. Our results indicate that no significant increases in Ce were observed in the basolateral medium (Fig. 6C), since Ce values were below the detection limit of the technique (0.01 μg). To test the methodology, the apical medium was also evaluated. In this case, the absolute amount (μg) of Ce in the apical chamber was elevated observing a concentration-related effect (Fig. 6D). In this case, the effects of three concentrations (10, 25 and 100 μg/mL) were evaluated. Finally, taking advantage of the ability of metal NPs to reflect polarized light, the basolateral medium was also analyzed by confocal microscopy. As observed, despite the previous reported negative results, confocal microscopy showed green spots corresponding to CeO2NPs in the basolateral medium after exposure (Fig. 6F). No signal was detected in the basolateral medium of the untreated cells (Fig. 6E).
Fig. 4. Percentage of LY in the basolateral (BL) chamber after treatment with CeO2NPs during 24 h. Data represented as mean ± SEM. Results were analyzed according to a oneway ANOVA with a Tukey post-test.
observed in green. This technique, by using a specific software, allows covering nuclei and NPs with a mask that allows locating structures in a three-dimensional space (Fig. 5 B,D). The results show that the undifferentiated cells present a much lower thickness, because they are not polarized, and do not express microvilli in the apical part. In these cells, CeO2NPs were able to internalize within the cell, reaching the nucleus (Fig. 5A and B). Nevertheless, in the differentiated state, practically all CeO2NPs were found attached to the apical membrane (Fig. 5C and D), and uptake was
Fig. 5. Confocal images from undifferentiated (A, B) and differentiated (C, D) Caco-2 cells, treated with 100 μg/mL of CeO2NPs. Cellular uptake was measured after 24 h of treatment. Nucleuses are stained in blue, cell membranes in red, and CeO2NPs in green. Arrows indicate the localization of CeO2NPs in cells. In A and B lateral and bottom boxes correspond to cut views. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
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Fig. 6. Translocation studies of CeO2NPs through Caco-2 monolayer. The basolateral extract was analyzed after 24 h of CeO2NPs exposure by using TEM: A (image) and B (TEM + EDX); by using ICP-MS: C (μg of Ce in the basolateral chamber) and D (μg of Ce in apical chamber); and by using confocal microscopy: E (control), and F (CeO2NPs). Results are represented as mean ± SEM and were analyzed according to a one-way ANOVA with a Tukey post-test.
observed in Fig. 7 no increases in DNA breaks were detected after our exposure conditions (A). Nevertheless, high and significant increases in the percentage of DNA in tail were observed when MMS (0.5 mM) was used as a positive control, supporting the suitability of the assay. In addition, no oxidative DNA damage was observed when FPG enzyme was used (Fig. 7B). It should be remembered that FPG enzyme detects oxidized bases and induces single-strand breaks after their excision. This means that no significant induction of oxidized bases was produced after 24 h of exposure (Fig. 7B), but the contrary since lower levels of oxidative damage were detected, suggesting an antioxidant effect. High and significant effects were observed after potassium bromate treatment, used as a positive pro-oxidant agent.
This would confirm the sensitivity of the technique showing that, in spite of the low uptake, some CeO2NPs were able to cross the monolayer. 3.6. Genotoxic and oxidative DNA damage induction Among the different potential harmful effects induced by NPs exposure, DNA damage is of crucial relevance. To assess general genotoxic damage resulting from DNA breaks, and to evaluate the oxidative damage induced on DNA bases, the alkaline comet assay, supplemented with FPG, was carried out. The effects of three concentrations (10, 25 and 100 μg/mL) were assayed in exposures lasting for 24 h. The percentage of DNA in tail was used as the biomarker of genotoxic effect. As 43
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Fig. 7. Analysis of genotoxic (A) and oxidative DNA damage (B) after 24 h of CeO2NPs exposure using the comet assay. Three drops per replicate were used per concentration. Results were analyzed according to a one-way ANOVA with a Tukey post-test. Data represented as mean ± SEM. *P < 0.05; ***P < 0.001.
4. Discussion
junctions, studies on the stability of this barrier are needed. In our Caco2 model, TEER values for the stabilised barrier were 475.28 ± 45.97 Ω/cm2 at the end of 21 days of differentiation. This value is similar to the TEER values reported by different authors [24] what means that our model of barrier was well constituted. Furthermore, permeability of the Caco-2 monolayer was evaluated by measuring the passive crossing of small hydrophilic molecules, as LY, through the paracellular space [33]. Its passage is easily detectable and provides a sensitive and reliable technique to confirm the integrity of the barrier [34]. In our study, 24 h of incubation with CeO2NPs did not alter the integrity or the permeability of the intestinal epithelium, regardless of the concentration. These negative findings agree with those obtained by other members of the NANoREG consortium [35]; this means that CeO2NPs exposure (under our experimental conditions) do not disrupt our model of Caco-2 monolayer. In addition to the studies on the potential alteration of the integrity of the barrier, it is also important to investigate whether NMs are able to internalize into Caco-2 monolayer or, even, if they are able to cross it. Confocal microscopy is a technique that allows the detection of metallic NMs such as cerium, because of their ability to reflect polarized light, although it does not distinguish between aggregates and individual NMs [36]. According to that, by using confocal microscopy as a new approach, we have been able to demonstrate that CeO2NPs strongly internalize in undifferentiated Caco-2 cells but few in differentiated Caco-2 cells. It is important to emphasize that this methodology permits to distinguish if NMs are located on the surface of the epithelia or if they are internalized, either in the cytoplasm or in the nucleus, due to differential staining between different cellular components (nucleus and plasmatic membrane) [37,38]. In our study, CeO2NPs were observed in both, inside cytoplasm and nucleus of undifferentiated Caco-2 cells. Nevertheless, they were mainly found outside cells, attached to plasmatic membrane, in differentiated Caco2 cells. This difference of internalization could be due to small intestinelike structure reached by differentiated cells. A more complex cell structure and morphology could complicate the penetration of NPs inside cells. Low, but relevant internalization of CeO2NPs inside differentiated Caco-2 cells were observed by confocal microscopy after 24 h of exposure [39], contrasting with our poor observed uptake. This discrepancy can be explained by methodological differences between studies, as Gaiser et al. [39] worked with 14 days-old Caco-2 monolayers and 14.9 ± 7.4 nm CeO2NPs, while we used 21 days-old Caco-2 monolayers and our NPs size were approximately 70.33 nm. Thus, a low
It is largely known that Caco-2 cells have the capability to differentiate spontaneously to enterocytes, when they arrive at confluence. In parallel, they acquire several characteristics associated with the physical and metabolic barrier of the intestinal epithelium such as cell polarity, formation of microvilli, presence of tight-junctions, expression of digestive enzymes in the brush border, secretion, and assembly of a matrix [20]. The European Center for the Validation of Alternative Methods (ECVAM) pre-validated the Caco-2 monolayer as a useful model to determine the effects of acute toxicity, and for the assessment of active transport of drugs and chemicals [21,22]; but not for evaluating the passive crossing of NPs. In spite of that, all the indicated characteristics make this model suitable to be used to determine potential translocation through intestinal barrier of ingested nanomaterials [23]. In our study no toxic effects of CeO2NPs in Caco-2 cells were observed, in neither undifferentiated nor differentiated cells. These results are in agreement with those obtained by Fisichella et al. [24] who found that CeO2NPs, did not induce cytotoxic effects on undifferentiated Caco-2 cells after 24 h or 72 h exposure, even at high concentrations (170 μg/mL). Although not statistically significant, an increase on cell viability was observed after CeO2NPs exposure to both cell states. This result would agree with those describing in vitro stimulation of cell proliferation by exposing primary mouse bone marrow stromal cells (BMSCs) [25], and primary mouse embryonic fibroblast [26] to cerium oxide nanoparticles. Despite the stimulation mechanisms are not totally clears, CeO2NPs could stimulate cell proliferation through reduction of intracellular levels of ROS by modulating the expression level of the major antioxidant enzymes [26]. Nevertheless, toxicity effects of CeO2NPs are controversial due to its reported prooxidant [27,28] or anti-oxidant role [29,30]. In general, it is assumed that the observed differences in toxicity of NPs depend on multiple factors, including: the manufacturer, the composition of the NPs, their shape and size, their ability to form aggregates, the method of preparation, and the dispersion protocol used [31,32]. In addition, the cells used in the study or even their clones, as in the case of the Caco-2 cell line [19], can also modulate the response to toxic agents. Epithelia are formed by cells that are attached to each other by tight-junctions. These junctions guarantee the integrity of the epithelium avoiding the crossing of undesirable compounds. Since the presence of NPs could alter the integrity of the barrier, by acting on tight44
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not suppose an important risk for human beings. Additionally, the resistance to NPs uptake showed by differentiated cells must be taken into consideration in the risk assessment of NPs exposure. In this way, data obtained in undifferentiated cells may overestimate the real risk of such exposures.
differentiated status and a more little NP diameter could enhance and trigger cell uptake. Nevertheless, and in spite of the great uptake of CeO2NPs in undifferentiated cells, no toxic effects were observed. Higher uptake in undifferentiated cells, with regard to differentiated cells, has also been found for TiO2NPs exposures [40]. Regarding these differences in cell uptake, and in order to avoid inconsistent results, we strongly suggest working with more physiologically realistic in vitro models as Caco-2 monolayers represent. Despite the apparent low internalization of CeO2NPs inside differentiated Caco-2 cells, NPs translocation was observed through the monolayer. Different techniques were used (EDX coupled to TEM, confocal microscopy, and ICP–MS) in order to detect NPs in the basolateral medium. Negative results were obtained by TEM-EDX and ICPMS, but confocal microscopy showed green spots in basolateral medium corresponding to CeO2NPs. These spots were not detected in the control (non-treated Caco-2 monolayer). This finding lead us to hypothesize that the main crossing route of CeO2NPs is intracellularly and that such NPs are readily uptake by the cells, remaining less than 24 h into the cytoplasm, as it was barely impossible to detect them into the cells. This result is surprisingly, due to the accepted high sensitivity of the ICP-MS methodology. Possibly the reflection of CeO2NPs made that, in spite of its low amount present in the basolateral chamber, the confocal microscopy is able to visualize them. Altogether, these results indicate the usefulness of confocal microscopy to detect metallic NPs like CeO2NPs. Moreover, more effort need to be done to further and exactly clarify the crossing mechanisms that CeO2NPs use to translocate through epithelial barriers. For instance, it could be interesting to use several time points enough close among them to specifically locate the CeO2NPs. To check changes at the transcriptional and translational level of tight-junction markers could be a useful tool to further demonstrate that the barrier integrity is not altered. At this point it should be indicated that TiO2NPs exposures up regulated the gene expression of impaired cell junction in others in vitro intestinal models, as a response to actively repair adverse effects in the barrier integrity [41]. The induction of oxidative stress is one of the common mechanisms of toxicity mediated by NMs, through reactive oxygen species (ROS) formation [42–44]. Nevertheless, the chemical composition of NMs, rather than size, seems to be the most determinant factor influencing ROS formation in exposed cells [45], which can damage DNA. In the present study, neither genotoxic nor oxidative DNA damage were produced by CeO2NPs in differentiated Caco-2. These negative results agree with other studies carried out by our group in BEAS-2B cells, where no increases in the levels of ROS were found after CeO2NPs exposure [46]. Such results would manifest the unique antioxidant properties of CeO2NPs, since its autoregenerative redox cycle can protect cells and tissues from oxidative stress induced damage [5,47]. Antioxidant properties of PEGylated CeO2NPs in HaCaT cells were reported showing that CeO2NPs were able to protect the cells from damage caused by the ROS resulting from the loss of cellular GSH production. Furthermore, exposure to PEGylated CeO2NPs did not induce any DNA damage; in fact, such pre-treated cells exhibited protection against nuclear fragmentation and micronucleus formation [48]. All this data would reinforce the view that CeO2NPs are not genotoxic, at least when the comet assay is used. Summing up, we can conclude that CeO2NPs are not toxic in Caco2 cells, independently of their state of differentiation. Furthermore, CeO2NPs exposure is not able to alter monolayer's integrity neither tight-junctions and, finally, CeO2NPs are not able to produce neither genotoxic nor oxidative DNA damage, as demonstrated by using the comet assay complemented with FPG enzyme. Although undifferentiated cells are able to uptake important amounts of CeO2NPs, differentiated cells present very low NP's internalization remaining mostly attached in the apical plasmatic membrane. In spite of this apparent low uptake in differentiated Caco-2 cells, some CeO2NPs are able to cross the monolayer, at least as visualized by confocal microscopy. All this together would reinforce the view that CeO2NPs exposure does
Conflicts of interest The authors report no conflict of interest. The authors alone are responsible for the content and writing of the paper. Acknowledgements This investigation has been partially supported by the Ministry of Economy and Competition (SAF2015-63519-R), and the EC-FP7NANoREG (Grant Agreement NMP4-LA-2013-310584). A. GarciaRodriguez and L. Vila were funded by postgraduate fellowships from the Universitat Autònoma de Barcelona and the Generalitat de Catalunya, respectively. Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.cbi.2018.01.018. References [1] G. Oberdörster, E. Oberdörster, J. Oberdörster, Nanotoxicology: an emerging discipline evolving from studies of ultrafine particles, Environ. Health Perspect. 113 (2005) 823–839. [2] H. Johnston, G. Pojana, S. Zuin, N.R. Jacobsen, P. Møller, S. Loft, M. SemmlerBehnke, C. McGuiness, D. Balharry, A. Marcomini, H. Wallin, W. Kreyling, K. Donaldson, L. Tran, V. Stone, Engineered nanomaterial risk. Lessons learnt from completed nanotoxicology studies: potential solutions to current and future challenges, Crit. Rev. Toxicol. 43 (2013) 1–20. [3] S. Das, J.M. Dowding, K.E. Klump, J.F. McGinnis, W. Self, S. Seal, Cerium oxide nanoparticles: applications and prospects in nanomedicine, Nanomedicine 8 (2013) 1483–1508. [4] C. Xu, X. Qu, Cerium oxide nanoparticle: a remarkably versatile rare earth nanomaterial for biological applications, NPG Asia Mater. 6 (2014) 1–16. [5] L. Rubio, B. Annangi, L. Vila, A. Hernández, R. Marcos, Antioxidant and anti-genotoxic properties of cerium oxide nanoparticles in a pulmonary-like cell system, Arch. Toxicol. 90 (2016) 269–278. [6] P.H. Hoet, I. Brüske-Hohlfeld, O.V. Salata, Nanoparticles -known and unknown health risks, J. Nanobiotechnol. 2 (2004) 12. [7] A. Kermanizadeh, D. Balharry, H. Wallin, S. Loft, P. Møller, Nanomaterial translocation–the biokinetics, tissue accumulation, toxicity and fate of materials in secondary organs–a review, Crit. Rev. Toxicol. 45 (2015) 837–872. [8] B. Srinivasan, A.R. Kolli, M.B. Esch, H.E. Abaci, M.L. Shuler, J.J. Hickman, TEER measurement techniques for in vitro barrier model systems, J. Lab. Autom. 20 (2015) 107–126. [9] C. Hilgendorf, G. Ahlin, A. Seithel, P. Artursson, A.L. Ungell, J. Karlsson, Expression of thirty-six drug transporter genes in human intestine, liver, kidney, and organotypic cell lines, Drug Metab. Dispos. 35 (2007) 1333–1340. [10] D.A. Volpe, Drug-permeability and transporter assays in Caco-2 and MDCK cell lines, Future Med. Chem. 3 (2011) 2063–2077. [11] S. Yamashita, T. Furubayashi, M. Kataoka, T. Sakane, H. Sezaki, H. Tokuda, Optimized conditions for prediction of intestinal drug permeability using Caco-2 cells, Eur. J. Pharm. Sci. 10 (2000) 195–204. [12] P. Artursson, K. Palm, K. Luthman, Caco-2 monolayers in experimental and theoretical predictions of drug transport, Adv. Drug Deliv. Rev. 46 (2001) 27–43. [13] A. Thubagere, B.M. Reinhard, Nanoparticle-induced apoptosis propagates through hydrogen-peroxide-mediated bystander killing: insights from a human intestinal epithelium in vitro model, ACS Nano 4 (2010) 3611–3622. [14] P. Ilina, S. Partti, J. Niklander, M. Ruponen, Y.R. Lou, M. Yliperttula, Effect of differentiation on endocytic profiles of endothelial and epithelial cell culture models, Exp. Cell Res. 332 (2015) 89–101. [15] Z.M. Song, N. Chen, J.H. Liu, H. Tang, X. Deng, W.S. Xi, K. Han, A. Cao, Y. Liu, H. Wang, Biological effect of food additive titanium dioxide nanoparticles on intestine: an in vitro study, J. Appl. Toxicol. 35 (2015) 1169–1178. [16] C. Singh, S. Friedrichs, G. Ceccone, N. Gibson, K.A. Jensen, M. Levin, H.G. Infante, D. Carlander, K. Rasmussen, Cerium Dioxide, NM-211, NM-212, NM-213. Characterization and Test Item Preparation, (2014) http://publications.jrc.ec. europa.eu/repository/bitstream/JRC89825/lbna26649enn.pdf. [17] Nanogenotox, (2011) http://www.nanogenotox.eu/files/PDF/Deliverables/ nanogeno tox%20deliverable%203_wp4_%20dispersion%20protocol.pdf. [18] B. Annangi, L. Rubio, M. Alaraby, J. Bach, R. Marcos, A. Hernández, Acute and
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