http://informahealthcare.com/nanISSN: 1743-5390 (print), 1743-5404 (electronic)

Nanotoxicology, Early Online: 1–11! 2014 Informa UK Ltd. DOI: 10.3109/17435390.2014.957252

ORIGINAL ARTICLE

Long-term exposures to low doses of titanium dioxide nanoparticlesinduce cell transformation, but not genotoxic damage in BEAS-2B cells

Gerard Vales1, Laura Rubio1, and Ricard Marcos1,2

1Grup de Mutagenesi, Departament de Genetica i de Microbiologia, Facultat de Biociencies, Universitat Autonoma de Barcelona, Bellaterra, Spain,

and 2CIBER Epidemiologıa y Salud Publica, ISCIII, Madrid, Spain

Abstract

There is a great interest in a better knowledge of the health effects caused by nanomaterialsexposures and, in particular to those induced by titanium dioxide nanoparticles (nano-TiO2) dueto its high use and increasing presence in the environment. To add new information on itspotential genotoxic/carcinogenic risk, we have carried out experiments using chronic exposures(up to 4 weeks), low doses, and the BEAS-2B cell line that, as a human bronchial epitheliumcells, can be considered a good cell target. Cell uptake has been assessed by transmissionelectron microscopy (TEM) and flow cytometry (FC); genotoxicity was evaluated using thecomet and the micronucleus (MN) assays; and cell-transforming ability was evaluated usingthe soft-agar assay to detect anchorage-independent cell growth. Results show an importantcell uptake at all the tested doses and sampling times used (except for 1 mg/mL and 24-hexposure). Nevertheless, no genotoxic effects were observed in the comet and in the MNassays. This lack of genotoxic effect agrees with the FC results showing no induction ofintracellular reactive oxygen species (ROS), the data from the comet assay with formamidopyr-imidine DNA glycosylase (FPG) enzyme showing no induction of oxidized bases, and the lackof induction of expression of heme-oxygenase (HO-1) gene both at the RNA and protein level.On the contrary, significant increases in the number of clones growing in an anchorage-independent way were observed. This study would indicate a potential carcinogenic riskassociated to nano-TiO2 exposure, not mediated by a genotoxic mechanism.

Keywords

Cell transformation, chronic exposure, comet,micronucleus, Titanium dioxide

History

Received 24 January 2014Revised 8 July 2014Accepted 8 August 2014Published online 19 September 2014

Introduction

Titanium dioxide nanoparticles (nano-TiO2) are widely used inindustrial and consumer products mainly due to their strongercatalytic activity, when compared with the traditional fineparticles. As indicated by the growing number of studies recentlypublished, there is a high interest to demonstrate the possiblechallenges that its exposure can suppose in terms of human health.In this context, a recent and interesting revision (Shi et al., 2013)point out many of the topics of interest of such approaches.

From the human health risk point of view, there are twoaspects that should be remarked. The first is the genotoxicpotential and the second is the carcinogenic risk that suchexposure can pose. No genotoxic effects have been recentlyreported after in-vivo exposures to nano-TiO2 (Xu et al., 2013),which would confirm other previous studies (Lindberg et al.,2012; Liu et al., 2006). Nevertheless, more conflictive arethe in vitro results where both positive (Ghosh et al., 2013;Kang et al., 2008; Prasad et al., 2013) and negative results(Bhattacharya et al., 2009; Hamzeh & Sunahara 2013) have beenreported. Perhaps, this would mean that there are differentmethodological approaches (protocols, dispersion media, cell

lines, methodologies, etc) that should be put under discussion aspossible causes inducing such conflictive data.

From our point of view, one of the weak points of most of thein vitro experimental data generated in the open literature, for bothnanomaterials and other chemicals, is that standard exposureconditions (short-term exposures to high doses) are far away fromthe expected human exposure situations. As consequence, theobtained results are conflictive in terms of future use in studiesof risk assessment. In spite of the methodological complexity ofthe chronic exposure in in-vitro approaches, some groups aremoving to such protocols with important success. Thus, in thestudy of Jacobsen et al. (2007) carbon black induced significantmutagenic effects in the FE1 Muta� Mouse lung epithelial cell lineafter eight repeated 72 h incubations. On the other hand, Huanget al. (2009) after fibroblasts exposure to nano-TiO2 for up12 weeks were able to demonstrate the induction of disturbance inthe cell cycle progression and duplicated genome segregation,leading to chromosomal instability and cell transformation. Long-term studies lasting for 3 months were not able to identifyimportant changes after nano-TiO2 exposure (with the exception ofthe apparition of nanotubular structures in the apical surface,contrarily what observed with the nano-ZnO exposures, wherechanges in cell morphology, loss of mitochondrial activity, andchanges in cell-cycle distribution were observed with increasedsubG1 phase (Kocbek et al., 2010)). Finally, in long-term exposureexperiments (3 weeks) using human mesenchymal stem cells(hMSC), no cytotoxic effects were observed although the presenceof nano-TiO2 persisted in the cytoplasm (Hackenberg et al., 2013).

Correspondence: Ricard Marcos, Grup de Mutagenesi, Departament deGenetica i de Microbiologia, Universitat Autonoma de Barcelona, EdificiCn, Campus de Bellaterra, 08193 Cerdanyola del Valles, Bellaterra,Spain. E-mail: ricard.marcos@uab.es

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One important advantage of using long-term exposures is thatcell dedifferentiation processes can also be determined, giving tosuch type of studies an extra value over the standard short-termassays, where genotoxicity is always the surrogate marker oftumorigenic processes. This approach is able to determine theexistence of non-genotoxic carcinogenic processes linked to thenanomaterial under study. In-vitro cell transformation assayshave been classically defined as the induction of certainphenotypic alterations leading to characteristics like tumorigeniccells (Barrett & Ts’o, 1978). Thus, alterations in cell morphology,disorganized patterns of colony formation and acquisitionof anchorage-independent growth are distinctive hallmarks ofsuch type of processes.

So far there are not specific epidemiological studies lookingspecifically for nano-TiO2 carcinogenicity on humans and, fromanimal data, the available studies are not adequate enough toclarify the possible mechanisms of action (Arora et al., 2012;Becker et al., 2011). Although some information exist indicatingthat nano-TiO2 may induce lung carcinogenesis involving M1P1aderived from alveolar macrophages (Xu et al., 2010), nopromoting effects have been observed in skin carcinogenesis inrats suggesting that probably nano-TiO2 cannot penetrate throughepidermis to reach underlying skin structures (Xu et al., 2011).

According to the importance of the potential risk of nano-TiO2

exposure on the respiratory system, we have chosen BEAS-2B asa cell model to evaluate their genotoxic and cell-transformingpotential following a 4 weeks long chronic exposure. Such chronicexposures can be useful to draw plausible conclusions aboutthe potential human health risk of NPs exposures (Hristozov et al.,2012). Cell uptake was assessed by TEM and flow cytometry,genotoxicity was evaluated by the comet and the micronucleus(MN) assay and cell transformation was measured according tothe induced ability of growing in a soft-agar matrix by promotinganchorage-independent growth.

Materials and methods

Cell cultures

BEAS-2B cells were provided by Dr. H. Norppa (Finnish Instituteof Occupational Health) and were grown as a monolayer intissue culture dishes with bronchial epithelial cell growthmedium (BEGM, Lonza, CA). Cultures were incubated at 37 �Cin a humidified atmosphere of 5% CO2.

Nanoparticles characterization, dispersion and cellexposure

Nano-TiO2 (NM102) was obtained from the Joint Research Center(Ispra, Italy). It is in anatase phase and the reported primaryparticle size was 21.7 ± 0.6 nm. For further characterization,transmission electron microscopy (TEM) was utilized to obtainthe NPs size and morphology on a JEOL JEM-2011 instrument.Furthermore, characterization of hydrodynamic size and zetapotential of nano-TiO2 dispersed in cell culture medium bydynamic light scattering (DLS) and laser Doppler velocimetry(LDV) methodologies, respectively, was performed on a MalvernZetasizer Nano-ZS zen3600 instrument.

For cell treatments, nano-TiO2 were pre-wetted in 0.5%absolute ethanol and afterwards dispersed in 0.05% bovineserum albumin (BSA) in MilliQ water, the nanoparticles in thedispersion medium were sonicated for 16 min to obtain a stockdispersion of 2.56 mg/mL according to the Nanogenotox protocol(Nanogenotox, 2011). BEAS-2B cells were exposed to theselected doses (1, 10 and 20mg/mL) up to 4 weeks. Cells werewashed twice with PBS, and fresh medium with the differentconcentrations of nano-TiO2 were added every 4 d and

sub-cultured weekly. Nano-TiO2 were newly prepared for eachsub-culture medium. No apparent agglomerations were observedduring the 4 d of sub-culturing. To determine the effects causedby the chronic exposure, cells were collected at various timeintervals depending on the cellular assay.

Cellular uptake measurement by transmission electronmicroscopy

BEAS-2B cells (exposed and unexposed) were fixed in 2.5% (v/v)glutaraldehyde (EM grade, Merck, Darmstadt, Germany) and 2%(w/v) paraformaldehyde (EMS, Hatfield, PA) in 0.1 M cacodylatebuffer (PB, Sigma-Aldrich, Steinheim, Germany), pH 7.4 andprocessed following conventional procedures, as described byRodrıguez-Carino et al. (2011). Samples were first post-fixed withosmium, dehydrated in acetone, later embedded in Epon, andfinally polymerized at 60 �C and cut with an ultramicrotome.Ultrathin sections were placed in copper grids and contrasted withuranyl acetate and Reynolds lead citrate solutions and thenobserved using a Jeol 1400 (Jeol LTD, Tokyo, Japan) TEMequipped with a CCD GATAN ES1000W Erlangshen camera(Mussa et al., 2012).

Intracellular ROS measurement

The intracellular generation of reactive oxygen species (ROS)was measured by flow cytometry (FC) after 24 h and 1 week ofnano-TiO2 exposure using the 6-carboxy-2,70- dichlorodihydro-fluorescein diacetate (DCFH-DA) assay (Toduka et al., 2012).Long-term treated cells (1 week treatment), were seeded intriplicate in 6 well plates at a density of 1� 105 cells/well. After48 h, cells were washed twice with PBS and incubated in 5-mMDCFH-DA in serum-free DMEM:F12 medium for 30 min at37 �C. Hydrogen peroxide (0.1 and 0.5 mM) was used as apositive control and added 15 min before trypsinization andcollection of the cells. The conversion of non-fluorescent DCFH-DA to DCF by action of cellular esterases and its posterioroxidation to its fluorescent form by the presence of intracellularROS was measured by Fluorescence-activated cell sorting (FACSCalibur) as mean of fluorescence intensity. The data wereanalyzed with Flowjo Ver. 7.6.5.

Total RNA extraction and real-time RT-PCR

The expression of IL-1�, IL-6, IL-8 and HO-1 were determinedafter 3 weeks of continuous exposure by real-time RT-PCR. TotalRNA was extracted using TRIzol Reagent (Invitrogen, Carlsbad,CA), and RNase-free DNase I (DNA-free� kit; Ambion, Austin,TX) was applied to remove DNA contamination. The first-strandcDNA synthesis was performed using 1 mg of total RNA and theOmniscript Reverse Transcription Kit (Qiagen, Valencia, CA)following the manufacturer instructions. The obtained cDNA wassubjected to real-time RT-PCR on a Lightcycler-480 using DFS-TaqDNA Polymerase (Bioron, Ludwigshafen, Germany) toqualitatively evaluate the expression of IL-1�, IL-6, IL-8 andHO-1 genes. Each 25mL of reaction volume contained 0.5 mL ofcDNA, 3 U of DSF-Taq DNA polymerase, 2.5 mL of 10�DSF-Taq Buffer, 1.5 mM MgCl2 (Bioron, Ludwigshafen, Germany) and200 nM of each primer pairs. The cycling parameters began with95�C for 5 min, then 45 cycles of 95 �C for 30 s, 61 �C for 30 s and72 �C for 45 s. The primer pairs used were: IL-1� fwd 50-AAACAGATGAAGTGCTCCTTCCA-30, IL-1� rev 50-GAGAACACCACTTGTTGCTCCA-30, IL-6 fwd 50-CCAGGAGCCCAGCTATGAACT-30, IL-6 rev 50-CCCCAGGGAGAAGGCAA-30, IL-8 fwd50-CTG GCC GTG GCT CTC TTG-30, IL-8rev50-CCTTGGCAAAACTGCACCTT-30, HO-1 fwd 50-CTCAAACCTCCAAAAGCC-30, HO-1 rev 50-TCAAAAACCACCCCAACCC-30.

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Total protein extraction and Western blot analysis

Additionally, the expression of HO-1 was also determined byWestern blot in the same time-point. RIPA lysis buffer was used tohomogenize the BEAS-2B samples. About 50 mg of protein samplewas run on a 10% SDS–PAGE gel and transferred to PVDFmembranes. After blocking the non-specific binding sites for 3 hwith 5% non-fat milk or 5% BSA, the membranes were incubatedovernight at 4 �C with primary monoclonal antibodies againsthemooxigenase-1 (Abcam, 1:2000 dilution). The membranes werewashed thrice with TBST during 15 min and afterwards incubatedwith HRP-conjugated secondary antibody (Santacruz, at a 1:2000dilution) for 1 h at room temperature. The membranes were thensubjected to three washes with TBST and visualized using anenhanced chemiluminescence system (ECL, Cell SignallingTechnologies, Danvers, MA). A relative quantification of proteinexpression was assessed using genome tools.

Comet assay

The induction of genotoxic and oxidative DNA damage toBEAS-2B cells chronically exposed to sub-toxic doses of nano-TiO2 were evaluated by using the alkaline comet assay with andwithout the addition of formamidopyrimidine DNA glycosylase(FPG) enzyme. The assay was performed after 24 h, 1 and 3weeks of the exposure. To process multiple samples at the sametime, hydrophilic Gelbond sheets were used (McNamee et al.,2000). Cells were first washed with ice-cold 0.2% ethylenedia-minetetraacetic acid (EDTA) in PBS, and then trypsinized andcentrifuged at 130� g for 8 min. The pellet was washed twice inRPMI medium and re-suspended in PBS with the objective ofobtaining about 17 500 cells/25 mL. Later, the cells were mixedwith 0.75% LMP at 37 �C (1:10) and dropped onto Gelbond film(10.5� 7.5 cm). Sixteen samples were analyzed per Gelbond,each sample represented by three drops (7 mL per drop), for atotal of 48 drops. Six identical Gelbond films with the samecomposition of samples were processed simultaneously in eachexperiment. Cells on Gelbond were lysed in ice-cold lysis bufferat 4 �C (2.5 M NaCl, 0.1 M Na2EDTA, 0.1 M Tris base, 1%Triton X-100, 1% lauryl sarcosinate, 10% DMSO), and pH 10overnight. Afterwards, Gelbonds with cells were washed twice(1� 5 min and 1� 50 min) in enzyme buffer at pH 8.0 (10 mMHEPES; 0.1 M KCl; 0.5 mM EDTA; 0.2 mg/mL BSA) at 4 �Cfollowed by 30 min incubation at 37 �C in only enzyme buffer(negative control), or in enzyme buffer containing FPG. Gelbondfilms were then incubated with electrophoresis buffer (0.3 MNaOH and 1 mM Na2EDTA, pH¼ 13.2) for 5 min. Afterrenewing the buffer, the Gelbonds were kept in it for 25 minto allow DNA unwinding and expression of alkali labile sites.Electrophoresis was performed during 20 min at 0.8 V/cm and300 mA at 4 �C. Afterwards, Gelbond films were rinsed in coldPBS for 15 min, then fixed in absolute ethanol for at least 2 h,air-dried overnight at room temperature, and stained for 20 minwith SYBR Gold (Molecular Probes; 495 nm excitation, 537 nmemission), 1/10 000 dilution of stock solution in TE buffer(10 mM Tris; 1 mM EDTA pH 7.5). Each Gelbond was cut intotwo same sized parts and mounted on an acrylic slide of52.5� 75� 3 mm. A coverslip of 74� 49 mm (IZASA,Barcelona, Spain) was applied on top of the drops effectivelysealing the samples. As a final step, the gels were observedusing an epifluorescent microscope at� 20 magnification inthe search of comets. The quantification of DNA damage in thecells was measured as the percentage of DNA in tail by theKomet 5.5 Image analysis system (Kinetic Imaging Ltd,Liverpool, UK). One hundred randomly selected comet imageswere analyzed per sample in each Gelbond. As a positivecontrol, 0.5 mM ethyl methane sulfonate was used.

Micronucleus assay

The cytokinesis-blocked micronucleus assay was performedusing the standard technique (Fenech, 2007), by adding Cyt-B(6 mg/mL). The assay was performed after 48 h, 1 and 3 weeks ofthe exposure. A total of 3 mL of the BEAS-2B cultures (500 000cells/mL) were set up in complete medium. Aliquots of 30mLnano-TiO2 were added to the cultures in order to reach the desiredconcentrations. All treatments were added at the beginning of theincubation, and cultures were kept for 48 h at 37�(C in a 5% CO2

atmosphere. Mitomycin C (MMC, 150 ng/mL) was used aspositive control. For each concentration, two replicates weremade. After incubation, cells were harvested, cultures werecentrifuged at 150� g for 8 min; then, the supernatant wasremoved and the cells were subjected to hypotonic treatment(5 mL KCl 0.075 M, 4 �C 7 min), and another centrifugation wascarried out. Cells were fixed with methanol/acetic acid (3:1 vol) atleast three times. In the last centrifugation, the supernatant waseliminated and the pellet was re-suspended and dropped ontoclean microscope slides (two drops of 20 mL each one).After drying, cells were stained with acridine orange bysubmerging the slides in a 1/30 dilution of acridine orangestock solution (0.1% w/v in distilled water) in buffer Sorensen for1 min. Afterwards, the slides are rinsed in Sorensen buffer3� 3 min and air dried in the dark. Then 1–2 drops of DAPIsolution (5 mg/mL DAPI in 2�SCC) are placed on the slides,covered with coverslips and incubated for 5 min. After rinsingand air dry in the dark, the slides were rehydrated with distilledwater and observed under an epifluorescent microscope at100�magnification.

All slides were coded before scoring, which was carried out bytwo persons, one per sub-culture. One thousand binucleated cellsper sub-culture were scored and classified, according to theirnumber of MN, to calculate the induction of MN. In addition, 500cells were scored to calculate the cytokinesis-block proliferationindex (CBPI) (Surralles et al., 1995). The induction of MN andthe calculation of CBPI were done for each replicate and thereported values correspond to the pooled data.

Soft-agar colony formation assay

A soft-agar assay (anchorage-independent colony formationassay) was performed after 4 weeks of continuous exposure to1, 10 or 20 mg/mL of nano-TiO2. Cells were trypsinized, thenwashed twice with PBS and passed through 30-mm filters toobtain single cell suspensions. Afterwards, 5� 103 cells/35-mmplate were mixed with 0.5 mL of 1�DMEM at 37 �C. Thismixture was added in 1:1 ratio of 1.2% bacto-agar (Difco) andBEGM gently layered onto precast 0.6% base agar medium platesin triplicate (equal amounts of 1.2% bacto-agar with 2�DMEMwere added). After 14 d of incubation, plates were stained with0.1% INT solution (2-p-iodophenyl-3-p-nitrophenyl-5-phenyltetrazolium chloride; Sigma Aldrich, St Louis, MO) and scannedat high resolution for image analysis using NIST’s IntegratedColony Enumerator (NICE) software (NIST, Boulder, CO).Colonies were classified as small (5200mm) or medium large(4200mm) sized. Three independent soft-agar assays wereperformed. No positive controls were run in parallel and thestatistical significance was assayed according the observed dose–response relationship.

Statistical analysis

For the Comet assay, an analysis of variance (ANOVA) wasperformed after a logarithmic transformation was applied to thereplicates of each sample. Tukey HSD test was used for the post-hoc analysis. ANOVA with Tukey HSD post-hoc test was also

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applied for Western blot and soft-agar assay. Finally, in themicronucleus assay, a two-sided Fisher test in a 2� 2 contingencytable was applied, comparing presence and absence of micro-nuclei in binucleated cells.

Results

Characterization of nano-TiO2

Although it was well characterized in the frame of theNanogenotox project (Nanogenotox, 2012), we have to proceedfor its characterization under our experimental conditions. Arepresentative TEM image of nano-TiO2 is showed in Figure 1(A)and the mean size distribution (Figure 1B), report values of20.99 ± 6.4 nm diameter calculated by measuring over 100particles in random fields of view. The average hydrodynamicradius and zeta potential of nano-TiO2 in culture medium were575.9 ± 8 nm and �19.5 ± 0.5 mV, respectively (Figure 1C). The

size of nanoTiO2 measured with TEM matched well with the dataobtained in the Nanogenotox characterization (21.7 ± 0.6 nm),DLS data revealed some aggregation or agglomeration ofnanoTiO2 in suspension.

Cellular uptake by TEM

To confirm the uptake of nano-TiO2 by BEAS-2B cells, under ourexperimental conditions, we carried out TEM analysis of cellsexposed under different time and dose conditions. Figure 2 showssome examples of TEM images from cells exposed to 20 mg/mL,and showing the internalization of nano-TiO2 already after short-time exposures (24 h, Figure 2A). Dose- and time-dependentcellular uptake was observed in BEAS-2B cells (result notshown). Nano-TiO2 both as nanoparticles and nano-aggregateswere mainly confined to vacuoles, although they were also presenton the surface of the nuclear membrane.

(A)

(C)

Averagediameter

(nm)

DLS LDV

PDI

Freq

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Zetapotential ζ

(mV)

0

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4

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8 10111213141516171819202122232425262728293032353648

Electrophoreticmobility

(µm cm V−1 s−1)

−1.53 ± 0.04575.9 ± 8 0.471 ± 0.021 −19.5 ± 0.5

Nano-TiO2 size (nm)

Viscosity(cP)

0.88 7

pH

Figure 1. (A) TEM image of nano-TiO2 in dry form. (B) Size distribution of nano-TiO2 over 100 particles. (C) nano-TiO2 average size and charge inexposure medium by pre-wetting with 0.5% volume and steric stabilization using sterile-filtered 0.05% w/v BSA. Data represented as mean ± standarddeviation.

Figure 2. TEM images from BEAS-2B cells treated with nano-TiO2. In this figure, examples of cells treated with 20 mg/mL of nano-TiO2 are presented.Image (A) shows internalization after 24 h treatment (�12 000, scale bar 2mm). Image (B) shows the results obtained after 1 week of treatment (�2500,scale bar 5 mm), while Image (C) presents the results obtained after 3 weeks of treatment (�4000, scale bar 5 mm). Nano-aggregates of nano-TiO2 invacuoles and in nucleus are observed. Arrows indicate some nanoparticles or nano-aggregates.

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Cell uptake and intracellular reactive oxygen speciesproduction by FACS

One important question to be elucidated was if nano-TiO2

exposure was able to generate intracellular ROS. Thus, usingFACS methodologies we determined the conversion of the non-

fluorescent DCFH-DA to fluorescent oxidized DCF in the cell.Our results showed that BEAS-2B cells exposed to different timeand dose conditions did not generate significant increases in thepercentage of intracellular ROS. Moreover the overall results arenot shown. Figure 3 shows (as example) that 20 mg/mL of nano-TiO2 does not induce intracellular ROS after 24 h (Figure 3C) and

Figure 3. Cell internalization of nano-TiO2 over time and percentage of ROS induction obtained by using flow cytometry methods. (A) FACS chartsof non-exposed BEAS-2B cells. (B) Shows the results obtained using H2O2 as a positive control. (C) Values observed after 24 h of treatment and(D) After 1 week of treatment with 20mg/mL of nano-TiO2 as examples. The upper and lower right-hand quadrant of each plot represents DCFH-DAfluorescence cells due to ROS generation whereas the events shown in upper and lower left-hand quadrant are non-fluorescent cells. (E) Graphicpresentation of the percentages of internalization after 24 h treatment (white columns) or 1 week treatment (gray columns). *p50.05; **p50.01;***p50.001.

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1 week (Figure 3D) of exposure. Our negative results obtainedwith nano-TiO2 exposures contrast with the results obtained in ourpositive control using H2O2 (Figure 3C) where high andsignificant percentages of ROS were obtained.

It is interesting to remark that this FACS methodology alsopermits to determine the uptake of nano-TiO2 by the BEAS-2Bcells as a measure of the scattering of the laser light afterinteracting with the cell (complexity) (Suzuki et al., 2007). In thiscase, Figure 3(E) clearly indicates that the levels of internalizationincrease with exposure conditions (time and dose). These resultswould confirm the data obtained by TEM.

Expression of IL-1b, IL-6, IL-8 and OH-1

The altered mRNA expression of IL-1�, IL-6 and IL-8 genes inBEAS-2B, after chronic exposure to nano-TiO2 during 3 weeks,is presented in Figure 4. Such expression was determined by real-time RT-PCR to demonstrate whether exposure to nano-TiO2

generated a continuous pro-inflammatory response. As observed,no significant increases in the expression were obtained with theexception of the IL-8 gene at the highest dose. Although no effectseems apparent for IL-1�, the expression of IL-6 and IL-8 show adose–response positive pattern, although only with IL-8 theincreased expression attain statistical significance.

To confirm the lack of oxidative stress induction by chronicnano-TiO2 exposure, the expression of the anti-oxidant HO-1marker was measured by determining changes in mRNA andprotein expression. As indicated in Figure 5, treatments withnano-TiO2 reduced the transcription of HO-1 that, although waspartially recovered at the highest doses of exposures, did not reachthe expression observed in the control. When the expression wasevaluated at the level of protein, Western blotting figures showedno changes with respect to control with a tendency to presentlower expression, mainly at the lower doses of exposure. Itmust be pointed out that HO-1 expression was only determinedat one time-point (3 weeks); nevertheless, and although someadaptation could take place, as a confirmatory value the lack of

expression agrees with the other end-points evaluated (DCFH-DAand comet assays).

Genotoxicity

Primary and oxidative DNA damage

The levels of primary and oxidative DNA damage induced bynano-TiO2 were measured over time after chronic and acuteexposures to 1, 10 and 20 mg/mL. No DNA breaks inductions weredetected after 24 h of acute exposure, when the damage wasmeasured as the % of DNA in tail by the alkaline comet assay.Also, when the comet assay was complemented with the use ofFPG enzyme, which detect oxidized bases and induce single-strand breaks after their excision, no significant effects wereobserved, although a tendency towards higher values wasobserved. Similarly, the follow-up study carried out after 1 and3 weeks of chronic exposure did not induce significant increasesin the levels of primary and oxidative DNA damage (Figure 6Band C). This lack of ability of nano-TiO2 to induce DNA oxidativedamage would confirm their lack of ability to induce intracellularROS, as observed in the FC experiments.

Chromosomal damage

The ability of nano-TiO2 to induce chromosomal damage wasmeasured using the micronucleus assay. This assay detects boththe induction of chromosome breaks and chromosome miss-segregation. Results (Figure 7) indicate that neither acute (24 h,Figure 7A) nor chronic treatments (1 and 3 weeks, Figure 7(B)and (C)) were able to induce significant increases in the frequencyof binucleated cells with micronuclei. On the contrary, 150 ng/mLmitomycin-C (positive control) induced significant increases inthe frequency of BNMN.

Figure 4. Relative mRNA expression compared to control of differ-ent interleukins after the continuous exposure to 1, 10 and 20 mg/mLof nano-TiO2 during 3 weeks. Results were obtained using real-timeRT-PCR. **p50.01.

Figure 5. Relative hemooxigenase-1 expression following real-timeRT-PCR and Western Blot approaches after the continuous exposure to1, 10 and 20mg/mL of nano-TiO2 during 3 weeks (A). Western blottingfigures showing the intensities of the 34 kD band corresponding to HO-1(B). 1, 2, 3 and 4 correspond to the negative control and the exposures to1, 10 and 20mg/mL of nano-TiO2 during 3 weeks, respectively. A-tubulinWestern blotting was performed to assess for the total amount of proteinsloaded on the gel.

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Assessment of acquired cancer phenotype by thesoft-agar assay

To determine whether the cells acquired an in-vitro cancerphenotype during nano-TiO2 long-term exposure, we assessedthe ability of BEAS-2B cells to form colonies on soft-agar.The promotion of anchorage-independent growth is considereda good biomarker of cell transformation processes linked to theacquisition of cancer phenotype. In our study, we have checkedthe effects after 4 weeks of treatment. As observed in Figure 8,a direct dose–effect relationship was observed when the totalnumber of colonies, as well as the number of medium-largesize colonies, was determined. In spite of the direct relationshipobserved, only the results obtained after evaluating the numberof medium-large colonies exposure to 20 mg/mL attain statis-tical significance. In summary, our results show that after 4weeks of exposure to low doses of nano-TiO2, there is asignificant dose-dependent increase, as indicated by theregression analysis, in the number of colonies growing onsoft-agar in BEAS-2B cells for both the total of colonies(r¼ 0.623, p¼ 0.031) and medium-large size colonies(r¼ 0.788, p¼ 0.002).

Discussion

Two important conclusions can be extracted from our study.The first is that nano-TiO2 exposure lasting for 4 weeks is ableto induce cell transformation in human bronchial epithelial cells(BEAS-2B). The second is that, according to our results, suchcell-transforming properties are generated by a non-genotoxicmechanism, since no genotoxic effects were observed in thecomet and the micronucleus assays.

It is interesting to remark that nano-TiO2 readily entered cellsas demonstrated in our study by using TEM and flow-cytometrymethods. Nano-TiO2 particles tend to form nano-aggregatesentering the cells via non-specific endocytosis process and ithas been shown a preferential uptake of small nano-aggregatessizes that coincide with their pristine size, showing anatase formsranging from 20 to 30 nm the highest uptake ability (Anderssonet al., 2011). These studies showed that the use of depth-profilingm-Raman microspectroscopy coupled with hyperspectral dataanalysis confirms the localization of nano-TiO2 particles insidethe cell allowing to discriminate particles inside the cell fromthose placed outside the cell membrane. Although these studieswere conducted in A549 lung epithelial cells, important uptake in

Figure 6. Genotoxic and oxidative DNA damage, as measured by the comet assay, after chronic exposure to nano-TiO2. Graphs show the resultsobtained after 24 h (A), 1 week (B) and 3 weeks (C) of treatments with the concentrations of 1, 10 and 20 mg/mL of nano-TiO2. White bars represent theresults obtained with the standard comet assay while gray bars represent the results obtained with the FPG-modified comet assay. ***p50.001. Positivecontrol (C+) correspond to 0.5 mM ethyl methane sulfonate (EMS).

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BEAS-2B cells has also been reported (Gibbs-Flournoy et al.,2011; Park et al., 2008).

Once nano-TiO2 enters the cell it can induce different types ofchemical and biological effects. Among the possible inducedeffects, different authors have reported the intracellular produc-tion of ROS by nano-TiO2 exposure. In fact, oxidative stress isthought to be a key mechanism related with the undesirablebiological effects of nanomaterials. Nevertheless, it must beindicated that in most of the reported studies high doses are usedto cause such type of effect. Positive ROS induction was obtainedin human fibroblasts, but only after exposures to 50mg/mL(Huang et al., 2009). In human hepatoma HepG2, high doses(250mg/mL) were needed to increase the production of ROS afterexposure to the rutile form of nano-TiO2 (Petkovic et al., 2011).Nevertheless, no intracellular production of ROS measured by theDCFH-DA assay has been recently reported in human pulmonaryfibroblast (Armand et al., 2013). This absence of intracellularROS induction was confirmed by the lack of HO-1 mRNA andprotein induction after exposure to different types of nano-TiO2,including rutile and anatase forms (Armand et al., 2013). Thesepublished results would agree with our data demonstrating thatthe lack of intracellular ROS induction correlates well with the noinduction of HO-1 that, although, show an under-expression at the

level of mRNA its protein level is equivalent to that observed inthe control. This would indicate that production of ROS is not aninherent property of nano-TiO2 but it can be affected by manyfactors including cell type, nanoparticle size, dispersion protocolsand agglomeration stage, among others (Andersson et al., 2011;Magdolenova et al., 2012a).

With respect to the ability of nano-TiO2 exposure to induce aninflammatory response, our results are not conclusive since onlysignificant increase in the expression of IL-8 was obtained. Thissignificant increase would agree with the results obtained in A549human lung epithelial cells (Singh et al., 2007), and in humanneutrophils (Goncalves et al., 2010), where significant increasesin the expression of IL-8 were observed after nano-TiO2 exposure.

Interestingly the lack of oxidative damage correlates with thelack of genotoxic effects observed. This lack effect has beenobserved at the level of primary DNA damage (comet) and at thelevel of chromosome damage (micronuclei). These two assayscover a wide range of genotoxic effects and would confirm that,under our conditions of testing, nano-TiO2 exposure does notincrease the levels of genetic damage in BEAS-2B cells. At thispoint it is interesting to remark the negative response obtained inthe comet assay when complemented with the use of FPG enzymewhich would confirm the lack of induction of oxidative damage

Figure 7. Chromosomal damage, as measured by the micronucleus assay, after chronic exposure to nano-TiO2. Graphs show the results obtained after24 h (A), 1 week (B) and 3 weeks (C) of treatments with the concentrations of 1, 10 and 20 mg/mL of nano-TiO2. Mitomycin-C (150 ng/mL) was used asa positive control. ***p50.001.

8 G. Vales et al. Nanotoxicology, Early Online: 1–11

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by nano-TiO2 exposure. It must be remembered that the alkalinecomet assay has proved to be a reliable and sensitive technique forthe study of low genotoxic DNA damage such as DNA strandbreaks and alkali-labile AP sites induced by environmentalcontaminants, including nanomaterials (Dusinska & Collins,2008; Magdolenova et al., 2012b). The generation of 8-OH-dGlesions can be easily measured by the use of formamidopyr-imidine DNA glycosylase (FPG) enzyme, as they are FPG-sensitive sites (Collins, 2004). In our case, the use of FPGalthough induce slights increases in the detected damage, nostatistically significance was attained.

The genotoxic potential of nano-TiO2 exposure is a contro-versial issue as recently reviewed (Shi et al., 2013). A disparity ofpositive and negative results in both in-vivo and in-vitroapproaches have been obtained, what can be attributed todifferences in the physicochemical characteristics of the nano-TiO2 used or the exposure metrics used by the investigators. InBEAS-2B cell, negative induction of DNA-breakage wasobserved in the comet assay (Bhattacharya et al., 2009), butthese results are contrary to those reported by other groups wherepositive response in the comet assay was found after treatmentswith rutile and anatase forms of nano-TiO2 (Falck et al., 2009).The same study found that only anatase form was able to produceslightly but significant increases in the frequency of micronuclei.Slight increases in the frequency of micronuclei were alsoreported in the study of Huang et al. (2009) using NIH 3TC cells.In this long-term exposure study, micronuclei only increase from6 (week 3) to 8% (week 11), but reporting total number ofmicronuclei without indication of the frequency of cells bearingmicronuclei, that is a better biological indicator, and withoutreporting positive controls. An overall analysis of the genotoxicitydata published on the genotoxic potential of nano-TiO2 indicates

that if it is genotoxic, it should be classified as weak genotoxic.This would explain the disparity of results where obtainingpositive or negative data would be influenced by stochastic/random processes related with the sampling size of theexperiments.

Although genotoxicity data are used as a putative endpoint ofcarcinogenic risk, carcinogenesis can result from both genotoxicand non-genotoxic mechanisms (Hernandez et al., 2009). Thus, inaddition to the genotoxic endpoints, many in-vitro approacheshave been proposed to detect the induction of cancer-likephenotypic changes (Vanparys et al., 2012), where anchorage-independent cell growth is considered one of the most importantcancer hallmarks biomarkers. Other biomarkers such as morpho-logical changes, secretion of metalloproteases, invasiveness,migration and inclusive angiogenesis are also included amongthe hallmark markers of carcinogenesis processes. In fact, goodassociations are often found among them as we have recentlyobserved between morphological changes, metalloproteasessecretion and anchorage-independent growth in front of nano-Co exposure (Annangi et al., 2014). BEAS-2B cell coloniesgrowing in the soft-agar matrix have shown carcinogenicproperties as demonstrated by its ability to form tumors in nudemice once injected subcutaneously (Sun et al., 2011). In addition,clones induced after chronic exposure to chromate present alteredgene expression with respect to the profiles observed in clonesobtained in untreated cultures. Thus, genes related to cell-to-celljunction were up-regulated while genes associated with theinteraction between cells and their extracellular matrices weredown-regulated. Additionally, expression of genes involved in cellproliferation and apoptosis were also changed (Sun et al., 2011).This does indicates that clones obtained in the soft-agar assayhave important genetic changes involved in the induction of

Figure 8. Soft-agar results obtained after treatments with nano-TiO2 lasting for 4 weeks. A to D are representative 35-mm plaque pictures of thesoft-agar assay. Plaque pictures corresponding to negative control (A) and to 1, 10 and 20 mg/mL of nano-TiO2 doses (B–D). Graphical representationof the total number of colonies and the number of medium-large size colonies (E). *p50.05 (ANOVA test).

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malignant transformation. Data like this do reinforce the useful-ness of looking for anchorage-independent growth as a way tomeasure the carcinogenic potential of nanomaterials.

In spite of the transcendence of the carcinogenic processes, todate only a limited number of studies focused on carcinogenicpotential of engineered NPs have been published; mainly whenlong-term exposure and environmentally or occupationally rele-vant doses are taken into consideration. Our data revealed a dose-dependent increase in the formation of colonies, although only thehighest dose produces statistically significant increases. Thiswould indicate the relevance of long-term exposures of nano-TiO2

to acquire cancer-like phenotype. As indicated, anchorage-inde-pendent growth of cells in soft-agar is one of the hallmarkcharacteristics of cellular transformation, with normal cellstypically not capable of growth in semi-solid matrices. Thus,cell growth in three-dimensional scenario mimicry more accur-ately what occurs in the in vivo cellular environment.

Our result would confirm those previous reported by Huanget al. (2009) who demonstrated that long-term exposure offibroblast cells to sub-toxic doses of nano-TiO2 increasedanchorage-independent growth. On the other hand, prolongedexposure of keratinocytes to low doses of nano-TiO2 causedformation of nanotubular intercellular structures that may result intransformation of normal keratinocytes to tumor cells (Kocbeket al., 2010). Recent results have shown that different nano-TiO2,including rutile and anatase forms, were able to induce theexpression of metalloprotease 1 in human pulmonary fibroblasts(Armand et al., 2013), these effects being higher than thoseinduced by micrometric TiO2. Metalloproteases secretion isconsidered as an early biomarker of the carcinogenic effectsplaying crucial roles in tumor invasion, morphogenesis, angio-genesis and metastasis, and wound healing by remodellingextracellular matrix (Oum’hamed et al., 2004). Thus, all thisdata would confirm the cell-transforming potential associated tonano-TiO2 exposure.

Our results would indicate that long-term exposure to sub-toxic doses of nano-TiO2 can generate carcinogenic processes.Interestingly, these activities are not linked to the induction ofoxidative stress or genotoxic mechanisms.

Declaration of interest

Gerard Vales and Laura Rubio were supported by predoctoral fellowships(PIFs) from the Universitat Autonoma de Barcelona. This investigationhas been supported in part by the Generalitat de Catalunya (CIRIT,2009SGR-725) and the NanoGenotox (Grant Agreement n�2009 21 01),and NANoREG (Grant Agreement NMP4-LA-2013-310584) EU projects.The authors report no conflict of interest and are responsible for thecontent and writing of the article.

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