Brief exposure to nanosized and bulk titanium dioxide forms induces subtle changes in human D384 astrocytes

Toxicology Letters 254 (2016) 8–21

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Toxicology Letters journal homepage: www.elsevier.com/locate/toxlet

Brief exposure to nanosized and bulk titanium dioxide forms induces subtle changes in human D384 astrocytes Uliana De Simone, Davide Lonati, Anna Ronchi, Teresa Coccini* Laboratory of Clinical & Experimental Toxicology and Poison Control Center, Toxicology Unit, Salvatore Maugeri Foundation IRCCS, Scientific Institute of Pavia, Pavia, Italy

H I G H L I G H T S

   

TiO2NPs caused subtle effects in human astrocytes D384 cells at not cytotoxic doses. Oxidative stress and apoptotic mechanisms occurred after low dose exposure to TiO2NP. Comparatively, similar effects were observed when testing TiO2 bulk. Cellular checkpoint perturbations were associated with increasing intracellular Ti.

A R T I C L E I N F O

Article history: Received 19 February 2016 Received in revised form 2 May 2016 Accepted 2 May 2016 Available online 3 May 2016 Keywords: CNS In vitro Nanotoxicity TiO2 Molecular mechanism Safety

A B S T R A C T

Although nanosized-titanium dioxide particles (TiO2NPs)-containing products are constantly placed on the market, little is known about their possible impact on human health, even regarding to CNS effects. In this study, mechanistic pathways, by which TiO2NPs induce cellular damage and death, have been investigated in human (astrocytes-like) D384 cells and comparatively weighed against the effects produced by the bulk counterpart. Cellular signals evaluated by multiple set of in vitro tests after 24 h exposure to TiO2NP concentrations (0.5–125 mg/ml) were: ROS production, p-p53, p53, p21, Bax, Bcl2 and caspase 3. TiO2 cellular uptake was also estimated by both light microscopy and ICP-MS. ROS were generated starting at 1.5 mg/ml and further increased at the highest concentrations (31 mg/ ml). At the same low concentration, an increased expression of p-p53, p53, p21, Bax, and activated caspase3 were also observed. Parallely, Bcl-2 decreased along with TiO2NP concentration increase. Similar alterations were observed when testing TiO2 bulk: cellular checkpoint perturbations were associated with rising intracellular Ti. The present data demonstrated that low TiO2NP concentrations were capable, after 24 h, to induce subtle cellular perturbation in D384 cells after a single cell treatment, supporting the evidence that both oxidative stress and apoptotic mechanisms may occur in this type of CNS cells. ã 2016 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Titanium dioxide nanoparticles (TiO2NPs) are among the most highly manufactured in the world due to their high physical stability, anticorrosion and photocatalytic activity (Baan et al.,

* Corresponding author at: Laboratory of Clinical & Experimental Toxicology, IRCCS Salvatore Maugeri Foundation, Scientific Institute of Pavia, Via Maugeri 10, 27100 Pavia, Italy. E-mail address: [email protected] (T. Coccini). http://dx.doi.org/10.1016/j.toxlet.2016.05.006 0378-4274/ã 2016 Elsevier Ireland Ltd. All rights reserved.

2006; Shi et al., 2013). Various investigations have established that TiO2 is much more effective as a photocatalyst in the form of nanoparticles than in bulk powder. Moreover, the surface modification of TiO2NPs appears to be more beneficial than the modification of bulk TiO2 (Gupta and Tripathi, 2011). The clear different optical, catalytic, and electronic characteristics of TiO2NPs compared to TiO2 fine particles are determined by the variation in size, structure, shape, by the surface to volume ratio, the charge, agglomerate and aggregate formation, together with their insolubility in aqueous solutions (Rollerova et al., 2015). Thus, during these few years, TiO2NP has become the most widely used

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nanoparticle and has been produced in large industrial scale. They are in the top five NPs used in consumer products (Shukla et al., 2011). Some recent estimates of annual global nano TiO2 production range between 5000 and 6400 metric tones (Mueller and Nowack, 2008; Robichaud et al., 2009) and it is expected to continue to increase until at least 2025 (Hendren et al., 2011; Landsiedel et al., 2010; Robichaud et al., 2009; Weir et al., 2012). It has been suggested that by 2023, up to 50% TiO2 might be manufactured in nanoform. In association to the tremendous potential of nanosized TiO2 for a host of applications, it must be highlighted that the distinctive physicochemical properties of these nanomaterials, which impart them beneficial characteristics, differ from their bulk counterpart. These new properties likely influence bioactivity (Nel et al., 2006; Schwirn et al., 2014; Zhang et al., 2015) and hence toxicity. Adverse effects of TiO2NPs should be carefully evaluated together with the bulk TiO2 although the latter has been considered as having relatively low toxicity. Thus, understanding the health impact of TiO2NPs has become a priority for ensuring health protection (Sha et al., 2015). Since many sources of nanoscale TiO2 could result in human exposure, the enormous amounts of nanomaterial produced raise the possibilities of occupational and environmental exposures for example during manufacturing as well as a current use by inhalation and dermal exposures (Shi et al., 2013). Although pulmonary absorption of TiO2NPs following inhalation represents very important entry gate of TiO2NPs into human body in occupational environment, no human data are available so far. Nevertheless, experimental evidences exist (Song et al., 2015; Wang et al., 2008a,b) indicating TiO2NPs ability to enter the brain, thus representing a realistic risk factor for both chronic and accidental exposure with the consequent needs for more detailed investigation on CNS (central nervous system). Several possible mechanisms have been suggested by which NPs can pass through the blood-brain barrier (BBB) (Cupaioli et al., 2014; Song et al., 2015). Specifically, an endo-lysosomal pathway has been demonstrated for TiO2NPs in in vitro BBB model of human cerebral endothelial cells (hCMEC) after 4- or 24-h exposure (Halamoda Kenzaoui et al., 2012; Ye et al., 2013) or transcytosis after 5-day exposure (Halamoda Kenzaoui et al., 2012). It has also been shown that TiO2NPs (as rutile form with hydrophobic and hydrophilic surface modifications) can transform benign cells, namely QR-32 fibrosarcoma cells (poorly tumorigenic and nonmetastatic cells) into aggressive metastatic tumor cells, through the generation of reactive oxygen species—ROS (Onuma et al., 2009). Recently we have shown that anatase TiO2NPs produced cytotoxicity in in vitro human CNS cell lines (both glial D384 and neuronal SH-SY5Y) by applying a cell-based screening platform. Effects were evident after not only short-term (4–48 h) but even prolonged (several days) exposure to low concentrations (0.1–1.5 mg/ml) (Coccini et al., 2015). NP-induced perturbations of cellular homeostatic mechanisms might act as basis of different pathophysiological processes depending upon the concentration and duration of exposure (Nel et al., 2006). Apoptosis, necrosis, cell senescence, and autophagy are possible cellular responses to persistent stress. Signals potentially leading to cell death are initiated from molecular sensors found in different organelles and subcellular structures. These sensory pathways converge to form a highly intricate network that produces a cellular “decision” to persist or die. Unraveling these organellespecific cell death regulatory mechanisms, and how they interface with pathways triggered by other subcellular components, is a field of intense research (for a review, see Galluzzi et al., 2014). Either an excess or a reduced apoptotic process is involved in many pathological conditions including neurodegenerative disorders and carcinogenesis (Shi, 2002).

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It has been suggested that the NP-induced biological responses may be linked to their accumulation within cells (Bartneck et al., 2010; Oh et al., 2010) leading to oxidative stress and oxidative DNA damage in the nuclear compartment (Bhattacharya et al., 2009; Trouiller et al., 2009). ROS play a dual role in the fate of cell i.e., causing cell death as well as acting as second messengers to induce an adaptive cell response (Suzuki and Forman, 1997). Indeed, oxidative stress has been shown to induce cell death by a variety of mechanisms (Baumgartner et al., 2007; Kannan and Jain, 2000; Zamzami et al., 1995). A hierarchical model for NP toxicity also describes the possibility of higher oxidative stress levels leading to cell death induction (Nel et al., 2006). Different types of NPs have been shown to induce oxidative stress (Gurr et al., 2005; Hussain et al., 2009, 2010; Limbach et al., 2007) but the role of oxidative stress in NP induced cell death has not yet been completely elaborated. Astrocytes, the most abundant type of glia, have a primary role to support neuron network in the CNS. Astrocytes have a variety of important functions such as supplying of metabolic nutrients to neurons and also protecting the brain against oxidative stress and metal toxicity (Eroglu and Barres, 2010; Hirrlinger and Dringen, 2010; Parpura et al., 2012; Tiffany-Castiglioni et al., 2011). It is also well recognized the particular role of astrocytes in several neurodegenerative diseases, being more likely the cell type initially affected during pathogenesis (Maragakis and Rothstein, 2006). Several studies have also reported that the rate of nanoparticle translocation into the brain can be significantly increased under certain pathological conditions, such as infection, meningitis, systemic inflammation, etc. (Sharma and Sharma, 2007; Sharma et al., 2010). Our recent findings have shown human astrocytes sensitivity to TiO2NP treatment (Coccini et al., 2015), in particular 25–50% decrease of cell viability was observed at concentrations ranging from 31 to 250 mg/ml after 24h-exposure. Based on these cytotoxicity results, the present study was designed to investigate whether single short-term exposure to low concentrations (0.5– 62 mg/ml) of TiO2NP were able to alter specific “metabolic checkpoints” relevant to cell fate for understanding the molecular mechanism/pathways implicated in the astrocytes toxicity. Moreover, the study aimed at clarifying differences or similarity between the toxicity of bare TiO2 nanoparticulates and their bulk counterpart. The metabolic signals were evaluated by multiple set of in vitro tests in D384 human astrocytoma cells, and included: reactive oxygen species (ROS) production, Bax (pro-apoptotic protein), Bcl-2 (considered as anti-apoptotic protein), p-p53 (phosphorylated ser 15 of p53 as a transcription factor and activator of several genes expression such as WAF1/CIP1 encoding for p21), p53 (a tumor suppressor of cell death, as well as controller of several protein expression), p21 (cyclindependent kinase 1 inhibitor – a cell cycle inhibitor), and caspase 3 (activated in the apoptotic cell both by extrinsic (death ligand) and intrinsic (mitochondrial) pathways). These effects were assessed in relation to the titanium (Ti) associated with astrocyte cells by ICP-MS measurement. 2. Materials and methods 2.1. Chemicals All reagents and chemicals for cell cultures, titanium (IV) oxide (TiO2), QuantiProTM BCA Assay Kit and Fluoroshield were purchased from Sigma-Aldrich (Milan, Italy). 65% Acid Nitric (HNO3) was purchased from VWR International PBI (Milan, Italy). The primary rabbit monoclonal antibody against Bax, Bcl-2,

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caspase 3, p21, p53, p-p53, beta Actin Cytoskeleton were purchased from Abcam (Prodotti Gianni, Milan, Italy), Alexa 488-labeled antibodies, dihydroethidium and Hoechst 33258 dyes were purchased from Molecular Probes (Life Technologies, Monza, Italy). 2.2. Titanium dioxide particles and their characteristics Titanium oxide nanoparticles (anatase isoform; # 5430MR) were purchased from Nanostructured & Amorphous Materials, Inc. (Houston, USA). Physico-chemical characteristics of the TiO2NP nanopowder provided by Company are shown in Fig. 1A–C. 2.3. TiO2NP and TiO2 bulk stock suspensions TiO2NP stock suspension was prepared following an optimized dispersion protocol developed by Guiot and Spalla (2013) involving a pH adjustment and addition of bovine serum albumin (BSA 10.24 mg/ml). Preparation details are described in our previous manuscript (Coccini et al., 2015). These stock suspensions were diluted in culture medium to prepare the selected concentrations to be used for cell treatments. The TiO2NP stock suspension (2.5 mg/ml; Fig. 1D and E) and TiO2NPs dispersed in culture medium (at the concentration of 31 mg/ml) after 24 h (Fig. 1F and G) were analyzed in order to measure the size of the nanoparticles and the zeta potential by dynamic light scattering (DLS) (by N.A.M. Srl, NANO-Analysis & Materials, Vigevano, Italy). The same dispersion protocol was also used to make TiO2 bulk suspension. Fresh solutions of test materials were prepared shortly before each experiment.

2.4. Astrocyte cells culture and treatments Human astrocytoma cells (D384 clonal cell line was established from Balmforth et al., 1986) were used to evaluate the effect of 24 h exposure to TiO2NP and TiO2 bulk. D384 cells were cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% heat-inactivated FBS, 2 mM L-glutamine, 50 IU/ml penicillin, 50 mg/ml streptomycin and 1% (v/v) sodium pyruvate. Cells were maintained at 37  C in a humidified atmosphere (95% air/5% CO2). Cells at about 80% confluence were split 3 times per week to ensure health and growth as well as the cell performing assays. D384 cells were incubated with increasing concentrations of TiO2NPs or TiO2 bulk (0.5–125 mg/ml) for 24 h to (i) investigate whether single short-term exposure to TiO2NP was able to alter specific metabolic checkpoints essential for the astrocyte cell fate, and (ii) clarify the differences/similarities between TiO2NP and TiO2 bulk form. The selected concentrations and the time point (24 h) of the present study were chosen based on our previous results. The latter demonstrated that TiO2NPs induced cytotoxicity in human cultured astrocytes after short-term (4, 24, 48 h) and prolonged exposure (several days) by traditional tests evaluation measuring cell death and cellular functions, namely MTT assay (mitochondrial function), double calcein AM/PI staining (membrane integrity) and clonogenic test (growth and cell proliferation). The lowest TiO2NP effective concentrations in decreasing cell viability (targeted the mitochondrial function) and compromising cell proliferative capacity were 31 mg/ml after 24 h exposure and 0.2 mg/ml after several days exposure, respectively (Coccini et al., 2015).

Fig. 1. TiO2NP physico-chemical properties. (A) TEM micrograph showing the morphology and sizes of 15 nm of the row nanomaterial (data provided by the Company). (B) Xray diffraction analysis confirmed titanium dioxide nanoparticles (data provided by the Company). (C) Physico-chemical properties of TiO2NP nanopowder (data provided by the Company). (D) Size distribution of TiO2NP stock suspension (2.5 mg/ml) by DLS measurements. (E) Physico-chemical properties of TiO2NP stock suspension. (F) Size distribution of TiO2NPs in D384 culture medium at the concentration of 31 mg/ml after 24 h, by DLS measurements. (G) Physico-chemical properties of TiO2NP suspension at the concentration of 31 mg/ml (from Coccini et al., 2015).

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For light microscopy visualization of TiO2 cellular uptake, ICPMS quantification and western blots, D384 cells were seeded in petri-dish (100 mm) at the cell density of 2.5  105/ml cells and kept for 24 h at 37  C. Experimental setup details were: 20 mm of height, 55 cm2 of growth area/petri-dish, 10 ml of working volume/ petri-dish, 1 ml treatment/volume petri-dish. For ROS determination and immunofluorescence detection of apoptotic signaling, D384 cells were cultured on coverslips at the density of 2  105 cells in 6-well plates and kept for 24 h at 37  C. Experimental setup details were: 16.8 ml of total volume/well, 9.5 cm2 of growth area/well, 2 ml of working volume/well, 200 ml treatment volume/well. 2.5. Light microscopy visualization of TiO2 cellular uptake To visualize the presence or absence of TiO2NP or TiO2 bulk into the D384, the cells were observed by light microscopy. Specifically, after 24-h attachment, cells were treated with TiO2NP or TiO2 bulk suspensions at the following concentrations: 0.5, 15, 31, 62, 125 mg/ ml for 24 h. At the end of the exposure, the cells were washed twice with PBS in order to remove unattached particles, followed by fixation in 4% paraformaldehyde for 20 min at room temperature (r.t.) and in 70% ethanol over night at 20  C. The cells were then washed twice with PBS and three times with sterile water and they were let dry at environmental conditions. The cells were examined under a CX41 Olympus light microscope using 100X oil immersion objective lens and combined with a digital camera (Infinity2). Digital photographs were taken and stored on the PC. 2.6. Quantification of TiO2 associated with cells by ICP-MS For Ti content determination, D384 cells were exposed to TiO2NP or TiO2 bulk at nominal mass concentrations of 1.5, 15, 31, 62 and 125 mg/ml for 24 h at 37  C and thereafter the medium was removed and cells washed three times with PBS (4 ml/petri-dish), then scrapered, and D384 pellets collected by centrifugation (1100 rpm for 8 min at 24  C). Samples of TiO2NP and TiO2 bulk media were stored at +4  C while the cell pellets were stored at 20  C for subsequent analysis of the Ti content. The quantification of Ti was expressed as a percentage of the administered concentration associated to the cell pellet or measured into the medium. Measurements of Titanium (representing the TiO2 uptake) were carried out using an Inductively Coupled Plasma Mass Spectrometer (ELAN 6100 DRCII ICP-MS; PerkinElmer SCIEX Instruments, Concord, Ontario, Canada) equipped with a dynamic reaction cell, a quadrupole mass filter, cyclonic spray chamber with a concentric nebulizer and AS 90 plus auto-sampler (PerkinElmer). The instrumental parameters were: radiofrequency power, 1350 W; plasma argon flow rate, 14 l/min; nebulizer argon flow rate, 0.9 l/ min; auxiliary argon flow rate, 1 l/min; acquisition time, 600 ms; dwell time, 300 ms; sweeps reading, 2; number of replicates, 10 (other ICP-MS parameters are presented in the Supplementary material (Table S1)). The radiofrequency and gas flow were optimized to obtain good sensitivity, a low production of oxides (<2%) and doubly charged ions (1%). Before the analysis, samples underwent a pre-treatment in microwave to reduce interferences caused by matrix; the use of a microwave assures rapidity of execution and a valid control of accidental pollution phenomena. Digestions were carried out in a microwave sample preparation system CEM (Corp., Matthews, NC, USA) Model MARS-Xpress equipped with a 1400 W magnetron that is adjustable in 1% increments and operating at a microwave frequency of 2455 MHz. The samples (cellular pellet and culture media,1 ml) were added to the digestion vessels with 2 ml HNO3 and 0.1 ml HF. The

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temperature was ramped (5 min) from 20  C to 100  C and held constant for 5 min. Subsequently, the temperature was increased to 160  C (ramping lasted 5 min) and held constant for 15 min. After complete digestion and cooling, the samples were transferred to 50 ml graduated polypropylene tubes and diluted to volume with deionized water. High purity water was produced starting from distilled water using a Milli-Q TM deionizing system (Millipore, Bedford, MA USA). HNO3 (65% m/v) and HF (40% m/v) were Suprapur reagents (E. Merck, Darmstadt, FRG). Calibration was performed by “standard addition” method. Standard solutions were prepared from 1000 mg/l titanium calibration standard (E. Merck, Darmstadt, FRG), by dilution with water containing the same amount of acids as the samples. An internal standard (IS) of Indium was added to all the samples to monitor and to correct for instrumental fluctuations. The Ti detection limit (LOD), calculated as 3 SD of the blank signal (n = 5), was 0.007 mg/l and the limit of quantification (LOQ), defined as 10 SD of the blank, was 0.024 mg/l. Recoveries ranged between 89.2 and 100.5% for solutions containing from 5 to 100 mg/l Ti. Linear calibration curve was generated from 1 and 100 mg/l with coefficients of variation (CV) ranging from 1 to 9.5%. 2.7. Detection of ROS by dihydroethidium (DHE) ROS generated within the cells were detected using dihydroethidium (DHE), a cell permeable fluorescent dye, as described by Shaikh and Nicholson (2008). In fact, intracellular ROS oxidize DHE into ethidium bromide which intercalates with the nuclear DNA and is thus detectable as a red fluorescence within the nucleus. The latter was counterstained with Hoechst 33258 blue fluorescence dye. Nuclear ethidium bromide red fluorescence intensity is proportional to ROS amount into cytoplasm. After cell treatment with TiO2NP or TiO2 bulk for 24 h, the D384 cells were incubated with DHE (5 mM) at 37  C for 30 min. Then, cells were washed in PBS and fixed in 4% paraformaldehyde for 20 min at r.t., washed again with PBS, and treated with 12 mg/ml Hoechst 33258 for 15 min at 37  C. Finally, cells were washed again with PBS and coverslips were mounted with Fluoroshield and let dry at environmental conditions in dark. All images were collected with the digital camera (Infinity2) combined with a fluorescence microscope (CX41 Olympus), provided by EPI LED Cassette (FRAEN, Settimo Milanese (MI), Italy), the measurement conditions were: 470 nm excitation (T% = 40), 505 nm dichroic beamsplitter, 510 nm long pass filter. The fluorescence images were taken using 100 oil immersion objective lens. 2.8. Apoptotic pathway: immunofluorescence detection of Bax and activated caspase 3 D384 cells were exposed to increasing concentrations of TiO2NPs or TiO2 bulk (0.5–62 mg/ml) for 24 h at 37  C. The protocol of immunofluorescence was previously described (De Simone et al., 2013). Briefly, the cells were fixed (4% paraformaldehyde for 20 min at r.t. then 70% ethanol over night at 20  C), rehydrated (in PBS) and blocked with 1% BSA, then incubated with polyclonal antibodies recognizing Bax (dilution 1:200 in PBS) or caspase 3 (dilution 1:200 in PBS). After washing with PBS, the cells were stained with secondary antibody (Alexa 488-labeled; dilution 1:100 in PBS), washed again with PBS. The nucleus was counterstained with 1 mg/ ml propidium iodide. Finally the coverslips were mounted with Fluoroshield and let dry at environmental (dark) conditions. Cells were examined with CX41 Olympus fluorescence microscope using same parameters as described in the previous paragraph. The red fluorescence of the nucleus was improved processing the images by the GIMP GNU Image Manipulation Program. The

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green background was reduced and the levels of contrast and brightness were adjusted to optimize the red fluorescence of the nucleus produced by propidium iodide. In order to verify whether the internalization of TiO2NPs into cells might cause false positive staining, increasing concentrations (from 1.5 to 62 mg/ml) of TiO2NPs or TiO2 bulk were added to culture medium without cells and the immunofluerescence staining protocol (specific either for Bax or activated caspase 3 protein) was applied similarly to that employed in presence of the astrocyte cells. 2.9. Western blots After TiO2NP and TiO2 bulk 24-h treatments (1.5, 15, 31, 62 mg/ ml), D384 cells were washed with PBS, lysed by protein lysis buffer plus protease inhibitor cocktail (Sigma, Milan, Italy) for 30 min on ice, then scraped and centrifuged at 13,000 rpm for 15 min at 4  C. Protein concentrations were determined using the BCA method (Sigma, Milan, Italy) and then the supernatant samples stored at 20  C for subsequent analysis of the protein expression. The proteins were mixed with equal volume of Laemmli buffer 2X, boiled for 5 min at 95  C and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on a 10% gel. Subsequently, the proteins were electrophoretically transferred to nitrocellulose membranes (45 mm; Bio-Rad). The membranes were blocked with 5% non-fat dry milk solution in Tris Buffered Saline with Tween 20 (TBST) overnight at 4  C. Following, membranes were incubated with individual primary antibody (namely p53, p-p53, p21, caspase 3, Bax and Bcl-2, and with antib-actin to confirm equal loading; dilution 1:1000 in non-fat dry milk solution) for 90 min at r.t. After washing, the blots were incubated with HRP conjugated anti-rabbit Ig-G (dilution 1:5000 in TBST) for 1 h at r.t. The protein band detections were performed using ECL reagents (BioRad, Segrate, Italy) by ChemiDocTM Imaging System (BioRad, Segrate, Italy). The band intensity was calculated using Image LabTM 5.1 software (BioRad, Segrate, Italy). The immunoblots were normalized with beta-actin and processed by ImageJ software for the densitometric analysis. Specifically: the contrast was adjusted such that the bands were clearly visible on the blot image. Then, the protein bands were selected by drawing a tight boundary around them and analyzed by: analyze > Gels on the ImageJ toolbar. The quantification of protein bands reflected the relative amounts as a ratio of each area of protein band (obtained after increasing TiO2 concentrations) relative to the area of control band (C). The experiment, comparing the two treatment conditions (TiO2NP and TiO2 bulk), was repeated at least three times with similar results. 2.10. Statistical analysis Each experiment (i.e., immunofluorescent assays, light microscopy, ROS evaluation) was performed in triplicated and repeated at least three times. ICP-MS data are expressed as means  SD. Densitometric was evaluated by one-way ANOVA followed by Tukey’s test. A value of P < 0.05 was considered statistically significant.

Fig. 1A–C shows information provided by the Nanostructured & Amorphous Materials, Inc. (Houston, USA) Company. The TEM image of the nanopowder displays an essentially spherical shape and the average diameter was estimated about 15 nm (Fig. 1A). The diffraction peaks (2u = 25 , 2 = 38 , 2 = 48 , 2 = 54 and 2 = 63 ), shown in Fig. 1B, corresponds to the anatase isoform. Fig. 1C summarizes the physico-chemical properties of TiO2NP nanopowder. At 24 h, the dynamic light scattering (DLS) observation of the TiO2NP stock suspension evidenced a wide range of particle size distribution from 10 to 1000 nm due to the aggregation or agglomeration. This value was evaluated as intensity% (Fig. 1D). The mean hydrodynamic diameter and the polydispersity index (PDI) were 52 nm and 0.444, respectively, the positive zeta potential (ZP) (0.35 mV) was indicative of a weak surface positive charge and the low value revealed a poor stability over time (Fig. 1E). When TiO2NPs were further diluted in DMEM medium to be used for cell culture, at 31 mg/ml concentration, the size distribution of the TiO2NPs after 24 h showed a mean hydrodynamic diameter of 356 nm and PDI of 0.289, and again a low zeta potential (0.95 mV) indicative of a poor stability over time (Fig. 1F and G). 3.2. Visualization of TiO2 cellular internalization TiO2NPs are well known to be very difficult to disperse as single objects. For this study, about 15 nm anatase TiO2NPs were dispersed to a final size, in DMEM medium used for the cell culture, of 356 nm, using a dispersion protocol optimized (as indicated in Section 2,Guiot and Spalla, 2013), and these clusters were easily visualized as high-density objects, under light microscopy, particularly inside cells. Fig. 2 shows a panel of representative randomly selected microscopic fields by light microscopy of D384 cells treated with increasing concentrations of TiO2NPs or TiO2 bulk (from 0.5 to 125 mg/ml after 24 h). After exposure to TiO2NP and TiO2 bulk, there was a clear evidence of concentration-dependent accumulation of particles (black bodies) inside the D384 cells. In particular, TiO2NPs and TiO2 bulk were up-taken into the cytoplasm rapidly after treatments (already after 4 h, data not shown) as well as after 24 h exposure. TiO2 (nano and bulk form) agglomerates could be easily distinguished as black bodies localized around the perinuclear region (Fig. 2), apparently TiO2 (nano and bulk form) did not enter the nucleus. The particles appeared to aggregate into the cytoplasm: fine aggregates were visible at the low concentrations of TiO2, while cells treated with high concentrations showed small and large aggregates all over the cytoplasm. Indeed, several studies have demonstrated, by cytofluorimetry and dark microscopy that TiO2NP moved into the cytoplasm, but not the nucleus (Zucker et al., 2010). TiO2 also appeared to be lying on the cells: the discrimination between particles attached to outside cell membrane from those inside was possible by changing the light microscope focus (Fig. 2: high magnification pictures). Similarly discrimination was also reported by Allouni et al. (2012) in TiO2NP-treated fibroblasts. The accumulation of both TiO2 types occurred without cell morphology changes with cells remaining adhered to the culture dishes throughout (Fig. 2). 3.3. Quantification of TiO2 associated with cells by ICP-MS

3. Results 3.1. TiO2NP physico-chemical properties Fig. 1 summarizes the characteristics of TiO2NP nanopowder as well as TiO2NP suspensions.

ICP-MS experiments were carried out to examine the abundance of TiO2NPs or TiO2 bulk in D384 cells calculated as the percent of cells associated with Ti after increasing concentrations of TiO2 NP or bulk. The results obtained are shown in Fig. 3. The values (%) of Ti content associated to D384 cellular pellet raised

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Fig. 2. Visualization of TiO2 cellular uptake. Representative images of randomly selected microscopic fields by light microscopy of D384 cells treated with increasing concentrations of TiO2NPs or TiO2 bulk (0.5–125 mg/ml) after 24 h. Concentration-dependent accumulation of particles (black bodies) inside of the D384 cells of both treatment types (TiO2NPs and TiO2 bulk). At the concentration of 0.5 mg/ml and 1.5 mg/ml, TiO2 (nano and bulk form) was detected in very few cells and between adjacent cells (white arrows), while at the higher concentrations of TiO2NPs and TiO2 bulk (15–125 mg/ml) black bodies were visible mainly around the perinuclear region. The magnification (3) is corresponding to the areas indicated by the orange arrows. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

with increasing TiO2NPs or TiO2 bulk treatment concentrations, meanwhile Ti percentage diminished in culture medium (Fig. 3). In particular after 24 h, the abundance of TiO2NPs associated with cellular pellet ranged between 50% at the lowest concentration of 1.5 mg/ml and 87% at the higher concentrations tested of 62 and 125 mg/ml. Similarly, TiO2 bulk accumulation by cells was 50–74% at the same range of concentrations (i.e., 1.5–125 mg/ml; Fig. 3). In control medium and cells the Ti amount was below the LOD. 3.4. ROS generation by TiO2NP and TiO2 bulk treatments

Fig. 3. Quantification of TiO2 associated with cells. Bar charts display Ti quantification by ICP-MS express as percent (%) of the nominal concentration associated to the cell pellet or measured into the medium after 24h-exposure to TiO2NPs ( ) and TiO2 bulk ( ) (1.5–125 mg/ml). Concentration-dependent association of particles in cellular pellet in both TiO2NPs and TiO2 bulk treatment groups: the abundance of TiO2NPs associated with cellular pellet ranged between 50% at the lowest concentration of 1.5 mg/ml and 87% at the higher concentrations tested of 62 and 125 mg/ml. Similarly, TiO2 bulk associated to cells was 50–74% at the same ranging concentrations (i.e., 1.5–125 mg/ml). Data (n = 3) are expressed as mean  SD.

D384 cells were exposed to various low TiO2 (nano or bulk form) concentrations (0.5–62 mg/ml) for 24 h. The ability of TiO2NPs to induce intracellular oxidant production in D384 cells was assessed using DHE fluorescent nuclear dye, whose fluorescence intensity in the nucleus corresponds to the amount of ROS generation inside the cells. In Fig. 4 is reported a panel of representative randomly selected microscopic fields by fluorescence microscopy. Qualitative analysis indicated that there was a correlation between concentration and oxidative stress. In particular, the effect was evidenced starting at 1.5 mg/ml TiO2NPs concentration: ROS were generated as fluorescent red spots in the nucleus that appeared markedly more evident at the highest concentrations (31–62 mg/ml) (Fig. 4). No effect was observed at 0.5 mg/ml. A concentration-dependent fluorescent intensity profile of the dots in D384 nucleus was also observed for TiO2 bulk, although the red nuclear spots were detected starting at 15 mg/ml concentration as shown in Fig. 4.

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Fig. 4. ROS evaluation. Representative images of randomly selected fluorescence microscopic fields of D384 cells treated with increasing concentrations of TiO2NPs or TiO2 bulk (0.5–62 mg/ml) after 24 h. DHE fluorescent dye (red) and Hoechst 33258 stain (blue) were used to identify the detection of ROS and the nucleus, respectively. TiO2NPs and TiO2 bulk showed a similar pattern: red dots in the nucleus indicate the accumulation of oxidized DHE by ROS, the fluorescent intensity of the dots is equivalent to the relative levels of ROS present in the D384 cells. Scale bar: 100 mm, insert scale bar: 200 mm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

3.5. Apoptotic pathway: immunofluorescence detection of Bax and activated caspase 3 Figs. 5 and 6 display the immunofluorescence detection of Bax and activated caspase 3, respectively, as a panel of representative randomly selected microscopic fields of D384 cells treated with increasing concentrations of TiO2NPs and TiO2 bulk (0.5–62 mg/ ml). D384 cells exposed to TiO2NPs showed an increasing gradual presence of Bax and activated caspase 3 (by proteolytic cleaved) as indicated by brilliant green intracellular spots directly observed at 1.5 mg/ml. The fluorescent intensity of the green dots was more strongly detected at the highest concentration of 62 mg/ml (Figs. 5 and 6, respectively). Similar patterns for both Bax and caspase 3 were demonstrated after TiO2 bulk treatment (Figs. 5 and 6, respectively). Again, 0.5 mg/ml was devoid of any effect. Supplementary materials (Figs. 5S and 6S) have been added showing immunofluorescent staining for propidium iodide (red nucleus), Bax/caspase 3 antibody (green) and their merge. A dark images were obtained by fluorescence microscope when culture medium (without cells) were supplemented with increasing concentration (1.5–62 mg/ml) of TiO2NP or bulk and immunofluorescence assay protocols (specific of either Bax or caspase 3) were carried out. These data clearly indicate that there were not interference between antibodies and TiO2 (in both nanoparticulate and bulk forms), and therefore, the fluorescence observed in cell preparations was independent from the NPs themselves. 3.6. Molecular mechanism of apoptosis by Western Blot To identify the intracellular mechanisms by which TiO2NPs induce the astrocyte cell death we measured the expression of Ser15-phosphorylation-p53 (p-p53), p53, p21, Bax, Bcl-2, and caspase 3 by western blot. Fig. 7 shows representative western blot analysis and relative densitometric quantification of each protein expression in human astrocytes. D384 cells were exposed to increasing concentrations (from 1.5 to 62 mg/ml) of TiO2NPs as well as TiO2 bulk for 24 h. The results demonstrated an increase concentration-dependent (5–35%) of

Ser15-phosphorylation-p53 (a DNA damage checkpoint) after TiO2NPs treatment. In addition, Western blot analysis also demonstrated that TiO2NPs induced expression increases of p53 (54–64%), as well as p21 (36–66%) and Bax (38–70%) which were the downstream proteins of p53 in regulating cell cycle and apoptosis (Fig. 7). Caspase 3, that plays a key role in the intrinsic apoptotic pathway of cells, was also increased (49–61%) by 24 h TiO2NP treatment (Fig. 7). Parallely, Bcl-2 (anti-apoptotic) was found to be reduced (7–39%) with TiO2NP treatment. Similar expression protein patterns were observed after TiO2 bulk treatment (Fig. 7). 4. Discussion Uncertainties still exists in the understanding of the relationship between toxic mechanism of NPs and their cellular uptake. With regard to TiO2NPs, both their attachment to cellular membranes as well as their internalization have been shown to interfere with cell function and to be relevant in the assessment of cytotoxicity (Allouni et al., 2012). In the present study, we have investigated the putative cell death mechanisms underlying the TiO2NPs-induced toxicity in human (astrocytes-like) D384 cells and comparatively weighed against the effect produced by the bulk counterpart of TiO2. Cellular uptake of TiO2NPs was also evaluated by both light microscopy visualization and ICP-MS measurement. Our data clearly support the evidence that both oxidative stress as well as apoptotic mechanisms occurred after low concentration exposure to TiO2NP in this type of CNS cell. The major findings indicate that low TiO2NP concentrations (from 1.5 to 62 mg/ml) were capable, already after 24 h, to induce subtle cellular perturbation in D384 cells altering check points involved in oxidative stress namely increase of ROS production, DNA damage and apoptotic markers, in particular: (i) ROS were generated in cells at 1.5 mg/ml concentration and further increased at the highest concentrations (31–62 mg/ ml). At the same low concentration (1.5 mg/ml) an increased

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Fig. 5. Immunofluorescence analysis of Bax protein expression. Analysis of Bax in D384 cells after 24 h incubation with increasing concentrations of TiO2NPs and TiO2 bulk (0.5–62 mg/ml). The presence of Bax protein was indicated by green spots starting at the concentration of 1.5 mg/ml in both TiO2NPs and TiO2 bulk treatment groups. Scale bar: 100 mm, insert scale bar: 200 mm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

expression of apoptotic markers, such as p-p53, p53, p21, Bax, and caspase 3 activation, was also observed that became more markedly evident at the highest concentration of 62 mg/ml. Parallely, Bcl-2 decreased with increasing TiO2NP concentration. Notably, the lowest effective concentrations, namely 1.5 and 15 mg/ml, were not cytotoxic based on MTT (mitochondrial function) results previously observed after short-term TiO2NP treatment (Coccini et al., 2015). (ii) Comparatively, similar effects were observed when testing TiO2 bulk. (iii) The no observed adverse effect level for these endpoints was 0.5 mg/ml. (iv) All effects were associated with increasing intracellular/ membrane attached Ti concentration. TiO2 (nanoparticulate and bulk form) associated with D384 cells was observed in a concentration-dependent manner (from 1.5 to 125 mg/ml) as evidenced by both light field microscopy and ICP-MS measurement. The microscopy images indicated that the TiO2NP aggregation was dependent on the concentration of

particles added to culture medium, and tended to be present in the perinuclear areas. Similar evidence was also observed for the TiO2 bulk. ICP-MS measurements showed concentration-dependent increase of Ti associated to astrocytes after both types of treatment (TiO2NP and TiO2 bulk) with parallel small amount of Ti content in the culture medium. The precise determination of NP content in biological samples is a critical issue for evaluating its potential effects on health. Up to day, no standard method is currently available for establishing the degree of cellular uptake of nanoparticles. Neither is there an established and reliable procedure for sample preparation which effectively differentiates attached vs. internalized NPs (Allouni et al., 2012). ICP-MS is widely regarded as a powerful technique for the quantification of internalized NPs elemental composition (Marquis et al., 2009) although this technique is unable to discriminate between NPs that are inside the cells from those that are attached to the cell membrane. A recent study (Ye et al., 2013) showed, in human capillary microvascular endothelial cells, that TiO2NPs were associated with the cell membrane and some were internalized inside vesicles

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Fig. 6. Immunofluorescence analysis of activated caspase 3 in D384 cells. Cells were incubated with increasing concentrations of TiO2NPs and TiO2 bulk (0.5–62 mg/ml) for 24 h. Activation of caspase 3 was indicated by green spots starting at the concentration of 1.5 mg/ml in both TiO2NPs and TiO2 bulk treatment groups. Scale bar: 100 mm, insert scale bar: 200 mm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

(likely clathrin-coated pits). Since the typical intracellular locations included endosomes, multivesicular bodies and lysosomes, the authors concluded that TiO2NPs were mainly trafficked along the endo-lysosomal pathway. Few in vitro studies on different CNS cell types (both from animals and humans) have been carried out to detect the toxic mechanism of TiO2NPs, and even more limited are especially those using cells of human brain-derived origin (see Table 1). Specifically, oxidative stress, apoptosis and cell cycle alteration, due to TiO2NPs were shown to be involved in the toxic mechanisms: effects were caused by low concentration exposure with the lowest effective TiO2NP concentrations being about 4 and 20 mg/ml in animal microglia (i.e., N9 and BV2) cells (Long et al., 2006, 2007; XiaoBo et al., 2009) and about 10 mg/ml in human astrocyte-like (U87) cells (Lai et al., 2008). The latter findings also indicated that either TiO2 nanoparticles or microparticles, irrespective of their sizes, induced apoptosis. A remarkable observation arising from all these in vitro studies is the evidence that brain effects were detectable not only at low

concentrations but even shortly (3–24 h) after just a single exposure. Evidence of a great relevance if translated in a more realistic situation of long-term exposure for potential chronic effects. Evaluation of these in vitro data by comparison with our results indicates that D384 cells are highly susceptible to TiO2NP exposure, since the critical concentration capable to induce effects was as low as 1.5 mg/ml after 24 h exposure give as a single hit. It should be emphasized that the in vivo cumulative exposure to nanosized TiO2 was shown to induce pathological responses in brain. For example, titanium was found to accumulate in the brains (0.100–0.500 mg/g vs 0.03 mg/g background Ti Levels) of mice injected daily into abdominal cavity for 14 days with nano-TiO2 particles (5 and 150 mg/kg: about 0.15–4.5 mg/mouse), and produced a reduction in brain weight, suggesting a causal relationship between nano-TiO2 and brain damage (Liu et al., 2009). A recent study also shows a significant increase in the bioaccumulation of the nano-TiO2 in the brain of rats treated intravenously, once a week for 4 weeks, with 5, 25, and 50 mg/kg of TiO2NPs. The concentrations of Ti were 2.28  0.37, 4.92  0.22, and

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Fig. 7. Western blot analysis. Evaluation of the expression levels by western blot analysis of: transcription factor (phosphorylated p53: p-p53), tumor suppressor (p53), cell cycle inhibitor (p21), pro-apoptotic (Bax), anti-apoptotic (Bcl-2), apoptotic (caspase 3) proteins in D384 cells induced by increasing concentrations of TiO2NPs or TiO2 bulk (1.5–62 mg/ml) after 24 h. b-actin was used as internal control to monitor for equal loading. Bar charts display the densitometric analyses. Representative data from three independent experiments are shown. Statistical analysis was performed by one-way ANOVA followed by Tukey’s test, *p < 0.05 vs. control.

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Table 1 Lowest concentrations of TiO2NP capable to induce toxicity on in vitro CNS cells. Cell lines

Exp. Time & Concentration

Mouse microglial cells (N9)

Concentration: 4, 8, 16, 32, 64, 125 mg/ ml ExpTime: 24 h

- Mouse microglial cells (BV2) - Rat mesencephalic neurons (N27) - Primary brain cultures of rat striatum

Main Results after TiO2NP exposure -

Morphologic changes: 4 mg/ml after 24 h TiO2NPs intracellular uptake: 8 mg/ml after 24 h Viability decrease: 16 mg/ml after 24 h Apoptosis: 16 mg/ml after 24h

Ref. XiaoBo et al. (2009)

N27

Long et al. (2007)

- Increased intracellular ATP levels: 80 mg/ml after 1 h and continued over 48 h (40 mg/ ml) - Increases in caspase 3/7 activity: 40 mg/ml after 24–48 h - TiO2NPs intracellular uptake: 20 mg/ml after 3 h Primary brain striatum cells (Rat) - Neuronal loss: 5 mg/ml after 6 h

Primary microglia (rat)

Ø 20 nm Concentration: 250, 500 mg/ml Exp. Time: 24–48 h

- Microglia activation, up-regulation of iNOS expression, TNF-alpha activation: 500 and 250 mg/ml, respectively, after 24 h - Up-regulation of MCP-1 and MIP-1alfa, NF-kB Activation: 500 mg/ml after 24 h

Xue et al. (2012)

Primary cultured hippocampal neurons (rat)

Ø 6 nm Concentration: 5, 15, 30, 40, 50 mg/ml Exp. Time: 6–48 h Ø 20 nm Concentration: 1, 25, 50, 100, 200 mg/ ml Exp. Time: 6, 24 h

- Decrease viability: at 5 mg/ml after 48 h (IC50 = 32.35 mg/ml) - Induction apoptosis (tunel, cytochrome c, caspase 3, Bax): 5 mg/ml

Sheng et al. (2015)

Rat PC12 cells

Human (U373) and rat (C6) glial cells

Ø 4–200 nm Concentration: 20 mg/cm2 Exp.Time: 2–48 h

- Viability decrease: 50 mg/ml after 24 h; Cell membrane damage: 25 mg/ml after 24 h Wu et al. (2010) - ROS generation: 100 mg/ml after 6 h; SOD and GSH Intracellular decrease: 50 mg/ml Liu et al. (2010) after 24 h; MDA intracellular increase: 100 mg/ml after 24 h - Changes of mitochondrial membrane potential: 100 mg/ ml after 24 h - Apoptosis/necrosis: 100 mg/ml after 24 h; and 25 mg/ml expression alterations of p21 GADD45, Bax, Bcl-2, after 24 h - Accumulation of G2/M phase: 100 mg/ml after 24 h - Oxidative stress in both cell types: ROS and lipid peroxidation production - Antioxidant Expression enzyme alterations (catalase, GPx, SOD2) in both cell lines - Mitochondrial depolarization in both cell types (effect: C6 > U373)

Huerta-García et al. (2014)

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Ø 30 nm BV2 Long et al. (2006) Concentration: 2.5, 5, 10, 20, 40, 60, 80, - ROS production: 20 mg/ml after 24 h 100, 120 mg/ml - Increases in mitochondrial’s membrane potential: 80 mg/ml after 20 min Exp. Time: 1, 6, 18, - TiO2NPs intracellular uptake: 2.5 mg/ml after 6 h 24, 48 h - Increases in caspase 3/7 activity: 40 mg/ml after 6 h - Reduced nuclear staining: 100 mg/ml after 24 h and 2.5 mg/ml after 48 h - Up-regul. inflammatory, apoptotic, cell cycling pathways; & down-regul. energy metabol: 20 mg/ml after 3 h

Lai et al. (2008) Viability decrease: 10 mg/ml after 48 h (IC50 = 34 mg/ml after 48 h) Cell damage: 50 mg/ml after 48 h (25% in LDH release) Morphological alterations: 1 mg/ml after 48 h Apoptosis/necrosis (annexin): 10 mg/ml after 48 h Ø 25 nm Concentration: 1, 10, 50, 100 mg/ml Exp.Time: 48 h Human astrocytoma (U87)

-

Ø 20 nm Concentration: 20, 80, 120, 150 mg/ml Exp.Time:3,6,24 h Human neuroblastoma (SH-SY5Y)

- TiO2NPs intracellular uptake: 80 mg/ml after 3 h Valdiglesias et al. (2013) - Cell cycle alterations: 80 mg/ml after 6 h - Apoptosis (annexin), mitochondrial membrane potential alterations and genotoxicity:  80 mg/ml after 3 h

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7.25  0.82 mg/g (ppm), respectively (Meena et al., 2015), and were accompanied by enhancement of brain ROS followed by a cascade of reactions including lipid peroxidation, DNA damage and reduction in the activities of key antioxidative enzymes (SOD, GPx, and CAT) involved in the oxidative defense mechanism. A significant increase in caspase 3 activity (at 50 mg/kg dose), DNA fragmentation and apoptosis at the highest doses (25 and 50 mg/ kg) was also reported. Other studies using protocols more realistic for an environmental exposure simulation (i.e., low dose of TiO2NP given by intranasal administration, i.n.) also described several brain pathological changes. In particular, reduction of hippocampal CA1 neurons, and increased oxidative stress and cytokine levels, associated to a high content of TiO2 in the brain ranging from 0.130 to 0.300 mg/ml (significantly higher than 0.050 mg/ml background levels), were observed after i.n. instillation of TiO2NP (0.5 mg/mouse; particle size: 80–155 nm) every other day for 30 days (Wang et al., 2008a,b). Similar brain Ti levels (0.500 mg/ml) were also reported in mice exposed to TiO2NP by whole-body inhalation (8 h/day, for 3 weeks at steady concentration of 6.34  0.22 mg/m3), although peculiar analogous high Ti levels were also measured in control unexposed animals (0.400 mg/ml) (Yin et al., 2014). On the basis of these in vivo results, the in vitro critical concentrations observed in the present investigation may be of relevant matter if related to the existing in vivo animal data on Ti levels detected in brain regions after i.n. administration of low repeated doses of TiO2NPs that were associated with brain damage. The detailed mechanistic pathways by which nanoparticles induce cellular damage or death are not yet fully elucidated. We observed an increased production of ROS in D384 cells after TiO2NPs using DHE, a cell permeable fluorescent dye that is oxidized by ROS present in the cytoplasm to ethidium bromide, a nucleic acid stain. The red fluorescence intensity in the nucleus corresponds to the amount of ROS inside the cells (Bucana et al., 1986; Perticarari et al., 1994; Shimoni et al., 2006). Thus, elevated ROS following nano-TiO2 treatment, as observed in these human derived astrocytes, may induce apoptosis in brain cells since oxidative damage to DNA and membranes has shown to induce the apoptosis. In particular, there are evidences suggesting that p53 plays a critical role in the cellular response to ROS-induced DNA damage and apoptosis in neurons and glial cells (Galluzzi et al., 2009; Mantovani et al., 2015). In general, p53 exists in an inactive state at low concentrations in cells, activated only when cells have undergone stress, resulting in the accumulation of p53 (Holly and St-Clair, 2009) and blocking the progression of cell cycle or cell death. p53 is regulated by intracellular redox state which in turn induces apoptosis by activation of certain targets like p21, Bax, and caspase 3 (Galluzzi et al., 2009). Indeed, our study demonstrated that p-p53, p53, p21 and Bax proteins were over expressed and Bcl-2 reduced, and caspase 3 activated already at the low TiO2NP dose of 1.5 mg/ml after 24 h exposure in D384 cells. Bax is an important pro-apoptotic factor that is involved in mitochondrial apoptosis. Following a death signal, Bax is translocated from the cytosol to the outer mitochondrial membrane where it promotes the release of different apoptogenic factors, such as Cytochrome c, via membrane permeabilization. The mitochondrial-mediated apoptotic pathway is controlled by pro-apoptotic (such as Bax) and anti-apoptotic (such as Bcl-2) members which play a central role to decide the cell fate. Caspase 3 is an important executioner caspase of apoptosis which mediate (at least part of) the catabolic processes that characterize end-stage apoptosis. In vivo study conducted by Ma et al. (2010) on ICR mice showed that oxidative stress induced by TiO2NPs played a significant role in induction of brain injuries. Similarly, Meena et al. (2015) demonstrated in rats that oxidative stress and injury of the brain

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occurred as TiO2NPs appeared to trigger a cascade of reactions such as inflammation, lipid peroxidation, alteration of neurotransmitter levels, as well as the increase of caspase 3 activity, increase expression of p53 and Bax protein and decrease of Bcl-2. Numerous mitochondrial membrane permeabilization regulators are implicated in cell loss also following acute neuronal injury (Galluzzi et al., 2009) and in the present study we demonstrated that some of them (i.e., p-p53, p53, p21, Bax, Bcl-2, caspase 3) other than ROS production (a condition of probably apoptosis trigger) were altered after brief exposure to TiO2NPs. Our data also indicate that the effects caused by TiO2NP were comparable to those caused by the TiO2 bulk counterpart when applying similar protocol of exposure time and concentrations. The International Agency for Research on Cancer (IARC) performed the last reassessment of TiO2 cancer potential in 2010 (IARC, 2010) and classified TiO2 as a human carcinogen group 2B, because there was enough evidence that nano-TiO2 may cause lung cancer by exposure through inhalation. TiO2 is also regulate in workplace by several agencies NIOSH (2011), OSHA (2002) and ACGIH (2001) that have set occupational exposure limits. Regarding to nanoparticulated TiO2 regulation, so far, only United States NIOSH (2011) proposed a recommended exposure limit (REL) for TiO2NPs at 0.3 mg/m3, which was about 10 times lower than the REL for TiO2FPs (FP: fine particles). These recommendations are based on using chronic inhalation studies in rats to predict lung tumor risks in humans. The nanoparticle REL reflects NIOSH’s greater concern for the potential carcinogenicity of ultrafine TiO2 particles as particle size decreases, the surface area increases (for equal mass), and the tumor potency increases per mass unit of dose. 5. Conclusions This study was warranted since there is an increasing interest in the potential toxicity of nanoparticles in the CNS, and there are very few studies of TiO2NP toxicity in glial cells (three times more abundant in brain than neuronal cells, and central to neuronal function regulation). Our results demonstrated that TiO2NPs cause subtle effects at low concentrations (i.e., 1.5–15 mg/ml) shortly (24 h) after a single treatment. Despite TiO2NPs have been policed (at least in occupational setting), there are still many concerns related to their small size and their potential toxic effects after inhalation. Human data related to absorption through inhalation of TiO2NPs are currently not available, however, data from rodent studies (see Shi et al., 2013 for review) show cerebral accumulation of Ti and adverse effects following repeated TiO2NPs exposure. This evidence corroborates the helpfulness of the in vitro assays for cellular TiO2NP toxicity evaluation, including those presently discussed, in that, the in vivo relevance is an essential criterion for accepting the in vitro assay utility (Han et al., 2012; Sayes et al., 2007). As a whole, the epidemiological studies imply that occupational exposures to TiO2 fine particles (or total dust) are not associated with increasing risk of cancers. Unfortunately, epidemiological studies of adverse health effects induced by TiO2NPs alone are lacking (Shi et al., 2013). Conflict of interest The authors declare that there is no conflict of interests regarding the publication of this paper.

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