Mechanisms of TiO2 nanoparticle-induced neuronal apoptosis in rat primary cultured hippocampal neurons Lei Sheng,1* Yuguan Ze,1* Ling Wang,2* Xiaohong Yu,1 Jie Hong,1 Xiaoyang Zhao,1 Xiao Ze,1 Dong Liu,1 Bingqing Xu,1 Yunting Zhu,1 Yi Long,1 Anan Lin,1 Chi Zhang,1 Yue Zhao,1 Fashui Hong1 1 2

Medical College of Soochow University, Suzhou 215123, China Library of Soochow University, Suzhou 215021, China

Received 1 June 2014; revised 16 June 2014; accepted 30 June 2014 Published online 00 Month 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.a.35263 Abstract: Exposure to titanium dioxide nanoparticles (TiO2 NPs) has been demonstrated to decrease learning and memory of animals. However, whether the impacts of these NPs on the recognition function are involved in hippocamal neuron damages is poorly understood. In this study, primary cultured hippocampal neurons from one-day-old fetal SpragueDawley rats were exposed to 5, 15, or 30 mg/mL TiO2 NPs for 24 h, we investigated cell viability, ultrastructure, and mitochondrial membrane potential (MMP), calcium homeostasis, oxidative stress, antioxidant capacity, apoptotic signaling pathway associated with the primary cultured hippocamal neuron apoptosis. Our findings showed that TiO2 NP treatment resulted in reduction of cell viability, promoted lactate dehydrogenase release, apoptosis, and increased neuron apoptotic rate in a dose-dependent manner. Furthermore,

TiO2 NPs led to [Ca21]i elevation, and MMP reduction, upregulated protein expression of cytochrome c, Bax, caspase3, glucose-regulated protein 78, C/EBP homologous protein and caspase-12, and down-regulated bcl-2 expression in the primary cultured hippocampal neurons. These findings suggested that hippocampal neuron apoptosis caused by TiO2 NPs may be associated with mitochondria-mediated signal pathway and endoplasmic reticulum-mediated signal pathC 2014 Wiley Periodicals, Inc. J Biomed Mater Res Part A: way. V 00A:000–000, 2014.

Key Words: titanium dioxide nanoparticles, primary cultured hippocampal neurons, calcium overload, mitochondria-mediated signal pathway, endoplasmic reticulum-mediated signal pathway

How to cite this article: Sheng L, Ze Y, Wang L, Yu X, Hong J, Zhao X, Ze X, Liu D, Xu B, Zhu Y, Long Y, Lin A, Zhang C, Zhao Y, Hong F. 2014. Mechanisms of TiO2 nanoparticle-induced neuronal apoptosis in rat primary cultured hippocampal neurons. J Biomed Mater Res Part A 2014:00A:000–000.

INTRODUCTION

Engineered nanoparticles with dimensions of approximately 1–100 nm exhibit novel physicochemical properties and functions, which make them extremely attractive for various areas in applications including medical science, agriculture, food, industry, and military sectors.1,2 Among many kinds of nanomaterials, titanium dioxide nanoparticles (TiO2 NPs) are widely applied in water, soil, and air decontamination and as whitening ingredient in the cosmetics, food additive, and paint industries. Its potential entry through dermal, ingestion, and inhalation routes exhibits an exposure risk to human beings.1 Moreover, TiO2 NPs can enter the central nervous system via the olfactory pathway and damaged the brain neurons and may be associated with neurodegenerative diseases.1,3–6 Therefore, the primary task of present research was to clarify the molecular mechanisms underlying the neurotoxicity of TiO2 NPs to animals and humans. In recent years, it was reported that various nanomaterials can induce neurotoxicity and cell death in vitro. For instance, the single-walled carbon nanotubes have been

shown to not only have a significant cytotoxicity on PC12 cells, induce lactate dehydrogenase (LDH) leakage and change cell morphology, but also result in overproduction of reactive oxygen species (ROS), increase level of lipid peroxidation and decrease mitochondrial membrane potential (MMP) and the antioxidant capacity in a dose-dependent manner, thereby causing neurons apoptosis.7 Moreover, TiO2 NPs have been shown to decrease the cell viability, increase LDH activity, and induce intracellular overproduction of ROS and the apoptosis of PC12 cells.8,9 In addition, TiO2 NPs could also stimulate brain microglia to produce excessive ROS through the oxidative burst, and interfere with mitochondrial energy production in vitro.10,11 All these researches indicated that NPs can induce neurotoxicity in vitro. Importantly, numerous studies in vivo demonstrated that nanoparticles can cross the blood–brain barrier12 and enter the central nervous system, ultimately cause lesion of brain in the exposed animals.3–6,13–19 Especially, TiO2 NPs can be translocated into the mouse hippocampus, and resulted in

*These authors contributed equally to this work. Correspondence to: F. Hong; e-mail: [email protected]

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hippocampal injury15,18,19 and impairment of spatial recognition memory in mice.14–19 Hippocampus as the first region of the brain to have damage with TiO2 NPs belongs to the limbic system of the brain and is related to the long-term memory and spatial navigation.15,20 Our previous studies have shown that the obvious reduction of spatial recognition memory in mice treated with TiO2 NPs is associated with hippocampal injury.15,18,19 Therefore, we hypothesize that decreased spatial recognition memory caused by TiO2 NPs may be involved in the hippocampal neuron damage and its developmental suppression during animal development. However, a few studies have been carried out to clarify the mechanism of TiO2 NPs on hippocampal neuron injury during animal development. In the present study, we selected fetal rat primary hippocampal neurons which retain neurodevelopment characteristics and its growth is associated with learning and memory, to explore the mechanism of action of TiO2 NPs without the influence of nutrition, endocrine status, and other variables,1 and focused on exploring whether TiO2 NPs would cause primary cultured hippocampal neuron apoptosis and activate signal pathways of apoptosis. MATERIALS AND METHODS

Chemicals Anatase TiO2 NPs were prepared via controlled hydrolysis of titanium tetrabutoxide. Details of the synthesis and characterization of TiO2 NPs were described in our previous reports.21 The particle size of TiO2 NPs suspended in neurobasal medium solution following incubation (5 mg/L) was determined using a TecnaiG220 transmission electron microscope (TEM) (FEI, USA) operating at 100 kV, respectively. In brief, particles were deposited in suspension onto carbon film TEM grids, and allowed to dry in air. The mean particle size was determined by measuring > 100 randomly sampled individual particles. X-ray-diffraction (XRD) patterns of TiO2 NPs were obtained at room temperature with a chargecoupled device (CCD) diffractometer (Mercury 3 Versatile CCD Detector; Rigaku Corporation, Tokyo, Japan) using Nifiltered Cu Ka radiation. The surface area of each sample was determined by Brunauer–Emmett–Teller (BET) adsorption measurements on a Micromeritics ASCORBIC ACIDP 2020M1C instrument (Micromeritics, USA). The average aggregate or agglomerate size of the TiO2 NPs after incubation in neurobasal medium solution (5 mg/L) was measured by dynamic light scattering (DLS) using a Zeta PALS 1 BI-90 Plus (Brookhaven Instruments, USA) at a wavelength of 659 nm. The scattering angle was fixed at 90 . Cell culture All animal experiments were approved by the Animal Care and Use Committee of Soochow University. Primary hippocampal neurons were prepared from the brains of new Sprague–Dawley rats about one day. The hippocampus were isolated from the brain and treated with 0.25% trypsin at 37 C for 10 min. Cells were suspended with Neurobasal medium (Gibco, NY, USA) containing 2% B27 supplement (Gibco, NY, USA) and 1% glutamine (Gibco, NY, USA) and

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were plated at a density 1.0–5.03105 cells/cm2 on poly-LLysine-coated cell culture clusters in a humidified 5% CO2 atmosphere at 37 C. Afterwards half of the medium was changed twice a week. The cultured neurons were identified by immunocytochemistry with antibody against neurone specific enolase (NSE), which is the marker for neurons. The culture with more than 95% neurons was used in the following experiments. MTT assay Cultured primary hippocampal neuron viability was determined by 3-(4, 5-Dimethyl-thiazol-2-yl)-2, 5-diphenyltetrazoliu-m bromide (MTT) assay. The neurons were plated in 96-well cell culture clusters at a density of 1 3 105 cells/mL in 100 lL medium. On the third day, neurons were cultured in medium with various concentrations (0, 5, 15, 30, 40, or 50 lg/mL) TiO2 NPs for 6, 12, 24, or 48 h, respectively. Ten lL of MTT was added into each well. Following incubation with 5% CO2 at 37 C for 4 h, 100 lL Formazan were added and the culture plate was shaken for 10 min. The optical density (OD) of each well was measured at 570 nm with a microplate Reader (Varioskan Flash, Thermo Electron, Finland). The results were calculated by the formula: S 5 (OD treated well2OD blank)/(OD control well2OD blank)3100%. Measurement of membrane integrity LDH leakage, which is another measurement of cell viability by detecting the activity of LDH released into the incubation medium when cellular membranes were damaged, was determined using a commercial LDH Kit (Genmed Scientifics, USA). At the end of various treatments, medium was mixed with nicotinamide adenine dinucleotide (NADH) solution and sodium pyruvate solution. Both agents were dissolved in a potassium phosphate buffer. The mixed solution was immediately assayed via a microplate Reader (Varioskan Flash, Thermo Electron, Finland) by monitoring the conversion of NADH to NAD at 440 nm at 37 C, coupled with the reduction of pyruvate to lactate. Observation of neuron ultrastructure Cultured primary hippocampal neurons were fixed in a fresh solution of 0.1M sodium cacodylate buffer containing 2.5% glutaraldehyde and 2% formaldehyde followed by a 2 h fixation period at 4 C with 1% osmium tetroxide in 50 mM sodium cacodylate (pH 7.2–7.4). Staining was performed overnight with 0.5% aqueous uranyl acetate. The specimens were dehydrated in a graded series of ethanol (75%, 85%, 95%, and 100%), and embedded in Epon 812. Ultrathin sections were obtained, contrasted with uranyl acetate and lead citrate, and observed with a HITACHI H600 TEM (HITACHI, Japan). Neuron apoptosis was determined based on the changes in nuclear morphology (e.g., chromatin condensation and fragmentation). TUNEL assay Cultured primary hippocampal neurons were plated on a poly-L-Lysine-coated glass coverslip at a density of 1 3 105

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were monitored by LSCM (Bio-Rad) at 488 nm excitation wavelength. Data were then analyzed by image analysis software (Bio-Rad) and expressed by intensity of Fluo-3/AM.

FIGURE 1. The (101) X-ray diffraction peak of anatase TiO2 NPs. The average grain size was about 5 nm by the calculation of Scherrer’s equation.

cells/cm2, and fixed in 4% paraformaldehyde in phosphatebuffered saline (PBS) at room temperature for 25 min. After washed with PBS, the cells were permeabilized in 0.2% Triton X-100 in PBS for 5 min, and covered with 100 lL of equilibration buffer at room temperature for 5–10 min. DNA strand breaks were labeled with fluorescein-12-dUTP using the recombinant terminal deoxy nucleotidyl transferase (rTdT) in equilibration buffer at 37 C for 1 h, avoiding exposure to light. The negative control was incubated with an incubation buffer but without rTdT enzyme. The reaction was terminated with 23SSC (300 mM sodium chloride and 30 mM sodium citrate, pH 7.4). After washed with PBS, the neurons were stained with 100 mL diaminobenzidine (DAB) at room temperature in the dark for 15 min.22 Apoptotic (TUNEL-positive) cells werediaminobenzidinedetected as localized bright brown cells by using Leica SP8 confocal microscopy (Bio-Rad, Hercules, CA, USA). Data were expressed as the ratio of apoptotic cells to total cells. Detection of MMP MMP by MMP assay kit with 5, 50 , 6, 60 -tetrachloro-1, 10 3, 30 tetrathylbenzimidazolyl-carbocyanine iodide (JC-1) was measured using the Leica SP8 laser scanning confocal microscope (LCMS) (Bio-Rad, Hercules, CA, USA). JC-1 is a convenient voltage sensitive probe to monitor MMP.23 Cells containing forming J-aggregates have high MMP, and show red fluorescence. Cells with low MMP are those in which JC-1 maintains (or reacquires) monomeric form, and show green fluorescence.24 The relative proportions of red and green fluorescence used to measure the ratio of mitochondrial depolarization. Measurement of intracellular Ca21 levels The [Ca21]i was monitored using the membrane-permeable Ca21-sensitive fluorescent dye Fluo-3 acetoxymethyl ester (AM). The Fluo-3/AM is converted to Fluo-3 upon deacetylation within the cells, and Fluo-3 increases green fluorescence upon Ca21 binding. Primary hippocampal neurons were incubated with 100 mL Fluo-3/AM solution (10 mg/ mL) at 37 C for 45 min. Microscopic images were captured with a Leica SP8 confocal system (Bio-Rad, Hercules, CA, USA). The intensity of Ca21 fluorescence and distribution

Assay of apoptotic cytokine protein expression Cultured primary hippocampal neurons were harvested and resuspended in 0.1 mol/L icecold PBS. Total protein from the cell suspensions from treated neurons and control neurons was extracted using Cell Lysis Kits (GENMED SCIENTIFICS, USA), centrifuged at 1, 0003g at 4 C for 10 min, and quantified using BCA protein assay kits (GENMED SCIENTIFICS, USA). To determine cytochrome c, caspase-3, caspase-12, Bax, Bcl-2, glucose-regulated protein 78 (GRP78), and C/EBP homologous protein (CHOP) levels, an enzymelinked immunosorbent assay (ELISA) was performed using commercial kits (R&D Systems, USA) that are selective for each respective protein. Manufacturer’s instruction was followed. The absorbance was measured on a microplate reader at 450 nm (Varioskan Flash, Thermo Electron, Finland), and the concentrations of cytochrome c, caspase-3, caspase-12, Bax, Bcl-2, GRP78, and CHOP levels were calculated from a standard curve for each sample. Statistical analysis Data were represented as mean 6 SD obtained from at least three separate experiments. Statistical analyses were performed by a SPSS 19.0 software (Chicago, IL, USA) and statistical comparisons were analyzed using one way ANOVA followed by Tukey’s HSD post hoc test. Differences were considered statistically significant when the p-value was less than 0.05. RESULTS

TiO2 NPs characteristics XRD measurements suggested that TiO2 NPs showed the anatase structure (Fig. 1), and the average particle size calculated from the broadening of the (101) XRD peak of anatase was approximately 5.5 nm using Scherrer’s equation. TEM showed that the average size of TiO2 NPs suspended in neurobasal medium ranged from 5–6 nm (Fig. 2), which was consistent with the XRD results. The surface area of the TiO2 NPs was 174.8 m2/g. To investigate the dispersion and stability of the suspensions of TiO2 NPs, we determined the aggregated size and f potential of TiO2 NPs in neurobasal medium. After incubation, the mean hydrodynamic diameter of TiO2 NPs in neurobasal medium ranged between 200 and 420 nm (mainly 300 nm), as measured by DLS (Fig. 3), which indicated that the majority of TiO2 NPs were clustered and aggregated in neurobasal medium. In addition, the f potential was 8.53 mV. Purities of primary hippocampal neurons The purities of the primary hippocampal neuron cultures were 95% (n 5 200 each), as determined by immunostaining with NSE. Cell viability As shown in Figure 4, cell viabilities were slightly decreased with the concentrations from 5 to 40 mg/mL for 6 h, but

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FIGURE 4. Effects of TiO2 NPs on neuron viability. *p < 0.05, **p < 0.01, and ***p < 0.01. Values represent means 6 SD (N 5 5). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] FIGURE 2. Transmission electron microscope images of anatase TiO2 NPs. TEM images showed that the sizes of the TiO2 NP suspended in neurobasal medium solvent were distributed from 5 to 6 nm.

only 50 mg/mL TiO2 NPs showed statistical significantly different (p < 0.05). When neurons were treated with TiO2 NPs for 12 h, significant decreases of cell viabilities were observed at the dose of 40 and 50 mg/mL (p < 0.05). After exposure to TiO2 NPs for 24 and 48 h, significant cytotoxicity of TiO2 NPs was observed at the concentrations from 5 to 50 mg/mL compared with control (p < 0.05). It was shown that cell viability decreased as a function of both time and dose by TiO2 NPs. When exposed to TiO2 NPs for 24 and 48 h, the values of IC50 were 42.97 and 32.35 mg/mL, respectively. Therefore, primary cultured hippocampal neurons were treated with 0, 5, 15, or 30 lg/mL TiO2 NPs for 24 h for following experiments. LDH activity Changes of LDH activity of primary cultured hippocampal neurons caused by TiO2 NP treatments for 24 h are presented in Figure 5. With increasing TiO2 NP concentration, LDH activities were greatly increased (p < 0.05), suggesting 19.54%, 36.13%, and 62.07% elevation, as compared with the control.

FIGURE 3. Hydrodynamic diameter distribution of TiO2 NPs in neurobasal medium solvent using DLS characterization. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary. com.]

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Observation of ultrastructure Changes to the primary cultured hippocampal neuron ultrastructure are presented in Figure 6. As shown, primary hippocampal neuron of the control group contained nuclei with homogeneous chromatin [Fig. 6(a)]; however, ultrastructure of primary hippocampal neuron treated with TiO2 NPs indicated a typical apoptosis, including significant mitochondrial swelling, carina disappearance, nucleus shrinkage, anomalous nuclear membrane, chromatin marginalization and dilation of endoplasmic reticulum (ER) [Fig. 6(b–d)]. To confirm further effects of TiO2 NPs on primary cultured neuron apoptosis, we examined apoptotic rate using TUNEL method and is shown in Figure 7. With increasing TiO2 NP concentration, primary neuron apoptotic rate were significantly increased (p < 0.05), equal to 2.51-, 3.14-, and 4.25-fold of the control value, respectively. MMP reduction To confirm further effects of TiO2 NPs on primary cultured hippocampal neuron mitochondrial injury, we assayed MMP in primary neuron under confocal fluorescence microscopy

FIGURE 5. Effects of TiO2 NPs on cell toxicity. *p < 0.05, **p < 0.01, and ***p < 0.01. Values represent means 6 SD (N 5 5).

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FIGURE 6. Ultrastructure of hippocampal neuron caused by TiO2 NPs (N 5 5). Control (10, 0003); 5 mg/mL TiO2 NP-treated group (80003); 15 mg/mL TiO2 NP-treated group (80003); 30 mg/mL TiO2 NP-treated group (80003). Yellow arrow indicates mitochondrial swelling and carina disappearance; green arrow indicates chromatin marginalization; red arrow indicates dilation of endoplasmic reticulum. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

and are shown in Figure 8. MMP in three TiO2 NP-treated groups were significantly lower than that in the control (p < 0.05), and equal to 84.2%, 60%, and 38% of control value, respectively. Ca21 concentration To confirm further effects of TiO2 NPs on neuron apoptosis, we assayed intracellular Ca21 concentrations in the primary cultured hippocampal neurons under confocal fluorescence microscopy and are shown in Figure 9. Cytoplasmic Ca21 concentrations in three TiO2 NP-treated groups were significantly higher than that in the control (p < 0.05), and equal to 1.55-, 2.66-, and 3.76-fold of control value, respectively. Apoptotic cytokine expression To confirm the role of apoptosis-related cytokines in the TiO2 NP-induced primary cultured neuron apoptosis, ELISA was used to demonstrate the changes of these cytokine expressions in the TiO2 NPs-treated neurons (Table I). In Table I, we observed that cytochrome c, caspase-3, Bax, caspase-12, GRP78, and CHOP expression in the treated neurons were significantly (p < 0.05), whereas the level of Bcl-2 expression was significantly inhibited by TiO2 NPs (p < 0.05).

DISCUSSION

Recently, the wider range of nanomaterial applications will almost certainly result in the increased potential hazards for organisms.1,2 Particularly, it was reported that TiO2 NPs impaired learning and memory of mice.14–19 Based on the previous studies, we hypothesize that TiO2 NPs have caused neuronal damage during animal development and we confirmed the hypothesis in the current study. The system of primary cultured rat hippocampal neurons subjected to TiO2 NPs was used as a model of neurodegenerative disease in this experiment. In this study, the degree of injury in primary cultured rat hippocampal neurons exposed to TiO2 NPs was examined using MTT activity and LDH release. Our data showed that treatments with TiO2 NPs from 5 to 50 mg/mL concentrations attenuated viability in a concentration-dependent manner or in a time-dependent manner (Fig. 4) and increased LDH release in a concentration-dependent manner (Fig. 5). These experimental findings indicated that TiO2 NPs do have cytotoxicity in primary cultured hippocampal neuronal cells and damaged their membrane integrity, which are similar to previous reports in PC12 cells8,9 and microglia.10,11,25,26 In the present study, however, cytotoxicity in primary cultured rat hippocampal neurons caused by

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the previous reports.8,9 It had been demonstrated that TiO2 NP (or aggregate) size determines if a NP enters the cellular environment though ROS-producing phagocytosis, through endocytosis, or some undefined mechanisms, while f potential of a NP not only affects its aggregation in solution and its behavior in an electric or ionic field, but also determines its interactions with specific biological receptors.8,10,11,27–31 Apoptosis is an active process with specific morphological changes, which was characterized chromatin condensation, nuclear DNA fragmentation, cell shrinkage, plasma membrane blebbing, and apoptotic body formation.32,33 In our study, we observed typical apoptotic features and

FIGURE 7. Effects of TiO2 NPs on neuron apoptotic rate. (a) Representative TUNEL photograms for apoptosis in the control and TiO2 NPtreated neurons. (b) Apoptotic rate in the control and TiO2 NP-treated neurons. *p < 0.05, **p < 0.01, and ***p < 0.001. Values represent means 6 SD (N 5 5). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

treatments with TiO2 NP concentrations (5–50 mg/mL) was more significant than that in PC12 cells by treatments with TiO2 NP concentrations (10–100, 25–200 mg/mL).8,9 It implied that primary hippocampal neurons seem to be very sensitive. In addition, the biological interactions of TiO2 NPs are involved in physical properties including specific surface area, crystal shape, f potential, and aggregate size.8,10,11,47–51 For valid interpretation of nanotoxicity data, we determined these NP physical properties in neurobasal medium that parallel the biological exposures. Our data suggested that TiO2 NP size, crystal shape, f potential, and aggregation could significantly affect its interaction with primary cultured hippocampal neurons and caused severe toxicity as compared to

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FIGURE 8. Changes of mitochondrial membrane potential in the hippocampal neurons caused by TiO2 NPs treatment. (a) Representative confocal images for mitochondrial injury in the neuron of control and TiO2 NP-treated neuron under confocal microscopy. (b) The levels of membrane potential are indicated by red fluorescence intensity versus green fluorescence intensity in neuron of control and TiO2 NP-treated neuron. *p < 0.05, **p < 0.01, and ***p < 0.001. Values represent means 6 SD (N5 5). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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FIGURE 9. Effect of TiO2 NPs on intracellular Ca21 concentration in the hippocampal neuron of rat. (a) Representative confocal images for Ca21 concentrations in the neuron of control and TiO2 NP-treated neuron under confocal microscopy. The levels of intracellular Ca21 are indicated by green. (b) Fluorescence intensity of representative intracellular Ca21 levels in neuron of control and TiO2 NP-treated neuron. *p < 0.05, **p < 0.01, and ***p < 0.001. Values represent means 6 SD (N 55). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

dilation of ER on the rat primary cultured hippocampal neurons caused by TiO2 NP treatments (Figs. 6 and 7). Previous reports in our laboratory had demonstrated that TiO2 NP

exposure resulted in hippocampal cell apoptosis in mice.15,16 Mitochondria act as final arbiters of life and death of the cell as these organelles not only are required to produce ATP but also can trigger apoptosis or necrosis.34 TiO2 NPs may cause depletion of the materials necessary to produce high energy phosphate and strongly affect the electron transport chain in mitochondrial. In the current study, therefore, we also conducted the MMP determination on rat primary cultured hippocampal neurons, suggesting greatly reductions in a concentration-dependent manner (Fig. 8). MMP can reflect performance of the electron transport chain and indicate a pathological disorder of this system.35 Mitochondrial membrane depolarization and subsequent cytochrome c release from inner membrane are supposed to be determining factors in the final step to apoptosis.36 Our data showed that TiO2 NPs significantly increased cytochrome c expression in rat primary cultured hippocampal neurons (Table I). Breakdown of Dwm is characteristic of early apoptosis. Mitochondrial dysfunction may be an early feature of rat primary cultured hippocampal neuron under TiO2 NP-induced toxicity. Mitochondria are susceptible targets of ROS-mediated damage.37 Hippocampal neurons injured by TiO2 NP may be involved in early apoptosis, and the oxidative stress properties of TiO2 NPs may contribute to the reduction of MMP. Calcium is an important second messenger that involves in neurotransmitter release and signal transduction. Alterations in intracellular Ca21 concentrations have been demonstrated to trigger a number of intracellular events, including apoptosis.38–40 Particularly, elevation of [Ca21]i leads to destabilization of the neuronal cell structure and causes cell damage, eventually cell death.41 To further explore the mechanisms of TiO2 NP-induced injury, we evaluated [Ca21]i overload. Our results showed TiO2 NPs significantly increased [Ca21]i concentrations (Fig. 9), which in turn resulted in apoptosis of primary cultured hippocampal neurons. Ca deposition or Ca21 overload and neurotransmitter reduction in mouse brain have been observed, and were associated with decreased learning and memory in mice.14 It is well known that TiO2 NPs have more biological activities to produce ROS and could be phagocytized by neurons and microglia, which then released ROS.7–11 After TiO2 NP treatment, Ca21 overload and thus may generate large amounts of ROS, making the dynamic balance between the oxidation and antioxidant in the primary cultured

TABLE I. Effects of TiO2 NPs on Expressions of Apoptosis-Related Cytokines in Neuron TiO2 NPs (mg/mL) Index Cytochrome c (ng/mL) Caspase-3 (ng/mL) Bax (ng/L) Bcl-2 (mg/mL) Caspase-12 (ng/mL) GRP78 (ng/L) CHOP (pg/mL)

0

5

15

30

16.75 6 1.52 13.11 6 1. 61 7.79 6 0.39 15.88 6 0.79 5.38 6 0.27 51.35 6 2.57 118.44 6 5.92

25.84 6 2.26* 28.51 6 2.45* 10.39 6 0.72* 14.51 6 0.73 5.94 6 0.30 59.3 6 2.97* 127.07 6 6.35

37.23 6 3.18** 37.35 6 3.91** 16.88 6 1.14** 12.74 6 0.64** 7.31 6 0.37** 65.44 6 3.27** 148.44 6 7.42**

63.22 6 7.75*** 54.61 6 4.75*** 22.08 6 1.65*** 10.37 6 0.52*** 8.73 6 0.44*** 70.1 6 3.51*** 166.32 6 8.32***

*p < 0.05, **p < 0.01, and *** p < 0.001. Values represent means 6 SD(N 5 5).

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hippocampal neurons to be destroyed. The previous studies in our laboratory have also demonstrated that TiO2 NP exposure resulted in overproduction of ROS and lipid peroxidation, reductions of the antioxidative enzymes including SOD, CAT, and GSH-Px as well as antioxidants like GSH and ascorbic acid (AsA) in mouse brain or hippocampus.13,15–17 The mitochondria are not only the major source of intracellular ROS generation, but also an important target for the damaging effects of ROS.42,43 Excessive ROS production can increase the permeability of mitochondrial membrane, decrease the MMP and contribute to the cytochrome c release from the mitochondria into the cytoplasm and then active downstream caspases, such as caspase-3 and eventually result in apoptosis.42,43 Furthermore, the mitochondriamediated apoptosis pathway is controlled by members of the Bcl-2 family consisting of pro-apoptotic (such as Bax) and anti-apoptotic members (such as Bcl-2), which play a central regulatory role to decide the fate of the cells.44 In our study, we observed TiO2 NP-induced apoptosis was accompanied by the increased level of intracellular Ca21, loss of MMP, and down-regulation of anti-apoptotic protein Bcl-2 level with concurrent up-regulation in cytochrome c, pro-apoptotic protein levels of Bax and caspase-3 in hippocampal cultured neurons (Table I), and this response occurred in a concentration-dependent manner. Hu et al. also found that hippocampal apoptosis was closely involved in increased expression of cytochrome c, Bax and caspase-3, and decreased Bcl-2 expression in mouse hippocampus following exposure to TiO2 NPs.15 The previous studies in vivo have indicated that TiO2 NPs induced ultrastructural changes of mouse hippocampal nerve cells, particularly the rough ER extended and free ribosome increased.5,6 In the present study, we also observed the dilation of ER in rat primary cultured hippocampal neurons treated with TiO2 NPs (Fig. 6). The ER is an organelle that plays an important role in proper folding and sorting of proteins, Ca21 homeostasis and cell death signal activation.45–48 In addition, recent studies suggested that certain pathological stress conditions such as oxidative stress and depletion of intracellular Ca21 stores can disrupt homeostasis and lead to accumulation of misfolded proteins in the ER, a condition referred to as ER stress.49,50 When ER stress is excessively severe for a long period, the unfolded protein response ultimately initiates ER-specific GRP78 expression. Then, the caspase-12 considered as critical in ER stress-induced apoptosis is activated during ER stress response.51 Furthermore, the ER stress associated CHOP is also up-regulated.49 Our data suggested that increased expression of GRP78, CHOP, and caspase-12 protein was associated with apoptosis on rat primary cultured hippocampal neurons in response to TiO2 NP treatments in a concentration-dependent manner (Table I). Therefore, these findings demonstrated that TiO2 NPs induced ER stress which mediated apoptosis on the primary cultured hippocampal neurons. In summary, we report a novel finding concerning the toxic effects of TiO2 NPs on rat primary cultured hippocampal neurons. Such toxic effects may be mediated by its

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action on apoptosis, oxidative stress, destabilization of MMP, the intracellular Ca21 elevation, and increased expression of apoptotic proteins. Primary hippocampal neuron apoptosis caused by TiO2 NPs may be involved in mitochondriamediated signal pathway and ER-mediated signal pathway. Furthermore, TiO2 NP exposure may influence hippocampal neuron development during animal development, in turn decrease learning and memory. ACKNOWLEDGMENTS

This work was supported by National Natural Science Foundation of China (grant No. 81273036, 30901218), A Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions, and the National Bringing New Ideas Foundation of Student of Soochow University (grant No. 201310285040Z). REFERENCES 1. Win-Shwe TT, Fujimaki H. Nanoparticles and Neurotoxicity. Int J Mol Sci 2011;12:6267–6280. 2. Suh WH, Suslick KS, Stucky GD, Suh YH. Nanotechnology, nanotoxicology, and neuroscience. Prog Neurobiol 2009;87:133–170. 3. Kreyling WG, Semmler M, Erbe F, Mayer P, Takenaka S, Schulz H, € rster G, Ziesenis A. Translocation of ultrafine insoluble Oberdo iridium particles from lung epithelium to extrapulmonary organs is size dependent but very low. J Toxicol Environ Health Part A 2002;65:1513–1530. € rster G, Sharp Z, Atudorei V, Elder A, Gelein R, Kreyling 4. Oberdo W, Cox C. Translocation of inhaled ultrafine particles to the brain. Inhal Toxicol 2004;16:437–445. 5. Wang JX, Chen CY, Liu Y, Jiao F, Li W, Lao F, Li YF, Li B, Ge CC, Zhou GQ, Gao YX, Zhao YL, Chai ZF. Potential neurological lesion after nasal instillation of TiO2 nanoparticles in the anatase and rutile crystal phases. Toxicol Lett 2008;183:72–80. 6. Wang JX, Liu Y, Jiao F, Lao F, Li W, Gu YQ, Li YF, Ge CC, Zhou GQ, Li B, Zhao YL, Chai ZF, Chen CY. Time-dependent translocation and potential impairment on central nervous system by intranasally instilled TiO2 nanoparticles. Toxicol 2008;254:82–90. 7. Wang JY, Sun PP, Bao YM, Liu JW, An LJ. Cytotoxicity of singlewalled carbon nanotubes on PC12 cells. Toxicol in Vitro 2011;25: 242–250. 8. Liu SC, Xu LJ, Zhang T, Ren GG, Yang Z. Oxidative stress and apoptosis induced by nanosized titanium dioxide in PC12 cells. Toxicology 2010;267:172–177. 9. Wu J, Sun J, Xue Y. Involvement of JNK and P53 activation in G2/M cell cycle arrest and apoptosis induced by titanium dioxide nanoparticles in neuron cells. Toxicol Lett 2010;199:269–276. 10. Long TC, Saleh N, Tilton RD, Lowry G, Veronesi B. Titanium dioxide (P25) produces reactive oxygen species in immortalized brain microglia (BV2): Implications for nanoparticle neurotoxicity. Environ Sci Technol 2006;40:4346–4352. 11. Long TC, Tajuba J, Sama P, Saleh N, Swartz C, Parker J, Hester S, Lowry GV, Veronesi B. Nanosize titanium dioxide stimulates reactive oxygen species in brain microglia and damages neurons in vitro. Environ Health Perspect 2007;115:1631–1637. 12. Lockman PR, Koziara JM, Mumper RJ, Allen DD. Nanoparticle surface charges alter blood–brain barrier integrity and permeability. J Drug Target 2004;12:635–41. 13. Ma LL, Liu J, Li N, Wang J, Duan YM, Yan JY, Liu HT, Wang H, Hong FS. Oxidative stress in the brain of mice caused by translocated nanoparticulate TiO2 delivered to the abdominal cavity. Biomaterials 2010;31:99–105. 14. Hu RP, Gong XL, Duan YM, Li N, Che Y, Cui YL, Zhou M, Liu C, Wang H, Hong FS. Neurotoxicological effects and the impairment of spatial recognition memory in mice caused by exposure to TiO2 nanoparticles. Biomaterials 2010;31:8043–8050. 15. Hu RP, Zheng L, Zhang T, Cui YL, Gao GD, Cheng Z, Chen J, Tang M, Hong FS. Molecular mechanism of hippocampal apoptosis of mice following exposure to titanium dioxide nanoparticles. J Hazard Mater 2011;191:32–40.

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