Titanium dioxide nanoparticles induce mouse hippocampal neuron apoptosis via oxidative stress- and calcium imbalance-mediated endoplasmic reticulum stress

Environmental Toxicology and Pharmacology 63 (2018) 6–15

Contents lists available at ScienceDirect

Environmental Toxicology and Pharmacology journal homepage: www.elsevier.com/locate/etap

Titanium dioxide nanoparticles induce mouse hippocampal neuron apoptosis via oxidative stress- and calcium imbalance-mediated endoplasmic reticulum stress

T

Qiong He, Xuejiao Zhou, Yang Liu, Wenfeng Gou, Jiahui Cui, Zengqiang Li, Yingliang Wu, ⁎ Daiying Zuo Department of Pharmacology, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenyang, Liaoning 110016, PR China

A R T I C LE I N FO

A B S T R A C T

Keywords: TiO2-NPs HT22 Cells Neurotoxicity Endoplasmic reticulum stress Oxidative stress Apoptosis

The purpose of this study was to explore the potential neurotoxicity and the underlying mechanism of titanium dioxide nanoparticles (TiO2-NPs) to mouse hippocampal neuron HT22 cells. We found that TiO2-NPs had concentration-dependent and time-dependent cytotoxicities to HT22 cells by the MTT assay. Propidium iodide (PI) staining with FACScan flow cytometry proved that TiO2-NPs dose-dependently increased the apoptosis rate in HT22 cells, and the apoptotic features were observed by Hochest 33258 and AO/EB staining. The levels of calcium (Ca2+) and reactive oxygen species (ROS) were significantly increased in TiO2-NPs-treated cells. Further studies by western blot and real-time QPCR proved that the protein and mRNA levels of GRP78, IRE-1α, ATF6, CHOP and caspase-12 were up-regulated after TiO2-NPs treatment, which indicates that TiO2-NPs-induced cytotoxicity is related to endoplasmic reticulum stress (ERS). Apoptosis-related protein cleaved caspase-3 and proapoptotic protein Bax expression levels were up-regulated, and the anti-apoptotic protein Bcl-2 expression level was down-regulated in TiO2-NPs-treated cells. The antioxidant N-acetyl-L-cysteine (NAC) can significantly reduce TiO2-NPs-induced ERS characterized by the down-regulation of GRP78 and cleaved caspase-12 levels, which indicates that oxidative stress is participated in TiO2-NPs-induced ERS. Our study suggests that TiO2-NPsinduced apoptosis in HT22 cells is through oxidative stress- and calcium imbalance-mediated ERS.

1. Introduction Due to the excellent physicochemical properties of large specific surface area, small particle size and efficient drug loading, titanium dioxide nanoparticles (TiO2-NPs) have been widely applied in industry, agriculture, medicine, food and other fields (Oberdörster et al., 2005). However, the widespread applications of TiO2-NPs have given rise to new cognition and attention. They may in advertently cause human infection and environmental pollution. Thus, in recent years, numerous in vitro and in vivo researches have been implemented to probe into the potential toxic properties of TiO2-NPs (Coccini et al., 2015; Numano et al., 2014; Younes et al., 2015). TiO2-NPs may be absorbed via oral

inhalation, food digestion and skin penetration, and then redistributed to other tissues (such as the brain, lung, liver, heart, etc.) through the circulation system, which may cause organ damage (Fabian et al., 2008; Liu et al., 2014). Significantly, in vivo studies have shown that TiO2NPs can directly reach to the olfactory bulb by breathing and then transfer to other brain tissue (Wang et al., 2008), or directly across the blood-brain barrier (BBB) into the central nervous system (Tsyganova et al., 2014) by destroying the structure and function of the BBB (Brun et al., 2012), which will affect most of the cells within the nervous system including neurons and glial cells. TiO2-NPs-induced inflammation, apoptosis and decrease of antioxidant capacity can lead to cell death, central nervous system disorders, and even lead to

Abbreviations: AO/EB, acridine orange/ethidium bromide; ATF6, activating transcription factor 6; BBB, blood-brain barrier; BSA, bovine serum; Ca2+, calcium; DAPI, 4,6-diamino-2-pheno-lindol dihydrochloride; DCF, 2’,7’-dichlorofluorescein; DCFH-DA, 2’,7’-dichlorodihydrofluorescein diacetate; DLS, dynamic light scattering; DMEM, Dulbecco’s modified eagle medium; ER, endoplasmic reticulum; ERS, endoplasmic reticulum stress; FBS, fetal bovine serum; GRP78, glucose regulated protein of molecular weight 78 (also called BiP); IRE1, inositol-requiring kinase/endoribonuclease 1; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NAC, N-acetyl-L-cysteine; PERK, protein kinase activated by double-stranded RNA (PKR)-like ER kinase; PI, propidium iodide; ROS, reactive oxygen species; TEM, transmission electron microscopy; TiO2-NPs, titanium dioxide nanoparticles; UPR, unfolded protein response; XRD, X ray diffraction ⁎ Corresponding author. E-mail address: [email protected] (D. Zuo). https://doi.org/10.1016/j.etap.2018.08.003 Received 10 February 2018; Received in revised form 2 July 2018; Accepted 3 August 2018 Available online 10 August 2018 1382-6689/ © 2018 Elsevier B.V. All rights reserved.

Environmental Toxicology and Pharmacology 63 (2018) 6–15

Q. He et al.

dissolved in culture solution or water at 200 μg/ml, ultrasonicated for 30 min, and then mixed using a shaker for TEM/DLS characterization. TiO2-NPs powder was used for XRD detection.

neurodegenerative diseases (Huang et al., 2015; Meena et al., 2015; Ze et al., 2014). Endoplasmic reticulum (ER) is a multi-functional, highly dynamic organelle, which accommodates protein synthesis, folding and secretion in eukaryotic cells (Cao et al., 2017). Many factors, such as oxidative stress (Liang et al., 2016), ER calcium (Ca2+) metabolism disorder (Song et al., 2016), excessive synthesis of cholesterol (Barbero-Camps et al., 2014) and protein misfolding, may disrupt ER homeostasis and lead to ER stress (ERS), and then induce the unfolded protein response (UPR) to adapt the changes (Ron and Walter, 2007). UPR activates three signaling proteins named as inositol-requiring kinase/endoribonuclease 1 (IRE1) (Lee et al., 2002), activating transcription factor 6 (ATF6) (Nadanaka et al., 2007) and protein kinase activated by double-stranded RNA (PKR)-like ER kinase (PERK) (Wek and Cavener, 2007), which are responsible for the ERS and the transmission of signals. If the adjustment of UPR does not control the ERS, the cells will enter to the apoptotic pathway. Sustained ERS may induce apoptosis through a variety of mechanisms including up-regulation of CHOP expression (Yamaguchi and Wang, 2004), activation of ER pro-apoptotic cysteine protease caspase-12 (Nakagawa et al., 2000), and phosphorylated IRE-1α-mediated c-Jun-N-terminal kinase (JNK) activation (Yang et al., 2006). Although TiO2-NPs-induced neurotoxicity has been reported in some experimental animals or cell cultures, the underlying mechanisms remain unclear. Therefore, further studies are still essential to focus on a deeper insight into the molecular mechanisms behind the observed effects. Since ERS plays an important role in cell injury, and to our knowledge, there is no study investigating the relationship between TiO2-NPs-induced neural cell apoptosis and ERS, mouse hippocampal neuron HT22 cells were used in this study to probe into the role of ERS in TiO2-NPs-induced neural cell apoptosis. We hypothesized that oxidative-mediated ERS might be involved in TiO2-NPs-induced neurotoxicity.

2.3. Cell culture Mouse hippocampal neuron cell line HT22 was purchased from JENNIO Biological Technology (Guangzhou, China). HT22 cells were cultured at 37 °C in DMEM complemented with 10% FBS, 100 mg/ml streptomycin, and 100 U/ml penicillin in humidified atmosphere containing 5% CO2. All of the TiO2-NPs solutions were ultrasonicated for 30 min, and then mixed using a shaker before incubation with the cells. 2.4. Cell viability assay The cell viability was evaluated by the MTT assay. HT22 cells were treated with TiO2-NPs at different doses (12.5, 25, 50, 100 and 200 μg/ ml). After incubation for 24 h, 20 μl of MTT (5.0 mg/ml) was added to each well and incubated for 4 h. The absorbance at 492 nm was determined by microplate reader (Multiskan MK3, Thermo, USA). The doses of TiO2-NPs used in this study were based on our preliminary experiments and the references (Sheng et al., 2015; Wu et al., 2010). 2.5. Observation of cellular morphology HT22 cells were seeded onto 6-well plate at a density of 2 × 105 cells per well. After incubation with the TiO2-NPs for 24 h, the cells were stained with Hoechst 33258 solution for 20 min. Then the morphological changes of nuclei were observed with fluorescence microscopy (Olympus, Tokyo, Japan). 2.6. Detection of morphological apoptotic cells

2. Materials and methods Apoptotic morphological changes of HT22 cells were detected by AO/EB staining. HT22 cells were plated onto 6-well plate. After being cultured for 24 h, HT22 cells were treated with TiO2-NPs for 24 h. Then each well was added the AO/EB mixed solution 20 μl (AO:EB = 1:1). After 5 min, the cells were washed three times with PBS, observed and photographed with a fluorescent microscope.

2.1. Materials and reagents TiO2-NPs (anatase) with 99% purity and diameter of 50 nm were obtained from Hao Tian Nano Technology Co., Ltd. (Shanghai, China). Fetal bovine serum (FBS) and dulbecco’s modified eagle medium (DMEM) were obtained from GIBCO (USA). Propidium iodide (PI), 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), acridine orange/ethidium bromide (AO/EB) and 4, 6-diamino-2-phenolindol dihydrochloride (DAPI) were purchased from Sigma (USA). Ca2+ kit, 2’,7’-dichlorodihydrofluorescein diacetate (DCFH-DA) and Nacetyl-L-cysteine (NAC) were purchased from Beyotime (Nanjing, China). Albumin from bovine serum (BSA) was purchased from Biosharp (Hefei, China). Rabbit anti-CHOP, -GRP78 -ATF6, -PERK, -pPERK, -IRE-1α, -p-IRE-1α IgG were purchased from Bioss (Beijing, China). Rabbit anti-caspase-3 and -caspase-12 IgG were purchased from Proteintech (Wuhan, China) and Santa Cruz Biotechnology (USA) respectively. Rabbit anti-Bax, -Bcl-2, horseradish peroxidase conjugated and FITC conjugated secondary antibodies were purchased from Santa Cruz Biotechnology (USA). Rabbit anti-cleaved caspase-3 was obtained from Cell Signaling Technology (USA). Trizol reagent was purchased from Qiagen (Germany). HiFiScript cDNA Synthesis Kit and Greenstar qPCR Master Mix were obtained from Cwbio (China) and Bioneer (South Korea) respectively.

2.7. Detection of sub-G1 hypodiploid cells Flow cytometry was used to analyze the amount of apoptotic sub-G1 hypodiploid cells. HT22 cells were treated with TiO2-NPs at various doses and incubated for 24 h. Then harvested cells were washed with PBS for three times, fixed in ice-cold 70% ethanol overnight, and stained with PI at 4 °C for 1 h. Then, the cells were tested with FACScan flow cytometry (Becton–Dickinson, Franklin Lakes, NJ, USA). 2.8. Intracellular reactive oxygen species (ROS) measurement Intracellular esterase can hydrolyze the stable, nonpolar compound DCFH-DA to DCFH. ROS in the cells can oxidize DCHF to highly fluorescent compound 2’,7’-dichlorofluorescein (DCF). The fluorescence intensity can be used to detect the generation of ROS in cells by confocal microscopy (Swift and Sarvazyan, 2000). In this study, the cells were seeded at 2 × 105 cells/well in 6-well plates with cover glass in each well. The cells were incubated with DCFH-DA for another 30 min, after treatment with TiO2-NPs. The images were observed and photographed using confocal microscopy (Nikon C2, Tokyo, Japan) at a maximum excitation wavelength of 480 nm and a maximum emission wavelength of 525 nm, and the fluorescence intensity was measured with Image J software.

2.2. Characterization of TiO2-NPs To characterize TiO2-NPs, transmission electron microscopy (TEM) (Hitachi H-600, Hitachi, Japan), X ray diffraction (XRD) (D/max 2000 X, Rigaku, Japan) and dynamic light scattering (DLS) (Zetasizer Nano ZS90, Malvern, UK) were applied. For these measures, TiO2-NPs were 7

Environmental Toxicology and Pharmacology 63 (2018) 6–15

Q. He et al.

Fig. 1. Characterization of TiO2-NPs. (A) Transmission electron microscope images of TiO2-NPs. Scale bar = 100 nm. (B) X-ray diffraction pattern of TiO2-NPs.

2.9. Intracellular Ca2+ measurement

2.13. Western blot analysis

Fluo-4/acetoxymethyl ester (AM) was used to monitor the intracellular Ca2+ levels. In the cells, Fluo-4/AM can be converted to Fluo-4 upon deacetylation, and Ca2+ can bind with Fluo-4 to increase green fluorescence. Cells were incubated with 1 ml Fluo-4/AM solution (10 mg/ml) for 30 min at 37 °C after treatment with TiO2-NPs. Microscopic images were achieved with confocal microscopy (Nikon C2, Tokyo, Japan). Fluorescence intensity was measured with Image J software.

After incubation with TiO2-NPs, the cells were harvested. A protein assay reagent was used to determine the protein content of isolated supernatant. Equivalent amounts of protein (40 mg) were separated by electrophoresis on 8–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After that, the protein was transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore Bedford, MA, USA). 5% skimmed milk was used to soak the membranes. Then primary antibodies were incubated with target antigens at 4 °C overnight, and the next day, target antigens were incubated with a horseradish peroxidase (HRP)-conjugated secondary antibodies (1:3000) for 2 h. The enhanced chemiluminescence was used to visualize proteins. Densitometry analysis was detected using Image J software.

2.10. ER structure by transmission electron microscopy HT22 cells were incubated with TiO2-NPs (200 μg/ml) for 24 h. The cultured cells were digested with trypsin, centrifuged, and fixed with 2.5% glutaraldehyde. Then, ultrathin sections (1 μm) were examined by TEM (Hitachi, Tokyo, Japan).

2.14. Statistical analysis The data were expressed as the mean ± SD from at least three representative tests, unless otherwise stated. Statistical significance was assessed by the one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison tests and two-way ANOVA using SPSS software 19.0. p-value < 0.05 was considered to be statistically significant.

2.11. Analysis of ER stress markers by Real-time QPCR The mRNA levels of ER stress-related genes were determined by HiFiScript cDNA Synthesis Kit and Greenstar qPCR Master Mix, and detected with real-time QPCR System (Stratagene M × 3000P). Total RNA was extracted from HT22 cells with Trizol reagent (Qiagen, Germany). The primer sequences were as follows: GRP78 5’- GAAGG AGGATGTGGGCACG-3’ and 5’-CGCATCGCCAATCAGACG-3’; CHOP 5’AGTCCCTGCCTTTCACCTT-3’ and 5’-GCTTTGGGATGTGCGTGTG-3’; Caspase-12 5’-TGGAAGGTAGGCAAGACT-3’ and 5’-ATAGTGGGCATCT GGGTC-3’; ATF6 5’-ACCTGTTCTTCCTCTGAAATCCAA-3’ and 5’-AGGA CAGAGAAACAAGCTCGG-3’; IRE-1α 5’-GCGATGGACTGGTGGTAACT3’ and 5’-TGCACCACCTTTCTCAGGAC-3’; PERK 5’-CCGCAAGAAGGAC CCTATCC-3’ and 5’-GAGTTTCAGACTCCTTCCGCT-3’; GAPDH 5’-GTAT GACTCCACTCACGGCAA-3’ and 5’-CACCAGTAGACTCCACGACA-3’.

3. Results 3.1. Characterization of TiO2-NPs As shown in Fig. 1A, TiO2-NPs had spherical shape in the cell culture medium, the average TEM diameter of TiO2-NPs was 46.19 ± 7.54 nm and the surface of TiO2-NPs was smooth. XRD analysis showed that TiO2-NPs had the amorphous nature (Fig. 1B). The indexes of TiO2-NPs detected by DLS and TEM are presented in Table 1. DSL results proved that TiO2-NPs formed small agglomerates in the culture medium. Hydrodynamic sizes of TiO2-NPs in DMEM and water were 653.4 nm and 387.4 nm, respectively.

2.12. Immunofluorescence staining The cells were seeded on 24-well plates at 2 × 104 cells/well. After incubation with TiO2-NPs for 24 h, the cells were fixed with 4% formaldehyde for 30 min, and washed three times with PBS and then the cells were permeabilized with 0.1% (v/v) Triton X-100 for 5 min. After the cells were blocked with BSA for 30 min, the primary CHOP antibody, which was diluted (1:100) with BSA, was incubated with cells at 4 °C overnight. The next day, the cells were washed three times with PBS, and then the cells were incubated with FITC-conjugated antirabbit secondary antibody at 37 °C for 2 h. Then, the cells were washed three times with PBS and stained with DAPI for 30 min. Immunofluorescence was detected with a fluorescent microscope.

Table 1 Characterization of TiO2-NPs.

8

Characterization

Method

Condition

Results

Size Hydrodynamic size Zeta Potential Hydrodynamic size Zeta Potential

TEM DLS DLS DLS DLS

Dry Water Water DMEM DMEM

46.19 ± 7.54 nm 387.4 nm 19.2 mV 653.4 nm −17 mV

Environmental Toxicology and Pharmacology 63 (2018) 6–15

Q. He et al.

3.3. TiO2-NPs induce apoptosis in HT22 cells In this study, HT22 cells were used to explore if TiO2-NPs could induce cell apoptosis and the underlying mechanism. As depicted in Fig. 3A, after incubation for 24 h with the TiO2-NPs (50, 100 and 200 μg/ml), HT22 cells exhibited obvious morphological changes characterized by nuclear condensation and fragmentation, which suggests the occurrence of apoptosis in HT22 cells. In order to discriminate the live cells from dead ones, AO/EB staining was applied. AO is a vital dye which can penetrate normal cell membrane, and appear as distinct spots in green color. While EB stains the early apoptotic cells in yellow color and the late apoptotic cells that have lost membrane integrity in orange color. In our study, nuclei in HT22 cells of the control group were stained in green with uniform shape by AO. Some nuclei were stained by EB in HT22 cells after TiO2NPs treatment, showing distinct orange or yellow fluorescence, which indicates that TiO2-NPs damaged cell membrane integrity and might induce HT22 cell apoptosis (Fig. 3B). Therefore, apoptosis was further analyzed with the level of sub-G1 hypodiploid DNA by flow cytometry. As shown in Fig. 3C and D, incubation with TiO2-NPs resulted in marked and dose-dependent increase of accumulation of sub-G1 phase cells.

Fig. 2. Inhibition ratio of HT22 cells after being treated with TiO2-NPs (12.5, 25, 50, 100 and 200 μg/ml) for 6, 12, 24 or 48 h (n = 3).

3.2. Cytotoxicity of TiO2-NPs HT22 cells were treated with TiO2-NPs (50 nm) for 6, 12, 24 and 48 h at 12.5, 25, 50, 100 and 200 μg/ml. Then, the cytotoxicity of TiO2NPs was investigated by the MTT assay. As shown in Fig. 2, TiO2-NPs exhibited potent cytotoxicity against HT22 cells in dose-dependent and time-dependent manners. The two-way ANOVA indicates that both concentration and time had significant effects on the inhibition rate (p < 0.01), and the concentration × time also had statistical significance (p < 0.05), which indicates that positive interactions might exist between concentration and time.

3.4. TiO2-NPs elevate intracellular ROS and Ca2+ levels To further investigate the potential mechanisms of neurotoxicity, we assayed the intracellular ROS and Ca2+ levels in HT22 cells under confocal microscopy after treatment with TiO2-NPs at 50, 100 and 200 μg/ml. In the three TiO2-NPs-treated groups, intracellular ROS levels were significantly increased (Fig. 4A and B), which suggests that

Fig. 3. Apoptotic changes of HT22 cells after exposure of TiO2-NPs. (A) In Hoechst 33258 staining images, nuclear condensation and fragmentation were observed. Scale bar = 50 μm. (B) In AO/EB staining images, normal cells were in green, and cells in yellow and orange represente early and late apoptotic stages, respectively. Scale bar = 50 μm. (C) TiO2-NPs (50, 100 and 200 μg/ml) induced apoptosis in a concentration-dependent manner in HT22 cells. Apoptotic cells with hypodiploid DNA were determined by FACS with PI staining. (D) Quantified apoptotic ratio (n = 3, **p < 0.01 vs control group) (For interpretation of the references to color in text, the reader is referred to the web version of this article). 9

Environmental Toxicology and Pharmacology 63 (2018) 6–15

Q. He et al.

Fig. 4. Alteration of intracellular ROS and Ca2+ levels in HT22 cells after incubation with TiO2-NPs (50, 100 and 200 μg/ml) for 24 h. (A) Intracellular ROS levels were evaluated by DCFH-DA (green). Fluorescence images were acquired by confocal microscope. (B) Quantitative analysis of intracellular ROS levels in HT22 cells. (C) Intracellular Ca2+ levels were detected by Fluo-4/AM (green) under confocal microscopy. (D) Quantitative analysis of intracellular Ca2+ levels in HT22 cells (n = 3, *p < 0.05, **p < 0.01 vs control group) (For interpretation of the references to color in text, the reader is referred to the web version of this article).

caspase-3 was increased after TiO2-NPs treatment (Fig. 7A and B). We also found that HT22 cells exposure to TiO2-NPs decreased the expression level of Bcl-2 and increased the expression level of Bax (Fig. 7A and B).

TiO2-NPs could induce oxidative stress in HT22 cells. Our results also proved that TiO2-NPs significantly increased the intracellular Ca2+ concentrations (Fig. 4C and D). 3.5. TiO2-NPs damage the ER and induce ERS in HT22 cells

3.7. NAC attenuates TiO2-NPs-induced ERS and apoptosis In the interest of exploring the change of ER, the ultrastructure changes of ER were observed with TEM after HT 22 cells being treated with TiO2-NPs (200 μg/ml) for 24 h. As shown in Fig. 5A, the ER became swollen and vacuolated after treatment with TiO2-NPs compared with the control group. To further explore the role of ERS in the damage of HT22 cells induced by TiO2-NPs, ERS-related mRNA levels were analyzed by real-time QPCR. After TiO2-NPs treatment, the mRNA levels of GRP78, IRE-1α, ATF6, CHOP and caspase-12 were dose-dependently up-regulated. However, the PERK mRNA level remained essentially unchanged (Fig. 5B). To further prove if ERS is involved in the mechanism of TiO2-NPsinduced apoptosis, ERS-related proteins including IRE-1α, p-IRE-1α, ATF6, PERK, p-PERK, GRP78, and cleaved caspase-12 were tested by western blot, and CHOP protein was detected by immunofluorescence. Fig. 6A and B showed that TiO2-NPs significantly up-regulated the expression of GRP78, ATF6 and p-IRE1α. Our results also proved that TiO2-NPs up-regulated CHOP expression (Fig. 6C), and the level of cleaved caspase-12 was also significantly increased after TiO2-NPs treatment (Fig. 6A and B)

To confirm the role of oxidative stress in ERS-induced apoptosis, antioxidant NAC was applied. NAC (5 mM) was incubated with HT22 cells for 2 h before exposure to TiO2-NPs (200 μg/ml) for 24 h. We found that NAC decreased the accumulation of ROS in HT22 cells (Fig. 8A and B). NAC pretreatment also reversed TiO2-NPs-induced elevation of GRP78, cleaved caspase-12 and cleaved caspase-3 levels (Fig. 8C and D). 4. Discussion To date, the mechanisms of TiO2-NPs-induced cytotoxicity are still not completely clear. The toxic effects of TiO2-NPs with different physicochemical characteristics on the brain are not the same (Liu et al., 2013). Therefore, it is necessary to further explore the underlying neurotoxicity of TiO2-NPs with specific characteristics. Firstly, we used TEM, XRD and DLS techniques to achieve the characterization of TiO2NPs. TEM result showed that TiO2-NPs had spherical shape in cell culture medium, the average TEM diameter of TiO2-NPs was 46.19 ± 7.54 nm and the surface of TiO2-NPs was smooth. Hydrodynamic sizes of TiO2-NPs in DMEM and water were 653.4 nm and 387.4 nm, respectively. The slight decrease in the hydrodynamic sizes obtained in water compared to the sizes in the culture medium might be owing to some agglomeration of TiO2-NPs in DMEM. Hippocampus, a major component of the brain in humans and other mammals, belongs to the limbic system, and plays an important role in

3.6. TiO2-NPs induce apoptosis through ERS in HT22 cells To further prove if TiO2-NPs-induced apoptotic pathway was related with ERS, the apoptosis-related proteins in the downstream of ER were detected. Our data showed that the expression levels of pro-caspase-3 and pro-casepase-9 were decreased and the expression level of cleaved 10

Environmental Toxicology and Pharmacology 63 (2018) 6–15

Q. He et al.

Fig. 5. Morphological changes of ER and ERS-related mRNA levels after treatment with TiO2-NPs. (A) HT22 cells were treated with TiO2-NPs (200 μg/ml) for 24 h, the ER became swollen and vacuolated, as the pointing of red arrows. White arrows mean normal ER. (B) Effect of TiO2-NPs on the mRNA levels of GRP78, IRE-1α, ATF6, PERK, CHOP and caspase-12 in HT22 cells (n = 3, *p < 0.05, **p < 0.01 vs control group) (For interpretation of the references to color in text, the reader is referred to the web version of this article).

Sheng et al., 2015). In this study, AO/EB and Hoechst 33258 staining showed that TiO2-NPs damaged cell membrane integrity and might induce HT22 cell apoptosis. Meanwhile, incubation with TiO2-NPs resulted in marked and dose-dependent increase of accumulation of subG1 phase cells, which further proved that TiO2-NPs induced apoptosis in HT22 cells. Studies have shown that nanomaterials can induce the production of ROS in vivo. ROS is associated closely with inflammation, oxidative stress, organelle damage and other cell damage (Du et al., 2012; Lu et al., 2015). Previous studies have indicated that TiO2-NPs were able to induce cytotoxicity by increasing the accumulation of ROS to disrupt redox homeostasis (Du et al., 2012; Lu et al., 2015). In the present study, intracellular ROS levels in the TiO2-NPs-treated groups were significantly increased, which suggests that TiO2-NPs could induce oxidative stress in HT22 cells. Ca2+, a very important second messenger, is involved in neurotransmitter release and signal transduction. Changes of intracellular Ca2+ concentrations can cause a series of cellular responses, particularly cell instability and then cell damage (Palop and Mucke, 2010). In order to investigate the potential mechanism of HT22 cell injury induced by TiO2-NPs, we examined the intracellular Ca2+ levels. Our

long-term memory and spatial navigation (Hu et al., 2011). TiO2-NPs can easily enter the body through inhalation, cross the BBB and accumulate in the brain, especially in the cortex and hippocampus. Some in vivo studies have indicated that TiO2-NPs exposure affected animal behaviors. Intragastric or intranasal administration of TiO2-NPs damaged the spatial recognition memory, as indicated by the Y-maze or Morris water maze tests (Hu et al., 2011; Ze et al., 2014). HT22 murine hippocampal neuronal cell line possesses essential properties of functional cholinergic neurons, and they can be used as an in vitro model for studies relevant to hippocampal function and memory formation and dysfunction (Liu et al., 2009). HT22 cells were also widely used to investigate neurotoxicity (Shim and Kwon, 2012; Wu et al., 2015). Therefore, HT22 cells were used in our study to explore the underlying mechanism of TiO2 NPs-induced neurotoxicity. Our data showed that TiO2-NPs exhibited potent cytotoxicity against HT22 cells in dose-dependent and time-dependent manners. Therefore, it is certainly worth to probe into the mechanism involving in the TiO2-NPs-induced cytotoxicity. Apoptosis, a programmed cell death, is usually caused by physiological or pathological factors. Previous studies reported that exposure to TiO2-NPs could cause apoptosis in different cells (Lu et al., 2011; 11

Environmental Toxicology and Pharmacology 63 (2018) 6–15

Q. He et al.

Fig. 6. TiO2-NPs induce ERS in HT22 cells. (A) After TiO2-NPs treatment, the protein expression levels of GRP78, ATF6, IRE-1α, p-IRE-1α, PERK, p-PERK and cleaved caspase-12 were elevated using western blot analysis. (B) Densitometric evaluation of ERS-related proteins (n = 3, *p < 0.05, **p < 0.01 vs control group). (C) Images of HT22 cells incubation with TiO2-NPs for 24 h and stained with CHOP (green) and DAPI (blue) (For interpretation of the references to color in text, the reader is referred to the web version of this article).

expression of CHOP and caspase-12, and induces apoptosis (Gorman et al., 2012). In the present study, after TiO2-NPs treatment, the mRNA levels of GRP78, IRE-1α, ATF6, CHOP and caspase-12 were dose-dependently up-regulated. These results indicate that TiO2-NPs might induce ERS in HT22 cells. Molecular chaperone GRP78 is the major regulator of ERS, and studies have shown that various damage to cells and microenvironment, including drug intervention, can increase the expression of GRP78 and aggravate ERS (Healy et al., 2009; Luo and Lee, 2012). In fact, the significant increase of GRP78 has been identified as a marker of ERS in cells (Zhang and Zhang, 2010). Upon ERS, GRP78 is sequestered away to make the transmembrane proteins regulate their UPR through their respective signaling pathways. IRE-1α signaling pathway can activate the ER response element gene, which is the main regulatory mechanism to coordinate with the ER folding function (Jäger et al., 2012). ATF6 can activate the expression of protein folding, secretion, and other related genes to regulate ERS (Yu et al., 2016). Our results showed that TiO2-NPs significantly up-regulated the expression of GRP78, ATF6 and p-IRE1α, which further suggests that ERS is involved in TiO2-NPs-induced apoptosis in HT22 cells.

results proved that TiO2-NPs could significantly increase the intracellular Ca2+ concentrations. Disorder of intracellular Ca2+ levels might cause HT22 cell apoptosis. ER is one of the important organs that regulates the stability of the intracellular environment and sensitivity to oxidative stress (Cao et al., 2017). Moreover, ERS is closely associated with the destruction of cellular Ca2+ homeostasis. Meanwhile, Ca2+ can regulate the expression of ER molecular chaperone (Song et al., 2016). Therefore, we supposed that the elevated intracellular ROS and Ca2+ levels may affect the ER. Ultrastructure study by TEM showed that the ER became swollen and vacuolated compared to the control group after treatment with TiO2-NPs. The expansion of ER lumen is considered to accommodate the increasing components, particularly which are being synthesized to manage ERS (Xiang et al., 2017). The result indicates that TiO2-NPs could damage the ER of HT22 cells. Mild and short-term ER damage leads to accumulation and aggregation of unfolded and misfolded proteins in the ER, initiating UPR to restore the normal function. However, when the ERS is severe and persistent, the UPR eventually activates GRP78, up-regulates the 12

Environmental Toxicology and Pharmacology 63 (2018) 6–15

Q. He et al.

that CHOP and caspase-12 participated in TiO2-NPs-induced neuronal apoptosis. In order to further detect the way of ERS-mediated apoptosis, we analyzed the expression level of CHOP and caspase-12. Our results showed that TiO2-NPs up-regulated CHOP expression, and the level of cleaved caspase-12 was significantly increased after TiO2-NPs treatment, which were similar with the changes of them at mRNA levels. These results indicated that TiO2-NPs-induced apoptosis was related to ERS in HT22 cells. Caspase-12 is a resident protein of ER, and studies have shown that caspase-12 participates in ERS-mediated apoptosis (Chen et al., 2015; Huang et al., 2014). Activation of caspase-3 is an important marker of apoptosis (Hitomi et al., 2004). It has been reported that cleaved caspase-12 can activate caspase-3-induced apoptosis (Zhang et al., 2016). We have proved that the expression of CHOP in HT22 cells was significantly increased after TiO2-NPs treatment, and it has been shown that CHOP can inhibit the expression of Bcl-2 (Mccullough et al., 2001), so we detected the expression levels of Bax and Bcl-2. We found that HT22 cells exposure to TiO2-NPs decreased the expression level of Bcl-2 and increased the expression level of Bax. Bcl-2 protein family is considered to be a key regulator of mitochondrial pathway-mediated apoptosis, so we speculate that TiO2-NPs may not only induce ERSmediated apoptosis, but also further induce apoptosis through mitochondrial pathways (Fig. 9), which requires further demonstration. Antioxidant NAC pretreatment decreased the accumulation of ROS in HT22 cells and reversed TiO2-NPs-induced elevation of GRP78, cleaved caspase-12 and cleaved caspase-3 levels, indicating that TiO2NPs-induced oxidative stress played a vital role in the induction of ERS and apoptosis. Based on our results, antioxidants might be an important measure to reduce the damage of TiO2-NPs.

Fig. 7. TiO2-NPs induce apoptosis in HT22 cells. (A) Western blot analysis of apoptosis-related proteins. (B) Densitometric evaluation of apoptosis-related proteins (n = 3, *p < 0.05, **p < 0.01 vs control group).

CHOP is a member of the bZIP transcription factor C/EBP family. In the normal state, CHOP is in a low expression state, and when the ERS occurs, the three main ERS pathways including IRE-1α, ATF6 and PERK pathways can activate CHOP at the same time (Cao et al., 2017; Pluquet et al., 2015). Caspase-12 is a unique caspase of ER and participates in apoptosis in the ERS (Moserova and Kralova, 2012). We hypothesized

5. Conclusion In conclusion, our data provide new evidence for the mechanism of TiO2-NPs-induced toxicity in HT22 cells. A brief summary of our study

Fig. 8. Effects of NAC on TiO2-NPs-induced intracellular ROS accumulation, ERS and apoptosis. (A) Fluorescence images of cells stained by DCFH-DA (green) were acquired by confocal microscope. (B) Quantitative analysis of the percentage of ROS-positive cells at each group. (C) Western blot analysis of GRP78, cleaved caspase3 and cleaved caspase-12 proteins in HT22 cells. (D) Densitometric evaluation of western blot (n = 3, **p < 0.01 vs HT22 control group, #p < 0.05, ##p < 0.01, TiO2-NPs + NAC vs TiO2-NPs) (For interpretation of the references to color in text, the reader is referred to the web version of this article). 13

Environmental Toxicology and Pharmacology 63 (2018) 6–15

Q. He et al.

Hu, R., Zheng, L., Zhang, T., Gao, G., Cui, Y., Cheng, Z., Cheng, J., Hong, M., Tang, M., Hong, F., 2011. Molecular mechanism of hippocampal apoptosis of mice following exposure to titanium dioxide nanoparticles. J. Hazard. Mater. 191, 32–40. Huang, Y., Li, X., Wang, Y., Wang, H., Huang, C., Li, J., 2014. Endoplasmic reticulum stress-induced hepatic stellate cell apoptosis through calcium-mediated JNK/P38 MAPK and Calpain/Caspase-12 pathways. Mol. Cell. Biochem. 394, 1–12. Huang, C.L., Hsiao, I.L., Lin, H.C., Wang, C.F., Huang, Y.J., Chuang, C.Y., 2015. Silver nanoparticles affect on gene expression of inflammatory and neurodegenerative responses in mouse brain neural cells. Environ. Res. 136, 253–263. Jäger, R., Bertrand, M.J.M., Gorman, A.M., Vandenabeele, P., Samali, Afshin, 2012. The unfolded protein response at the crossroads of cellular life and death during endoplasmic reticulum stress. Biol. Cell 104, 259–270. Lee, K., Tirasophon, W., Shen, X., Michalak, M., Prywes, R., Okada, T., Yoshida, H., Mori, K., Kaufman, R.J., 2002. IRE1-mediated unconventional mRNA splicing and S2Pmediated ATF6 cleavage merge to regulate XBP1 in signaling the unfolded protein response. Genes Dev. 16, 452–466. Liang, Z., Liu, R., Zhao, D., Wang, L., Sun, M., Wang, M., Song, L., 2016. Ammonia exposure induces oxidative stress, endoplasmic reticulum stress and apoptosis in hepatopancreas of pacific white shrimp (Litopenaeus vannamei). Fish Shellfish Immunol. 54, 523–528. Liu, J., Li, L., Suo, W.Z., 2009. HT22 hippocampal neuronal cell line possesses functional cholinergic properties. Life Sci. 84, 267–271. Liu, Y., Xu, Z., Li, X., 2013. Cytotoxicity of titanium dioxide nanoparticles in rat neuroglia cells. Brain Inj. 27, 934–939. Liu, H., Ma, L., Zhao, J., Liu, J., Yan, J., Ruan, J., Hong, F., 2014. Biochemical toxicity of nano-anatase TiO2 particles in mice. Biol. Trace Elem. Res. 129, 170–180. Lu, T., Sha, B., Gao, W., Wang, S., Xu, F., 2011. Cytotoxicity of titanium dioxide nanoparticles differs in four liver cells from human and rat. Compos. Part B Eng. 42, 2136–2144. Lu, T.H., Su, C., Tang, F.C., Chen, C.H., Yen, C.C., Fang, K.M., kI, Lee, Hung, D.Z., Chen, Y.W., 2015. Chloroacetic acid triggers apoptosis in neuronal cells via a reactive oxygen species-induced endoplasmic reticulum stress signaling pathway. Chem. Biol. Interact. 225, 1–12. Luo, B., Lee, A.S., 2012. The critical roles of endoplasmic reticulum chaperones and unfolded protein response in tumorigenesis and anticancer therapies. Oncogene 32, 805–818. Mccullough, K.D., Martindale, J.L., Klotz, L.O., Aw, T.Y., Holbrook, N.J., 2001. Gadd153 sensitizes cells to endoplasmic reticulum stress by down-regulating bcl2 and perturbing the cellular redox state. Mol. Cell. Biol. 21, 1249. Meena, R., Kumar, S., Paulraj, R., 2015. Titanium oxide (TiO2) nanoparticles in induction of apoptosis and inflammatory response in brain. J. Nanoparticle Res. 17, 1–14. Moserova, I., Kralova, J., 2012. Role of ER stress response in photodynamic therapy: ROS generated in different subcellular compartments trigger diverse cell death pathways. PLoS One 7 32972–32972. Nadanaka, S., Okada, T., Yoshida, H., Mori, K., 2007. Role of disulfide bridges formed in the luminal domain of ATF6 in sensing endoplasmic reticulum stress. Mol. Cell. Biol. 27, 1027–1043. Nakagawa, T., Zhu, H., Morishima, N., Li, E., Xu, J., Yankner, B.A., Yuan, J., 2000. Caspase-12 mediates endoplasmic-reticulum-specific apoptosis and cytotoxicity by amyloid-beta. Nature 403, 98–103. Numano, T., Xu, J., Futakuchi, M., Fukamachi, K., Alexander, D.B., Furukawa, F., 2014. Comparative study of toxic effects of anatase and rutile type nanosized titanium dioxide particles in vivo and in vitro. Asian Pac. J. Cancer Prev. 15, 929. Oberdörster, G., Oberdörster, E., Oberdörster, J., 2005. Nanotoxicology: an emerging discipline evolving from studies of ultrafine particles. Environ. Health Perspect. 113, 823–839. Palop, J.J., Mucke, L., 2010. Amyloid-b induced neuronal dysfunction in Alzheimer’s disease: from synapses toward neural networks. Nat. Neurosci. 13, 812–818. Pluquet, O., Pourtier, A., Abbadie, C., 2015. The unfolded protein response and cellular senescence. A review in the theme: cellular mechanisms of endoplasmic reticulum stress signaling in health and disease. Am. J. Physiol. Cell Physiol. 308, C415–425. Ron, D., Walter, P., 2007. Signal integration in the endoplasmic reticulum unfolded protein response. Nat. Rev. Mol. Cell Biol. 8, 519–529. 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., 2015. Mechanisms of TiO2 nanoparticleinduced neuronal apoptosis in rat primary cultured hippocampal neurons. J. Biomed. Mater. Res. A 103, 1141–1149. Shim, S., Kwon, J., 2012. Effects of [6]-shogaol on cholinergic signaling in HT22 cells following neuronal damage induced by hydrogen peroxide. Food Chem. Toxicol. 50, 1454–1459. Song, Y.F., Huang, C., Shi, X., Pan, Y.X., Liu, X., Luo, Z., 2016. Endoplasmic reticulum stress and dysregulation of calcium homeostasis mediate Cu-induced alteration in hepatic lipid metabolism of javelin goby Synechogobius hasta. Aquat. Toxicol. 175, 20–29. Swift, L.M., Sarvazyan, N., 2000. Localization of dichlorofluorescin in cardiac myocytes: implications for assessment of oxidative stress. AJP Heart Circ. Physiol. 278, H982–H990. Tsyganova, N.A., Khairullin, R.M., Terentyuk, G.S., 2014. Penetration of pegylated gold nanoparticles through rat placental barrier. Bull. Exp. Biol. Med. 157, 383–385. Wang, J., Chen, C., Liu, Y., Jiao, F., Li, W., Lao, F., Li, Y., Li, B., Ge, C., Zhou, G., Gao, Y., Zhao, Y., Chai, Z., 2008. Potential neurological lesion after nasal instillation of TiO2 nanoparticles in the anatase and rutile crystal phases. Toxicol. Lett. 182, 72–80. Wek, R.C., Cavener, D.R., 2007. Translational control and the unfolded protein response. Antioxid. Redox Signal. 9, 2357–2371. Wu, J., Sun, J., Xue, Y., 2010. Involvement of JNK and P53 activation in G2/M cell cycle arrest and apoptosis induced by titanium dioxide nanoparticles in neuron cells.

Fig. 9. A schematic illustration of possible mechanism of TiO2-NPs-induced toxicity in HT22 cells.

is outlined in Fig. 9 TiO2-NPs treatment could impair HT22 cell membrane integrity, disrupt the homeostasis of ROS and Ca2+, activate UPR, and then trigger ERS to induce apoptosis. Our results indicate that TiO2NPs-induced apoptosis in HT22 cells was at least partly through oxidative stress- and calcium imbalance-mediated ERS. These data suggest that the safe widespread use of TiO2-NPs requires intensive study of their toxicities. Conflict of interest None. Acknowledgements This study was supported by grants from Natural Science Foundation of Liaoning Province of China (201602704), National Natural Science Foundation of China (81403023) and Program for Liaoning Excellent Talents in University (LJQ2015111). References Barbero-Camps, E., Fernández, A., Baulies, A., Martinez, L., Fernández-Checa, J.C., Colell, A., 2014. Endoplasmic reticulum stress mediates amyloid β neurotoxicity via mitochondrial cholesterol trafficking. Am. J. Pathol. 184, 2066. Brun, E., Carrière, M., Mabondzo, A., 2012. In vitro evidence of dysregulation of bloodbrain barrier function after acute and repeated/long-term exposure to TiO2 nanoparticles. Biomaterials 33, 886–896. Cao, Y., Long, J., Liu, L., He, T., Jiang, L., Zhao, C., Li, Z., 2017. A review of endoplasmic reticulum (ER) stress and nanoparticle (NP) exposure. Life Sci. 186, 33–42. Chen, Y., Tang, Y., Xiang, Y., Xie, Y.Q., Huang, X.H., Zhang, Y.C., 2015. Shengmai injection improved doxorubicin-induced cardiomyopathy by alleviating myocardial endoplasmic reticulum stress and caspase-12 dependent apoptosis. Biomed Res. Int. 2015, 952671. Coccini, T., Grandi, S., Lonati, D., Locatelli, C., De, S.U., 2015. Comparative cellular toxicity of titanium dioxide nanoparticles on human astrocyte and neuronal cells after acute and prolonged exposure. Neurotoxicology 48, 77–89. Du, H., Zhu, X., Fan, C., Xu, S., Wang, Y., Zhou, Y., 2012. Oxidative damage and OGG1 expression induced by a combined effect of titanium dioxide nanoparticles and lead acetate in human hepatocytes. Environ. Toxicol. 27, 590–597. Fabian, E., Landsiedel, R., Ma-Hock, L., Wiench, K., Wohlleben, W., van Ravenzwaay, B., 2008. Tissue distribution and toxicity of intravenously administered titanium dioxide nanoparticles in rats. Arch. Toxicol. 82, 151–157. Gorman, A.M., Healy, S.J.M., Jäger, R., Samali, A., 2012. Stress management at the er: regulators of er stress-induced apoptosis. Pharmacol. Ther. 134, 306–316. Healy, S.J.M., Gorman, A.M., Mousavi-Shafaei, P., Gupta, S., Samali, A., 2009. Targeting the endoplasmic reticulum-stress response as an anticancer strategy. Eur. J. Pharmacol. 625, 234–246. Hitomi, J., Katayama, T., Taniguchi, M., Honda, A., Imaizumi, K., Tohyama, M., 2004. Apoptosis induced by endoplasmic reticulum stress depends on activation of caspase3 via caspase-12. Neurosci. Lett. 357, 127–130.

14

Environmental Toxicology and Pharmacology 63 (2018) 6–15

Q. He et al.

and pathophysiological examination. Environ. Sci. Pollut. Res. – Int. 22, 8728–8737. Yu, Y., Sun, G., Luo, Y., Wang, M., Chen, R., Zhang, J., Ai, Q., Xing, N., Sun, X., 2016. Cardioprotective effects of Notoginsenoside R1 against ischemia/reperfusion injuries by regulating oxidative stress- and endoplasmic reticulum stress- related signaling pathways. Sci. Rep. 6, 21730. Ze, Y., Sheng, L., Zhao, X., Ze, X., Wang, X., Zhou, Q., Liu, J., Yuan, Y., Gui, S., Sang, X., Sun, Q., Hong, J., Yu, X., Wang, L., Li, B., Hong, F., 2014. Neurotoxic characteristics of spatial recognition damage of the hippocampus in mice following subchronic peroral exposure to TiO2 nanoparticles. J. Hazard. Mater. 264, 219–229. Zhang, Q., Liu, J., Chen, S., Liu, J., Liu, L., Liu, G., Wang, F., Jiang, W., Zhang, C., Wang, S., Yuan, X., 2016. Caspase-12 is involved in stretch-induced apoptosis mediated endoplasmic reticulum stress. Apoptosis 21, 432–442. Zhang, L.H., Zhang, X., 2010. Roles of GRP78 in physiology and cancer. J. Cell. Biochem. 110, 1299–1305.

Toxicol. Lett. 199, 269–276. Wu, M., Jia, J., Lei, C., Ji, L., Chen, X., Sang, H., Xiong, L., 2015. Cannabinoid receptor CB1 is involved in nicotine-induced protection against Abeta1-42 neurotoxicity in HT22 cells. J. Mol. Neurosci. 55, 778–787. Xiang, C., Wang, Y., Zhang, H., Han, F., 2017. The role of endoplasmic reticulum stress in neurodegenerative disease. Apoptosis 22, 1–26. Yamaguchi, H., Wang, H., 2004. CHOP is involved in endoplasmic reticulum stress -induced apoptosis by enhancing DR5 expression in human carcinoma cells. J. Biol. Chem. 279, 45495–45502. Yang, Qingfeng, Kim, Y.-S., Lin, Y., 2006. Tumour necrosis factor receptor 1 mediates endoplasmic reticulum stress-induced activation of the MAP kinase JNK. EMBO Rep. 7, 622–627. Younes, N.R., Amara, S., Mrad, I., Ben-Slama, I., Jeljeli, M., Omri, K., 2015. Subacute toxicity of titanium dioxide (TiO2) nanoparticles in male rats: emotional behavior

15