Chinese Science Bulletin

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Citation: Li X B, Xu S Q, Zhang Z R, et al. Apoptosis induced by titanium dioxide nanoparticles in cultured murine microglia N9 cells. Chinese Sci Bull, 2009, 54:

3830―3836, doi: 10.1007/s11434-009-0548-x

Apoptosis induced by titanium dioxide nanoparticles in cultured murine microglia N9 cells

LI XiaoBo1, XU ShunQing2, ZHANG ZhiRen3 & Hermann J Schluesener3

1 School of Public Health, Southeast University, Nanjing 210009, China; 2 Key Laboratory of Environment and Heath of the Ministry of Education, Institute of Environmental Medicine, School of Public Health, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China;

3 Institute of Brain Research, University of Tuebingen, Calwer Str. 3, D-72076 Tuebingen, Germany

Owing to the rapidly increasing output of nano-scale titanium dioxide (TiO2) particles, their potential risk for central nerve system (CNS) has elicited much concern recently. Microglia is the resident macrophage in CNS and essential for the homeostasis of the CNS microenvironment. They are sup-posed to response to nanoparticles depositing in the brain tissues. Therefore, we investigated the cy-totoxic effects of TiO2 NPs on microglia N9 cells in vitro. Results of propidium iodide/fluorescein di-acetate (PI/FDA) double staining and MTT test clearly showed that TiO2 NPs more efficiently affected the viability of microglia N9 cells. Further Hoechst 33258 staining and flow cytometric analysis proved that nano-scale but not normal scale TiO2 induced apoptosis in vitro. These data suggest that TiO2 NPs can elicit apoptosis of N9 cells in vitro and thus present a potential risk for CNS.

apoptosis, nanoparticles, titanium dioxide, N9 cells

Manufactured nanoparticles (NPs) of titanium dioxide (TiO2) have been widely used in recent years. Their po-tential toxicity provides a growing concern for human health. Besides their small size, NPs display properties as greater surface area, high reactive activities and other unique physicochemical properties, which pose potential applications for diagnosis and therapy. However, data from several research groups indicated that TiO2 NPs had distinct risks to human health[1–3] compared with their normal size counterparts. The toxicity of TiO2 NPs focused on the inhalation exposure and lungs seemed to be the main target organ in the previous studies. Animal studies showed that the inhaled TiO2 NPs could deposit in lung tissues, induce the inflammatory response and pathological changes of lung tissues[4]. The TiO2 NPs were also found accumulated in the liver, kidney, spleen with the related toxicity except the lung which means that TiO2 NPs could be transported to the other organ after uptake by the gastrointestinal and respiratory tracts. Recently, more studies concern about neurotoxicity, be-

cause it is proved that TiO2 NPs could pass through the blood brain barrier (BBB)[5].

BBB is a metabolic or cellular structure in the central nervous system (CNS) which allows the passage of sub-stances between the bloodstream and the neural tissue selectively to strictly regulate the composition of the fluid microenvironment of the brain. Even a slight change in the brain fluid microenvironment may lead to alter brain function[6]. Under normal conditions, non-lipid-soluble NPs are not allowed to cross the BBB. However, there are still two possible routes for their en-try into the CNS. For inhaled NPs, olfactory nerve is the most viable pathway for the transport because of the close proximity of olfactory mucosa and bulb[7,8]. A few days following exposure, their concentrations in the ol-factory bulb and other brain regions (e.g. hippocampus, Received April 13, 2009; accepted June 6, 2009 Doi: 10.1007/s11434-009-0548-x †Corresponding author (email: lixiaobo1007@hotmail.com) Supported by the German Academic Exchange Service (DAAD) and Chinese Schol-arship Council (CSC)

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cerebral cortex and cerebellum) increased[5,7]. After na-sal exposure to manganese (Mn) oxide NPs, the concen-trations of Mn in the olfactory bulb, striatum, frontal and other brain regions increased[7]. The other route is through the direct cell membrane toxicity to endothelial cells[9,10]. After exposure to different sized TiO2 particles by oral, the concentrations of Ti in mice brains of TiO2 NPs exposure group were significantly higher than those in the controls[11]. Since accumulated data showed that NPs could facilitate delivery of drugs to the brain[12,13], or even had the capacity to bypass the BBB[14] them-selves, information about interactions between NPs and CNS are urgently required.

Federici et al.[15] reported that responses of oxidative stress in rainbow trout were induced after exposure to TiO2 NPs. Wang et al.[8] showed that after nasal instilla-tion of TiO2 NPs, morphological changes of neurons and increased GFAP-positive astrocytes in mice brain were observed. Further, oxidative stress occurred and exces-sive glutamic acid and nitric oxide were released in whole brain of exposed mice. In the CNS, the number of non-neural cells far exceeds the number of neurons. The non-neural cells, such as microglia, astrocytes and en-dothelial cells are essential to maintain the normal func-tion of neurons. Microglial cells are the CNS counterpart of tissue macrophages, occupy 12% of the CNS cell population and involved in many different CNS dis-eases[16]. They participate in immune responses in the CNS, mediate brain inflammatory states and are respon-sible for removal of dead cells, debris, as well as invad-ing pathogens. Phagocytosis of “foreign” materials by microglia is the first defense in the brain, so it is impor-tant to know the effects of NPs on microglia. In the pre-sent study, we evaluated the cytotoxic effects of TiO2 nanoparticles using N9 cell line derived from murine microglia cells. Suspensions of nanoparticles were pre-pared at concentrations covering a range from 0 to 125 μg/mL, based on other in vitro systems[1,11,17].

1 Materials and methods 1.1 Materials

Commercial TiO2 NPs and non-nano TiO2 particles were purchased from Riedel-dehaeun, Germany. Microglial cell line N9 was obtained from ATCC (American Type Culture Collection). RPMI-1640 media, fetal calf serum (FCS) and RNase A were purchased from GIBCO Invi-trogen. Thiazolyl blue tetrazolium bromide (MTT),

fluorescein diacetate (FDA), propidium iodide (PI) and bisbenzimide H 33258 (Hoechst 33258) were purchased from Sigma-Aldrich (St. Louis, MO, USA).

1.2 Cell culture and NPs treatment

Microglial N9 cell lines were cultured in RPM1 1640 medium with 10% heat-inactivated FCS, 100 U/mL penicillin and 100 pg/mL streptomycin. Cells were grown and maintained in 28-cm2 cell culture flasks at 37℃ in a 5% CO2 humidified incubator. The TiO2 NPs suspension was prepared using the culture media and dispersed for 20 min by using a sonicator (Bandelin Sonopuls HD70, Germany) to prevent aggregation. The cells were treated with various concentrations of particles.

1.3 Cell morphology

Cells were plated into a 35 mm tissue culture plate at a density of 2×105 cells (in 2 mL growth medium). After overnight growth, supernatants from the culture plates were aspirated out and fresh medium containing TiO2 NPs in desired concentrations (4, 8, 16, 32, 64 and 125 μg/mL) were added. Phosphate-buffered saline (PBS, pH 7.4) and non-nano TiO2 particles (125 μg/mL) were used as control. Following incubation for 24 h, cells were washed with PBS and the morphological changes were observed under an inverted phase-contrast micro-scope at 200×magnification.

1.4 Cell viability test

Following incubation with nanoparticle suspension at desired concentrations (4, 8, 16, 32, 64 and 125 μg/mL), using PBS and non-nano TiO2 particles (125 μg/mL) as control groups, supernatants of N9 cells cultured in chamber slides were removed, then 200 µL fresh media, 4 µL PI (0.2 mg/mL) and 6 µL FDA (0.5 mg/mL) were added, and cells were incubated for 3 min at room tem-perature. After rinsing once with PBS, cells were exam-ined under the fluorescent microscope immediately.

Cell viability was also semi-quantified by the MTT assay. Cells were seeded on 96-well plates with 5×103 cells in 100 μL media per well. After a 16 h stabilization of the cells, 100 μL nanoparticle suspension (8, 16, 32, 64, 125 and 250 μg/mL concentrations) was added to the cell medium, to make final concentrations of 4, 8, 16, 32, 64 and 125 μg/mL, and using PBS and non-nano TiO2 particles (125 μg/mL) as control groups. Cells were ex-posed to the NPs for 24 h. At the end of exposure, 20 μL of MTT solution (5 mg/mL) was added and the cells

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were incubated for 4 h at 37℃. Cells were treated with 150 μL of dimethylsulfoxide (DMSO) and absorbance was measured at 540 nm using the microplate spectro-photometer system (MWG-Biotech, Germany). The vi-ability of the treated groups was expressed as a percent-age of non-treated control groups, which was assumed to be 100%.

1.5 Hoechst 33258 staining for chromosome con-densation

To evaluate chromosome condensation, Hoechst 33258 staining was performed. Cultured N9 cells in chamber slides with nanoparticle suspension (16, 64 and 125 μg/mL) were fixed by 4% paraformaldehyde (PFA) for 45 min at room temperature, and washed with PBS/0.1% Triton X-100 for 5 min. Then the Hoechst 33258 solu-tion (2.5 μg/μL Hoechst 33258 in PBS/0.1% Triton X-100) was added to cultured cells, followed by incuba-tion for 5 min in the dark at room temperature. PBS and non-nano TiO2 were used as control. The images of the nucleus were made by a fluorescence microscope (Nikon, Tokyo, Japan) with a DAPI filter.

1.6 Flow cytometric analysis of cell cycle

Apoptosis rate was further measured by flow cytometry. After co-culture with 16 or 125 μg/mL TiO2 NPs, using PBS as control, about 2×106 cells were pelleted by spin-ning at 1000 r/min, 4℃ for 5 min, then resuspended in 1 mL of cold PBS, and fixed by adding 4 mL of absolute ethanol at −20℃ for 40 min. After fixation, cells were washed and resuspended in 1 mL PBS, then 100 µL of 200 µg/mL DNase-free, RNase A was added and incu-bate at 37℃ for 30 min. Following adding 100 µL of 1 mg/mL PI and incubating at room temperature for 10 min, samples were analyzed by flow cytometry.

1.7 Statistical analysis

Statistical analysis was performed by one-way analysis of variance followed by Dunnett’s multiple comparison tests (SPSS12.0). For all statistical analyses, significance levels were set at P <0.05.

2 Results

2.1 Morphologic changes

After exposure to TiO2 NPs for 24 h, N9 cells were ob-served under a phage-contrast microscope. Rinsing with PBS gently is necessary to remove excessive NPs. For

unexposed (Figure 1(a)) and non-nano TiO2 exposed groups (Figure 1(b)), cells were regularly in spindle shape, while hypertrophied, ameboid cells were more easily found in cells exposed to 4, 8, 16, 32, 64 or 125 μg/mL TiO2 NPs (Figure 1(c)－(h)). This transformation from spindle shape to activated ameboid morphology indicates their response to pathological insults[18]. NPs were up-taken into the cytoplasm rapidly after treatment and after 24 h exposure, aggregated nanoparticles resided mainly around the peri-region of the nuclear membrane, not in the cytoplasmic region as arrows shown in Figure 1(d)－(h). Meanwhile, the cell density of nanoparticle-treated groups decreased with the increasing of concentration of TiO2 NPs, suggesting the reduced cell proliferation or cell death after 24 h exposure.

2.2 Cytotoxicity

FDA and PI double staining were used to observe the viability of N9 cells. FDA is a non-fluorescent derivative of fluorescein, it can be transported across cell mem-branes and deacetylated by nonspecific esterases. Re-sultant fluorescein accumulates within cells and allows direct visualization by fluorescent microscopy. There-fore, FDA can indicate the cell metabolism and only living cells acquire the FDA stain with a mean green fluorescence[19]. In addition, the cationic dye PI enters the cells only if the cellular membrane is permeable or damaged. Compared with control group (Figure 2(a)) and non-nano TiO2 treated group (Figure 2(b)), mem-brane permeability of N9 cells to PI increased when ex-posed to TiO2 NPs with 4, 8, 16, 32, 64 and 125 μg/mL individually (Figure 2(c)―(h)), which suggested that the normal function of cells is destroyed by NPs.

The FDA and PI double staining gives no quantitative measure of cell viability but is used to estimate the number of viable cells. Furthermore, to examine the toxic effects of TiO2 NPs, N9 cells were incubated with different concentrations (4, 8, 16, 32, 64 and 125 μg/mL) of NPs and 125 μg/mL non-nano TiO2 particles. Fur-thermore, cell viability was semi-quantified by MTT analysis. As shown in Figure 3, the decrease in N9 cell viability became more prominent with increasing con-centrations of TiO2 NPs, and a 70% cell viability was seen in cultures treated with 16 μg/mL NPs, while the high concentration (125 μg/mL) of non-nano TiO2 parti-cles did not have significant effects on cell viability.

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Figure 1 Phase-contrast micrographs of N9 cells. (a) Cells treated with PBS; (b) cells treated with 125 μg/mL non-nano-TiO2; (c)–(f) cells treated with 4, 8, 16 and 32 μg/mL TiO2 NPs (×200); (g) and (h) cells treated with 64 and 125 μg/mL TiO2 NPs for 24 h (×400).

Figure 2 FDA and PI double staining of N9 cells. (a) Cells treated with PBS; (b) cells were treated with 125 μg/mL non-nano TiO2; (c)－(h) cells were treated with 4, 8, 16, 32, 64 and 125 μg/mL TiO2 NPs for 24 h, stained with FDA and PI, then observed with fluorescent microscopy. PI enters the cells when the cellular membrane is permeable or damaged (×200).

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Figure 3 Effects of TiO2 NPs on the viability of N9 cells. Cell vi-ability was assessed by MTT assays, and results are presented as a percentage of control group viability. Results represent the means of three separate experiments, and error bars represent the stan-dard error of the mean. * and # show that 16, 32, 64,125 μg/mL nano-TiO2 treated groups showed statistically significant differences from the control group and non-nano TiO2 group by the Turkey’s test (P < 0.05), respectively.

2.3 Apoptosis of cells

The morphologic changes of nuclear of N9 cells treated with non-nano TiO2 particles, or 16, 64, 125 μg/mL TiO2 NPs for 24 h were observed by Hoechst 33258 staining. In the control and 125 μg/mL non-nano TiO2 parti-cles-treated group, nuclei of N9 cells were round and homogeneously stained (Figure 4(a) and (b)); however, condensed or fragmented nuclei were present in many TiO2 NPs-treated cells (Figure 4(c)－(e)), which are different from evenly stained nuclei of living cells.

As the process of Hoechst 33258 staining may lose part of the dead cells, changes in DNA content during treatment with different doses of TiO2 NPs were moni-tored by flow cytometry. The cultured cells and super-natant were gently collected and treated as previously described. The apoptotic sub-G1 peaks were observed when the N9 cells were cultured with 16 and 125 µg/mL TiO2 NPs seperately for 24 h. (Figure 5). Apoptosis rate was determined by evaluating the percentage of events accumulated in the pre-G0/G1 position. After treated with 16 and 125 µg/mL TiO2 NPs for 24 h, apoptosis rates of N9 cells were accumulated to 11.69% and 87.37% respectively (Figure 5(b) and (c)). Significant

Figure 4 Nuclei condensation in TiO2 NPs treated cells. (a) Cells were treated with PBS; (b) cells were treated with 125 μg/mL non-nano TiO2 particles; (c)－(e) cells were treated with 16, 64, 125 μg/mL TiO2 NPs for 24 h, stained with Hoechst 33258, then observed with fluorescent microscopy. Arrowheads in (c)－ (e) indicate nuclei condensation observed in TiO2 NPs-treated cul-tures (×200).

difference can be seen at the 16 μg/mL TiO2--treated group (Figure 5(d)).

4 Discussion

Microglial cells constitute 12% of the cells in the central nervous system (CNS). The brain and spinal cord are separated from the rest of body by BBB, which prevents most infections from reaching the vulnerable nervous tissue. In the case where infectious agents are directly introduced to the brain or cross the BBB, microglia re-spond to invading pathogens or other inflammatory sig-nals before they damage the sensitive neural tissue. Working as tissue macrophages, microglia also secrete inflammatory cytokines and toxic mediators which may amplify the inflammation[20]. Dysfunction of microglia is related to different pathologic processes, such as Alz-heimer’s disease[21] and brain lesions[22].

TiO2 NPs are poorly soluble particulates and widely used in industrial products, such as cosmetics and phar-maceuticals[23,24]. Therefore, potential widespread expo-sure may occur during both manufacturing and applica-

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Figure 5 TiO2 NPs induced apoptosis in N9 cells. (a)－(c) Representative of flow cytometric histogram of N9 cells treated for 24 h with fresh medium containing with PBS, 16 and 125 μg/mL TiO2 NPs respectively. The quantitative data from three independent experiments are shown in (d).*, P<0.05 vs N9 cells treated by PBS.

tion. Accumulated data have explored the deposit and adverse health effects of TiO2 NPs in brains after respi-ratory or gastrointestinal exposure. As for the study of nano-neurotoxity, most research focused on the biomar- kers of oxidative stress, activity of nitric oxide synthases and concentration of nitric oxide[1,8,25]. Since microglia may release NO and induce oxidative stress responses, the study on the interaction between microglia and TiO2 NPs may be helpful to understand the mechanism of nano-neurotoxicity. The murine microglia N9 cell line was chosen for the current study.

It is suggested that TiO2 particles induce apoptosis or necrosis in different types of cells, such as mesenchymal stem cells[26], lymphoblastoid cells [11], human bronchial epithelial cells[1], and fibroblasts[27]. Such information is essential for the optimal design of nano TiO2 involved medicine and implantable devices. Apoptosis is charac-terized by cellular and nuclear shrinkage and condensa-tion followed by nuclear fragmentation, cellular budding, and formation of apoptotic bodies[28]. Necrosis is char-acterized by cellular swelling, karyolysis, and rupture of the plasma membrane and release of the cytoplasmic constituents[29]. Apoptosis and necrosis, the two main modes of cell death, have different biological signifi-cance and can be distinguished by simple and reliable screening tests. These methods include membrane per-

meability to nuclei staining (i.e. PI and Hoechst) and other flow cytometry-based methods, such as measure-ment of forward scatter (FS), side scatter (SS) and DNA quantification by PI. As one of the toxic mechanisms of NPs, the apoptosis of target cells seems to be most widely studied.

In this study, cell morphology altered in most of the NPs-treated groups after 24 h incubation and the distri-bution of NPs in cells is similar to Park’s report[1]. Meanwhile, the cell density decreased with the increas-ing of NPs’ concentration and MTT analysis showed the same result. We also presented evidence of apoptosis in N9 cells induced by this nano-materials based on mor-phologic changes in cellular nuclei, membrane imper-meability to PI and flow cytometric analysis.

After Hoechst 33258 staining, cells were scored as apoptotic by their nuclei present chromatin condensation, nuclear beading or nuclei fragmenting into smaller structures[30]. The results of flow cytometry were in ac-cordance with that of the MTT. It was revealed that there was the dose-dependent effect of apoptosis in the cells exposed to nano-TiO2 and the significant difference observed in 16 μg/mL TiO2 NPs-treated groups. These data were similar to what reported by other research groups[1,3,17]. The oxidative stress was one of the mecha-nisms involved in apoptosis. Distribution of nanoparti-

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cles in cell substructure, especially mitochondria was reported [31], and the dysfunctions of mitochondria are related to oxidative stress and cell apoptosis. Whether this mechanism contributes to the N9 cell apoptosis in our study is still needed to be further investigated.

In summary, when comparing with non-nano TiO2 particles, the TiO2 nanoparticles display a distinct cy-totoxicity with the decreasing of cell viability and the induction of apoptosis in cultured murine microglia N9

cells. With the aggregation of TiO2 NPs in the perinu-clei region, permeability of cellular membrane to PI increased and cell viability decreased. Low concentra-tion (16 μg/mL) of TiO2 NPs showed capability to in-duce apoptosis which might lead to dysfunction of mi-croglia. For TiO2 NPs may deposit in different brain regions and interact with microglia, this brain-cell damage suggested potential adverse biological re-sponses in brain.

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