Time-dependent translocation and potential impairment on central nervous system by intranasally instilled TiO2 nanoparticles

Toxicology 254 (2008) 82–90

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Time-dependent translocation and potential impairment on central nervous system by intranasally instilled TiO2 nanoparticles Jiangxue Wang a,b , Ying Liu a,b , Fang Jiao a,b , Fang Lao a,b , Wei Li a,b , Yiqun Gu c , Yufeng Li a,b , Cuicui Ge a,b , Guoqiang Zhou a,b , Bai Li a,b , Yuliang Zhao a,b,∗ , Zhifang Chai a,b , Chunying Chen a,b,∗∗ a Laboratory for Bio-Environmental Effects of Nanomaterials and Nanosafety and Key Lab of Nuclear Analytical Techniques, Institute of High Energy Physics, Chinese Academy of Sciences, Beijing 100049, PR China b National Center for Nanoscience and Technology, Beijing 100190, PR China c Maternity Hospital of Haidian District, Beijing 100080, China

a r t i c l e

i n f o

Article history: Received 12 August 2008 Received in revised form 14 September 2008 Accepted 15 September 2008 Available online 25 September 2008 Keywords: TiO2 nanomaterials Translocation Neurotoxicology Redox status Proinflammatory cytokines Immune response

a b s t r a c t Nanoparticles can be administered via nasal, oral, intraocular, intratracheal (pulmonary toxicity), tail vein and other routes. Here, we focus on the time-dependent translocation and potential damage of TiO2 nanoparticles on central nervous system (CNS) through intranasal instillation. Size and structural properties are important to assess biological effects of TiO2 nanoparticles. In present study, female mice were intranasally instilled with two types of well-characterized TiO2 nanoparticles (i.e. 80 nm, rutile and 155 nm, anatase; purity > 99%) every other day. Pure water instilled mice were served as controls. The brain tissues were collected and evaluated for accumulation and distribution of TiO2 , histopathology, oxidative stress, and inflammatory markers at post-instillation time points of 2, 10, 20 and 30 days. The titanium contents in the sub-brain regions including olfactory bulb, cerebral cortex, hippocampus, and cerebellum were determined by inductively coupled plasma mass spectrometry (ICP-MS). Results indicated that the instilled TiO2 directly entered the brain through olfactory bulb in the whole exposure period, especially deposited in the hippocampus region. After exposure for 30 days, the pathological changes were observed in the hippocampus and olfactory bulb using Nissl staining and transmission electron microscope. The oxidative damage expressed as lipid peroxidation increased significantly, in particular in the exposed group of anatase TiO2 particles at 30 days postexposure. Exposure to anatase TiO2 particles also produced higher inflammation responses, in association with the significantly increased tumor necrosis factor alpha (TNF-␣) and interleukin (IL-1␤) levels. We conclude that subtle differences in responses to anatase TiO2 particles versus the rutile ones could be related to crystal structure. Thus, based on these results, rutile ultrafine-TiO2 particles are expected to have a little lower risk potential for producing adverse effects on central nervous system. Although understanding the mechanisms requires further investigation, the present results suggest that we should pay attention to potential risk of occupational exposure for largescaled production of TiO2 nanoparticles. © 2008 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Because of the good physicochemical properties, nanoscale TiO2 was widely used in the fields of paints, waste water treatment, sterilization, cosmetics, food additive, bio-medical ceramic and

∗ Corresponding author at: Laboratory for Bio-Environmental Effects of Nanomaterials, Institute of High Energy Physics, Chinese Academy of Sciences, Beijing 100049, PR China. Tel.: +86 10 88233191; fax: +86 10 88233191. ∗∗ Corresponding author at: Laboratory for Bio-Environmental Effects of Nanomaterials and Nanosafety, National Center for NanoScience and Technology, Beijing 100190, PR China. Tel.: +86 10 82545560; fax: +86 10 62656765. E-mail addresses: [email protected] (Y. Zhao), [email protected] (C. Chen). 0300-483X/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.tox.2008.09.014

implanted biomaterials and so on. However, the unique characteristics of nanoparticles, such as the small size, large surface area per mass and high reactivity raises great concern on the adverse effects of TiO2 particles on ecological system and human health (Oberdörster et al., 2005; Warheit et al., 2007a). In the aquatic system, experimental study (Lovern et al., 2007) showed that TiO2 exposure caused slightly changes in Daphnia magna including hopping frequency, feeding appendage, postabdominal curling movement and heart rate, in contrast, significant increases in hopping frequency, appendage movement and heart rate were observed after exposure to nano-C60 suspension. Additionally, Federici et al. (2007) reported the changes of Cu and Zn levels in the brain and the decreases of some enzyme activities in the gills and intestine of rainbow trout after exposure to TiO2 nanoparticles.

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Animal studies have revealed that the inhaled TiO2 nanoparticles can readily deposit in lung tissue and induce the inflammatory response, the increased neutrophils, the progressively fibroproliferative lesions and epithelial metaplasia in lung alveoli by analyzing the components of bronchoalveolar lavage fluid and pathological changes of lung tissues (Bermudez et al., 2004; Oberdörster et al., 2000; Osier and Oberdörster, 1997; Warheit et al., 2005). In vivo, the intraperitoneally injected and orally ingested TiO2 nanoparticles would cause transcytosis across epithelial and endothelial cells into the blood circulation, respectively, and can be entrapped in the reticular-endothelial system that would further induce certain lesions to the tissues with macrophagic activity such as lung, liver and spleen (Olmedo et al., 2002; Wang et al., 2007b). Recently, International Agency for Research on Cancer (IARC) have classified pigment-grade TiO2 as possible carcinogen to human beings (group 2B) after testing the ultrafine TiO2 on animals by different administration methods (Baan et al., 2006). Under the ultraviolet (UV) radiation, nanoscale TiO2 can produce reactive free radicals including hydroxyl and superoxide anion radicals on its surface because of the electron–hole formation, and exert a strong oxidizing ability. It was reported that when the human skin fibroblasts or calf thymus DNA were co-cultured with TiO2 nanoparticles, hydroxyl radicals were produced and damaged C-8 bits of DNA under the UVA irradiation (Wamer et al., 1997). The lightilluminated TiO2 nanoparticles could not only photocatalyze DNA oxidative damage, but also cell apoptosis both in vitro and in human cells (Hidaka et al., 1997; Nakagawa et al., 1997). Recently, Long et al. (2006) reported that nanoscale TiO2 (P25) particles not only stimulated brain microglia to produce reactive oxygen species (ROS) through the oxidative burst, but also interfered with mitochondrial energy production in vitro. There are several pioneer studies reported the potential neuronal uptake and translocation of inhaled particulates and pathogens to the brain (Dorman et al., 2004; Elder et al., 2006; Oberdörster et al., 2004). Oberdörster et al. (2005) have reviewed in detail that the olfactory nerve is the most viable pathway for the transport of intranasally inhaled/instilled particles because of the close proximity of olfactory mucosa and bulb. Both in vivo and in vitro studies have shown that the physicochemical properties such as the particle size, charge and surface chemistry would endow nanoparticles the ability for their transcytosis or endocytosis across epithelial, endothelial and macrophage cell membranes and preferential location in lysosome or mitochondria (Li et al., 2008; Sayes et al., 2004; Smith and Helenius, 2004). There exist a lot of lipids, especially phospholipid and glycolipid in the biomembrane of nerve cells in brain. Brain cephaline and sphingoglycolipid are the important parts of the lipid complexes that provide sheathing for nerve cells, promote healthy brain and neurotransmitter metabolism, and improve memory, cognition and overall mental performance. They are more prone to be attacked by ROS and result in the physiological dysfunction of neurons and brain oxidative injury (Matés, 2000; Barnham et al., 2004). In our pilot study, the intranasally instilled TiO2 nanoparticles could be translocated into the central nervous system (CNS) of mice via the olfactory nerve tract, and accumulated in the olfactory nerve layer, olfactory ventricle, cerebral cortex, thalamus and CA1 and CA3 regions of hippocampus detected by synchrotron radiation X-ray fluorescence analysis (SRXRF) and inductively coupled plasma mass spectrometry (ICP-MS) (Wang et al., 2007a; Wang et al., 2008). Based on these results, the time-dependent translocation of intranasally instilled nanoscale TiO2 particles to the brain tissue was therefore determined using ICP-MS in this study. The morphology analysis in the olfactory bulb and brain was detected by Nissl staining and transmission electron microscopy (TEM). Oxidative damage and cytokines are the most important factors as responses

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to resist the infection and/or inflammation. Therefore, the oxidative response and activation of proinflammatory cytokines were assayed to prove the injury of intranasal instilled nanoparticles on CNS. 2. Materials and methods 2.1. Materials Nanoscale TiO2 (Hangzhou Dayang Nanotechnology Co. Ltd., 80 nm) and commercial fine TiO2 (Zhonglian Chemical Medicine Co., 155 nm) particles without any coating were used in this study. The properties such as purity, size, surface area, crystal profile and structure state of TiO2 were well characterized previously (Wang et al., 2008). In brief, the 80 nm TiO2 particles are rutile profile with the average sizes of 71.4 ± 23.5 nm, and the 155 nm TiO2 particles are anatase profile with the average size of 155.0 ± 33.0 nm. Their surface areas increase with the reduction of particle size. But their pore diameters are almost the same, which are 16.6 nm and 16.7 nm for the 80 nm and 155 nm TiO2 nanoparticles, respectively. Their TiO2 contents are more than 99% as confirmed by ICP-MS and X-ray fluorescence spectroscopy. A MilliQ water system (Millipore, Bedford, MA, USA) was used to prepare the ultra pure water. All of other reagents were at least of analytical grade. 2.2. General experimental design The aim of this study is time-course assessments of any observed effects on two crystalline TiO2 nanoparticles. We firstly evaluate time-dependent translocation of inhaled TiO2 particles into murine brain; secondly evaluate histopathological changes, the antioxidative and immune responses of murine brain tissue to the repeatedly instilled TiO2 particles. Thus, two separate animal experiments were carried out. The animals (six mice per group for each time point) were used for determining Ti content in the different brain regions (olfactory bulb, cerebral cortex, hippocampus, cerebellum, brain stem and rest of brain) after intranasal instilling TiO2 nanoparticles for different intervals. The second experiment (10 mice per group) was designed for assessing the other endpoints in the whole brain of exposed mice. 2.3. Animals CD-1(ICR) female mice (Beijing Vitalriver Experimental Animal Technology Co. Ltd., body weights of 19–22 g) were housed in stainless steel cages. The standard conditions (20 ± 2 ◦ C room temperature, 60 ± 10% relative humidity) were maintained with a 12 h light/dark cycle for mice. Distilled water and sterilized food for mice were available ad libitum. All procedures used in this experiment were compliant with the local ethics committee. Animals were acclimated to this environment for 5 days prior to treatment. All animals were randomly divided into four groups: control group and two experimental groups (80 and 155 nm group). TiO2 nanoparticles were dispersed in Milli-Q water, ultrasonicated for 15–20 min, and vortexed for 2 min just before nasal instillation of each mouse to prevent from the aggregation and to keep the maximum dispersed state of particles in water. TiO2 suspensions were instilled into the nasal cavity using micro-syringe every other day (about 500 ␮g per mice), and the Milli-Q water was taken as control. At post-instillation time points of 2, 10, 20 and 30 days (i.e. 1 day after 1, 5, 10 and 15 times for instillation, respectively), animals were anaesthetized by ether. The sub-brain regions of interest including olfactory bulb, cerebral cortex, hippocampus and cerebellum were divided from six murine brains in each group for determining Ti contents. Another six murine brains were homogenized to assay enzyme activities and cytokines. The remaining four brain tissues were used for histopathological examination and TEM observations. 2.4. Serum biomarker assays The serum was harvested to evaluate the hepatic function with levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST). Renal function was determined by blood urea nitrogen (BUN) and creatinine (Cr). The total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C) were assayed for evaluating the cholesterol level using a Biochemical Autoanalyzer (Type 7170, Hitachi, Japan). 2.5. Assay of enzymatic activities in brain The six brain tissues per group were weighed and minced and transferred into a centrifuge tube. The 1:9 (w/v) volume of cold 0.1 mol/L phosphate buffer (0.1 mol/L Na2 HPO4 , 0.1 mol/L KH2 PO4 , 0.1 mmol/L PMSF, pH 7.4) was added, and the mixtures were homogenized by a ultrasonic cell disruptor (Sonics vibra cell, VCX105) for 8 s × 4 times at 4 ◦ C. An aliquot of 200–300 ␮L homogenates was taken out to determine Ti content. The remainder was centrifuged at 14,000 × g for 5 min in 4 ◦ C, collecting the supernatants to assay some oxidative biomarkers. The activities of glutathione peroxidase (GSH-Px), glutathione-S-transferase (GST), superoxidase

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dismutase (SOD), and the levels of reduced glutathione (GSH) and lipid peroxidation (marked as malondiadehyde (MDA)) in brain extracts were examined according to the method previously described (Wang et al., 2006). Briefly, the activity of GSHPx was assayed by determination of the reduced GSH in the homogenate and GST was measured spectrophotometrically by the standard substrate, i.e. 1-chloro-2,4dinitrobenzene (CDNB) conjugated with GSH (Nanjing Jiancheng Bioengineering Institute, Jiangsu, China). The lipid peroxidation product of MDA generated in the brain was measured by thiobarbituric acid (TBA) reactivity using the commercial colorimetric assay kit (Nanjing Jiancheng Bioengineering Institute, Jiangsu, China). Protein concentrations were determined according to the Bradford’s method (1976), using bovine serum albumin as a standard. 2.6. Determination of Ti content Ti contents in whole brain homogenate and sub-brain regions of mice in every group were determined using ICP-MS (Thermo Elemental X7, Thermo Electron Co.) as described in Wang et al. (2007b). 2.7. Histopathological examination After exposure for 30 days, the tissues/organs, such as heart, liver, spleen, kidneys, lung and brain, were excised out and weighed accurately for tissue coefficient. A part of tissues/organs was cut and immediately fixed in a 10% formalin solution. The histopathological tests were performed using standard laboratory procedures. Briefly, the tissues were embedded in paraffin blocks, then sectioned into 5 ␮m slice and mounted onto the glass slides. After hematoxylin–eosin (HE) staining, the sections were observed and the photos were taken using optical microscope (Nikon U-III Multi-point Sensor System, USA). The identity and analysis of the pathology sections were blind to the pathologist. 2.8. Ultrastructure of hippocampus by transmission electron microscopy The fresh olfactory bulb and hippocampus samples were immersed in 2.5% glutaraldehyde at 4 ◦ C. After washing with phosphate buffer solution sufficiently, they were fixed with 1% osmium tetroxide, dehydrated in a graded series of ethanol, and embedded in araldite, polymerized for 24 h at 37 ◦ C. Ultrathin sections (50 nm) were cut with ultramicrotome (LKB-V, Sweden), contrasted with uranyl acetate and lead citrate, and observed with TEM (H-600, Hitachi). 2.9. Measurement of cytokines TNF-␣ in the serum and TNF-␣, IL-1␤, and IL-6 in the brain of mice after exposure to TiO2 particles for different intervals were analyzed by enzyme linked immunosorbent assay (ELISA) kits that is specific for mouse (Jingmei Biotech Co. Ltd. Shenzhen, China). The assays were performed strictly according to the manufacturer’s instructions. Photometric measurements were conducted at 450 nm using Opsys MR 96-well microplate reader (DYNEX, USA). The detection limit of each assay was 15 pg/mL for TNF-␣, 4 pg/mL for IL-1␤ and IL-6. 2.10. Statistical analysis Results were expressed as mean ± standard deviation (S.D.). Multigroup comparisons were evaluated by student’s t-test or one-way analysis of variance (ANOVA) followed by pairwise comparisons using the Student–Newman–Keuls test, as appropriate. In post-hoc tests, the Dunnett’s test was used to compare the differences between the 80 and the 155 nm groups. The statistical significance was considered at p < 0.05.

3. Results 3.1. Ti content in each brain region The Ti contents in the olfactory bulb, hippocampus, cerebral cortex and cerebellum were determined at post-instillation time points of 2, 10, 20 and 30 days and are shown in Fig. 1. The concentrations of most brain parts were significantly higher than any of the controls for all postexposure time periods. In the olfactory bulb, Ti contents increase gradually with time. In the hippocampus, Ti contents show significantly increase after exposure for 2 days, and keep for 10 and 20 days of exposure. However, after 30 days, it reaches the highest points in the hippocampus if compared to other parts of brain. Ti contents in the cerebral cortex show an increase from the time point of 10 days, and then keep at similar level until 30 days. In the cerebellum, Ti contents show a persistent increase with the prolonged exposure time. In the case of Ti levels in sub-brain regions

measured at 30 days of exposure, the ranking of TiO2 deposition is hippocampus > olfactory bulb > cerebellum > cerebral cortex. 3.2. Serum biomarkers Table 1 shows the change of serum biomarkers in the mice treated with 80 nm and 155 nm nanoscale TiO2 at the time points of 2, 10, 20 and 30 days to evaluate the injury of intranasal instilled TiO2 nanoparticles on the viscera. Generally, most of these serum parameters do not show any statistic values. Only at 10 days, the ALT and AST levels in the mice treated with 80 nm TiO2 particles decrease comparing with the control. From the cholesterol level in the serum, TC level is not influenced in each group. Meanwhile, the ALP activity increased significantly (p < 0.05) at second day exposed to 155 nm particles. After 30 days exposure, all biomarkers are recovered and similar to the control, which indicates the low toxicity of repeated instillation of TiO2 nanoparticles at the tested dosage. 3.3. Antioxidative responses of brain The time-course changes of activities of GSH-Px, GST and SOD and GSH level are presented in Fig. 2. Surprisingly, there are no obvious changes of these four markers at the time point of 30 days. However, there are the significantly increased GSH-Px, GST and SOD activities and GSH levels in the 80 nm group (p < 0.05) at 10 days postexposure. Subsequently, at 20 and 30 day, they decreased to the normal level (Fig. 2). MDA, a marker for lipid peroxidation, shows an obvious elevation with time course in three experimental groups. At the 30 days exposure, MDA contents are significantly higher (p < 0.05) in the brain compared with the control (Fig. 3). The 155 nm TiO2 particles produced much higher MDA level than 80 nm ones but the significant difference was not detected between them. 3.4. Pathology changes in tissues After exposure to TiO2 , the coefficient of each tissue to body weight is no different from the control except that the coefficient of kidneys in the 80 nm group is significantly higher than the control (data not shown). Accordingly, the histopathological examinations also show the severe atrophy of renal glomerulus, infiltration and dwindle of interstitially inflammatory cells in the lumen of Bowman’s capsules in the kidneys (data not shown). However, there is no pathological change for the heart, liver and spleen. The pathological changes of olfactory bulb and brain are also examined using Nissl staining method. No apparent difference is found in cerebral cortex and cerebellum between exposed and normal mice. However, to our surprise, the increased numbers of neuron cells in the olfactory bulb and the irregular arrangement of neuron cells in the olfactory nerve layers were observed as shown in Fig. 4. Enlarged and elongated pyramidal cell soma and the decreased number of Nissl body were only found in the CA1 region of hippocampus after exposure to the different TiO2 nanoparticles (Wang et al., 2008). Subsequently, the ultrastructure of neurons in the olfactory bulb was photographed by TEM. Fig. 5 shows the ultrastructure micrographs of olfactory bulb of brain after intranasally instilled TiO2 nanoparticles for 30 days. The obvious condensation of chromatin distributing over the fringe of nuclei and small amount of increased mitochondria were observed in the olfactory bulbs of the 80 and 155 nm groups. Further, the ultrastructure of neurons in CA1 region of hippocampus was photographed by TEM. As shown in Fig. 6, hippocampal nerve cells were degenerating, in association with nucleus membrane crinkling like wave shape and chromatin condensing with high electron density after exposure for 30 days.

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Fig. 1. Ti content in the olfactory bulb (A), cerebral cortex (B), hippocampus (C) and cerebellum (D) of mice (n = 6) intranasally instilled 80 and 155 nm TiO2 particles on the time point of 2, 10, 20 and 30 days.

The crimpled and decreased mitochondria and increased rough endoplasmic reticulum and free ribosome were also observed in the cytoplasm when three to four sections from each group were observed and counted (Fig. 6). All these results imply that the function of neurons in the hippocampus would be greatly injured resulting from the intervention of deposited TiO2 nanoparticles.

3.5. Immune responses of brain The proinflammatory cytokines TNF-␣, IL-1␤ and IL-6 are often produced by the activated monocytes and macrophages. There was no significant change for TNF-␣ level in the serum (Fig. 7A). However, in the brain, the significantly higher TNF-␣ and

Table 1 Serum biochemical parameters in the mice exposed to 80 and 155 nm TiO2 nanoparticles at the time points of 2, 10, 20 and 30 days (n = 6). Groups

ALT (U/L)

2 days Control 80 nm 155 nm

14.0 ± 3.6 18.5 ± 3.4* 13.2 ± 3.5

97.2 ± 21.3 105 ± 26 92.2 ± 15.5

10 days Control 80 nm 155 nm

21.7 ± 5.6 13.8 ± 2.6* 16.2 ± 4.4*

20 days Control 80 nm 155 nm 30 days Control 80 nm 155 nm *

AST (U/L)

BUN (mmol/L)

Cr (␮mol/L)

HDL-C (mmol/L)

TC (mmol/L)

LDL-C (mmol/L)

111 ± 15 122 ± 28 153 ± 32*

7.2 ± 0.9 7.7 ± 0.6 8.1 ± 0.9

60.6 ± 9.3 60.4 ± 7.4 59.6 ± 7.1

2.1 ± 0.2 2.3 ± 0.3 2.2 ± 0.3

2.6 ± 0.3 2.6 ± 0.3 2.6 ± 0.3

0.44 ± 0.08 0.38 ± 0.09 0.38 ± 0.04

86.6 ± 7.9 73.8 ± 9.5* 102 ± 28

122 ± 17 116 ± 18 103 ± 15

7.1 ± 0.6 6.6 ± 0.5 7.5 ± 0.6

53.5 ± 3.5 52.0 ± 2.5 51.3 ± 4.3

2.2 ± 0.3 2.1 ± 0.2 2.1 ± 0.3

2.6 ± 0.3 2.5 ± 0.2 2.6 ± 0.4

0.38 ± 0.11 0.42 ± 0.07 0.30 ± 0.10

22.3 ± 3.9 23.7 ± 5.0 20.0 ± 3.7

99.7 ± 5.8 125.3 ± 39.7 95.2 ± 13.0

91.0 ± 7.6 118.2 ± 28.9 110.8 ± 20.8

8.1 ± 0.8 7.8 ± 0.9 8.3 ± 1.3

51.0 ± 4.3 58.2 ± 6.6 52.4 ± 2.3

2.4 ± 0.3 2.2 ± 0.3 2.4 ± 0.4

2.9 ± 0.3 2.6 ± 0.3 2.8 ± 0.4

0.28 ± 0.08 0.20 ± 0.07 0.27 ± 0.07

23.5 ± 4.2 20.0 ± 3.3 22.4 ± 6.2

101.5 ± 14.5 93.5 ± 17.6 108.8 ± 19.4

88.7 ± 6.9 81.0 ± 14.0 95.2 ± 19.9

8.2 ± 1.1 7.4 ± 0.7 8.2 ± 0.9

66.0 ± 12.5 60.0 ± 8.1 60.3 ± 13.4

2.3 ± 0.5 2.5 ± 0.3 2.4 ± 0.3

2.5 ± 0.5 2.8 ± 0.3 2.6 ± 0.3

0.24 ± 0.05 0.36 ± 0.12 0.29 ± 0.13

significantly different from control group.

ALP (U/L)

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Fig. 2. Changes of GSH-Px (A), GST (B), GSH (C), and SOD (D) in the brain of mice (n = 6) intranasally instilled 80 and 155 nm TiO2 particles on the time point of 2, 10, 20 and 30 days. * , significantly different from control group. + , significantly different from 155 nm group.

IL-1␤ levels (p < 0.05) were induced by exposure to 155 nm TiO2 (Figs. 7B and 8A). In the 80 nm group, TNF-␣ levels show slightly elevated expression (p > 0.05) both in the serum and brain tissue (Fig. 7).

Fig. 3. MDA levels in the brain of mice (n = 6) intranasally instilled 80 and 155 nm TiO2 particles for 2, 10, 20 and 30 days. * p < 0.05 significantly different from the control group.

4. Discussion 4.1. Time-dependent translocation of intranasally instilled TiO2 nanoparticles Generally speaking, the mechanism for deposition of inhaled particles in the respiratory system includes the diffusion, inertial impaction, gravitational settling and interception (Oberdörster et al., 2005b). Based on a predictive mathematical model, about 90% 10-nm particles deposit in the tracheobronchial and alveolar regions of the respiratory tract, only 10% deposit in the nasopharyngeal (ICRP, 1994). Because of the special intricate network of sensory nerve endings between the nose and olfactory bulb, the inhaled/instilled drugs (or virus, nanoparticles) are transported directly to the brain via the olfactory nerve tract for treating CNS disorders, thereby circumventing the blood–brain barrier (Talegaonkar and Mishra, 2004). In this study, the intranasal instilled TiO2 nanoparticles were firstly translocated to the olfactory bulb and further to the hippocampus at the second day postexposure. With the prolonged time, Ti content in the olfactory bulb continuously increased and the translocation to the cerebral cortex and cerebellum occurred under the diffusion mechanism, even to other organs such as lung and kidneys. At the end of exposure, the irregular arrangement of neuron cells in the olfactory nerve layers and the dispersed arrangement and loss of neurons in the CA1 region of hippocampus were induced by the deposited

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Fig. 4. Nissl staining of olfactory bulb in the mice intranasally instilled 80 nm (A), 155 nm (B) TiO2 nanoparticles and control group (C). Arrows indicate the irregular arrangement of neuron cells in the olfactory nerve layers.

Fig. 5. Ultrastructure of olfactory bulb after intranasally instilled TiO2 nanoparticles for 30 days. (A) Control group (10,000×); (B) 80 nm group (10,000×); and (C) 155 nm group (8000×). Nu: Nucleus.

Fig. 6. Ultrastructure of hippocampus after intranasally instilled TiO2 nanoparticles for 30 days. (A) Control group (10,000×); (B) 80 nm group (10,000×); and (C) 155 nm group (10,000×). ER: endoplasmic reticulum, MT: Mitochondria, Nu: Nucleus.

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Fig. 7. Levels of TNF-␣ in the serum (A) and brain (B) of mice (n = 6) intranasally instilled 80 and 155 nm TiO2 particles for 30 days. * p < 0.05 significantly different from the control group.

Fig. 8. Levels of IL-1␤ (A) and IL-6 (B) in the brain of mice (n = 6) intranasally instilled 80 and 155 nm TiO2 particles for 30 days. * p < 0.05 significantly different from the control group.

4.2. Oxidative damage in murine brain

TiO2 . All results indicate that a lot of intranasal instilled TiO2 first entered into the olfactory bulb and then deposited in the hippocampus and CNS as the time extended to induce the pathology change and the abnormal arrangement of neurons, but only a little diffusing to the lung tissue. Several studies (Bermudez et al., 2004; Warheit et al., 2007b) have reported the severe pathological damage of lung in the rats after exposure to TiO2 nanoparticles through inhalation or intratracheal instillation. However, similar studies have also shown that the olfactory neuronal pathway is an important route for translocating inhaled nanoparticles to the central nervous system bypass the blood–brain barrier and that this can result in inflammatory changes (Dorman et al., 2001, 2004; Elder et al., 2006; Oberdörster et al., 2004). Inhaled elemental carbon particles (13 C; 35 nm, count median diameter) can accumulate in rat olfactory bulb after whole body inhalation (Oberdörster et al., 2004). Inhalation of Mn oxide nanoparticles aerosols for 12 days also resulted in significant increases (∼3.5-fold) in olfactory bulb Mn content; the increase in liver Mn content was much higher than the increase in lung tissue. In present study, Ti accumulated in olfactory bulbs quickly for only once exposure when both nares were administrated (Fig. 1). Furthermore, Ti accumulation increased in the hippocampus with time postexposure and exposure duration. A small, but insignificant, amount of Ti also accumulated in the cerebral cortex and cerebellum. Consistent with the previous reports, thus, this neuronal translocation pathway should be taken in consideration for the health risk assessment of nanoparticles.

The special character of small size and large surface area per mass render nanoparticles more biological activity. The phagocytosis of nanoscale TiO2 by embryo cell, neurons and microglia and oxidative damage related with ROS (• OH and O2 •− ) production by these aggregates have been previously reported (Dunford et al., 1995; Long et al., 2006, 2007; Wang et al., 2008). The slightly changed SOD activity and highly elevated MDA level in the brain indicated that the deposited TiO2 particles in the brain could induce certain impairment. Similarly, particulate matters in polluted air were reported to increase inflammatory biomarkers in mouse brain (Champion and Mitragotri, 2006). In the cell membrane of brain, the abundant lipids, polysaccharides and proteins are the targets of ROS. It should be emphasized that the free-radical attack at the membrane lipids would result in the accumulation of a complex mixture of products at the cell surface, consisting of the initial lipids and their fragmentation products, as well as peroxidation products of both former and latter. The superoxide radical (O2 •− ) produced by TiO2 can get across the cell membranes and form the highly reactive • OH by interaction with transition metal ions (Ahsan et al., 2003), and the • OH radical attacks membrane phospholipids in a free radical process, resulting in phospholipid peroxidation (MDA elevation) and loss of membrane integrity (upregulated LDH activation, Blaisdell, 2002). In the brain microglia cells, TiO2 nanoparticles preferentially mobilize to mitochondria and deposit in it (Long et al., 2006) to influence the energy metabolism and respiration function. Consistently, present in vivo study also shows the ultrastructural changes such as the crimpled and decreased mitochondria, many rough endoplasmic reticulum and increased free ribosome in the cytoplasm

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in the CA1 region of hippocampus as indicated from TEM images (Fig. 6).

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(Nos. 10490180 and 20751001) and the Knowledge Innovation Program of Chinese Academy of Sciences.

4.3. Inflammatory responses in murine brain References The inflammatory event is, in most cases, a chronic long-lasting and low-grade systemic autoimmunity, which is the tier-2 stage for oxidative stress response pathway associating with the induction of transcriptional factor NF-␬B (Nel et al., 2006). In the current study, as a response to oxidative stress, proinflammatory cytokines of TNF-␣, IL-1␤ and IL-6 were highly secreted in the brain of mice after exposure to TiO2 particles for 30 days. Elder et al. (2006) also reported that the intranasal instillation of manganese oxide stimulated TNF-␣ mRNA increase and the elevation of macrophage inflammatory protein-2, neuronal cell adhesion molecule mRNA in the olfactory bulb and brain regions. The immune cytokines are synthesized by the activated microglia, astrocyte, monocyte, macrophages and neutrophil in response to foreign stimuli, such as TiO2 , ultrafine particles, Ti and Ti alloys particulate wear debris, (Campbell et al., 2005; Vallés et al., 2006; Vermes et al., 2001). In lung tissue, the elevated releases of cytokines have been also reported in mammalians exposed to ultrafine particles (Rao et al., 2005; Inoue et al., 2005). It is demonstrated that there is a further increase in such inflammation in neurodegenerative disorders (Rothwell and Relton, 1993; Venters et al., 1999). The median timeweighted average exposure of workers (cumulative exposure index divided by duration of exposure) was estimated to be 10 mg/m3 at European and four US TiO2 manufacturing plants (Boffetta et al., 2004). There was no any carcinogenic effect associated with workplace exposure to TiO2 . In this mouse model, although there are no significant changes of serum biochemical parameters, the significantly increased release of proinflammatory cytokines indicate the neuron cells (including microglia and astrocytes) were activated in the brain of mice exposed to 155 nm nanoparticles, which might be related with some neurodegenerative diseases. In conclusion, time-dependent translocation and potential impairment on central nervous system by intranasally instilled TiO2 nanoparticles were assessed. Intranasal instillation of either rutile or anatase TiO2 nanoparticles produced the sustained accumulation in brain tissues especially deposit in the hippocampus during whole exposure, which indicate that the TiO2 nanoparticles can enter the brain via the olfactory bulb. After exposure for 30 days, no obvious changes were observed in heart, liver, spleen and lung except the kidneys and brain. In the brain, the irregular arrangement of neurons in the olfactory bulb and hippocampus were detected. Further, the deposited TiO2 particles induced the oxidative damage as elevated MDA levels and the ultrastructural changes of neurons in the hippocampus. The immune responses (increased TNF-␣ and IL-1␤ levels) were activated in murine brain to counteract the attack of TiO2 particles. However, the effects of nanoparticle properties on the immune system are still being explored. Although understanding the mechanisms requires further investigation, the present results suggest we should pay attention to potential risk of occupational exposure for large-scaled production of TiO2 nanoparticles. Conflict of interest None declared. Acknowledgements We thank financial support from the 973 programme (No. 2006CB705603), the National Natural Science Foundation of China

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