Nanosized titanium dioxide enhanced inflammatory responses in the septic brain of mouse

Neuroscience 165 (2010) 445– 454

NANOSIZED TITANIUM DIOXIDE ENHANCED INFLAMMATORY RESPONSES IN THE SEPTIC BRAIN OF MOUSE J. A. SHIN,a E. J. LEE,b S. M. SEO,a H. S. KIM,b J. L. KANGc AND E. M. PARKa*

particles is increasing, as is the risk to human of intentional or accidental exposure to nanoparticles (Stone et al., 2007). While it is the small size of nanoparticles that makes them unique and useful, this same quality also underlies their potential to interfere with human health. Although there is a consensus for the urgent need to evaluate the biosafety of nanoparticles, the study of nanoparticle toxicity has fallen behind their rapid introduction to industry (Stone et al., 2007; Papp et al., 2008). Because of its whitening and photocatalytic effects, titanium dioxide (TiO2) is used widely in the production of everyday products such as paper, cosmetics and food (Powell and Kanarek, 2006). Nanosized TiO2 is also used in sunscreens, toothpastes, surface coatings, and in water and air remediation. Bulk TiO2 has been regarded as safe and has been used as a negative control in toxicity studies (Tran et al., 2000; Hext et al., 2005), but recent studies have shown that nanosized TiO2 acts differently from its bulk counterpart and causes adverse health effects. The effects of two different sizes of TiO2 on the lung were compared in inhalation studies. Ultrafine (21 nm) but not fine (⬍1 ␮m) particles caused lung damage, inflammation and fibrosis, suggesting that smaller size is more toxic to biologic systems (Zhang et al., 1998; Oberdorster et al., 2000, 2005; Bermudez et al., 2002, 2004; Dick et al., 2003). The mechanism underlying the toxicity of nanoparticles has been repeatedly suggested as inflammation through oxidative stress. For example, ultrafine TiO2 was shown to induce reactive oxygen species (ROS), followed by cytokine production through nuclear factor-␬B (NF-␬B) or mitogen-activated protein (MAP) kinase (Nel et al., 2006; Kang et al., 2008). The potential adverse effects of ultrafine TiO2 on the brain have been also studied in microglial cell culture as well as in vivo. Microglia reacted to ultrafine TiO2 and produced ROS, which caused neuronal cell death in the striatum (Long et al., 2006, 2007). Repetitive exposure of ultrafine TiO2 by various routes of administration induced pathologic changes and resulted in brain damage (Wang et al., 2008a,b; Chen et al., 2009; Liu et al., 2009). These studies showed that nanoparticles can enter the brain and imply that brain may be susceptible to nanoparticle-induced oxidative stress and inflammation not only in normal population, but particularly in individuals prone to inflammation or with pre-existing brain diseases. Because inflammation is one of the major pathologic findings in patients with many neurological diseases, the possibility of toxic effects of ultrafine TiO2 should be investigated in these individuals. Furthermore engineered nanoparticles can be applied to patients with brain diseases as tools for

a Department of Pharmacology, Ewha Medical Research Institute, School of Medicine, Ewha Womans University, Seoul, Republic of Korea b Department of Molecular Medicine, Ewha Medical Research Institute, School of Medicine, Ewha Womans University, Seoul, Republic of Korea c Department of Physiology, Ewha Medical Research Institute, School of Medicine, Ewha Womans University, Seoul, Republic of Korea

Abstract—Nanosized titanium dioxide (TiO2) is used widely in various everyday products and can be applied to the medical field for diagnostic or therapeutic tools. However, its neurobiological responses have not been defined completely in the brain. To evaluate the acute inflammatory response to TiO2 particles of two different sizes in normal and septic brains, male C57BL/6 mice were given intraperitoneal injections of fine (<1 ␮m) or ultrafine (21 nm) TiO2, 30 min after vehicle or lipopolysaccaride (LPS). In the normal brain, neither fine nor ultrafine TiO2 induced inflammation. However, in the brains of LPS-exposed mice, ultrafine TiO2 significantly elevated proinflammatory cytokine interleukin-1␤ (IL-1␤) and tumor necrosis factor-␣ (TNF-␣) mRNAs, and IL-1␤ protein levels. Also ultrafine TiO2 increased the levels of reactive oxygen species and activated microglia 24 h after LPS challenge. In BV2 microglial cells stimulated with LPS, ultrafine TiO2 enhanced TNF-␣ production and augmented nuclear factor-kB binding activity. These findings suggest that nanosized TiO2 promotes an exaggerated neuroinflammatory responses by enhancing microglial activation in the pre-inflamed brain, in part. © 2010 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: nanoparticle, ultrafine TiO2, sepsis, LPS, microglia, neuroinflammation.

The development of nanotechnology provides a variety of possibilities for the application of engineered nanoparticles in the industrial and medical fields (Gwinn and Vallyathan, 2006; Powell and Kanarek, 2006). The production of nano*Correspondence to: E. M. Park, Department of Pharmacology, School of Medicine, Ewha Womans University, 911-1 Mok6dong, Yangcheon-gu, Seoul, 158-710, Republic of Korea. Tel: ⫹82-22650-5743; fax: ⫹82-2-2653-8891. E-mail address: [email protected] (E. M. Park). Abbreviations: ANOVA, analysis of variance; BBB, blood– brain barrier; DMEM, Dulbecco’s Modified Eagle’s Medium; DPPC, dipalmitoyl phosphatidylcholine; ELISA, enzyme-linked immunosorbent assay; EMSA, electrophoretic mobility shift assay; IL-1␤, interleukin-1␤; iNOS, inducible nitric oxide synthase; LPS, lipopolysaccharide; MAP kinase, mitogen-activated protein kinase; NF-␬B, nuclear factor-␬B; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; ROS, reactive oxygen species; SDS, sodium dodecyl sulfate; TBS, Tris– buffered saline; TiO2, titanium dioxide; TNF-␣, tumor necrosis factor-␣.

0306-4522/10 $ - see front matter © 2010 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2009.10.057

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diagnosis and treatment (Stone et al., 2007; Lopez et al., 2008). Systemic administration of lipopolysaccharide (LPS) is an acute model of infection and induces breakdown of blood– brain barrier (BBB), ROS production, and inflammatory responses in the brain (Semmler et al., 2005; Noble et al., 2007; Qin et al., 2007). We investigated the acute effects of TiO2 using two different sizes, fine and ultrafine, on the normal and septic brains. In addition, the effect of TiO2 on microglial reactivity was investigated in BV2 microglial cell cultures. We found that ultrafine TiO2 aggravated the inflammatory responses in the septic but not in normal brain, and microglia stimulated with LPS and ultrafine TiO2 showed enhanced inflammatory responses.

EXPERIMENTAL PROCEDURES Particles and preparation Fine TiO2 (Titanium (IV) oxide; Sigma-Aldrich, Atlanta, GA, USA) was 100% rutile and had a particle size of 1 ␮m. Ultrafine TiO2 (Aeroxide® TiO2 P 25, Evonik Degussa Corporation, Piscataway, NJ, USA) was a mixture of 80% anatase and 20% rutile forms and had an average primary particle diameter of 21 nm with surface area 50 m2/g. Each particulate sample was sieved in a Vibratory Sieve Shaker (JISICO, Seoul, Republic of Korea) for 10 min at a vibration level 3 to break apart large clumps. Three different sieve sizes (1.18-mm, 250-␮m, 45-␮m openings) were used successively in the sieving process (Kang et al., 2008). The particle samples were sterilized by heating for 2 h at 160 °C in a dry oven, and then the particles (5 mg/ml, respectively) were suspended in a dispersion medium of Ca⫹2-and Mg⫹2-free phosphate-buffered saline (PBS), containing 10 ␮g/ml dipalmitoyl phosphatidylcholine (DPPC) and 0.6 mg/ml bovine serum albumin (Sager et al., 2007). Suspensions were sonicated for 10 min using a Cole-palmer probe sonicator (Cole-palmer instrument, Vernon Hills, IL, USA) at a duty cycle setting of 10% and an output setting of one. Particle suspension and sonication were performed just before the preparation was given to an animal.

Animals and treatments Experiments were performed in male C57BL/6 mice (age 10 –11 weeks, weighing 24 –26 g, Koatech, Pyeongtaek, Gyeonggi-do, Republic of Korea). Mice were acclimatized for 1 week before the experiments in an animal room under a 12 h light/dark cycle at 22⫾2 °C. All procedures were approved by the Institutional Animal Care and Use Committee at the Medical School of Ewha Womans University and conformed to international guidelines on the ethical use of animals. The number of animals used for the study was minimized to reduce animal suffering. The experimental protocol is shown in Fig. 1. LPS (Escherichia coli serotype 055:B5, Sigma Chemical Co., St Louis, MO, USA) was dissolved in saline and administered at a dose of 5 mg/kg, i.p. in a volume of 0.2 ml/mouse. Fine or ultrafine TiO2 was administered at a dose of approximately 40 mg/kg (1 mg/mouse) in a volume of 5 ml/kg. Mice were divided randomly into six groups. Mice in three groups served as vehicle controls for LPS injections. These mice received an injection of the LPS vehicle (normal saline, 0.2 ml/mouse, i.p.), followed 30 min later by an injection of TiO2 dispersion medium (control⫹vehicle), fine TiO2 (control⫹fine), or ultrafine TiO2 (control⫹ultrafine). Mice in the three LPS-treated groups received an injection of LPS, followed 30 min later by an injection of dispersion medium (LPS⫹vehicle), fine TiO2 (LPS⫹fine), or ultrafine TiO2 (LPS⫹ultrafine). The dose of systemic LPS that induced brain inflammation and microglial activation was based on the study of Qin et al. (2007). The dose

Fig. 1. Experimental protocol for in vivo study. Control and LPS groups were subdivided according to vehicle, fine TiO2 and ultrafine TiO2 treatments. Levels of cytokines and ROS production were measured between 2 and 24 h, and OX42 (CD11b) immunoreactivity was examined 24 h after vehicle (normal saline) or LPS treatment.

and route of administration of TiO2 used in the current study was adopted from the previous study using TiO2 nanoparticles as drug carriers to treat glioblastoma multiforme in the brain (Lopez et al., 2008).

RNA isolation and real-time PCR RNA was isolated from cortical and hippocampal tissue from one hemisphere of mouse brain and subjected to quantitative real-time polymerase chain reaction (PCR), 2 h after treatment with normal saline or LPS. Total RNA was prepared using Trizol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA). Tissues were homogenized by sonication in 0.5 ml Trizol, and RNA was purified using chloroform/isopropanol precipitation and dissolved in diethylpyrocarbonate-treated water (iNtRON Biotechnology, Seongnam, Gyeonggi-do, Republic of Korea). First strand synthesis was performed with 1 ␮g of total RNA from each sample and 1 ␮l of oligo (dT)15 (Bioneer, Daejeon, Republic of Korea), using a Power cDNA Synthesis kit (iNtRON Biotechnology). A 2-␮l aliquot of diluted cDNA (1:10) was amplified by SYBR® Green PCR Master Mix (Applied Biosystems, Forster City, CA, USA) to a final volume of 20 ␮l. A PCR reaction was performed using an ABI Prism 7000 sequence detector (Applied Biosystems). PCR cycles consisted of initial denaturation at 95 °C for 5 min, followed by 40 cycles of 95 °C for 30 s, 52 °C for 30 s, and 72 °C for 45 s. The primers for cyclophilin (forward: 5=-TGAAGTTGTCCACAGTCAGC-3=; reverse: 5=-TTCATCTGCACTGCCAAGAC-3=), interleukin-1␤ (IL-1␤, forward: 5=-AATGATCTGTTCTTTGAGGCTGAC-3=; reverse: 5=CGAGATGCTGCTGTGA GATTTGAAG-3=), tumor necrosis factor-␣ (TNF-␣, forward: 5=-GAACTGGCAGAAGAGGCACTC-3=; reverse: 5=-AGGGTCTGGGCCATAGAACTG-3=) and inducible nitric oxide synthase (iNOS, forward: 5=-GGAAGAGGAACAACACTGCTGGT-3=; reverse: 5=-GAACTGAGGGTACATGCTGGAGC3=) were purchased from Bioneer. The cycling threshold (Ct) values of IL-1␤, TNF-␣ and iNOS were normalized to that of cyclophilin and relative expression levels were calculated by the 2⌬⌬Ct method (Livak and Schmittgen, 2001). All samples were run in triplicate.

Western blot hybridization The cortical and hippocampal tissues from each animal were subjected to simultaneous Western blot hybridization. The tissues obtained from one hemisphere was lysed in a sodium dodecyl

J. A. Shin et al. / Neuroscience 165 (2010) 445– 454 sulfate (SDS) buffer (62 mM Tris–HCl, 1 mM ethylenediamine tetraacetic acid, 2% SDS, pH 6.8 –7.0) containing one tablet of a protease inhibitor cocktail (Complete Mini, Boehringer Mannheim, Mannheim, Germany) in 10 ml of solubilizing buffer, incubated for 20 min on ice, and centrifuged at 13,000 rpm for 10 min at 4 °C. Protein concentration from the supernatant was determined (BioRad Laboratories, Hercules, CA, USA), and 100 ␮g of protein was loaded for SDS-polyacrylamide gel electrophoresis. Proteins were transferred onto polyvinylidine difluoride (Amersham Phamarcia Biotech Inc., NJ, USA) membranes using an electroblotting apparatus. Membranes were blocked in Tris– buffered saline (TBS) containing 0.1% Tween-20 and 5% dry milk for 1 h, incubated overnight with IL-1␤ (1:100, R&D, Minneapolis, MN, USA) antibody, and washed three times (30 min each) with TBS containing 0.1% Tween-20. The membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h each and washed three times (30 min each) with TBS containing 0.1% Tween-20. Protein bands were visualized with the ECL Plus Western blotting detection system (Amersham Pharmacia Biotech). Each membrane was re-blotted using an antibody stripping solution (Chemicon International, Temecula, CA, USA) according to the manufacturer’s instruction to visualize actin (1:1000; Santa Cruz biotechnology, Santa Cruz, CA, USA). For quantification, densities of IL-1␤ were normalized by corresponding blots for actin.

Quantification of ROS production ROS production was determined using in vivo hydroethidine microfluorography (Kondo et al., 1997), as described previously (Cho et al., 2005). Cell-permeable hydroethidine is a fluorescent dye that is oxidized to ethidium by superoxide (Carter et al., 1994; Bindokas et al., 1996). Ethidium intercalates into DNA and labels cells with red fluorescence. The fluorescence signal attributable to ethidium reflects the cumulative ROS production during the period between hydroethidine administration and brain removal (Cho et al., 2005). Hydroethidine (10 mg/kg, Sigma-Aldrich) was injected into the tail vein and allowed to circulate for 3 h. Animals were sacrificed under isofluorane anesthesia and brains were removed and frozen. Serial coronal sections (20 ␮m thickness) of the brain were collected at 600-␮m intervals in a cryostat. Images of each section were viewed and captured using a digital camera with a fluorescence microscope equipped with a custom filter set (Axiovert200, Carl Zeiss, Jena, Germany), and ROS production was analyzed using imaging software (Axiovision LE 4.1, Carl Zeiss). After subtraction of the camera dark current, pixel intensities for ethidium signals were assessed in predefined areas of the cortex and hippocampus (Cho et al., 2005). Fluorescence intensity was measured in four serial sections per animal (Fig. 4A, rostrocaudal levels, ⫺1.5, ⫺2.1, ⫺2.7, and ⫺3.3 mm from bregma). The sum of the fluorescence intensity for each region was divided by the total number of pixels analyzed and expressed as relative fluorescence units (Manabe et al., 2004; Cho et al., 2005).

Immunofluorescence staining Animals were anesthetized with sodium pentobarbital (120 mg/kg, i.p.) and perfused transcardially with saline followed by 4% cold formaldehyde in 0.1 M sodium phosphate buffer (pH 7.2). The brains were removed and incubated overnight in fixatives and stored in a 30% sucrose solution. Serial coronal brain sections (30 ␮m thickness, 600-␮m interval) were collected in a cryostat in regions containing the dorsal hippocampus (between ⫺2.0 and ⫺3.0 mm from bregma). Brain sections were incubated in TBS containing 0.1% Triton X-100, 5% normal serum and 1% bovine serum albumin for 1 h and subsequently incubated with OX42 antibody (rat-anti-CD11b, 1:500, Serotec, Raleigh, NC, USA). On the following day, the secondary antibody conjugated fluorescein isothiocyanate (goat-anti-rat, 1:200, Serotec) was applied to sec-

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tions for 1 h. TBS was used to wash sections between all steps. The sections were mounted with Vectashield mounting medium (Vector Laboratories, Burlingame, CA, USA), and fluorescence microcopy images were obtained (Axiovert 200).

Microglial cell cultures All reagents used for cell culture, including penicillin/streptomycin, trypsin and Dulbecco’s modified Eagle’s medium (DMEM), were purchased from Gibco BRL (Grand Island, NY, USA). The immortalized murine BV2 cell line that exhibits both the phenotypic and functional properties of reactive microglia cells (Bocchini et al., 1992) was grown and maintained in DMEM supplemented with 10% fetal bovine serum (Hyclone Laboratories Inc., Logan, UT, USA), streptomycin and penicillin. Cultures were treated either singly with LPS (100 ng/ml), fine TiO2 or ultrafine TiO2 or were treated simultaneously with the indicated combinations (co-treatment).

Measurement of TNF-␣ levels in microglial cell cultures Microglial cells (1⫻105 cells/well in a 24-well plate) were treated with TiO2 or LPS (100 ng/ml) singly or in combination in the presence of serum. The supernatants of the cultured microglia were collected 24 h after LPS or TiO2 treatment, and the concentrations of TNF-␣ were measured by enzyme-linked immunosorbent assay (ELISA) using monoclonal antibodies according to the procedure recommended by the supplier (PharMingen, San Diego, CA, USA). The serum in the media did not interfere with the assay.

Nuclear extract preparation and electrophoretic mobility shift assay (EMSA) Nuclear extracts were prepared from microglia treated with TiO2 or LPS as described previously (Woo et al., 2003). Double stranded DNA oligonucleotides containing the NF-␬B consensus sequences (Promega, Madison, WI, USA) were end-labeled using T4 polynucleotide kinase (New England Biolabs, Beverly, MA, USA) in the presence of [␥-32P]ATP. Five micrograms of the nuclear proteins were incubated with 32P-labeled NF-␬B probe for 30 min on ice and resolved on a 5% acrylamide gel as previously described (Woo et al., 2003).

Statistical analysis The data from in vivo experiments are expressed as mean⫾SE. Multiple comparisons were evaluated by the analysis of variance (ANOVA) followed by post hoc Fisher’s PLSD tests using the program StatView (StatView version 5, SAS Institute, Cary, NC, USA). Differences were considered significant at P⬍0.05. All in vitro experiments were performed with triplicate samples and repeated at least three times unless otherwise stated, and the data are presented as means⫾SD. Statistical comparisons between groups were performed using one-way ANOVA followed by Student’s t-test. A value of P⬍0.05 was considered significant.

RESULTS Ultrafine TiO2 increases proinflammatory cytokine mRNA levels in the inflamed brain To determine whether nanoparticles of TiO2 induce or enhance proinflammatory cytokine production in the normal or septic brain, mice were treated with normal saline or LPS, followed 30 min later by TiO2 administration. The mRNA levels of IL-1␤, TNF-␣, and iNOS, which play major

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Fig. 2. Levels of proinflammatory cytokines mRNA in the brain exposed to fine or ultrafine TiO2. (A) Representative figures of IL-1␤, TNF-␣ and iNOS mRNA expression by reverse transcription-PCR technique in the cortex and hippocampus, 2 h after treatment with normal saline or LPS (5 mg/kg, i.p.). Vehicle (Veh, 0.2 ml/mouse), fine (fTiO2) or ultrafine TiO2 (ufTiO2) particles (1 mg/mouse, i.p., each) were given 30 min after normal saline or LPS treatment. (B) Quantitative changes of IL-1␤, TNF-␣ and iNOS mRNA by real-time PCR in the cortex and hippocampus 2 h after treatment with normal saline or LPS (n⫽4 in each group). Values are expressed as the ratio versus control⫹vehicle group (given a nominal value of 1) and represent mean⫾SE. * P⬍0.05 from all control groups; # P⬍0.05 from LPS⫹Veh and LPS⫹fine TiO2 groups; ⽧ P⬍0.05 from control⫹vehicle and control⫹fine TiO2 groups (ANOVA with post hoc Fisher’s PLSD test).

roles in neuroinflammation, were examined in the cortex and hippocampus 2 h after normal saline or LPS injection. The time points selected to examine cytokine expression in this study were based on previous studies (Qin et al., 2007). In control groups, neither fine nor ultrafine TiO2 significantly influenced cytokine levels (Fig. 2B). In response to LPS treatment, cytokine levels were increased but were not affected by fine TiO2. Ultrafine TiO2, however, markedly increased IL-1␤ (3.2-fold and 3.0-fold in the cortex and hippocampus, respectively) and TNF-␤ (2.7-fold and 3.0-fold in the cortex and hippocampus, respectively) levels compared to the LPS⫹vehicle group. The level of iNOS was not significantly induced by LPS, but was in-

creased by ultrafine TiO2 (6.3-fold in the cortex and 6.2fold in the hippocampus compared to the control⫹vehicle group). These findings suggest that genes of proinflammatory cytokines are not affected by fine or ultrafine TiO2 in the normal brain, but ultrafine TiO2 induces or enhances the gene expression levels of proinflammatory cytokines in the septic brain. Increased expression of IL-1␤ protein by ultrafine TiO2 in the inflamed brain To determine the extent to which cytokine genes are translated, we examined the protein level of IL-␤, for which we

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Fig. 3. IL-1␤ protein levels in the cortex and hippocampus exposed to ultrafine TiO2. Representative blots and quantitative levels of IL-1␤ protein in the cortex at 2 h (A), 6 h (B) and 24 h (C), and in the hippocampus at 6 h (D) after treatment with normal saline or LPS (5 mg/kg, i.p., n⫽3 in each group). Vehicle (Veh, 0.2 ml/mouse) or ultrafine TiO2 (ufTiO2) particles (1 mg/mouse, i.p.) were given 30 min after normal saline or LPS treatment. Values are expressed as ratio versus control⫹vehicle group (given a nominal value of 1) and represent mean⫾SE. * P⬍0.05 from control⫹vehicle group; # P⬍0.05 from LPS⫹vehicle group (ANOVA with post hoc Fisher’s PLSD test).

found the greatest increase in mRNA level induced by ultrafine TiO2 (Fig. 2B), at 2 h 6 h, and 24 h after normal saline or LPS treatment in the cortex. In response to LPS treatment alone, IL-␤ protein expression was detectable at 6 h, but not at 2 or 24 h (Fig. 3A–C). However, in brains from mice treated with ultrafine TiO2, IL-␤ protein was detectable at 2 h, and there was a marked increase in IL-␤ protein 6 h (4.8-fold, Fig. 3B) compared to the LPS⫹ vehicle group. We also checked IL-␤ level in the hippocampus at 6 h. Again, IL-␤ protein expression was significantly increased by ultrafine TiO2 in LPS stimulated brain (1.7fold, Fig. 3D). IL-␤ was not detectable in the brains of mice exposed to ultrafine TiO2 only at any time points examined. These findings show that ultrafine TiO2 enhances the expression of cytokines at both mRNA and protein levels in the septic brain. Enhanced ROS production by ultrafine TiO2 in the inflamed brain Nanosized TiO2 is known to generate ROS when tested in cells (Afaq et al., 1998; Gurr et al., 2005; Long et al., 2006; Kang et al., 2008). Systemic LPS is also known to generate ROS in the brain (Noble et al., 2007). To determine whether ultrafine TiO2 induces or enhances ROS in the normal or septic brains, ROS production was examined by hydroethidine at 3 and 24 h after the administration of

normal saline or LPS. ROS was not increased by ultrafine TiO2 in the brains of mice treated with saline only. In brains from LPS-treated mice, ROS production was significantly increased at 3 h, falling somewhat at 24 h in both cortex and hippocampus (Fig. 4A, B). In LPS-treated mice, ultrafine TiO2 had different effects on ROS production at different time points. ROS production was not changed by ultrafine TiO2 at 3 h but the level was significantly enhanced at 24 h in the cortex and hippocampus of septic brain. These findings suggest that ultrafine TiO2 augments ROS generation at later time point in the septic brain. Increased activation of microglia by ultrafine TiO2 in the inflamed brain Microglia, resident mononuclear phagocytes in the brain, play an important role in neuroinflammation (GonzalezScarano and Baltuch, 1999). LPS activates microglia, thereby stimulating the production of pro-inflammatory cytokines and ROS (Wang et al., 2004; Park et al., 2007). Therefore, the next experiment was done to determine whether microglial activation in the brain was increased by ultrafine TiO2 24 h after the administration of normal saline or LPS, a time point corresponding to increased production of ROS in response to ultrafine TiO2 in the septic brain (Fig. 4). OX-42 (CD11b) antibody was used to detect microglial activation, showing ramified, rounded and ameboid

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Fig. 4. ROS production in the brain exposed to ultrafine TiO2 assessed by hydropethidine microfluorography. (A) Regions of the cortex and hippocampus from which the ROS signal was measured (upper panel). Representative figures of ROS signals in the cortex and hippocampus 24 h after treatment with normal saline or LPS (5 mg/kg, i.p., lower panels). Vehicle (Veh, 0.2 ml/mouse) or ultrafine TiO2 (ufTiO2) particles (1 mg/mouse, i.p.) were given 30 min after treatment with normal saline or LPS. (B) Quantitative analysis of ROS production in the cortex and hippocampus 3 and 24 h after treatment with normal saline or LPS (5 mg/kg, i.p.; n⫽3, in control groups; n⫽6, in LPS groups). Values represent mean⫾SE. * P⬍0.05 from control⫹vehicle group; # P⬍0.05 from LPS⫹vehicle group (ANOVA with post hoc Fisher’s PLSD test). For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.

shape (Akiyama and McGeer, 1990; Park et al., 2009). Microglial activation was not detected in the brains of normal mice (Fig. 5), but the expression of OX-42 was increased in the cortex and hippocampus of LPS-treated mice. After exposure to ultrafine TiO2, the expression and the number of activated form microglia were further enhanced in the septic brain (Fig. 5, arrows). The findings indicate that ultrafine TiO2 augments the stimulatory actions of LPS on microglia.

Ultrafine TiO2 augments TNF-␣ production in LPS-stimulated microglial cells To investigate the direct effects of TiO2 particles on microglia, BV2 microglial cells were stimulated with LPS (100 ng/ml) in the presence or absence of TiO2, and the effect of TiO2 on the level of TNF-␣, a critical mediator of neuroinflammation and neurotoxicity, was determined. As shown in Fig. 6A, BV2 cells exposed to ultrafine TiO2 only

Fig. 5. OX-42 (CD11b) expression in the cortex and hippocampus exposed to ultrafine TiO2. The number of ramified CD11b positive cells (green) with thick and densely stained processes was markedly increased in the brain of the LPS⫹ultrafine TiO2 group compared to control and LPS⫹vehicle groups at 24 h after LPS treatment (5 mg/kg), especially in the cortex (arrows). Vehicle (Veh, 0.2 ml/mouse) or ultrafine TiO2 (ufTiO2) particles (1 mg/mouse, i.p.) were given 30 min after normal saline or LPS treatment. Representative images were obtained from one set of experiments, and three experiments were done independently. Scale bar, 100 ␮m. For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.

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LPS-induced TNF-␣ release by about 25%. However, higher concentrations did not further augment TNF-␣ production. In contrast to ultrafine TiO2, fine TiO2 had no significant effect on TNF-␣ production in BV2 cells, either with or without LPS treatment. The results suggest that ultrafine TiO2 aggravates the LPS-induced inflammatory response in microglia. Ultrafine TiO2 augments LPS-induced NF-␬B DNA binding activity in microglial cells NF-␬B is an important upstream regulator of various cytokines as well as iNOS gene expression in microglia (Pahl, 1999). Therefore, we examined the effect of ultrafine TiO2 on NF-␬B activity. As shown in Fig. 6C, stimulation of BV2 cells with LPS resulted in strong NF-␬B binding, which was significantly augmented in cells treated with ultrafine TiO2. Therefore, NF-␬B may be an important mediator of the inflammatory activity of ultrafine TiO2 in LPS-stimulated microglia.

DISCUSSION

Fig. 6. Effects of TiO2 on TNF-␣ production and NF-␬B activity in LPS-stimulated BV2 microglial cells. BV2 microglial cells were incubated with ultrafine (UF, 25–200 ␮g/ml) or fine (F, 25–200 ␮g/ml) TiO2 in the absence (A) or presence of LPS (100 ng/ml) (B). After 24 h, the supernatants were collected, and the amount of TNF-␣ was measured as described in Experimemtal Procedures. The data are expressed as the mean⫾SD of four independent experiments. * P⬍0.05, significantly different from the value in cells treated with LPS alone (B, Cont). (C) EMSA for NF-␬B DNA binding activity. Nuclear extracts were prepared from BV2 cells 3 h after exposure of ultrafine TiO2 (uf, 25 and 100 ␮g/ml) with or without LPS (100 ng/ml). The upper arrow indicates a DNA-protein complex of NF-␬B. “F” indicates free probe. Fold induction of NF-␬B binding, representative of three experiments, is shown.

failed to produce significant levels of TNF-␣, while the production of TNF-␣ in BV2 cells exposed to LPS was markedly enhanced by exposure to ultrafine TiO2 (Fig. 6B, see the value of the ordinate compared to that of Fig. 6A). Ultrafine TiO2 at a concentration of 25 ␮g/ml increased the

The present study shows that a single exposure to either fine or ultrafine (nanosized) particles of TiO2 is not sufficient to evoke an inflammatory response in the brains of normal, healthy mice. However, exposure to nanosized particles of TiO2 aggravated neuroinflammation in LPStreated mice, enhancing production of proinflammatory cytokines and ROS, and increasing activation of microglia. Also in vitro data showed that microglia activated by the combination of LPS and ultrafine TiO2 increased their expression of TNF-␣ and NF-␬B, suggesting that microglial NF-␬B may contribute to the effects of ultrafine TiO2 in the septic brain. Several recent studies have reported the effects of a single or cumulative exposure to nanosized TiO2 particles on the brains of healthy rodents. After a single oral (2 g/kg) or i.v. (5 mg/kg and 1.8 mg/mouse) dose of nanosized TiO2 (20 –30 nm), TiO2 was not detected in the brain, and no pathological signs were observed when brains were examined for up to 1 month after exposure (Wang et al., 2007; Warheit et al., 2007; Fabian et al., 2008; Sugibayashi et al., 2008). However, mice exposed to high i.p. doses of nanosized TiO2 (324 to 2592 mg/kg; particle size ⬃100 nm) exhibited passive behavior, tremor and lethargy, indications of a CNS effect, during the first 2 days following exposure at all tested doses (Chen et al., 2009). Half of the animals that received 1944 and 2592 mg/kg died a week after treatment. Therefore, a high dose of nanosized TiO2 can cause adverse effects in the brain, depending on the route of administration. The dose of TiO2 used in this study corresponds to 40 mg/kg (1 mg/mouse, average weight 25 g), which was used in the study for therapeutic drug carriers to treat brain tumor (Lopez et al., 2008), and we did not observe any significant inflammatory response in either the brains of mice or in BV2 cells exposed to ultrafine TiO2, up to 24 h after exposure. Although a single exposure to nanosized TiO2 at low to medium doses can be safe for a normal individual (Fabian et al., 2008), our

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results cannot exclude potential for chronic effects of TiO2 on the brain since the fate of TiO2 particles after entering the brain has not been defined. It should be emphasized that the cumulative exposure to nanosized TiO2 induces a pathological responses in the normal brain. For example, titanium was found to accumulate in the brains of mice injected daily for 14 days with nano-TiO2 particles (5 and 50 mg/kg, i.p.), accompanied by a reduction in brain weight, suggesting a causal relationship between nano-TiO2 and brain damage (Liu et al., 2009). Also, administration of an intranasal instillation of TiO2 (0.5 mg/mouse; particle size, 80 nm and 155 nm) every other day for 30 days, investigators found reduction of hippocampal CA1 neurons, and increased oxidative stress and cytokine levels, and a high content of TiO2 in the brain (Wang et al., 2008a,b). In addition, increased levels of titanium were found in the brains after continuous dermal exposure to TiO2 (21 nm, 400 ␮g per cm2) for 60 days, indicating that nano-TiO2 particles can penetrate the BBB after chronic dermal exposure, although no pathological changes were found (Wu et al., in press). Therefore, in contrast to a single exposure, subchronic or chronic exposure to nanoparticles may cause pathological responses in the normal brain. This hypothesis is supported by epidemiologic studies showing a relationship between ultrafine particulates in polluted air and neurodegenerative changes, including inflammation, degenerating neurons, non-neuritic plaque and neurofibrillary tangles in the brains of Mexican subjects (Calderon-Garciduenas et al., 2002; Peters et al., 2006). Furthermore, controlled studies have also shown that subchronic exposure of inhaled concentrated ultrafine airborne particulates increased inflammatory responses in the brains of asthmatic and apolipoprotein E knockout mice, in vivo models that are susceptible to inflammation and oxidative stress, respectively (Campbell et al., 2005; Kleinman et al., 2008). Another study observed that pre-exposure to metallic nanoparticles (Cu, Ag or Al, 50 mg/kg, i.p.) for 7 days enhances BBB disruption, edema formation, and pathological brain changes induced by whole-body hyperthermia (Sharma and Sharma, 2007). These studies support the view that nanoparticles aggravate pathologic responses in the susceptible subject. All animals tested in previous studies were exposed to nanoparticles for more than 7 days, and it is unknown whether a single exposure could enhance the adverse responses in animals prone to inflammation or those with ongoing brain inflammation. This possibility should be tested before nanoparticles are applied to the patients as diagnostic or therapeutic tools, because inflammation is involved in many neurological diseases. The present study is, to our knowledge, the first report of the potential effects of single-dose, nanosized TiO2 on the stressed brain. We used a sepsis model induced by peripheral administration of LPS. Systemic LPS increases ROS and pro-inflammatory cytokines in the brain during the acute stage (Semmler et al., 2005; Noble et al., 2007; Qin et al., 2007). Accordingly, we showed increases in ROS production and cytokine mRNA levels in response to LPS in both the cortex and hippocampus, regions respon-

sible for cognitive function, memory, and learning. These increases were markedly enhanced by ultrafine TiO2. Interestingly, ROS production was decreased at 24 h in the LPS⫹vehicle group, but it was further enhanced in the LPS⫹ultrafine TiO2 group (Fig. 4). Accompanied by an increase in activated microglial cells at 24 h (Fig. 5), ultrafine TiO2 might not only enhance but also sustain inflammatory responses induced by LPS in the brain. These enhanced and sustained responses may eventually lead to more damage to the brain since the dose of systemic LPS used in the study has been shown to result in neuronal death with persistent microglial activation and neuroinflammation (Qin et al., 2007). One possible mechanism for the aggravated inflammatory response by ultrafine TiO2 may result from the breakdown of the BBB caused by LPS (Bohatschek et al., 2001; Xaio et al., 2001; Descamps et al., 2003). An increased influx of nanoparticles with the recruitment of neutrophils to the brain at sites of openings in the BBB may aggravate inflammatory responses via oxidative stress, similar to the previous suggestion that BBB breakdown was associated neurotoxic effects of nanomaterials (Sharma and Sharma, 2007). It appears that stimulated microglia play a role in this exaggerated inflammatory response. Long et al. (2006, 2007) showed that ultrafine TiO2 itself, induced the release of ROS and cytokines from non-stimulated microglia. However, we did not observe any significant responses of microglia in resting state to ultrafine TiO2 in either in vivo or in vitro experiments (Figs. 5 and 6). The differences may relate to differences in the sensitivity of detection methods and time points of observations. Instead, the responses of microglia, as assessed by increases in the number of activated microglia, TNF-␣ production, and NK-␬B binding activity, were more significant when they were exposed to combination of LPS with ultrafine TiO2 (Fig. 6). It has been known that LPS-stimulated microglia express proinflammatory cytokines via NF-␬B and MAP kinases, and that inflammation induced by nanoparticles is initiated by the same signaling cascades (Bhat et al., 1998; Park et al., 2007, 2009; Papp et al., 2008). Therefore, LPS and ultrafine TiO2 may act synergistically to activate microglia, resulting in enhanced TNF-␣ production with increased NK-␬B binding activity. Interestingly, these effects of ultrafine TiO2 were not dose-dependent, suggesting that once microglia are activated, a certain threshold nanoparticle level can be sufficient to enhance the inflammatory responses. The enhanced NK-␬B mediated inflammatory response was observed not only in the brain, but also in lungs exposed to i.p.-administered ultrafine TiO2 after systemic LPS administration (Moon et al., in press), indicating that exposure of nanoparticles to individuals with systemic infection may increase the risk of multiorgan damages. These results have important clinical implications, that is, that exposure to nanoparticles can aggravate neuroinflammation in patients with one of several brain diseases with underlying microglia activation such as Parkinson’s disease, Alzheimer’s disease, and multiple sclerosis (Wyss-Coray and Mucke, 2002). Present data support further investigations of the toxicity of nanopar-

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ticles in animal models of brain diseases. In parallel, the clearance of nanoparticles from the brain should be determined to help clarify their chronic effects on the brain.

CONCLUSION In conclusion, a single systemic exposure to nanosized TiO2 did not induce toxic responses in the brains of normal mice, but existing inflammatory responses were exaggerated by ultrafine TiO2 in the septic brain during the acute period. Activated microglia may be partially involved in the enhanced neuroinflammation induced by nanosized TiO2. Altogether, this study suggests that subjects with brain diseases involving CNS inflammation are potentially susceptible to adverse effects from nanoparticles, and studies on the toxicity of nanomaterials in susceptible populations should be done prior to their application to humans. Acknowledgments—This work was partly supported by the special grant from Medical School of Ewha Womans University.

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(Accepted 25 October 2009) (Available online 3 November 2009)