Abstract
Animal Cells and Systems Vol. 15, No. 2, June 2011, 107114 Toxicoproteomic identification of TiO nanoparticle-induced protein expression changes in mouse brain Yu-Mi Jeon, Seul-Ki Park and Mi-Young Lee* Department of Medical Biotechnology, SoonChunHyang University, Asan, Chungnam, 336-600, Republic of Korea (Received 26 August 2010; received in revised form 17 October 2010; accepted 24 October 2010) A proteomic analysis of the proteins in mouse brain that were differentially expressed in response to TiO nanoparticles was conducted to better understand the molecular mechanism of TiO nanoparticle-induced brain toxicity at the protein level. A total of 990 proteins from mouse brain were resolved by two-dimensional gel electrophoresis. A comparative proteomic analysis revealed that the expression levels of 11 proteins were changed by more than 2-fold in response to TiO nanoparticles: eight proteins were upregulated and three were downregulated by TiO nanoparticles. In addition, the activities of several antioxidative enzymes and acetylcholine esterase were reduced in TiO nanoparticle-exposed mouse brain. The protein profile alterations seem to be due to an indirect effect of TiO nanoparticles, because TiO nanoparticles were not detected in the brain in this investigation. 2 2 Keywords: proteomics; TiO nanoparticles; mouse; brain Introduction kidneys (Liu et al. 2009). However, there remain some inconsistent results regarding the uptake and translo- Nanoparticles have been manufactured from many cation of the nanoparticles to the brain (Johnston different metals for diverse applications. In particular, et al. 2009). Moreover, there is little information on titanium dioxide (TiO ) nanoparticles are an important the mechanism of action of TiO nanoparticles and nanomaterial used in industry (Borm et al. 2006). the molecular pathogenesis of their toxic effects in the Nanoparticles of TiO have been used in ultraviolet brain with regard to protein expression levels. There- sunscreen blocks and in self-cleaning and anti-microbial fore, the aim of this work is to identify, via a materials owing to their photochemical characteristics. proteomic approach, the proteins that are differen- They have also been used as photocatalysts in the tially expressed in mouse brain following exposure to cosmetics and pharmaceutical industries, including TiO nanoparticles. applications in photodynamic therapy, food production and environmental decontamination of air, soil and water (Kaida et al. 2004; Choi et al. 2006). Although we Materials and methods benefit from the unusual characteristics of TiO nano- Animals and TiO nanoparticle exposure particles, they pose a safety risk to our health and environment. Recent data have indicated that the Seven-week-old male ICR mice, weighing 1920 g, toxicity of TiO nanoparticles may be related to their were obtained from Orient Bio (Seoul, Korea). The surface area, and not to their weight (Warheit et al. mice were acclimated for 2 days at 25928C and with a 2007; Park et al. 2008). normal day/night cycle before starting the experiment. TiO nanoparticles increase hydrogen peroxide and Commercial TiO nanoparticles (Sigma-Aldrich, St. nitric oxide production in human bronchial epithelial Louis, MO), consisting of the anatase crystallographic cells and induce micronuclei formation and apoptosis form with an average diameter B25 nm, were mixed in Syrian hamster embryo fibroblasts (Park et al. 2009). with 1 PBS. To avoid aggregation, the suspensions The cytotoxicity and reactive oxygen species generation were ultrasonicated for 10 min in sealed sterile tubes induced by TiO nanoparticles caused oxidative DNA 2 (Son et al. 2007; Shim and Om 2008). damage. Moreover, TiO nanoparticles caused muta- The male ICR mice were divided into a control and tions resulting in cell death or proliferative disorders a sample group (n6 for each). The sample group was due to a loss of cell cycle control (Kiss et al. 2008). exposed by intraperitoneal (i.p.) injection with 2.5 mg TiO nanoparticles were absorbed and distributed of TiO nanoparticles in 0.2 mL of endotoxin-free 2 2 to the lungs, lymph nodes, liver, red blood cells and normal PBS (Park et al. 2008). At 30 min after *Corresponding author. Email: miyoung@sch.ac.kr ISSN 1976-8354 print/ISSN 2151-2485 online # 2011 Korean Society for Integrative Biology DOI: 10.1080/19768354.2011.555144 http://www.informaworld.com 108 Y.-M. Jeon et al. injection, TiO nanoparticles were instilled nasally in Auburn, CA) with reversed-phase HPLC (MagicC18, the sample group (Chen et al. 2006). After 7 days, the 0.2 mm50 mm5 mm; Michrom Bio Resources). animals were anaesthetized with ether, and the brains Peptides were detected using an electrospray ioniza- were collected and stored at 708C. tion ion trap mass spectrometer (LCQ Deca XP; Thermo-Finnigan, Rockford IL) in positive ion mode, with a spray voltage of 2.5 kV and spray temperature of Sample preparation 1508C. The LCQ Deca XP automatically sets the Brain tissues were solubilized using tip-probe sonica- collision energy in LC-MS/MS mode. After acquiring full-scan mass spectra, three LC-MS/MS scans were tion in modified lysis buffer (7 M urea, 2 M thiourea, 4.5% w/v CHAPS, 40 mM Tris, 100 mM DTE) acquired for the next three most intense ions, using containing a protease inhibitor cocktail. The solubi- dynamic exclusion (Yamashita et al. 2005). lized samples were clarified by centrifugation at 12,000g for 50 min at 158C. The supernatant Data analysis fractions were transferred to new tubes, divided into aliquots, and stored at 708C until analysis (Shim and Proteins were identified using the Turbo SEQUEST Lee 2008; Kim and Lee 2009). algorithm in the BioWorks 3.1 software package and the NCBI database (NIH, Bethesda, MD). The identi- fied peptides were further evaluated using the charge Two-dimensional gel electrophoresis (2-DE) state versus the cross-correlation number (Xcorr). The Isoelectric focusing (IEF) was performed with IPG Xcorr criteria for peptide evaluation were 2.0 for Drystrips on nonlinear pH gradient 310 (24 cm) on singly charged ions, 2.5 for doubly charged ions, and IPGphore (all GE Healthcare). Protein was solubilized 3.7 for triply charged ions. Only the best-matched in rehydration buffer (7 M urea, 2 M thiourea, 4.5% peptides were adopted (Yamashita et al. 2005). w/v CHAPS, 40 mM Tris, 100 mM DTE, 0.25% IPG buffer). Sample loading on IPG strips with a pH Western blotting gradient from 3 to 10 was performed with a total volume of 450 mL followed by focusing to a total of Brain tissues were homogenized in ice-cold lysis buffer 84 kVh at 208C. After isoelectric focusing, the strips for 30 min at 48C and then centrifuged at 12,000 g were soaked twice for 20 min each in equilibration for 30 min. Total proteins were denatured and resolved buffer (6 M urea, 2% w/v SDS, 20% v/v glycerol, 5 mM by 8% sodium dodecyl sulfatepolyacrylamide gel tributylphosphine, 2.5% acrylamide, 0.01% bromophe- electrophoresis. The proteins were transferred onto nol blue, 0.375 M Tris-HCl, pH 8.8). The second- polyvinylidene difluoride (PVDF) membranes, incu- bated overnight with individual antibody at 48C and dimension SDS-PAGE was performed in 816% gradient polyacrylamide gels without a stacking gel, then with horseradish peroxidase-conjugated anti- at 108C for 2 h at 5 mA/gel and then at 18 mA/gel mouse IgG for 1 h. The membranes were visualized (Bartkowiak et al. 2009; Choi et al. 2009; Jeon et al. using the ECL plus Western blotting detection system 2011; Park et al. 2010). Protein bands were visualized (Amersham Biosciences, Piscataway, NJ). by staining with Coomassie Brilliant Blue G-250. The stained gels were scanned on a flatbed scanner (Power- Antioxidative enzyme assays Look 1100; UMAX, Fremont, CA), and the data were analyzed using ImageMaster 2D Platinum 6.0 software Antioxidative enzyme assays were performed as in our (GE Healthcare, Fairfield, CT). earlier reports (Lee and Shin 2003). Catalase (CAT) activity was determined spectrophotometrically by measuring the decrease of absorbance at 240 nm due LC-MS/MS to H O decomposition. Superoxide dismutase (SOD) 2 2 The two-dimensional liquid chromatography mass activity was determined using the xanthine/xanthine spectroscopy (LC-MS/MS) system used in this study oxidase/nitroblue tetrazolium system. Inhibition of consisted of an autosampler, two capillary high- cytochrome c reduction by SOD was measured by performance liquid chromatography (HPLC) pumps, the reduction of nitroblue tetrazolium. Glutathione and an LCQ Deca XP Plus ion trap mass spectro- peroxidase (GPX) was determined by the method of meter (Thermo-Finnigan, San Jose, CA) with an Paglia and Valentine (1967). All determinations are electrospray ionization source (AMR, Tokyo, Japan). expressed as the mean9S.E. of three separate experi- Two-dimensional HPLC was performed using a ments. Acetylcholine esterase activity was evaluated by combination of SCX chromatography (Microtrap measuring the product of its reaction with the sub- SCH, 1 mm8mm12 mm; Michrom BioResources, strate acetylthiocholine iodide (ATC). The product, Animal Cells and Systems 109 thiocholine, was evaluated according to the method of 205 kDa, were detected by 2-DE followed by liquid Ellman with some modifications (Ellman et al. 1961). chromatography tandem mass spectrometry (LC-MS/ MS). A comparison of the 2-DE protein patterns from brains with and without TiO nanoparticle treatment Results allowed the identification of 11 protein spots that differed in expression level by more than 2-fold Proteomic analysis between the basal and treated conditions (Table 1). To study the TiO nanoparticle-induced alterations of Of these, eight proteins were upregulated and three protein expression in mouse brain, proteomic analysis were downregulated (Figure 2). The upregulated was performed using high-resolution 2-DE. Figure 1 proteins included a mitochondrial inner membrane shows the results of 2-DE of proteins from mouse protein, heat shock protein, coronin-1A, guanine brain, following exposure to TiO nanoparticles. More than 990 protein spots, with isoelectric points between nucleotide-binding protein, apolipoprotein A-1, hemo- 3 and 10 and relative molecular masses between 6.5 and globin beta, lactoylglutathione lyase and cytochrome Figure 1. Representative two-dimensional gel electrophoretic profiles of mouse brain by TiO nanoparticle treatment. Proteins on 2-DE gel were visualized by coomassie brilliant blue staining. 110 Y.-M. Jeon et al. Table 1. Identifications of altered proteins from mouse brain following TiO nanoparticle treatment. No. Identified Protein Annotation Score Cov% MW Change 1 Isoform 1 of mitochondrial inner membrane protien IPI00228150.1 260.34 42.54 83899.5 P 2 Isoform 1 of 60kDa heat shock protien, mitochondrial precursor IPI00308885.6 260.33 52.01 60955.1 P 3 T-complex protien 1 subunit beta IPI00320217.9 270.33 63.18 57476.9 o 4 Coronin-1A IPI00222600.4 80.30 24.73 50988.9 P 5 Septin-5 IPI00416280.3 88.26 27.57 42875.8 o 6 Guanine nucleotide-binding protien G(o) subunit alpha 2 IPI00115546.4 118.29 39.27 40036.2 P 7 Apolipoprotien A-1 precursor IPI00121209.1 106.23 39.77 30587.3 P 8 Hemoglobin, beta adult major chain IPI00110658.1 70.40 64.08 15202.2 P 9 Lactoylglutathione lyase IPI00321734.7 90.31 54.89 20809.5 P 10 Cytochrome b-c1 complex subunit IPI00133240.1 80.35 38.32 29367.4 P 11 Carbonic anhydrase 2 IPI00121534.11 20.23 12.70 29032.3 o b-c1 complex. The downregulated proteins were t- have revealed that the abundance of a protein is not complex protein 1, septin-5 and carbonic anhydrase strongly correlated with the level of the corresponding 2. These results were reproducible in three independent mRNA. Thus, proteomic analysis was performed using experiments. high-resolution two-dimensional electrophoresis (2- DE) and mass spectrometry to identify differentially expressed proteins in mouse brain after treatment with Validation by Western blotting TiO nanoparticles. To confirm the upregulation of heat shock protein and Spot 1, determined to be a mitochondrial inner hemoglobin beta expression by TiO , detected via membrane protein (mitofilin), was upregulated by TiO proteomics, we performed Western blotting analysis nanoparticles. This protein controls mitochondrial using specific antibodies (Figure 3). The results in- cristae morphology and is essential for normal mito- dicated that heat shock protein and hemoglobin beta chondrial function (John et al. 2005). A recent study of were expressed at considerably lower levels in normal cortical brain samples from fetuses with Down’s brain cells, but were present at markedly elevated levels syndrome showed a significant reduction in mitofilin. after TiO nanoparticle treatment. The result indicates Although the precise physiological role of this protein that Western blotting data were well consistent with the is unclear, its upregulation suggests that it is involved in proteomic data. enhancing mitochondrial function (Myung et al. 2003). The heat shock protein 60 (HSP60) level increased following TiO nanoparticle exposure. HSP60 is a Enzyme assays mitochondrial chaperone protein involved in mediating The activities of antioxidative enzymes, named super- the proper folding and assembly of mitochondrial oxide dismutase (SOD), catalase (CAT) and glu- proteins, especially in response to oxidative stressors. tathione peroxidase (GPX), and acetylcholine esterase Additionally, HSP60 has been proposed to be an were measured in TiO nanopaerticle-exposed mice as antiapoptotic protein (Lin et al. 2001). The loss of shown in Table 2. The activities of SOD, CAT and HSP60 function leads to increased misfolding and GPX in the brain were notably reduced after TiO aggregation of proteins, as well as increased vulner- nanoparticle exposure. Moreover, the acetylcholine ability to oxidative stress. The increased HSP60 level esterase activity, one of the indirect markers for brain induced by TiO nanoparticles may be linked to an damage, was also reduced in TiO nanoparticle- important defense mechanism that protects the mouse exposed mice. Acetylcholine esterase metabolizes brain from deleterious stress conditions by preventing acetylcholine to choline and acetyl CoA. irreversible protein aggregation (Boyd-Kimball et al. 2005). Septins are evolutionarily conserved proteins that Discussion have an essential function in cytokinesis and also Proteomics, a powerful tool for analyzing the expressed participate in apoptosis, vesicular transport, cytoskele- genome, has been used successfully to examine changes tal dynamics and cell polarity. Interestingly, septins are in global protein profiles in order to select specific abundant in the central nervous system, which is biomarkers at the protein level (Blackford et al. 1997; composed largely of postmitotic neurons. Septins are Xiao et al. 2003). While gene expression can be associated with many neurological diseases, including analyzed by measuring mRNA levels, numerous studies Parkinson’s and Alzheimer’s diseases (Tsang et al. Animal Cells and Systems 111 Figure 2. Up- and down-regulated proteins from mouse brain following TiO nanoparticle treatment. Protein expression levels were determined by relative intensity using image analysis. The data on all the bar charts represent means 9SEM from three individual 2DE gels in each experimental group. 2008). Septin 5 (spot 5) was found to be downregulated Guanine nucleotide-binding protein (spot 6), which by TiO nanoparticles. Septin 5, a parkin substrate, is a showed increased expression with TiO nanoparticle 2 2 vesicle- and membrane-associated protein that signifi- treatment, is integral to one of the most prevalent cantly inhibits exocytosis (Son et al. 2005). signaling systems in mammalian cells, regulating Figure 3. Western blot analysis of protein expression of the hemoglobin beta and heat shock protein 60 in mouse brain by TiO nanoparticle treatment. Band volumes in the western blots were normalized against b-action. 112 Y.-M. Jeon et al. Table 2. The activities of antioxidative enzymes and enzyme, is highly expressed in most organs; in brain, it acetylcholine esterase of mouse brain after TiO nanoparticle is located primarily in oligodendrocytes (Haapasalo treatment. Relative activity refers to comparison with the et al. 2007). This enzyme catalyzes the reversible control. reaction involving the hydration of carbon dioxide and dehydration of carbonic acid. A recent study has Enzyme Relative activity (%) revealed that CAII expression was regulated in en- Superoxide dismutase 39.1794.84 dothelial cells of melanoma neovessels and in esopha- Catalase 78.7191.18 geal, renal and lung cancer (Yoshiura et al. 2005). CAII Glutathione peroxidase 68.0993 is also expressed in several brain cancers, including Acetylcholine esterase 62.592.13 oligodendrogliomas and astrocytomas. The downregu- lation of CAII by TiO nanoparticle treatment suggests functions as diverse as sensory perception, cell growth that it may have an important functional role in brain and hormonal regulation (Roberts and Waelbroeck tumor metabolism (Haapasalo et al. 2007). 2004). We observed the intracellular localization of TiO Apolipoprotein A-1, the main apolipoprotein in the using light microscopy, to examine whether TiO central nervous system (Ito et al. 2006), is present in a nanoparticles enter and accumulate in the mouse brain. high-density lipoprotein complex in the cerebrospinal However, TiO nanoparticles were not observed in fluid and is thought to contribute to intercellular mouse brains exposed to TiO nanoparticles for up to 7 cholesterol transport in the brain (Dietschy and Turley days (data not shown), although recent studies have 2001). Some studies have proposed that systemically addressed the neurotoxicity of TiO nanoparticles, circulating apolipoprotein A-1 is transported across the which can pass through the bloodbrain barrier bloodbrain barrier (Ito et al. 2004). Apolipoprotein (Wang et al. 2007, 2008). Some reports about transport A-1 (spot 7) was upregulated in TiO nanoparticle- 2 of nanoparticles to the brain are often inconsistent. treated mouse brain. Actually TiO was retained in the liver, spleen, kidney Alpha and beta chains of hemoglobin in brain are and lung, not in the brain, when the biodistributions of known to be the precursors of numerous bioactive different-sized TiO were systematically examined peptides, some of which are the hemorphins, neokyo- (Johnston et al. 2009). The translocation rate of torphin and the hemopressins (Gelman et al. 2010). nanoparticles to the brain has been known to be very Moreover, hemoglobin expression in mouse brain was low, probably due to the very tight bloodbrain barrier. suggested to be upregulated in response to ischemia Moreover, the in vivo and in vitro response to and mutation, and the hemoglobin-derived peptides nanoparticles exhibited great diversity, with regard to could be endogenous signaling molecules within the nanoparticle sizes, aggregates, surface charge, composi- central nervous system, which could bind to CB1 tion and crystal form as well as cell type or organ cannabinoid receptors. Moreover, hemoglobin-induced target, exposure time and exposure route (Johnston cytotoxicity, caspase activation and oxidative stress et al. 2009; Sohaebuddin et al. 2010). Accordingly, were also reported (Gelman et al. 2010). Upregulation TiO nanoparticles seem to affect the protein profile of hemoglobin beta (spot 8) was found in TiO indirectly in the brain, because we could not detect nanoparticle-exposed mouse brain. TiO nanoparticles in the brain under the experimental Exposure to TiO nanoparticles increased the conditions used here. The investigations into the expression of the cytochrome b-c1 complex (spot 10), toxicity of TiO nanoparticle via bloodbrain barrier which is the most widely occurring electron transfer transport should be a focus of future experiment. complex capable of energy transduction. Cytochrome Various antioxidative enzymes were notably re- b-c1 complexes are found in the plasma membranes of duced in TiO -exposed mouse brain, probably suggest- phylogenetically diverse photosynthetic bacteria, and ing a severe oxidative stress state through reactive in the inner mitochondrial membrane of all eukaryotic oxygen species accumulation. Moreover, the reduction cells. In all of these species, the b-c1 complex transfers of acetylcholine esterase activity by TiO might suggest electrons from a low-potential quinol to a higher- an increase in cholinergic activity by raising the potential c-type cytochrome and links this electron acetylcholine level, and the elevated acetylcholine transfer to proton translocation (Trumpower 1990). continuously influences the receptor to cause choliner- Thus, upregulation of the cytochrome b-c1 complex gic nerve excitability in the TiO nanoparticle-exposed may enhance energy production in response to TiO brain (Ma et al. 2010). nanoparticle-induced toxicity. Our results identify proteins associated with brain The expression of carbonic anhydrase II (spot 11) neurotoxicity and provide a basis for understanding was reduced with exposure to TiO nanoparticles. how nanoparticles might indirectly influence protein Carbonic anhydrase isozyme II (CAII), a cytosolic biosynthesis and enzyme activities in brain. 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Journal
Animal Cells and Systems
– Taylor & Francis
Published: Jun 1, 2011
Keywords: proteomics; TiO 2 nanoparticles; mouse; brain
