research open access intragastric exposure to titanium

RESEARCH Open Access

Intragastric exposure to titanium dioxidenanoparticles induced nephrotoxicity in mice,assessed by physiological and gene expressionmodificationsSuxin Gui1†, Xuezi Sang1†, Lei Zheng1, Yuguan Ze1†, Xiaoyang Zhao1†, Lei Sheng1, Qingqing Sun1, Zhe Cheng1,Jie Cheng1, Renping Hu1, Ling Wang1, Fashui Hong1 and Meng Tang2,3*

Abstract

Background: Numerous studies have demonstrated that titanium dioxide nanoparticles (TiO2 NPs) inducednephrotoxicity in animals. However, the nephrotoxic multiple molecular mechanisms are not clearly understood.

Methods: Mice were exposed to 2.5, 5 and 10 mg/kg TiO2 NPs by intragastric administration for 90 consecutivedays, and their growth, element distribution, and oxidative stress in kidney as well as kidney gene expression profilewere investigated using whole-genome microarray analysis technique.

Results: Our findings suggest that TiO2 NPs resulted in significant reduction of renal glomerulus number, apoptosis,infiltration of inflammatory cells, tissue necrosis or disorganization of renal tubules, coupled with decreased bodyweight, increased kidney indices, unbalance of element distribution, production of reactive oxygen species andperoxidation of lipid, protein and DNA in mouse kidney tissue. Furthermore, microarray analysis showed significantalterations in the expression of 1, 246 genes in the 10 mg/kg TiO2 NPs-exposed kidney. Of the genes altered, 1006genes were associated with immune/inflammatory responses, apoptosis, biological processes, oxidative stress, iontransport, metabolic processes, the cell cycle, signal transduction, cell component, transcription, translation and celldifferentiation, respectively. Specifically, the vital up-regulation of Bcl6, Cfi and Cfd caused immune/ inflammatoryresponses, the significant alterations of Axud1, Cyp4a12a, Cyp4a12b, Cyp4a14, and Cyp2d9 expression resulted insevere oxidative stress, and great suppression of Birc5, Crap2, and Tfrc expression led to renal cell apoptosis.

Conclusions: Axud1, Bcl6, Cf1, Cfd, Cyp4a12a, Cyp4a12b, Cyp2d9, Birc5, Crap2, and Tfrc may be potential biomarkersof kidney toxicity caused by TiO2 NPs exposure.

Keywords: Titanium dioxide nanoparticles, Nephrotoxicity, Oxidative stress, Gene-expressed profile, Mice

BackgroundThe dynamically development of the nanotechnolo-gy industry has led to the wide-scale productionand application of nanomaterials. Among the variousnanomaterials, customarily titanium dioxide nanoparticles

(TiO2 NPs), owing to their high surface area to particlemass ratio and high reactivity, have been used as nontoxic,chemical inert and biocompatible pigment products orphotocatalysts in cosmetics, pharmaceuticals and paintindustries [1-7]. However, their attractive properties are thesource of reservations. The potential human toxicity andenvironmental impact of TiO2 NPs have attracted consider-able attention with their increased use in industrialapplications.Recently, published data indicated that the toxicity of

TiO2 NPs. Liu et al. had found that TiO2 NPs wereabsorbed and accumulated in the liver, lungs, brain,

* Correspondence: [email protected]†Equal contributors2Key Laboratory of Environmental Medicine and Engineering, Ministry ofEducation, School of Public Health, Southeast University, Nanjing 210009,China3Jiangsu key Laboratory for Biomaterials and Devices, Southeast University,Nanjing 210009, ChinaFull list of author information is available at the end of the article

© 2013 Gui et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the CreativeCommons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, andreproduction in any medium, provided the original work is properly cited.

Gui et al. Particle and Fibre Toxicology 2013, 10:4http://www.particleandfibretoxicology.com/content/10/1/4

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lymph nodes, and red blood cells [8]. Park et al. observedthat TiO2 NPs induced apoptosis and micronuclei forma-tion in Syrian hamster embryo fibroblasts and increasedthe production of nitric oxide and hydrogen peroxide inhuman bronchial epithelial cells [9]. Furthermore, anin vitro study showed that high concentration of TiO2

NPs caused renal proximal cell death [10]. TiO2 NPs werealso supposed to impair nephric functions and causenephric inflammation, which through reactive oxygen spe-cies (ROS) accumulation to reveal its toxicity [11]. Ourprevious study also demonstrated that exposure to TiO2

NPs induced nephric inflammation and nephric cellnecrosis [12]. We hypothesize that TiO2 NPs -inducedkidney damages in mice may have special biomarkers oftoxicity.Newly, a large body of in vivo animal model stud-

ies have shown the toxicologic characteristics whichcause striking changes of gene expression of somenanomaterials in kidney. For instance, curcumintreatment can alter the gene expression profile ofkidney in mice on endotoxin-induced renal inflam-mation [13]. In addition, nanocopper can result inwidespread renal proximal tubule necrosis and dramatic-ally gene expression alterations in rat kidney [14]. Further-more, a recent report found that proteins weredifferentially expressed in mouse kidney by exposure toTiO2 NPs [15]. However, the synergistic molecularmechanisms of multiple genes activated by TiO2 NP-induced renal toxicity in animals and humans remainunclear. In this study, mice were exposed to 2.5, 5 and 10mg/kg body weight (bw) TiO2 NPs for 90 consecutivedays, and their growth, element distribution, and oxidativestress as well as kidney gene expression profile wereinvestigated. Our findings suggested that exposure toTiO2 NPs resulted in histopathological changes, apoptosis,oxidative stress, and impairment of element balance inkidney with increased TiO2 NPs doses. Furthermore,microarray analysis showed marked alterations in the ex-pression of 1006 genes were associated with immune/in-flammatory responses, apoptosis, biological processes,oxidative stress, metabolic processes, the cell cycle, trans-port, signal transduction, cell component, transcription,translation, and cell differentiation in the 10 mg/kg TiO2

NPs-exposed kidney. Therefore, the application of TiO2

NPs should be carried out cautiously.

ResultsTiO2 NPs characteristicXRD measurements show that TiO2 NPs exhibit theanatase structure (Figure 1), and the average grain sizecalculated from the broadening of the (101) XRD peakof anatase was roughly 5.5 nm using the Scherrer’s equa-tion. TEM demonstrated that the average size of theparticles of powder (Figure 2a) and nanoparticles which

suspended in HPMC solvent after 12 h and 24 h incuba-tion ranged from 5—6 nm, respectively (Figure 2band c), which is consistent with the XRD results. Thevalue of the sample surface area was generally smallerthan the one estimated from the particle size, and itwould seem that the aggregation of the particles maycause such a decline (Table 1). To investigate the disper-sion and the stability of the suspensions of TiO2 NPs,we detected the aggregated size and the zeta potential ofTiO2 NPs in HPMC. After the 12 h and 24 h incubation,the mean hydrodynamic diameter of TiO2 NPs inHPMC solvent ranged between 208 and 330 nm (mostlybeing 294 nm), as measured by DLS (Figure 3a and b),which indicates that the majority of TiO2 NPs wereclustered and aggregated in solution. In addition, thezeta potential was 7.57 mV and 9.28 mV, respectively,and the particle characteristics for the TiO2 NPs used inthis study are summarized in Table 1. The leakage of Ti4+

ions from 12 h, 24 h and 48 h incubation of TiO2 NPsin HPMC solvent after centrifugation was measured byICP-MS. However, Ti4+ contents were not detected infiltrate, which are lower than the detection limit of 0.074ng/mL (not listed). Therefore, these results suggestedthat the Ti4+ ions leakage from TiO2 NPs is limited inHPMC incubation.

Body weight, coefficient of kidney and titaniumaccumulationTitanium accumulation, bw, and kidney indices of miceare listed in Table 2. As shown, an increased TiO2 NPsdose led to a gradual decrease in bw, whereas kidneyindices and titanium content were significantly increased(P < 0.05), indicating growth inhibition and kidneydamage in mice. These findings were confirmedby subsequent renal histological and ultrastructuralobservations and oxidative stress assays.

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Gui et al. Particle and Fibre Toxicology 2013, 10:4 of 16http://www.particleandfibretoxicology.com/content/10/1/4

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Mineral element contentsThe contents of mineral elements in kidney provideinsight into how mineral elements in the kidneys of miceresponded to treatment with TiO2 NPs. The mineralelements in the kidney, such as Ca, Na, K, Mg, Zn, Cuand Fe, were determined and listed in Table 3. It can beseen that with increased doses, TiO2 NPs exposure ledto marked increased Ca, K, Mg, Zn, and Cu contents,whereas Na, and Fe contents decreased (P < 0.05, Table 3).

Histopathological evaluation of kidneyFigure 4 presents the histopathological changes of kidneysin mice treated by TiO2 NPs exposure for 90 consecutivedays. Unexposed kidney did not suggest any histologicalchanges (Figure 4a), while those exposed to increasedTiO2 NPs concentrations exhibited severe pathologicalchanges, including significant reduction of renal glomeru-lus number, apoptosis or vacuolization, infiltration of

inflammatory cells, cell abscission on vessel wall as well astissue necrosis or disorganization of the renal tubules(Figure 4b, c and d), respectively. In addition, we alsoobserved significant black agglomerates in the 10 mg/kgbw TiO2 NPs exposed kidney (Figure4d). Confocal Ramanmicroscopy further showed a characteristic TiO2 NPs peakin the black agglomerate (148 cm-1), which furtherconfirmed the aggregation of TiO2 NPs in kidney (see thespectrum B in the Raman insets in Figure 4d). The resultsalso suggested that exposure to TiO2 NPs deposited in thekidney and resulted in mouse renal injury.

Nephric ultrastructure evaluationChanges to the nephric ultrastructure in mouse kidneyare presented in Figure 5. As shown, the untreatedmouse renal cells (control) contained round nucleuswith homogeneous chromatin (Figure 5a), whereas withincreased TiO2 NPs doses, the ultrastructure of renal

Figure 2 Transmission electron microscope image of anatase TiO2 NPs particles. (a) TiO2 NPs powder; (b) TiO2 NPs suspended in HPMCsolvent after incubation for 12 h; (c) TiO2 NPs suspended in HPMC solvent after incubation for 24 h. TEM images showed that the sizes of theTiO2 NPs powder or suspended in HPMC solvent for 12 h, 24 h were distributed from 5 to 6 nm, respectively.

Table 1 Characteristics of TiO2 NPs

Sample Crystllite size (nm) Phase Surface area (m2/g) Composition Zeta potential

TiO2 NPs 5.5 Anatase 174.8 Ti, O 7.57(a), 9.28(b)

(a) Zeta potential after the 12 h incubation in 0.05% w/v HPMC solvent;(b) Zeta potential after the 24 h incubation in 0.05% w/v HPMC solvent.


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cell from the TiO2 NPs-treated groups indicated a clas-sical morphology characteristic of apoptosis, includingmitochondria swelling, nuclear shrinkage, and chromatinmarginalization in the renal cell (Figure 5b, c, and d).In addition, black deposits were also observed in the10 mg/kg TiO2 NPs -exposed nephric cell via TEM(Figure 5d), Raman signals of TiO2 NPs was also ex-hibited via confocal Raman microscopy (Figure 5d).

Oxidative stressAlterations in ROS levels such as (O2

– and H2O2) in thekidney can be regarded as markers of adaptive responseof kidney to oxidative damage. As shown in Table 4, thelevels of both O2

– and H2O2 in mouse kidney followingexposure to TiO2 NPs significantly increased comparedwith control values (P <0.05, Table 4). To prove the effectsof TiO2 NPs on ROS generation, the levels of lipidperoxidation (MDA), protein peroxidation (carbonyl) andDNA peroxidation (8-OHdG) in mouse kidney wereevaluated and presented in Table 4. The great increases ofMDA, carbonyl and 8-OHdG in the TiO2 NPs -exposedkidney were also observed with increased TiO2 NPs doses(P < 0.05), suggesting that ROS accumulation led to lipid,protein, and DNA peroxidation in the kidney.

Change in the gene expression profileTreatment with high dose of 10 mg/kg bw of TiO2 NPsresulted in the most severe kidney damages, and thesetissues were used to detect gene expression profiles tofurther explore the mechanisms of kidney damages

induced by TiO2 NPs. Whole-genome expression profil-ing using mRNAs from pulmonary tissues of vehiclecontrol groups and those treated with 10 mg/kg bwof TiO2 NPs exposed groups for 90 consecutive dayswere analyzed with the Illumina Bead Chip. Comparedto the vehicle control group, 1, 246 genes of total genes(45, 000 genes) were found to be differentially expressedin the 10 mg/kg TiO2 NPs group (Additional file 1:Table S1), including 610 genes up-regulated and 636down-regulated. Using the ontology-driven clustering al-gorithm included with the PANTHER Gene ExpressionAnalysis Software (www.pantherdb.org/) as a tool forbiological themes analysis, indicating that the 1, 006genes among 1, 246 genes were associated with im-mune/inflammatory responses, apoptosis, biologicalprocesses, oxidative stress, metabolic processes, the cellcycle, ion transport, signal transduction, cell component,transcription, translation, and cell differentiation, an-other 240 genes function are unknown (Figure 6),respectively.

RT-PCRTo verify the accuracy of the microarray analysis, twenty-eight genes that demonstrated significantly different ex-pression patterns were further evaluated by qRT-PCR dueto their association with immune/inflammatory responses,apoptosis, oxidative stress, cell cycle, signal transductionand biological process. These 14 genes including Psmb5,Ngfrap1, Cycs, Tnfrsf12, Birc5, Fn1, Cd55, Cfi, Bub1b,Egr1, Nid1, Odc1, Cd34, and Apaf1 were up-regulated,

Figure 3 Hydrodynamic diameter distribution of TiO2 NPs in HPMC solvent using DLS characterization. (a) Incubation for 12 h;(b) Incubation for 24 h.

Table 2 Body weight, coefficient of kidney and titanium accumulation in mice kidney by intragastric administration ofTiO2 NPs for 90 consecutive days

Index TiO2 NPs (mg/kg bw)

0 2.5 5 10

Net increase of body weight (g) 22.55 ± 1.13a 17.59 ± 0.88b 14.22 ± 0.71c 12.05 ± 0.61d

Relative weight of kidney (mg/g) 10.07 ± 0.50a 11.58 ± 0.58b 13.31 ± 0.67c 15.69 ± 0.78d

Ti content (ng/g tissue) Not detected 105 ± 5a 193 ± 10b 366 ± 18bc

Different letters indicate significant differences between groups (p < 0.05). Values represent means ± SEM(N = 10).


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whereas 14 genes including Bcl2l1, Ccl19, Ccl21a, Bmp6,Cd74, Cfd, Cxcl12, C3, Bcl6, Cygb, Klf1, Txnip, andSerpinalb were down-regulated (Table 5). The qRT-PCRanalysis of all 28 genes displayed expression patterns com-parable with the microarray data (i.e., either up- or down-regulation; Additional file 1: Table S1).

DiscussionNPs were shown to attain the systemic circulation afteringestion, inhalation or intravenous injection. They candistribute to several organs like kidney, liver, spleen,heart, brain, and ovary [16-20]. The kidney has beenknown to eliminate harmful substances from the body,thus NPs assimilate in the systemic circulation can befiltered by renal clearance [21,22]. In this study, wefound that intragastric administration of 2.5, 5, and 10mg/kg bw of TiO2 NPs for 90 consecutive days inducedbw reduction, increased kidney indices, TiO2 NPs depos-ition (Table 2), renal inflammation, tissue necrosis ordisorganization of renal tubules (Figure 4), and renalapoptosis (Figure 5) in mouse kidney tissues coupledwith element unbalance (Table 3), and severe oxidativestress, significant production of O2

:– and H2O2, andperoxidation of lipids, proteins, and DNA (Table 4). Therenal damages and oxidative stress following exposure toTiO2 NPs may be involved in impaired immune functionand antioxidant capacity in mice and, thus, may beassociated with changed gene expression in renal tissue.Large-scale gene expression analysis provides an ap-proach to obtain a global view of the genomic changesand to gain insights into the detailed mechanisms be-hind the pathogenesis of various diseases [23]. To eluci-date the molecular mechanisms of kidney damages andidentify specific biomarkers induced by TiO2 NPs expos-ure, RNA microarray analysis of mouse kidney wasperformed to establish a global gene expression profileand identify toxicity-response genes in mice induced byexposure to 10 mg/kg bw of TiO2 NPs for 90 consecutivedays. Our analysis indicated that the expression levels of1, 246 genes were significantly changed and 1, 006 of thesegenes were involved in immune-inflammatory responses,oxidative stress, apoptosis, metabolism, the cell cycle,

signal transduction, and ion transport etc. The mainresults are discussed below.As we known, the development of kidney immune/

nflammatory responses is result from the interaction be-tween multifactor, multigene, multi-cell, multi-stage andinherent kidney cells, such as infiltration of inflamma-tory cells (Figure 4). The pathogenesis is involved in ex-pression alterations of immune/inflammation-relatedgenes. In this study, 36 genes linked to immune/inflam-matory responses were significantly altered by exposureto 10 mg/kg TiO2 NPs (Figure 6). Of these genes altered,29 genes were up-regulated and 7 genes were down-regulated. Ye et al. investigated that BCL-6 may regulatespecific T-cell-mediated responses and can control ger-minal centre formation as a transcriptional switch.Modification of expression of BCL-6 in lymphomaresults in the unnormal B cell proliferation and a de-regulation of germinal centre formation [24], while B cellis an immune cell, so the up-regulated of the differenti-ation of B cell triggers the immune responses in the kid-ney. In our data, Bcl6 gene was greatly increased with aDiffScore of 67.89 in the kidney (Additional file 1:Table S1), suggesting that TiO2 NPs disordered theprocess of B cell differentiation, thus interfering withimmune responses in mice. The inflammatory kidneydisease membranoproliferative glomerulonephritis typeII (MPGN2) is following the presence of complementC3. At the same time, complement factor I (cfi) canmodulate the activation of C3 through the alternativepathway. And the breakdown of activated C3 isregulated by factor I, the deficiency of factor I causes un-controlled C3 activation [25]. Our results showed thatc3 gene up-regulated with a Diffscore of 30.23 andcfi gene down-regulated with a DiffScore of −54.62following exposure to TiO2 NPs (Additional file 1: TableS1). The renal inflammation following exposure to TiO2

NPs was closely associated with overexpression of c3gene and decreased expression of cfi gene in the kidney.While, our result also showed that complement factor D(Cfd) gene was observably up-regulated with a DiffScoreof 52.09. Cfd is expressed in the kidney and plays acentral role in the activation of the alternative pathway

Table 3 Accumulation of metal elements in mouse kidney by intragastric administration of TiO2 NPs for 90 consecutivedays

TiO2 NPs (mg/kg bw)

Metal element contents (μg/g tissue)

Ca Na K Mg Zn Cu Fe

0 1054 ± 53a 3540 ± 177a 2383 ± 119a 138 ± 7a 9.88 ± 0.49a 1.986 ± 0.10a 33.26 ± 1.66a

2.5 1259 ± 63b 3083 ± 154b 3039 ± 152b 159 ± 8b 18.72 ± 0.94b 5.69 ± 0.28b 17.16 ± 0.86b

5 1486 ± 74c 2772 ± 139c 3866 ± 193c 215 ± 11c 31.89 ± 1.59c 10.27 ± 0.51c 9.81 ± 0.49c

10 1823 ± 91cd 2511 ± 125d 4839 ± 242d 300 ± 15d 47.88 ± 2.39d 18.48 ± 0.92d 2.326 ± 0.12d

Different letters indicate significant differences between groups (p < 0.05). Values represent means ± SEM(N = 5).


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as a serine protease [26]. So the significant increasedexpression of Cfd gene demonstrated that TiO2 NPs ex-posure affected renal biochemical functions in the kid-ney [12]. CXCL12 (stromal cell-derived factor-1) is notonly a unique homeostatic chemokine but also a potentsmall proinflammatory chemoattractant cytokines thatbinds primarily to CXC receptor 4 (CXCR4; CD184).As an inflammatory chemokine, CXCL12 has been

immunodetected not only in normal tissues but also inmany different inflammatory diseases [27]. In thepresent study, CXCL12 gene was up-regulated with aDiffScore of 28.02 after TiO2 NPs treatment, which wasassociated with infiltration of inflammatory cells in thekidney.The current study suggested that TiO2 NPs exposure

increased ROS significant production and led to

Figure 4 Histopathological observation of kidney caused by intragastric administration of TiO2 NPs for 90 consecutive days. (a) Control,(b) 2.5 mg/kg TiO2 NPs, (c) 5 mg/kg TiO2 NPs, (d) 10 mg/kg TiO2 NPs. Yellow arrows indicate apoptosis or vacuolization, green arrows indicatecell abscission, fatty degeneration or cell necrosis, green virtual circle indicates infiltration of inflammatory cells, blue arrows indicate tissuenecrosis or disorganization of renal tubules. Yellow virtual circle indicates TiO2 NPs aggregation. Arrow A spot is a representative cell that notengulfed the TiO2 NPs, while arrow B spot denotes a representative cell that loaded with TiO2 NPs. The right panels show the correspondingRaman spectra identifying the specific peaks at about 148 cm-1.


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peroxidation of lipids, proteins, and DNA in mouserenal tissue (Table 4), and caused renal cell apoptosis(Figure 5), which may be associated with alterations ofoxidative stress-related or apoptosis-related gene expres-sion. The overproduction of ROS has been shown to beclosely associated with the induction of apoptotic andnecrotic cell death in cell cultures [28]. This breaksdown the balance of the oxidative/antioxidative system

in the kidney, resulting in lipid peroxidation, whichincreased the permeability of mitochondrial membrane[11]. In our previous studies, TiO2 NPs were also shownto mediate apoptosis in the liver, spleen, brain, lung, andovary in mice through the induction of ROS [21,29-35].Meena et al. also showed that TiO2 NPs can induce oxi-dative stress which causes cell apoptosis in the kidney[36]. However, the apoptotic mechanism following TiO2

Figure 5 Ultrastructure of kidney in male mice caused by intragastric administration of TiO2 NPs for 90 consecutive days. (a) Control,(b) 2.5 mg/kg TiO2 NPs, (c) 5 mg/kg TiO2 NPs, (d) 10 mg/kg TiO2 NPs. Yellow arrows indicate nucleus shrinkage, chromatin marginalization,green arrows indicate mitochondria swelling, and red arrows show presence of TiO2 NPs. Arrow A spot is a representative cell that not engulfedthe TiO2 NPs, while arrow B spot denotes a representative cell that loaded with TiO2 NPs. The right panels show the corresponding Ramanspectra identifying the specific peaks at about 148 cm-1.


2.5 mgreen arrowthe TiO2 NPs, wspectra identify

5 Ultrastru ure/kg TiO2 NPs,

indicatehil

NPs -induced nephrotoxicity remains unclear. In thepresent study, our findings indicated that about 49 genesinvolved in oxidative stress and about 35 genes involvedin apoptosis were dramatically altered in the 10 mg/kgTiO2 NPs exposed kidney, in which 49 were up-regulated and 35 were down-regulated (Figure 6). Forexample, Cyp4a12a, Cyp4a12b, Axud1, Ccl19, andCcl21a genes were greatly up-regulated with DiffScoresof 38.54, 123.6, 60.66, 83.27, and 28.86, respectively;while Cyp24a1, Akrlc18, Birc5, and E2F1 genes weresignificantly down-regulated with DiffScores of −33.79, -56.24, -101.23, and −66 (Additional file 1: Table S1), re-spectively. As we know, the cytochrome P450 (CYP)is a gene superfamily of enzymes encodes many isoformsand reveals a variety of catalytic activity, regulatorymechanisms and substrates [37]. Cyp4a12a, and Cyp4a12bare members of Cyp4 family of cytochrome P450 proteinsand can hydroxylated arachidonic acid (AA) to 20-hydroxyeicosatetraenoic acid (20-HETE) effectively. Fur-thermore, Cyp4a12a and Cyp4a12b also effectively

transformed eicosapentaenoic acid (EPA) into 19/20-OH-and 17, 18-epoxy-EPA, which are the predominant 20-HETE synthases in mouse kidney [38]. The up-regulationof Cyp4a12a and Cyp4a12b genes following exposure toTiO2 NPs illustrated that these abnormal expression maycause the disorder of oxidation-reduction process involvedin 20-HETE production. The catabolic enzyme product ofCyp24a1 regulates the levels of hormonal 1, 25-dihydroxyvitamin D(3) (1, 25(OH)2D3) intracellular. Theregulation of expression of this enzyme is crucial to the bio-logical activity of 1, 25(OH)2D3 [39]. Therefore, down-regulated of Cyp24a1 gene following exposure to TiO2 NPssuggested may disrupt the metabolism of 1, 25(OH)2D3 inthe kidney. Aldo–keto reductases (AKRs) are members of alarge enzymes family that catalyze NADPH- and NADH-dependent oxidoreduction of a wide variety of substrates,including 20α-Hydroxysteroid dehydrogenase (20α-HSD)simple carbohydrates and steroid hormones [40,41]. It iswell-known that the AKR1C18 (20α-HSD) is a member ofthe AKR superfamily that catalyze the inactivation of

Cell differentiation 1.12%

Translation 1.69%

Apoptosis 2.81%

Immune/Inflammatory response 2.89%

Transcription 3.69%

Oxidative stress 3.93%

Transport 4.90%

Cell component 5.46%

Cell cycle 6.10%

Signal transduction 6.50%

Metabolic process 13.40%

Unknown 19.26%

Biological process 28.25%

0 50 100 150 200 250 300 350

Number of genes

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up-regulated genes

353

240

166

81

76

68

61

49

36

35

46

21

14

Figure 6 Functional categorization of 1246 genes. Genes were functionally classified based on the ontology-driven clustering approachof PANTHER.

Table 4 Oxidative stress in mouse kidney after intragastric administration of TiO2 NPs for 90 consecutive days

Oxidative stress TiO2 NPs (mg/kg BW)

0 2.5 5 10

O2.- (nmol/mg prot. min) 18 ± 0.9a 25 ± 1.23b 38 ± 1.91c 43 ± 2.15d

H2O2 (nmol/mg prot. min) 31 ± 1.55a 46 ± 2.28b 77 ± 3.845c 92 ± 4.6d

MDA (μmol/ mg prot) 1.02 ± 0.05a 1.97 ± 0.10b 3.05 ± 0.15c 4.88 ± 0.24d

Carbonyl (μmol/mg prot) 0.51 ± 0.03a 1.13 ± 0.06b 1.89 ± 0.09c 2.79 ± 0.14d

8-OHdG (mg/g tissue) 0.48 ± 0.02a 2.16 ± 0.11b 3.58 ± 0.18c 5.87 ± 0.29d

Different letters indicate significant differences between groups (p < 0.05). Values represent means ± SEM (N = 5).


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5.87 ± 0.29d

progesterone, which stereoselective converts progesteroneto its inactive metabolite 20α-hydroxy-4- pregnen-3-one(20α-HP) [40-43]. Down-regulation of Akrlc18 gene byTiO2 NPs exposure implied that TiO2 NPs may induce theactivation of progesterone, which affects renal physiologicalprocesses. Axud1 (cysteine-serine-rich nuclear protein-1)also known as Csrnp-1 is an immediate early gene whichstrongly caused as a response to IL-2 in mouse T cells [44].Overexpression of Axud1 conducts to apoptosis throughthe activation of the JNK pathway and inhibits mitosis[44]. In the study, significant increase of Axud1 gene ex-pression caused by TiO2 NPs promoted renal cell apop-tosis. Birc5 is a member of the inhibitor of apoptosis(IAP) gene family that encodes negative regulatoryproteins which block apoptotic cell death. What’s more,the functions of Birc5 (survivin) are to enhanceproliferation and survival of cells in the kidney [45].Whereas, Birc5 gene down-regulation following

exposure to TiO2 NPs may result in decreased survivalof cells, and renal cells apoptosis in the kidney (Figure 5).It was previously reported that the stimulation of DCswith CCR7 ligands CCL19 and CCL21 inhibits well-known apoptotic hallmarks of serum-deprived DCs, in-cluding increased membrane blebs and membranephosphatidylserine exposure, nuclear changes, and lossof mitochondria membrane potential [46]. In this study,we observed significant nucleus shrinkage, chromatinmarginalization and mitochondria swelling in renal cellfollowing exposure to TiO2 NPs (Figure 5). Ccl19 andCcl21a upregulation, however, may serve as a protectiverole for kidney following TiO2 NPs-induced apoptosis.In addition, E2F1 is suggested to induce apoptosis andactivation of p53-responsive target genes whichcoincides with an ability of E2F1 to induce accumulationof p53 protein. By affecting the accumulation of p53,E2F1 serves as a specific signal for the induction of

Table 5 RT-PCR validation of selected genes from microarray data

Function Gene ΔΔCt Fold Microarray

Apoptosis Apaf1 0.516327 0.699149555 0.5535156

Ngfrap1 0.15624 0.897360758 0.6713378

Cycs 0.34444 0.787613639 0.7045653

Tnfrsf12a 0.04954 0.966244365 0.655954

Bcl2l1 −1.131909 2.191485298 1.663064

Birc5 3.213009 0.107841995 0.10103

Fn1 0.470814 0.721557364 0.4940369

Ccl19 −2.490308 5.618978967 4.313047

Ccl21a −1.826821 3.547545038 2.850085

Immune/Inflammatory response Bmp6 −1.291201 2.447317025 40.56928

Cd55 0.54583 0.684997202 0.3824578

Cd74 −1.327117 2.509007877 1.832029

Cfd −1.861094 3.632830363 2.601335

Cfi 1.259912 0.417569429 0.3720603

Cxcl12 −1.913449 3.76708608 2.043431

C3 −1.580409 2.990546189 1.886813

Cd34 0.282201 0.822335491 0.4588617

Bcl6 −2.069258 4.196707749 3.073497

Oxidative stress Cygb −1.439087 2.711492162 1.646112

Gpx7 0.585512 0.666412792 0.3389097

Psmb5 0.057543 0.960899198 0.6914788

Cell cycle Klf1 −1.646997 3.131810673 2.100916

Bub1b 3.216187 0.1076047 0.2573011

Txnip −1.615053 3.063228525 1.648611

Signal transduction Egr1 0.348792 0.785241322 0.5866718

Nid1 0.918823 0.528940372 0.3838886

Biological process Odc1 1.576015 0.335407069 0.3957967

Serpinalb −2.035634 4.100028676 3.299695


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apoptosis [47]. Decreased expression of E2F1 gene mayalso be associated with a protective role for kidneyfollowing TiO2 NPs–induced nephrotoxicity.The equilibrium of various elements is essential for

immune integrity in the kidney and plays an importantrole in renal physiology. Our data indicated that TiO2

NPs exposure led to significant increases in Ca, K, Mg,Zn, and Cu concentrations, but decreased Na, and Feconcentrations in the kidney (Table 3). The changes ofthese elements can provide useful information on physi-ology and pathology of kidney. To further clarify themolecular mechanisms of mineral element unbalance,we analyzed microarray data and found significantalterations of related-gene in the kidney. Sri geneoverexpresses sorcin in K562 cells by gene transfection,which results in marked decrease of the level of cytosoliccalcium and increased the ability of cell to resistance toapoptosis [48]. Intracellular Ca2+ homeostasis plays animportant role in sustaining the biological functions ofthe cell and Ca2+overload may trigger apoptosis [49].In contrast, our results showed that Sri gene was down-regulated with a Diffscore of −16.77 by TiO2 NPs expo-sure (Additional file 1: Table S1), which resulted in asignificant Ca2+ overload in the kidney, thus leading torenal cell apoptosis. Slc10a6, also known as Soat, encodesprotein of SOAT [50]. The transport conducted by SOATis highly sodium dependence, indicating a symport trans-port with Na+ of the substrate [51]. In our data, Slc10a6overexpressed with a Diffscore of 25.48, therefore, theincreased Na+ concentration may be closely associatedwith Slc10a6 up-regulation in the kidney following ex-posure to TiO2 NPs. Establishing and maintaining highK+ and low Na+ in the cytoplasm are required for nor-mal resting membrane potentials and various cellularactivities. Therefore, the imbalance of Na+ and K+

caused by TiO2 NPs disturbed the ion homeostasisand cause a series of physiological disorders in thekidney. We also found that Cp gene was up-regulatedwith a Diffscore of 17.04, and Tfrc gene was dramati-cally down-regulated with a diffscore of −67.61 in thekidney (Additional file 1: Table S1). Ceruloplasmin (Cp),a copper-containing ferroxidase, is essential for body ironhomeostasis as selective iron overburden takes place inaceruloplasminemia. Copper is an essential metal cofac-tor for numerous cuproenzymes which can catalyze someimportant biochemical reactions [52]. And the stickingpoint in the molecular mechanisms associated with copper-iron hypothesis is the cuproenzyme Cp [53]. Therefore, theincreased Cu concentration and decreased Fe concentra-tion may correlate with the up-regulation of Cp gene ex-pression. In addition, Cu is a heavy metal, its overloadfollowing exposure to TiO2 NPs would lead to Cupoisoning in the kidney. Iron-restricted erythropoiesis is acommon clinical condition in patients with chronic kidney

disease. Iron status can be monitored by differentparameters such as ferritin, transferrin saturation etc.Transferrin receptors (TfRc) are the principal pathway bywhich various organ cells to obtain iron for physio-logical requirements [54]. The number of TfRc on thecell surface displays the requirement of iron, so thesynthesis of transferrin receptor is closely related to theiron requirements [55]. Decreased Fe level caused byTiO2 NPs may be also closely correlated to significantreduction of Tfrc gene expression, whereas Fe deficitwould aggravate renal anemia and decrease immunecapacity in the TiO2 NPs-exposed mice.

ConclusionsThe present study suggested that long-period exposureto TiO2 NPs resulted in severe kidney pathologicalchanges and apoptosis, coupled with unbalance of min-eral elements and severe oxidative stress. Furthermore,the nephrotoxicity following exposure to TiO2 NPs maybe closely related to significant alterations in theexpression of genes involved in immune/inflammatoryresponses, apoptosis, biological processes, oxidativestress, metabolic processes, the cell cycle, ion transport,signal transduction, cell component, transcription, trans-lation, and cell differentiation. Axud1, Bcl6, Cf1, Cfd,Cyp4a12a, Cyp4a12b, Cyp2d9, Birc5, Crap2, and Tfrcgenes may be potential biomarkers of kidney toxicitycaused by TiO2 NPs exposure. Therefore, the applicationof TiO2 NPs in food, toothpastes, cosmetics and medi-cine should be carried out cautiously.

MethodsPreparation and characterization of TiO2 NPsNanoparticles anatase TiO2 was prepared via controlledhydrolysis of titanium tetrabutoxide. The details of thesynthesis TiO2 NPs were previously described [34,56].Briefly, colloidal titanium dioxide was prepared via acontrolled hydrolysis of titanium tetrabutoxide. In a typ-ical experiment, 1 mL of Ti (OC4H9)4 was dissolved in20 mL of anhydrous isopropanol, and was addeddropwise to 50 mL of double-distilled water that wasadjusted to pH 1.5 with nitric acid under vigorousstirring at room temperature. The temperature of the so-lution was then raised to 60°C, and maintained for 6 hto promote better crystallization of TiO2 nanoparticles.Using a rotary evaporator, the resulting translucentcolloidal suspension was evaporated yielding a nano-crystalline powder. The obtained powder was washedthree times with isopropanol, and then dried at 50°Cuntil the evaporation of the solvent was complete. A0.5% w/v hydroxypropylmethylcellulose (HPMC) K4Mwas used as a suspending agent [57]. TiO2 powder wasdispersed onto the surface of 0.5% w/v HPMC solution,and then the suspending solutions containing TiO2


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particles were treated ultrasonically for 15–20 min andmechanically vibrated for 2 min or 3 min.The particle sizes of both the powder and nanoparticles

suspended in 0.5% w/v HPMC solution after incubationfor 12 h and 24 h (5 mg/L) were determined using aTecnaiG220 transmission electron microscope (TEM)(FEI Co., USA) operating at 100 kV, respectively. In brief,particles were deposited in suspension onto carbon filmTEM grids, and allowed to dry in air. The mean particlesize was determined by measuring > 100 randomly sampledindividual particles. X-ray-diffraction (XRD) patterns ofTiO2 NPs were obtained at room temperature with acharge-coupled device (CCD) diffractometer (Mercury 3Versatile CCD Detector; Rigaku Corporation, Tokyo, Japan)using Ni-filtered Cu Kα radiation. The surface area of eachsample was determined by Brunauer–Emmett–Teller(BET) adsorption measurements on a Micromeritics ASAP2020M+C instrument (Micromeritics Co., USA). Theaverage aggregate or agglomerate size of the TiO2 NPs afterincubation in 0.5% w/v HPMC solution for 12 h and 24 h(5 mg/L) was measured by dynamic light scattering (DLS)using a Zeta PALS + BI-90 Plus (Brookhaven InstrumentsCorp., USA) at a wavelength of 659 nm. The scatteringangle was fixed at 90°. The Ti4+ ions leakage from TiO2

NPs at time 0 and/or after 12, 24, 48 h of incubation in0.5% w/v HPMC was measured by Inductively coupledplasma-mass spectrometry (ICP-MS, Thermo ElementalX7, Thermo Electron Co., Finland) after sample wascentrifugated at 1,719 × g for 10 min and filtrated with a0.001 μmmembrane filter.

Animals and treatmentOne hundred and twenty male CD-1 (ImprintingControl Region) mice aged 5 weeks with an average bwof 23 ± 2 g were purchased from the Animal Center ofSoochow University (Jiangsu, China). All mice werehoused in stainless steel cages in a ventilated animal fa-cility with a temperature maintained at 24 ± 2°C andrelative humidity of 60 ± 10% under a 12-h light/darkcycle. Distilled water and sterilized food were availablead libitum. Prior to dosing, the mice were acclimated tothe environment for 5 days. All animals were handled inaccordance with the guidelines and protocols approvedby the Care and Use of Animals Committee of SoochowUniversity (Jiangsu, China). All procedures used in theanimal experiments conformed to the U.S. NationalInstitutes of Health Guide for the Care and Use ofLaboratory Animals [58].An HPMC concentration of 0.5% was used as a

suspending agent. TiO2 NPs powder was dispersed ontothe surface of 0.5% w/v HPMC, and then the suspendingsolutions containing TiO2 NPs were treated ultrasonic-ally for 30 min and mechanically vibrated for 5 min. Forthe experiment, the mice were randomly divided into

four groups (N = 30 each), including a control group(treated with 0.5% w/v HPMC) and three experimentalgroups (treated with 2.5, 5, and 10 mg/kg bw TiO2 NPs,respectively). About the dose selection in this study, weconsulted the report of World Health Organization in1969. According to the report, LD50 of TiO2 for rats islarger than 12,000 mg/kg bw after oral administration.In addition, the quantity of TiO2 nanoparticles does notexceed 1% by weight of the food according to the Fed-eral Regulations of US Government. The mice wereweighed and then the TiO2 NPs suspensions wereadministered by intragastric administration every day for90 days. All symptoms and deaths were carefullyrecorded daily. After the 90-day period, all mice wereweighed, anesthetized with ether, and then sacrificed.Blood samples were collected from the eye vein by rap-idly removing the eyeball and serum was collected bycentrifuging the blood samples at 1, 200 × g for 10 min.The kidneys were quickly removed and placed on ice,and the kidneys were dissected and frozen at −80°C.

Coefficient of kidneyAfter weighing the body and kidneys, the coefficients ofkidney mass to bw were calculated as the ratio of kidney(wet weight, mg) to bw (g).

Elemental content analysisThe frozen kidneys tissues were thawed and ~ 0.1 gsamples were weighed, digested, and analyzed for titan-ium, sodium, magnesium, potassium, calcium, zinc, andiron content content. Briefly, prior to elemental analysis,the kidney tissues were digested overnight with nitricacid (ultrapure grade). After adding 0.5 mL of H2O2, themixed solutions were incubated at 160°C in high-pressure reaction containers in an oven until thesamples were completely digested. Then, the solutionswere incubated at 120°C to remove any remaining nitricacid until the solutions were colorless and clear. Finally,the remaining solutions were diluted to 3 mL with 2%nitric acid. Inductively coupled plasma-mass spectrom-etry (Thermo Elemental X7; Thermo Electron Co.,Waltham, MA, USA) was used to determine the titan-ium, sodium, magnesium, potassium, calcium, zinc, andiron concentration. Indium (20 ng/mL) was chosen asan internal standard element. Data are expressed asnanograms per gram fresh tissue.

Histopathological evaluation of kidneyFor pathological studies, all histopathological examinationswere performed using standard laboratory procedures. Thekidneys were embedded in paraffin blocks, then sliced(5-μm thickness), and placed on glass slides. Afterhematoxylin–eosin staining, the stained sections wereevaluated by a histopathologist unaware of the treatments


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Table 6 Real time PCR primer pairs

Gene name Description Primer sequence Primer size (bp)

Refer-actin Mactin-F 50-GAGACCTTCAACACCCCAGC-30

Mactin-R 50-ATGTCACGCACGATTTCCC-30 263

Apaf1 mApaf1-F 5'-TAGCGGCTCATCTGTTCTGTAG-3'

mApaf1-R 5'-CCACTTGAAGACAAAAGACCAA-3' 87

Bcl2l1 mBcl2l1-F 5'- ATTTCCCATCCCGCTGTG-3'

mBcl2l1-R 5'-GGCTAAAAGCACCTCACTCAAT-3' 82

Bcl6 mBcl6-F 5'- TTTCAATGATGGACGGGTGT-3'

mBcl6-R 5'- ACGCAGAATGTGGGAGGAGT-3' 118

Birc5 mBirc5-F 5'-TCTAAGCCACGCATCCCA-3'

mBirc5-R 5'-CAATAGAGCAAAGCCACAAAAC-3' 150

Bmp6 mBmp6-F 5'-ATTAAATATCCCTGGGTTGAAAGAC-3'

mBmp6-R 5'-CTGGGAATGGAACCTGAAAGAG-3' 117

Bub1b mBub1b-F 5'-AATGGGTGGGGCTTTTGA-3'

mBub1b-R 5'-CCTGGCTGCTTGTCTTGC-3' 117

C3 mC3-F 5'-GGAGAAAAGCCCAACACCAG-3'

mC3-R 5'-GACAACCATAAACCACCATAGATTC-3' 148

Ccl19 mCcl19-F 5'-CCTCCTGATGCTCTGTCCCA-3'

mCcl19-R 5'-CGGTACCAAGCGGCTTTATT-3' 145

Ccl21a mCcl21a-F 5'-CACGGTCCAACTCACAGGC-3'

mCcl21a-R 5'-TTGAAGCAGGGCAAGGGT-3' 102

Cd34 mCd34-F 5'-CTCAGTCCCCTGGCAGATTC-3'

mCd34-R 5'-GGACCCCTGTTCTCCCCTTA-3' 147

Cd55 mCd55-F 5'-AAATCCAGGAGACCAACCAAC-3'

mCd55-R 5'-CTGTAGATGTTCTTATTGGATGACG-3' 113

Cd74 Cd74-F 5'-ACGGCAAATGAAGTCAGAACA-3'

Cd74-R 5'-AAGACTACTAATGGGTCAGAAATGG-3' 97

Cfd mCfd-F 5'-AGCAACCGCAGGGACACTT-3'

mCfd-R 5'-TTTGCCATTGCCACAGACG-3' 108

Cfi Cfi-F 5'-CCCGAGTTCCCAGGTGTTTA-3'

Cfi-R 5'-GAAGGAGGTCATAGCTTCAGACA-3' 112

Cxcl12 mCxcl12-F 5'-CCAGTCAGCCTGAGCTACCG-3'

mCxcl12-R 5'-TTCTTCAGCCGTGCAACAA-3' 128

Cycs mCycs-F 5'-CAACTCCGACTACAGCCACG-3'

mCycs-R 5'-GACACCACTATCACTCATTTCCCT-3' 134

Cygb mCygb-F 5'-GCTCAGTGCCCTGCATTCC-3'

mCygb-R 5'-CCGTGGAGACCAGGTAGATGAC-3' 120

Egr1 mEgr1-F 5'-TTACCTACTGAGTAGGCTGCAGTT-3'

mEgr1-R 5'-GCAATAGAGCGCATTCAATGT-3' 141

Fn1 mFn1-F 5'-TGAAGCAACGTGCTATGACGA-3'

mFn1-R 5'-GTTCAGCAGCCCCAGGTCTAC-3' 149

Klf1 mKlf1-F 5'-ACCACCAGATAAATCAACTCAAATG-3'

mKlf1-R 5'-ATAGTAACGACAACAATCCTAGCAGA-3' 146

Ngfrap1 mNgfrap1-F 5'-GCCTTTAATGACCCGTTTGTG-3'

mNgfrap1-R 5'-TCCATGCTAATGGGCAACACT-3' 147


Cygb

Egr1

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CACCA

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AG-3'

1

87

82

using light microscopy (U-III Multi-point Sensor System;Nikon, Tokyo, Japan).

Observation of kidney ultastructureThe kidneys were fixed in fresh 0.1 M sodium cacodylatebuffer containing 2.5% glutaraldehyde and 2% formalde-hyde followed by a 2 h fixation period at 4°C with 1% os-mium tetroxide in 50 mM sodium cacodylate (pH 7.2—7.4). Staining was performed overnight with 0.5% aque-ous uranyl acetate, then the specimens were dehydratedin a graded series of ethanol (75, 85, 95, and 100%) andembedded in Epon 812 resin. Ultrathin sections weremade, contrasted with uranyl acetate and lead citrate,and observed by TEM (model H600; Hitachi, Ltd.,Tokyo, Japan). Kidney apoptosis was determined basedon the changes in nuclear morphology (e.g., chromatincondensation and fragmentation).

Confocal Raman microscopy of kidney sectionsRaman analysis of renal glass or TEM slides wasperformed using backscattering geometry in a confocalconfiguration at room temperature in a HR-800 Ramanmicroscope system equipped with a 632.817 nm HeNelaser (JY Co., France). It has been previously reportedthat when the size of TiO2 NPs reached to 6 nm, theRaman spectral peak was 148.7 cm-1 [59]. Laser powerand resolution were approximately 20 mW and 0.3 cm-1,respectively, while the integration time was adjusted to1 s. The slides were scanned under the confocal Ramanmicroscope.

Oxidative stress assaySuperoxide ion (O2

·–) in the kidney tissues was measuredby monitoring the reduction of 3′-{1-[(phenylamino)carbonyl]-3, 4-tetrazolium}–bis (4-methoxy- 6-nitro)benzenesulfonic acid hydrate (XTT) in the presence of

O2·–, as described by Oliveira et al. [60]. The detection of

H2O2 in the kidney tissues was carried out by thexylenol orange assay [61].Lipid peroxidation of kidneys was determined as the

concentration of malondialdehyde (MDA) generated bythe thiobarbituric acid (TBA) reaction as described byBuege and Aust [62]. Protein oxidation of kidneys wasinvestigated according to the method of Fagan et al. bydetermining the carbonyl content [63]. DNA of kidneyswas extracted using DNeasy Tissue Mini Kit (NanjingJiancheng Bioengineering Institute, Jiangsu, China) asdescribed by the manufacturer. Formation of 8-OHdGwas determined using the 8-OHdG ELISA kit (Japan In-stitute for the Control of Aging, Haruoka, Japan). Thiskit provides a competitive immunoassay for quantitativemeasurement of the oxidative DNA adduct 8-OHdG. Itwas carefully performed according to manufacturer’sinstructions, and using a microplate varishaker-incubator,an automated microplate multi-reagent washer, and acomputerized microplate reader.

Microarray assayGene expression profiles of the kidney tissues isolatedfrom 5 mice in the control and TiO2 NPs-treated groupswere compared by microarray analysis using IlluminaBeadChip technology (Illumina Inc., USA). Total RNAwas isolated using the Ambion Illumina RNA AmplificationKit (cat no. 1755, Ambion, Inc., Austin, TX, USA)according to the manufacturer’s protocol, and stored at−80°C. RNA amplification is the standard method for pre-paring RNA samples for array analysis [64]. Total RNA wasthen submitted to Biostar Genechip, Inc. (Shanghai, China)to analyze RNA quality using a bioanalyzer and comple-mentary RNA (cRNA) was generated and labeled using theone-cycle target labeling method. cRNA from eachmouse was hybridized for 18 hrs at 55°C on Illumina

Table 6 Real time PCR primer pairs (Continued)

Nid1 mNid1-F 5'-ACCTCCTTTTCTTCTACTTTCACTG-3'

mNid1-R 5'-TCCAATTATTTAAGTAAAGACTCCCT-3' 122

Odc1 mOdc1-F 5'-TGCTGAGCAAGCGTTTGTAG-3'

mOdc1-R 5'-ATTCCCTGATGCCCAGTTATT-3' 107

Psmb5 mPsmb5-F 5'-GCTTCTGGGAGCGGTTGTT-3'

mPsmb5-R 5'-CATGTTAGCGAGCAGTTTGGA-3' 101

Serpina1b mSerpina1b-F 5'-TGAGTCCACTGGGCATCAC-3'

mSerpina1b-R 5'-GCTTCTGTTCCTGTCTCATCG-3' 136

Tnfrsf12a mTnfrsf12a-F 5'-CCAAGGACTGGGCTTAGAGTT-3'

mTnfrsf12a-R 5'-CCTTAGTATGGGTCGCTTTGTG-3' 114

Txnip mTxnip-F 5'-CCTGGGTGACATTCTACATTGA-3'

mTxnip-R 5'-TAAGGCTTAGTGAGCTTCCGAG-3' 141

PCR primers used in the gene expression analysis.


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HumanHT-12 v3.0 BeadChips, containing 45,200 probes(Illumina, Inc., San Diego, CA, USA), according to themanufacturer’sprotocolandsubsequently scanned with theIllumina BeadArray Reader 500. This program identifiesdifferentially expressed genes and establishes the biologicalsignificance based on the Gene Ontology Consortiumdatabase (http://www.geneontology.org/GO.doc.html). Dataanalyses were performed with GenomeStudio software ver-sion 2009 (Illumina Inc., San Diego, CA, USA), by compar-ing all values obtained at each time point against the0 hrs values. Data was normalized with the quantilenormalization algorithm, and genes were considered asdetected if the detection p-value was lower than 0.05. Stat-istical significance was calculated with the IlluminaDiffScore, a proprietary algorithm that uses the bead stand-ard deviation to build an error model. Only genes with aDiffScore ≤ −13 and ≥13, corresponding to a p-value of0.05, were considered as statistical significant [65,66].

Quantitative real-time PCR (qRT-PCR)The levels of mRNA expression of Apaf1, Bcl2l1,Bcl6, Bmp6, Birc5, Bub1b, C3, Ccl19, Ccl21a, Cd74,Odc1, Cd34, Cd55, Cfd, Cfi, Cxcl12, Cygb, Cycs, Egr1,Fn1 Klf1, Ngfrap1, Nid1, Psmb5, Serpinalb, Tnfrsf12,and Txnip in the mouse kidney were determinedusing real-time quantitative RT polymerase chain re-action (RT-PCR) [67-69]. Synthesized complimentaryDNA was generated by qRT-PCR with primersdesigned with Primer Express Software (AppliedBiosystems, Foster City, CA, USA) according to thesoftware guidelines, and PCR primer sequences arelisted Table 6. The quantitative real-time PCR wasperformed by SYBR Green method using the specialprimers (as shown in Table 6) by the AppliedBiosystems 7700 instrument (Applied Biosystems,USA).

Statistical analysisAll results are expressed as means ± standard errorof the mean(SEM). The significant differences wereexamined by unpaired Student's t-test using SPSS 19software (USA). A p-value <0.05 was considered asstatistically significant.

Additional file

Additional file 1: Table S1. Genes which related to apoptosis, oxidativestress, immune/inflammatory, biological process, translation, celldifferentiation, cell cycle, transcription, transport, metabolic process, cellcomponent and signal transduction altered significantly by intragastricadministration with TiO2 NPs for consecutive 3.

Competing interestsThe authors declare that they have no competing financial interests.

Authors’ contributionsConceived and designed the experiments: FH, MT, SG, XS, LZ, YZ, and XZ.Performed the experiments: FH, SG, XS, LZ, YZ, and XZ. Analyzed the data:FH, SG, XS, LZ, YZ, XZ, LS, QS, ZC, JC, RH, LW. Contributed reagents/materials/analysis tools: LS, QS, ZC, JC, RH, LW. Wrote the paper: FH, MT, SG, XS, LZ, YZ,and XZ. All authors read and approved the final manuscript.

AcknowledgementsThis work was supported by the National Natural Science Foundation ofChina (grant No. 81273036, 30901218, 81172697), A Project Funded by thePriority Academic Program Development of Jiangsu Higher EducationInstitutions, the Major State Basic Research Development Program of China(973 Program) (grant No. 2006CB705602), National Important Project onScientific Research of China (grant No. 2011CB933404) and the National IdeasFoundation of Student of Soochow University (grant No.111028534).

Author details1Medical College of Soochow University, Suzhou 215123, China. 2KeyLaboratory of Environmental Medicine and Engineering, Ministry ofEducation, School of Public Health, Southeast University, Nanjing 210009,China. 3Jiangsu key Laboratory for Biomaterials and Devices, SoutheastUniversity, Nanjing 210009, China.

Received: 26 November 2012 Accepted: 3 February 2013Published: 13 February 2013

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doi:10.1186/1743-8977-10-4Cite this article as: Gui et al.: Intragastric exposure to titanium dioxidenanoparticles induced nephrotoxicity in mice, assessed by physiologicaland gene expression modifications. Particle and Fibre Toxicology 201310:4.

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