NMR-based metabonomic study of the sub-acute toxicity of titanium dioxide nanoparticles in rats...

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NMR-based metabonomic study of the sub-acute toxicity of titanium dioxide nanoparticles in

rats after oral administration

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2010 Nanotechnology 21 125105

(httpiopscienceioporg0957-44842112125105)

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IOP PUBLISHING NANOTECHNOLOGY

Nanotechnology 21 (2010) 125105 (12pp) doi1010880957-44842112125105

NMR-based metabonomic study of thesub-acute toxicity of titanium dioxidenanoparticles in rats after oraladministrationQian Bu17 Guangyan Yan27 Pengchi Deng3 Feng Peng4Hongjun Lin1 Youzhi Xu1 Zhixing Cao1 Tian Zhou1 Aiqin Xue5Yanli Wang6 Xiaobo Cen28 and Ying-Lan Zhao18

1 State Key Laboratory of Biotherapy and Cancer Center West China Hospital West ChinaMedical School Sichuan University Chengdu 610041 Peoplersquos Republic of China2 National Chengdu Center for Safety Evaluation of Drugs State Key Laboratory ofBiotherapy West China Hospital West China Medical School Sichuan University Chengdu610041 Peoplersquos Republic of China3 Analytical amp Testing Center Sichuan University Chengdu 610041Peoplersquos Republic of China4 Department of Thoracic Oncology of Cancer Center and State Key Laboratory of BiotherapyWest China Hospital West China Medical School Sichuan University Chengdu 610041Peoplersquos Republic of China5 Institute of Bioengineering Zhejiang Sci-Tech University Road 2 Xiasha Hangzhou310018 Peoplersquos Republic of China6 Tianjin Childrenrsquos Hospital Tianjin 300074 Peoplersquos Republic of China

E-mail alancenxbsinacom

Received 25 November 2009 in final form 1 February 2010Published 5 March 2010Online at stacksioporgNano21125105

AbstractAs titanium dioxide nanoparticles (TiO2 NPs) are widely used commercially their potential toxicity on human healthhas attracted particular attention In the present study the oral toxicological effects of TiO2 NPs (dosed at 016 04and 1 g kgminus1 respectively) were investigated using conventional approaches and metabonomic analysis in Wistarrats Serum chemistry hematology and histopathology examinations were performed The urine and serum wereinvestigated by 1H nuclear magnetic resonance (NMR) using principal components and partial least squaresdiscriminant analysis The metabolic signature of urinalysis in TiO2 NP-treated rats showed increases in the levels oftaurine citrate hippurate histidine trimethylamine-N-oxide (TMAO) citrulline α-ketoglutaratephenylacetylglycine (PAG) and acetate moreover decreases in the levels of lactate betaine methionine threoninepyruvate 3-D-hydroxybutyrate (3-D-HB) choline and leucine were observed The metabonomics analysis of serumshowed increases in TMAO choline creatine phosphocholine and 3-D-HB as well as decreases in glutaminepyruvate glutamate acetoacetate glutathione and methionine after TiO2 NP treatment Aspartate aminotransferase(AST) creatine kinase (CK) and lactate dehydrogenase (LDH) were elevated and mitochondrial swelling in hearttissue was observed in TiO2 NP-treated rats These findings indicate that disturbances in energy and amino acidmetabolism and the gut microflora environment may be attributable to the slight injury to the liver and heart causedby TiO2 NPs Moreover the NMR-based metabolomic approach is a reliable and sensitive method to study thebiochemical effects of nanomaterials

(Some figures in this article are in colour only in the electronic version)

7 These authors contributed equally to this work8 Address for correspondence State Key Laboratory of Biotherapyand Cancer Center West China Hospital West China Medical School

Sichuan University 1 Keyuan Road 4 Gaopeng Street High TechnologicalDevelopment Zone Chengdu 610041 Peoplersquos Republic of China

0957-448410125105+12$3000 copy 2010 IOP Publishing Ltd Printed in the UK1

Nanotechnology 21 (2010) 125105 Q Bu et al

1 Introduction

Titanium dioxide nanoparticles (TiO2 NPs) belong to a poorlysoluble particulate group by virtue of their low solubility andtoxicity [1] Because of their high stability anticorrosionand photocatalysis TiO2 NPs are produced abundantly andare capable of a wide variety of applications in life scienceand industry such as in the productions of paints coatingsplastics skin care products foods water cleaning and somepharmaceuticals [2 3] The US Food and Drug Administrationestablished a regulation for TiO2 as a color additive infoods [4]

With the increasing development of nanotechnology thepotential impacts of nanoparticles on human health and theenvironment have become more important because of theirsmall size and large surface area [5 6] Earlier studiesindicated that TiO2 NPs can cause pulmonary inflammationfibrosis and DNA damage [7ndash9] Moreover the toxic effectsof TiO2 NPs are dose and size dependent Smaller TiO2 NPscause a greater pulmonary inflammatory response in rats andmice than larger particles [10] Chronic intratracheal exposuresof TiO2 NPs can be carcinogenic to humans resulting in benignlung tumors and emphysema-like lung injury [11] Mechanismstudies show that free radical production which exerts a strongoxidizing ability and catalyzes DNA damage may be involvedin the damage induced by TiO2 NPs [12]

Until now most studies on the toxicity of TiO2 NPs inmammals have focused on the pulmonary impact via inhalationor dermal exposure Because TiO2 NPs have been used asan additive in toothpaste and capsules oral ingestion of TiO2

NPs is a potential exposure route for the general populationHowever little information is available on the effects of TiO2

NPs through oral exposure To our knowledge there has onlybeen one report concerning oral TiO2 toxicity that a single oralexposure to 5 g kgminus1 TiO2 NPs results in hepatic injury andrenal lesion [13] Therefore it is necessary to investigate theoral repeated toxicity of TiO2 NPs in a mammalian model

Metabonomics a system approach for studying metabolicprofiles is able to provide information on drug toxicity diseaseprocesses and biochemical functions [14] It has been widelyused in the safety assessment of chemicals as a method foridentifying target organ toxicity and toxicological mechanismthrough urine and serum biomarkers [15ndash19] One selectivetechnique for metabonomics is nuclear magnetic resonance(NMR) spectroscopy coupled with pattern recognition (PR)methods It can offer rapid noninvasive toxicologicalinformation that is robust and reproducible with littleor no added technical resources to existing studies inchemical metabolism and toxicity required [20] Studieshave demonstrated that NMR spectroscopy techniques aresuitable for probing the mechanisms and evaluating the safetyof nanomaterials [21 22] For example the biochemicaleffects of nano-sized copper particles were analyzed by NMRspectroscopy and some sensitive biomarkers of nano-coppertoxicity were identified [22] These studies support the fact thatan integrated metabolomic approach is a promising method forrapid toxicological screening of nanomaterials

Figure 1 SEM photograph for 50 nm TiO2 NPs 100 000times

In the present study we applied an integrated metabo-nomic approach to investigate the oral repeated-dose toxic-ity of TiO2 NPs in rats The 1H NMR spectroscopic meth-ods with principal components analysis (PCA) and partial leastsquares discriminant analysis (PLS-DA) were used to analyzethe metabonomic profile of urine and serum Serum biochem-istry hematology and histopathology examinations were alsoperformed We found that disturbance in energy and aminoacid metabolism as well as gut microflora environment maybe attributable to the slight injury in the liver and heart causeby TiO2 NPs

2 Materials and methods

21 Materials

TiO2 NPs were purchased from Sigma Chemical Co (St LouisMO) Its characteristics are as follows a mixture of rutileand anatase nanoparticles dispersion lt50 nm particle size byx-ray diffraction (XRD) 10 wt in H2O Furthermore thesurface morphology of the TiO2 NPs was confirmed using aHitachi S-4800 scanning electron microscope (SEM) operatingat an acceleration voltage of 30 kV (figure 1) The samples forSEM assay were prepared by sprinkling the solution of TiO2

NPs onto double-sided sticky tape and mounting them on amicroscope stub

22 Animal handling and dosing

32 male (weight ranging 200plusmn 10 g) and 32 female Wistar rats(weight ranging 160 plusmn 10 g) were housed in polypropylenecages with stainless steel grids with commercial food andwater freely available The environmental conditions of theroom were well controlled (temperature of 22 plusmn 3 C relativehumidity of 50 plusmn 10 and a cycle of 12 h each lightdark)All animal experiments were carried out in accordance withthe guidelines established by the Association for Assessmentand Accreditation of Laboratory Animal Care (AAALAC)

After seven days of acclimatization rats were randomlyassigned to four groups with 16 rats in each group andintragastrically administrated with 0 016 04 and 1 g kgminus1

body weight (BW) TiO2 NPs respectively once a day for 14consecutive days and were euthanized on the 15th day

2


Figure 2 600 MHz presaturated 1H NMR spectra (δ80ndashδ05) of urine samples at various time points following oral administration of TiO2

NPs (1 g kgminus1 BW) (A) pre-dose (B) day 1 (C) day 3 (D) day 7 (E) day 14

23 Sample collection

When designing a metabonomic experiment all the factorsthat affect the baseline metabolic profile detected such asgender [23 24] must be controled So in the experimentsall the samples from male rats have been collected formetabonomic investigation

The body weight (BW) of rats was recorded every threedays Urine samples of each male rat were collected into ice-cooled vessels containing 1 sodium azide overnight (fromPM 400 to AM 700) on pre-dose day minus1 and on post-dosedays 1 3 7 and 14 respectively Blood samples from thesacrificed rats were allowed to clot at 4 C for 60 min Then allsamples were centrifuged (4 C 3000 rpm 10 min) to removeany precipitates and supernatants were stored at minus80 C untilmetabonomic analysis The other blood samples were usedfor clinical biochemical analysis and blood for hematologystudies was collected into tubes containing ethylene diaminetetra acetic acid (EDTA) anticoagulant Liver kidney brainadrenal gland spleen lung heart testis ovary and uterussamples were collected and used for histopathology

24 Transmission electron microscopy (TEM) examination

The heart brain kidney and liver were cut into 1 mm3 piecesand immediately fixed in 25 glutaraldehyde overnight thenthe samples were treated according to the general protocols forTEM study [25] The ultra-thin sections (70ndash100 nm) werestained with lead citrate and uranyl acetate and the specimenswere examined using an Hitachi H-7650 electron microscope

25 Clinical chemistry hematological analysis andhistopathology

Standard spectrophotometric methods on a Cobas Integra 400Plus Automatic Biochemistry Analyzer were used for themeasurement of the following serum parameters alanineaminotransferase (ALT) aspartate aminotransferase (AST)creatinine (CREA) cholesterol (CHOL) creatine kinase(CK) lactate dehydrogenase (LDH) total bili special (TB)triglyceride (TG)

Hematological parameters consisting of white blood cellcount (WBC) neutrophils (NEU) lymphocytes (LYM) mono-cytes (MONO) erythrocytes (RBC) hemoglobin (HGB) andplatelet count (PLT) were determined using a hematologi-cal autoanalyzer (Coulter T540 hematology system FullertonCA)

All tissue samples were fixed in 10 formalin andprocessed for light microscopy Samples were stained withhematoxylin and eosin and evaluated in a nonblinded fashion

26 1 H NMR spectroscopic measurement of urine

To minimize chemical shift variation due to differences in theurine pH 200 μl buffer solutions (02 M Na2HPO402 MNaH2PO4 pH 74) were added to 400 μl aliquots of the urinethe urinendashbuffer mixture was incubated for 5 min and thencentrifuged (4 C 14 000 rpm and 10 min) The supernatant(500 μl) was then pipetted into a 5 mm NMR tube togetherwith 50 μl of D2O containing 1 mg mlminus1 TSP-d4 TheD2O and TSP provided the deuterium lock signal for theNMR spectrometer and the chemical shift reference (δ00)respectively The urine samples were analyzed by 1H NMRspectroscopy at 60013 MHz using a Bruker Avance II 600

3


Figure 3 PCA analysis of 1H NMR spectra of urine samples (A)PCA score plots based on 1H NMR spectra of the urine trajectory foreach time point Key pre-dose day 1 day 3 day 7 day14 (B) PCA loading plots corresponding to (A)

spectrometer (Bruker Biospin Germany) at 300 K A one-dimensional spectrum was acquired by using a standard (1D)NOESY-presaturation (noesygppr) pulse sequence to suppressthe water signal with a relaxation delay of 2 s and mixingtime of 150 ms Sixty-four free induction decays (FID) wererepresented by 64 K data points with a spectral width of70028 Hz an acquisition time of 468 s and a total pulserecycle delay of 668 s The FIDs were weighted by anexponential function with a 05 Hz line-broadening factor priorto Fourier transformation (FT) The preparation of the urinefor NMR analysis was based on a protocol reported in theliterature [22]

27 1 H NMR spectroscopic measurement of serum

Serum samples (375 μl) were centrifuged (4 C 14 000 rpm10 min) to remove any precipitate The supernatant wasmixed with 200 μl D2O and then transferred into 5 mm NMRtubes Samples were analyzed by 1H NMR spectroscopy at60013 MHz using a Bruker Avance II 600 spectrometer Thewater-suppressed CarrndashPurcellndashMeiboomndashGill pulse (CPMG-pulse) sequence (Bruker Biospin Germany) was acquiredusing it Sixty-four FIDs were represented by 64 K data pointswith a spectral width of 70028 Hz an acquisition time of468 s and a total pulse recycle delay of 668 s The FIDswere weighted by an exponential function with a 05 Hz line-

broadening factor prior to FT All spectra were referenced tothe CH3 resonance of creatine at δ305

28 Data reduction and PR analysis of 1 H NMR spectra

All NMR spectra were manually rephased baseline correctedand then data-reduced to 225 integrated regions of equalwidth (004 ppm) corresponding to the region of δ95ndashδ05using MestRe-C23 software (httpqobrueuscesjsgroupMestRe-c) For urine spectra the region of δ62ndashδ46 wasremoved prior to statistical analysis to exclude the uncertaintyof the residual water signal and urea For serum CPMGspectra the region of δ51ndashδ47 was excluded to eliminatethe artifacts of the residual water resonance All remainingspectral segments were scaled to the total integrated area of thespectrum to reduce variation in concentration

The 1H NMR spectral data sets were imported into theSIMCA-P 110 (Version 10 Umetrics AB and Umea Sweden)software package separately The 1H-NMR spectral dataare always generated in enormous amounts Data subjectedto PCA or PLS-DA were prior mean centered This wasachieved by calculating the average value of each variable andsubtracting it from the data The scores plot is a summaryof the relationship between the observations (ie spectra)and can be used to establish any significant pattern in thedata the loadings plot is a similar summary of the variables(ie the spectral integrals) The loadings can be viewed asa means to interpret the pattern seen in the scores plot asthe two plots are complementary The trajectory map wasconstructed by connecting points which represent the averageposition of all samples in a particular group Next PLS-DA asupervised PR method was applied to maximize the separationbetween different groups [26 27] Finally the loadingplots or coefficient plots were used to identify the differentchemical shift from control and TiO2 NP treatment accordingto the default method of SIMCA-P 110 software Differentmetabolites have different chemical shifts and the far wc [1]from the zero line the bigger of variation We found that thevarious metabolites were assigned in the different chemicalshift from the human metabolome database (HMDB) [28] andsome metabonomic study papers [29ndash31]

29 Statistics

The data including serum clinical chemistry and hematolog-ical parameters are expressed as the mean plusmn SD Statisti-cal comparisons were performed using analysis of varianceANOVA followed by Studentrsquos t-test the SPSS for Windows115 software package was used The criterion for statisticalsignificance was set at P lt 005

3 Results

31 1 H NMR spectroscopic and PR analysis of urine

A series of changes in endogenous metabolite levels wereobserved in the 1H NMR spectra of urine samples collectedat various time points after intragastric administration of TiO2

NPs at a dose of 1 g kgminus1 (figure 2) PCA was performed on

4


Figure 4 600 MHz presaturated 1H NMR spectra (δ80ndashδ05) of urine samples from rats oral administered with TiO2 NPs (A) 1 g kgminus1 BW(B) 04 g kgminus1 BW (C) 016 g kgminus1 BW (D) control on day 14

the 1H NMR spectra of the urine trajectory and the scores plot(figure 3(A)) showed a clear separation for each time pointexcept for a partial overlap between day 1 and day 3 with amaximum shift reached on day 14 The prominent changes inendogenous urinary metabolite levels confirmed by inspectionof the original NMR spectra and the loading plots (figure 3(B))comprised decreases in the levels of pyruvate lactate betaineand choline Increases in α-ketoglutarate hippurate histidineTMAO taurine citrulline and acetate levels were detectedPAG and citrate in the 1 g kgminus1 group on day 14 increasedsignificantly (over 05-fold) whereas methionine and 3-D-HB decreased (lower 05-fold) as compared with the basallevel on day 0 The variations in the endogenous urinarymetabolite levels responsible for the separation of samples aresummarized in table 1

Typical 1H NMR spectra of urine samples at 14 days aftervarious doses of TiO2 NPs were administered are shown infigure 4 PCA the unsupervised PR method was initiallyapplied to analyze the urine NMR spectra However PCA ofthe urine NMR spectra only displayed a slight separation forthe first two principal components (PCs) with partial overlapbetween dosed groups and the control group (data not shown)and no significant separation between different dosed groupsand the control group was observed Therefore PLS-DAa supervised PR method was subsequently used to enhancethis separation and investigate the dose-dependent metaboliceffects of TiO2 NPs [32 33]

Shown in figure 5(A) are score plots obtained fromthe PLS-DA method on the first two LVs of the 1H NMRspectra from the control urine samples and TiO2 NP treatedsamples with accumulated R2X of 05 and R2Y of 0454

Table 1 Summary of the variations from urine trajectorymetabolites induced by oral administration of TiO2 NPs (Notevariations compared to control samples uarr indicates the relativeincrease in signal darr the relative decrease in signal mdash no change)

Metabolite Chemical shiftVariation followingnano-TiO2 treatment

Hippurate 784 764 756 396 uarrHistidine 728 uarrLactate 408 136 darrBetaine 388 darrTaurine 344 340 uarrTMAO 328 uarrCholine 320 darrCitrulline 276 uarrCitrate 264 256 uarrMethionine 260 darr3-D-HB 240 120 darrPyruvate 236 darrα-Ketoglutarate 244 300 uarrAcetate 192 uarrPAG 744 736 376 uarr

The corresponding loadings plot (figure 5(B)) revealed themetabolites which contributed to these differences Themetabolites discriminating this separation included taurine α-ketoglutarate and acetate whose levels increased and lactatethreonine pyruvate and leucine whose levels were decreasedCitrate in the 1 g kgminus1 group increased significantly (over05-fold) compared to the control group whereas methioninedecreased (lower 05-fold) on day 14 Detailed analysison the loadings plot indicated variations in the endogenousmetabolites in urine as summarized in table 2

5


Figure 5 PR of 1H NMR spectra of urine samples (A) PLS-DAscore plots derived from 1H NMR spectra of urine samples on day 14from TiO2 NP-treated rats at a dosage of 1 g kgminus1 BW 04 g kgminus1

BW 016 g kgminus1 BW and control (B) Coefficient plotsderived from 1H NMR spectra of urine

32 1 H NMR spectroscopic and PLS-DA of serum

Typical 1H NMR spectra of serum samples at 14 days afterTiO2 NP administration are shown in figure 6 PCA wasinitially applied to analyze the serum NMR spectra Similarto urine NMR spectra analysis no significant separationbetween different dosed groups was observed using PCA(data not shown) Therefore PLS-DA was applied for theclassification of the NMR data of serum samples We chosethe region (05ndash55 ppm) which contained most endogenousmetabolic signals in serum for PLS-DA calculation Thescore plots on the first two LVs of the 1H NMR spectrafrom control serum samples and TiO2 NP treatment serumsamples collected on day 14 are shown in figure 7(A)Obvious classifications were observed between the controlgroup and the dosed groups on day 14 with an accumulatedR2X of 0367 and R2Y of 0413 The correspondingloadings plot (figure 7(B)) revealed the metabolites whichcontributed to these differences Following TiO2 NP treatmentthere were increases in TMAO choline creatine 3-D-HBand phosphocholine together with decreases in glutamateacetoacetate glutathione and methionine Glutamine andpyruvate in the 1 g kgminus1 group decreased significantly (lower05-fold) on day 14 Detailed analysis on the loadings plotindicated variations in the endogenous metabolites in serum assummarized in table 3

Table 2 Summary of the variations from urine metabolites inducedby oral administration of TiO2 NPs on day 14 (Note variationscompared to control samples uarr indicates relative increase in signaldarr relative decrease in signal mdash no change)


Lactate 408 darrThreonine 360 darrTaurine 340 uarrMethionine 26 212 darrCitrate 256 264 uarrα-Ketoglutarate 244 300 uarrPyruvate 236 darrAcetate 192 uarra 128 darrLeucine 092 darra Unidentified

Table 3 Summary of the variations from serum metabolites inducedby oral administration of TiO2 NPs on day 14 (Note variationscompared to control samples uarr indicates relative increase in signaldarr relative decrease in signal mdash no change)


TMAO 328 uarrCholine 352 uarrCreatine 392 uarrPhosphocholine 324 uarrGlutamine 244 darrPyruvate 240 darrGlutamate 232 darrAcetoacetate 228 darrGlutathione 216 darrMethionine 212 darr3-D-HB 120 uarr

33 TEM inspection

The TEM examination showed mitochondrial swelling in TiO2

NP-treated heart tissue (figure 8) No apparent alterations inkidney brain and liver were observed in the TiO2 NP-treatedgroups on day 14 (data not shown)


Significant increase in the levels of the serum enzymes ASTCK and LDH were observed in a dose-dependent mannerafter the administration of TiO2 NPs to rats There were nosignificant changes in the levels of other parameters betweenthe control group and the dosed groups (table 4) Significantelevations in WBC LYM MONO and EOS levels weredetected (table 5) No apparent histopathological alterationsin liver kidney brain adrenal gland spleen lung heart testisovary and uterus were observed on TiO2 NPs treatment groups(figure 9) In addition TiO2 NPs had no marked effects onbody weight and relative organ weight in rats (data not shown)

6


Figure 6 600 MHz CPMG 1H NMR spectra (δ60ndashδ 05) of serum from rats orally administered with TiO2 NPs (A) 1 g kgminus1 BW(B) 04 g kgminus1 BW (C) 016 g kgminus1 BW (D) control on day 14

Table 4 Effect of TiO2 NPs on selected clinical chemistry parameters (Note data were presented as mean plusmn SD (N = 16) Statisticalanalysis was performed by one-way ANOVA followed by Dunnettrsquos test)

TiO2 NPs (g kgminus1)

Parameters Control 016 04 1

ALT (U lminus1) 4373 plusmn 612 4523 plusmn 508 4646 plusmn 404 4327 plusmn 375AST (U lminus1) 13749 plusmn 2045 16000 plusmn 2434a 18698 plusmn 2722a 17971 plusmn 1982a

CREA (μM) 1796 plusmn 308 1775 plusmn 254 1846 plusmn 304 1785 plusmn 315CHOL (Mm) 141 plusmn 012 141 plusmn 018 136 plusmn 026 155 plusmn 021CK (U lminus1) 87438 plusmn 27122 90541 plusmn 24250 124538 plusmn 21461a 139588 plusmn 21289a

LDH (U lminus1) 175625 plusmn 51211 217656 plusmn 52206b 289850 plusmn 51162a 301014 plusmn 43255a

TB (μM) 238 plusmn 102 249 plusmn 054 264 plusmn 057 237 plusmn 081TG (Mm) 034 plusmn 015 034 plusmn 006 035 plusmn 009 047 plusmn 022b

a p lt 001 for a significant difference from the control group b p lt 005

Table 5 Effect of TiO2 NPs on selected clinical hematology parameters (Note data were presented as mean plusmn SD (N = 16) Statisticalanalysis was performed by one-way ANOVA followed by Dunnettrsquos test)

TiO2 NPs treatment (g kgminus1)


WBC (109 lminus1) 363 plusmn 111 327 plusmn 051 352 plusmn 108 455 plusmn 097a

NEU (109 lminus1) 059 plusmn 022 061 plusmn 014 061 plusmn 021 064 plusmn 026LYM (109 lminus1) 286 plusmn 099 247 plusmn 044 269 plusmn 083 365 plusmn 079a

MONO (109 lminus1) 014 plusmn 006 015 plusmn 006 017 plusmn 008 021 plusmn 007a

RBC (109 lminus1) 754 plusmn 019 716 plusmn 035 726 plusmn 024 748 plusmn 027HGB (g lminus1) 149 plusmn 35 142 plusmn 61 143 plusmn 53 147 plusmn 52PLT (109 lminus1) 1079 plusmn 38 1024 plusmn 49 1031 plusmn 66 1067 plusmn 47

a p lt 001 for a significant difference from the control group

4 Discussion

NMR-based metabonomic methods can be used as a rapidanalytical tool for the study of biochemical variation inbiofluids [34ndash36] In the present study 1H NMR-based metabonomics methods coupled with serum chemistryhematological analysis and histopathology methods were usedto investigate the biochemical effects induced by TiO2 NPs

41 Effect of TiO2 NPs on clinical chemistry hematologicalanalysis histopathology and TEM inspection

TiO2 NPs can be absorbed across the gastrointestinal tract andpass through the mesentery lymph supply and lymph node tothe liver and spleen [37] In the present study increased levelsof serum enzymes AST CK and LDH were observed in TiO2

NP-treated rats in spite of the fact that no histopathological

7


Figure 7 PR of 1H NMR spectra of serum samples (A) PLS-DAscore plots derived from 1H NMR spectra of serum samples on day14 from TiO2 NP-treated rats at a dosage of 1 g kgminus1 BW 04 g kgminus1 BW 016 g kgminus1 BW and control (B) Coefficientplots derived from 1H NMR spectra of serum

changes were observed It is well known that the AST CKand LDH are often used as the markers of cardiovascularandor liver damage Generally increased LDH level indicatesmyocardial lesion when combined with CK and hepatocellulardamage when combined with AST and ALT enzymesTherefore our data suggest that cardiovascular andor liverdamage may occur after repeated oral administration of TiO2

NPs These findings are consistent with previous reports thatTiO2 NPs with sizes of 25 and 80 nm via gastrointestinaladministration elevate serum ALT AST and LDH [13] TheTEM examination revealed mitochondrial swelling in the hearttissue of treated rats which also supported those findings inserum chemistry Although a single oral administration of TiO2

NPs can cause histopathological alterations including hydropicdegeneration around the central vein and spotty necrosis ofhepatocytes as shown in a previous study [13] no apparenthistopathological alterations in liver heart kidney and brainwere observed after repeated TiO2 NP treatment in the presentstudy We presume that these observations may be due to thedifferences in the particle size dosage and dosing period

WBC and MONO of TiO2 NP-treated rats increasedsuggesting that TiO2 NPs may induce inflammation Thereis a link between oxidative stress and inflammation thatrecruited inflammatory leukocytes can generate oxidativestress augmented by oxidants via activation of oxidative stress-responsive transcription factors [38] Therefore increased

WBC and MONO resulting from administration of TiO2 NPsmay induce oxidative stress Previous studies also reported thatTiO2 NPs can produce free radicals exert a strong oxidizingability [39 40] and produce epithelial injury and oxidativestress in rodents [41ndash43] Moreover oxidative stress caninduce liver and heart injury Therefore we deduce thatinflammation induced by TiO2 NPs might produce oxidativestress which is responsible for TiO2 NP-induced injury in theliver and heart

42 TiO2 NP-induced energy and choline metabolismdisturbance

In order to exploit the toxic mechanism of TiO2 NPs weutilized the NMR-based metabonomic technique to study thedose-dependent alternations of metabolic profiles in urineplasma and the time-dependent metabolic trajectory variationsin urine Changes in a number of metabolites involved inenergy metabolism were observed in this work

We found that decreased pyruvate in urine and serumwas accompanied by elevated urinary excretion of Krebscycle intermediates citrate and α-ketoglutarate Theseresults indicate that the trend of pyruvate metabolism istowards the formation of acetyl-coenzyme A (acetyl-CoA) andconsequently results in an expanded Krebs cycle pool [18]It is known that when acetyl-CoA derived from glycolysisand lipid β-oxidation exceeds the capacity of the Krebs cycleit will produce ketone bodies acetoacetate 3-D-HB andacetone [44] In the preset study a TiO2 NP-induced rise inserum 3-D-HB was detected Normally ketone bodies aretransported from the liver to other tissues where they can bereconverted to acetyl-CoA to produce energy An impairmentof the Krebs cycle leads to increased release of ketone bodiesfrom the liver as fuel in other tissues Thus the changes of3-D-HB might offer a general decrease of energy metabolismMoreover excess acetyl-CoA can also be hydrolyzed to acetateby acetyl-CoA hydrolase (ACH EC 3121) [45] and urineacetate after TiO2 NP treatment Therefore the increases inserum 3-D-HB and urine acetate resulting from repeated TiO2

NP treatment reflect the onset of ketogenesis as a consequenceof perturbed energy metabolism

TiO2 NP treatment markedly increased serum cholineand phosphocholine levels It is well known thatcholine and phosphocholine are breakdown products ofphosphatidylcholine a major membrane constituent Theincreased level of TMAO a product of choline degradationwas also observed in serum and urine after TiO2 NP treatmentGenerally increased choline and TMAO are associated withcell membrane disruption [46] Moreover the oxidants such aslipid peroxidation (LPO) induced by TiO2 NPs may also impaircell integrity [47 48] Therefore TiO2 NP-induced elevationsof choline phosphocholine and lipoproteins in serum are likelycaused by membrane toxicity Consistently a previous studyshowed that TiO2 NP exposure leads to enhanced membranepermeability and loss of membrane integrity [49 50]

As discussed previously inflammation induced by TiO2

NPs could induce oxidative stress which is defined as animbalance between oxidants and antioxidants [51] The

8


Figure 8 TEM images of the heart tissue 14 days after oral administration of TiO2 NPs

Figure 9 Histological examination of tissues (100times) Hematoxylin and eosin stained sections of tissues from TiO2 NP-treated rats andcontrol rats

overproduction of LPO was observed after TiO2 NP treatmentmoreover a decrease in serum glutamine was also detectedThese results together with increased WBC and MONOsupport a notion that TiO2 NPs may induce oxidative stress

43 TiO2 NP-induced effect on gut microflora

Repeated exposure to TiO2 NPs via the oral route and alarge amount of TiO2 NPs observed in the intestine of treatedanimals implied that the intestinal brush border and mucosamight be damaged and the gut flora be disrupted Interestinglyurine PAG and hippurate were found to increase after TiO2

NP treatment The precursors of PAG and hippurate areproduced by gut bacteria [52 53] and PAG is the endproduct of phenylalanine metabolism in rodents [54] Ahigher concentration of PAG and hippurate may be indicativeof either drug-induced toxicity or a drug effect on the gutmicroflora [55] Actually altered levels of urine trajectory PAG

and hippurate have been documented in animals treated withantimicrobial drugs or food [56ndash58]

Besides gut microbiota the liver also plays an importantrole in the production of PAG and hippurate All of theirprecursors produced from gut microflora are assimilated by theintestinal tract and penetrate the systemic circulation into theliver through the portal vein [59] Then the precursor acids areconjugated with glycine to form PAG and hippurate in hepaticmitochondria [60] Further studies are needed to determinethe concentration change of PAG and hippurate caused bydisturbance of intestinal bacteria and liver function after oraladministration of TiO2 NPs

44 TiO2 NP-induced amino acid metabolism perturbation

Increased taurine concentration in urine has been related tohepatotoxic compound induced hepatocytes necrosis [61 62]In this study simple leakage of taurine seems an unlikely

9


explanation to account for the observed hypertaurinuriasince there is no clear evidence for liver necrosis detectedfrom histopathology The gradually increased levels ofurinary taurine after TiO2 NP treatment likely resulted fromaccelerated synthesis of its precursor cysteine due to increasedcholine levels Moreover the decrease in serum glutaminewas observed following TiO2 NP treatment whereas the raisedlevel of taurine was detected in urine Since glutamine is acompetitive metabolic product with taurine from cysteine thecontrary alterations in taurine and glutamine imply that thebiosynthesis of GSH from cysteine is blocked and the cysteineis in turn diverted to produce taurine which contributed to theobserved hypertaurinuria further

5 Conclusion

In summary NMR-PR analyses of rat serum and urinehighlight complex disturbances in the endogenous metabolitesprofiles TiO2 NPs can lead to energy metabolism aminoacid metabolism and gut flora disturbances which may beattributable to their toxicological effects on the liver and heartMoreover our findings support the fact that metabonomicanalysis is more sensitive than conventional histopathologyand serum chemistry analysis The established 1H NMR-technique-based metabonomics approach can be employed toinvestigate the toxicological effect of other nanomaterials andxenobiotics and provide a systematic and holistic view of theirbiochemical effects on an organism

Acknowledgments

The authors thank the Analytical amp Testing Center of SichuanUniversity for excellent technical assistance with the NMRanalysis during this experiment This work was supported bythe National 863 projects (2006AA03Z356) the National KeyBasic Research Program of China (2004CB518800) and theProject of the National Natural Sciences Foundation of China(20505006)

References

[1] Warheit D B Yuen I S Kelly D P Snajdr S andHartsky M A 1996 Subchronic inhalation of highconcentrations of low toxicity low solubility particulatesproduces sustained pulmonary inflammation and cellularproliferation Toxicol Lett 88 249ndash53

[2] Hayauchi Y 2005 A precise colour determination method fortabletsmdashan application of instrumental colour measurementin the pharmaceutical development Pharmeur Sci Notes2005 21ndash6

[3] Salthammer T and Fuhrmann F 2007 Photocatalytic surfacereactions on indoor wall paint Environ Sci Technol41 6573ndash8

[4] FDA 2002 Listing of color additives exempt fromcertificationin Title 21-Food and DrugsFood and DrugAdministration Code of Federal Regulations 21 CFR732575

[5] Handy R Ramsden C Smith T and Shaw B 2008 Toxicologyof dietary titanium dioxide nanoparticles to rainbow trout(Oncorhynchus mykiss) Comp Biochem Physiol A 84415ndash30

[6] Jemec A Drobne D Remskar M Sepcic K and Tisler T 2008Effects of ingested nano-sized titanium dioxide on terrestrialisopods (Porcellio scaber) Environ Toxicol Chem27 1904ndash14

[7] Zhang D D Hartsky M A and Warheit D B 2002 Time courseof quartz and TiO(2) particle-induced pulmonaryinflammation and neutrophil apoptotic responses in rats ExpLung Res 28 641ndash70

[8] Baan R A 2007 Carcinogenic hazards from inhaled carbonblack titanium dioxide and talc not containing asbestos orasbestiform fibers recent evaluations by an IARCMonographs Working Group Inhal Toxicol 19 213ndash28

[9] Rehn B Seiler F Rehn S Bruch J and Maier M 2003Investigations on the inflammatory and genotoxic lungeffects of two types of titanium dioxide untreated andsurface treated Toxicol Appl Pharmacol 189 84ndash95

[10] Oberdorster G Finkelstein J N Johnston C Gelein R Cox CBaggs R and Elder A C 2000 Acute pulmonary effects ofultrafine particles in rats and mice Res Rep Health Eff Inst96 5ndash86

[11] Liao C M Chiang Y H and Chio C P 2008 Model-basedassessment for human inhalation exposure risk to airbornenanofine titanium dioxide particles Sci Total Environ407 165ndash77

[12] Bhattacharya K Davoren M Boertz J Schins R PHoffmann E and Dopp E 2009 Titanium dioxidenanoparticles induce oxidative stress and DNA adductformation but not DNA breakage in human lung cells PartFibre Toxicol 6 17

[13] Wang J et al 2007 Acute toxicity and biodistribution ofdifferent sized titanium dioxide particles in mice after oraladministration Toxicol Lett 168 176ndash85

[14] Nicholson J K Connelly J Lindon J C and Holmes E 2002Metabonomics a platform for studying drug toxicity andgene function Nat Rev Drug Discov 1 153ndash61

[15] Park J C Hong Y S Kim Y J Yang J Y Kim E Y Kwack S JRyu do H Hwang G S and Lee B M 2009 A metabonomicstudy on the biochemical effects of doxorubicin in rats using(1)H-NMR spectroscopy J Toxicol Environ Health A72 374ndash84

[16] Shi C Wu C Q Cao A M Sheng H Z Yan X Z and Liao M Y2007 NMR-spectroscopy-based metabonomic approach tothe analysis of Bay41-4109 a novel anti-HBV compoundinduced hepatotoxicity in rats Toxicol Lett 173 161ndash7

[17] Wei L Liao P Wu H Li X Pei F Li W and Wu Y 2008Toxicological effects of cinnabar in rats by NMR-basedmetabolic profiling of urine and serum Toxicol ApplPharmacol 227 417ndash29

[18] Wei L Liao P Wu H Li X Pei F Li W and Wu Y 2009Metabolic profiling studies on the toxicological effects ofrealgar in rats by 1H NMR spectroscopy Toxicol ApplPharmacol 234 314ndash25

[19] Wang Q Jiang Y Wu C Zhao J Yu S Yuan B Yan X andLiao M 2006 Study of a novel indolin-2-ketone compoundZ24 induced hepatotoxicity by NMR-spectroscopy-basedmetabonomics of rat urine blood plasma and liver extractsToxicol Appl Pharmacol 215 71ndash82

[20] Keun H C 2006 Metabonomic modeling of drug toxicityPharmacol Ther 109 92ndash106

[21] Wang S Tang M Zhang T Huang M M Lei H Yang YLu M Y Kong L and Xue Y Y 2009 Metabonomic study ofplasma after intratracheally instilling titanium dioxidenanoparticles in rats Zhonghua Yu Fang Yi Xue Za Zhi 43399ndash403

[22] Lei R Wu C Yang B Ma H Shi C Wang Q Wang QYuan Y and Liao M 2008 Integrated metabolomic analysisof the nano-sized copper particle-induced hepatotoxicity andnephrotoxicity in rats a rapid in vivo screening method fornanotoxicity Toxicol Appl Pharmacol 232 292ndash301

10


[23] Bollard M E Holmes E Lindon J C Mitchell S CBranstetter D Zhang W and Nicholson J K 2001Investigations into biochemical changes due to diurnalvariation and estrus cycle in female rats usinghigh-resolution 1H NMR spectroscopy of urine and patternrecognition Anal Biochem 295 194ndash202

[24] Plumb R Granger J Stumpf C Wilson I Evans J andLenz E 2003 Metabonomic analysis of mouse urine byliquid-chromatography-time of flight mass spectrometry(LC-TOFMS) detection of strain diurnal and genderdifferences Analyst 128 819ndash23

[25] Mascorro J and Bozzola J 2007 Processing biological tissuesfor ultrastructural study Methods Mol Biol 369 19

[26] Wold S Trygg J Berglund A and Antti H 2001 Some recentdevelopments in PLS modeling Chemometrics Intell LabSyst 58 131ndash50

[27] Jiang N Yan X Zhou W Zhang Q Chen H Zhang Y andZhang X 2008 NMR-based metabonomic investigations intothe metabolic profile of the senescence-accelerated mouseJ Proteome Res 7 3678

[28] Wishart D Tzur D Knox C Eisner R Guo A Young NCheng D Jewell K Arndt D and Sawhney S 2007 HMDBthe human metabolome database Nucleic Acids Res35 D521


[30] Lei R Wu C Yang B Ma H Shi C Wang Q Yuan Y andLiao M 2008 Integrated metabolomic analysis of thenano-sized copper particle-induced hepatotoxicity andnephrotoxicity in rats a rapid in vivo screening method fornanotoxicity Toxicol Appl Pharmacol 232 292ndash301

[31] Li L Sun B Zhang Q Fang J Ma K Li Y Chen H Dong FGao Y and Li F 2008 Metabonomic study on the toxicity ofHei-Shun-Pian the processed lateral root of Aconitumcarmichaelii Debx (Ranunculaceae) J Ethnopharmacol116 561ndash8

[32] Zhou J Xu B Huang J Jia X Xue J Shi X Xiao L andLi W 2009 1H NMR-based metabonomic and patternrecognition analysis for detection of oral squamous cellcarcinoma Clin Chim Acta 401 8ndash13

[33] Martin J-C Canlet C Delplanque B Agnani G Lairon DGottardi G Bencharif K Gripois D Thaminy A andParis A 2009 1H NMR metabonomics can differentiate theearly atherogenic effect of dairy products in hyperlipidemichamsters Atherosclerosis 206 127ndash33

[34] Griffin J L and Bollard M E 2004 Metabonomics its potentialas a tool in toxicology for safety assessment and dataintegration Curr Drug Metab 5 389ndash98

[35] Coen M Holmes E Lindon J C and Nicholson J K 2008NMR-based metabolic profiling and metabonomicapproaches to problems in molecular toxicology Chem ResToxicol 21 9ndash27

[36] Bollard M E Stanley E G Lindon J C Nicholson J K andHolmes E 2005 NMR-based metabonomic approaches forevaluating physiological influences on biofluid compositionNMR Biomed 18 143ndash62

[37] Jani P U McCarthy D E and Florence A T 1994 Titaniumdioxide (rutile) particle uptake from the rat GI tract andtranslocation to systemic organs after oral administration IntJ Pharm 105 157ndash68

[38] Donaldson K Stone V Seaton A and MacNee W 2001Ambient particle inhalation and the cardiovascular systempotential mechanisms Environ Health Perspect109 (Suppl 4) 523ndash7

[39] Dalton J S Janes P A Jones N G Nicholson J A Hallam K Rand Allen G C 2002 Photocatalytic oxidation of NOx gasesusing TiO2 a surface spectroscopic approach EnvironmenPollution 120 415ndash22

[40] Cho I Park J and Kim Y 2005 Oxidative degradation andtoxicity reduction of trichloroethylene (TCE) in water usingTiO2solar light comparative study of TiO2 slurry andimmobilized systems J Environ Sci Health A 40 1033ndash44

[41] Warheit D B Brock W J Lee K P Webb T R andReed K L 2005 Comparative pulmonary toxicity inhalationand instillation studies with different TiO2 particleformulations impact of surface treatments on particletoxicity Toxicol Sci 88 514ndash24

[42] Warheit D B Webb T R Colvin V L Reed K L andSayes C M 2007 Pulmonary bioassay studies with nanoscaleand fine-quartz particles in rats toxicity is not dependentupon particle size but on surface characteristics Toxicol Sci95 270ndash80

[43] Bermudez E Mangum J B Wong B A Asgharian B Hext P MWarheit D B and Everitt J I 2004 Pulmonary responses ofmice rats and hamsters to subchronic inhalation of ultrafinetitanium dioxide particles Toxicol Sci 77 347ndash57

[44] McGarry J D and Foster D W 1980 Regulation of hepatic fattyacid oxidation and ketone body production Annu RevBiochem 49 395ndash420

[45] Knowles S E Jarrett I G Filsell O H and Ballard F J 1974Production and utilization of acetate in mammals BiochemJ 142 401ndash11

[46] Griffin J L Mann C J Scott J Shoulders C C andNicholson J K 2001 Choline containing metabolites duringcell transfection an insight into magnetic resonancespectroscopy detectable changes FEBS Lett 509 263ndash6

[47] Miura K Naganuma A Himeno S and Imura N 1995 Mercurytoxicity Handbook Experimental Pharmacology vol 115(New York Springer-Verlag) pp 163ndash87

[48] Byung P 1994 Cellular defense against damage from reactiveoxygen species Physiol Rev 74 139ndash62

[49] Huang Z Maness P-C Blake D M Wolfrum E JSmolinski S L and Jacoby W A 2000 Bactericidal mode oftitanium dioxide photocatalysis J Photochem Photobiol A130 163ndash70

[50] Gogniat G Thyssen M Denis M Pulgarin C and Dukan S 2006The bactericidal effect of TiO2 photocatalysis involvesadsorption onto catalyst and the loss of membrane integrityFEMS Microbiol Lett 258 18ndash24

[51] Valavanidis A Vlahogianni T Dassenakis M andScoullos M 2006 Molecular biomarkers of oxidative stress inaquatic organisms in relation to toxic environmentalpollutants Ecotoxicol Environ Saf 64 178ndash89

[52] Delaney J Neville W A Swain A Miles A Leonard M S andWaterfield C J 2004 Phenylacetylglycine a putativebiomarker of phospholipidosis its origins and relevance tophospholipid accumulation using amiodarone treated rats asa model Biomarkers 9 271ndash90

[53] Phipps A N Stewart J Wright B and Wilson I D 1998 Effect ofdiet on the urinary excretion of hippuric acid and otherdietary-derived aromatics in rat A complex interactionbetween diet gut microflora and substrate specificityXenobiotica 28 527ndash37

[54] James M O Smith R L Williams R T and Reidenberg M 1972The conjugation of phenylacetic acid in man sub-humanprimates and some non-primate species Proc R Soc B182 25ndash35

[55] Nicholls A W Mortishire-Smith R J and Nicholson J K 2003NMR spectroscopic-based metabonomic studies of urinarymetabolite variation in acclimatizing germ-free rats ChemRes Toxicol 16 1395ndash404

[56] Daykin C A Van Duynhoven J P Groenewegen A Dachtler MVan Amelsvoort J M and Mulder T P 2005 Nuclear magneticresonance spectroscopic based studies of the metabolism ofblack tea polyphenols in humans J Agric Food Chem53 1428ndash34

[57] Waters N J Waterfield C J Farrant R D Holmes E andNicholson J K 2006 Integrated metabonomic analysis ofbromobenzene-induced hepatotoxicity novel induction of5-oxoprolinosis J Proteome Res 5 1448ndash59

[58] Wang Y Tang H Nicholson J K Hylands P J Sampson J andHolmes E 2005 A metabonomic strategy for the detection of

11


the metabolic effects of chamomile (Matricaria recutita L)ingestion J Agric Food Chem 53 191ndash6

[59] Dumas M E et al 2006 Metabolic profiling reveals acontribution of gut microbiota to fatty liver phenotype ininsulin-resistant mice Proc Natl Acad Sci USA103 12511ndash6

[60] Kasuya F Yamaoka Y Osawa E Igarashi K and Fukui M 2000Difference of the liver and kidney in glycine conjugation ofortho-substituted benzoic acids Chem Biol Interact125 39ndash50

[61] Waterfield C J Turton J A Scales M D and Timbrell J A 1993Investigations into the effects of various hepatotoxiccompounds on urinary and liver taurine levels in rats ArchToxicol 67 244ndash54

[62] Clayton T A Lindon J C Everett J R Charuel C Hanton GLe Net J L Provost J P and Nicholson J K 2004Hepatotoxin-induced hypercreatinaemia andhypercreatinuria their relationship to one another to liverdamage and to weakened nutritional status Arch Toxicol78 86ndash96

12

1 Introduction


21 Materials




25 Clinical chemistry hematological analysis and histopathology

26 1H NMR spectroscopic measurement of urine

27 1H NMR spectroscopic measurement of serum

28 Data reduction and PR analysis of 1H NMR spectra

29 Statistics

3 Results

31 1H NMR spectroscopic and PR analysis of urine

32 1H NMR spectroscopic and PLS-DA of serum

33 TEM inspection


4 Discussion

41 Effect of TiO2 NPs on clinical chemistry hematological analysis histopathology and TEM inspection

42 TiO2 NP-induced energy and choline metabolism disturbance



5 Conclusion

Acknowledgments

References

IOP PUBLISHING NANOTECHNOLOGY

Nanotechnology 21 (2010) 125105 (12pp) doi1010880957-44842112125105

NMR-based metabonomic study of thesub-acute toxicity of titanium dioxidenanoparticles in rats after oraladministrationQian Bu17 Guangyan Yan27 Pengchi Deng3 Feng Peng4Hongjun Lin1 Youzhi Xu1 Zhixing Cao1 Tian Zhou1 Aiqin Xue5Yanli Wang6 Xiaobo Cen28 and Ying-Lan Zhao18

1 State Key Laboratory of Biotherapy and Cancer Center West China Hospital West ChinaMedical School Sichuan University Chengdu 610041 Peoplersquos Republic of China2 National Chengdu Center for Safety Evaluation of Drugs State Key Laboratory ofBiotherapy West China Hospital West China Medical School Sichuan University Chengdu610041 Peoplersquos Republic of China3 Analytical amp Testing Center Sichuan University Chengdu 610041Peoplersquos Republic of China4 Department of Thoracic Oncology of Cancer Center and State Key Laboratory of BiotherapyWest China Hospital West China Medical School Sichuan University Chengdu 610041Peoplersquos Republic of China5 Institute of Bioengineering Zhejiang Sci-Tech University Road 2 Xiasha Hangzhou310018 Peoplersquos Republic of China6 Tianjin Childrenrsquos Hospital Tianjin 300074 Peoplersquos Republic of China

E-mail alancenxbsinacom

Received 25 November 2009 in final form 1 February 2010Published 5 March 2010Online at stacksioporgNano21125105

AbstractAs titanium dioxide nanoparticles (TiO2 NPs) are widely used commercially their potential toxicity on human healthhas attracted particular attention In the present study the oral toxicological effects of TiO2 NPs (dosed at 016 04and 1 g kgminus1 respectively) were investigated using conventional approaches and metabonomic analysis in Wistarrats Serum chemistry hematology and histopathology examinations were performed The urine and serum wereinvestigated by 1H nuclear magnetic resonance (NMR) using principal components and partial least squaresdiscriminant analysis The metabolic signature of urinalysis in TiO2 NP-treated rats showed increases in the levels oftaurine citrate hippurate histidine trimethylamine-N-oxide (TMAO) citrulline α-ketoglutaratephenylacetylglycine (PAG) and acetate moreover decreases in the levels of lactate betaine methionine threoninepyruvate 3-D-hydroxybutyrate (3-D-HB) choline and leucine were observed The metabonomics analysis of serumshowed increases in TMAO choline creatine phosphocholine and 3-D-HB as well as decreases in glutaminepyruvate glutamate acetoacetate glutathione and methionine after TiO2 NP treatment Aspartate aminotransferase(AST) creatine kinase (CK) and lactate dehydrogenase (LDH) were elevated and mitochondrial swelling in hearttissue was observed in TiO2 NP-treated rats These findings indicate that disturbances in energy and amino acidmetabolism and the gut microflora environment may be attributable to the slight injury to the liver and heart causedby TiO2 NPs Moreover the NMR-based metabolomic approach is a reliable and sensitive method to study thebiochemical effects of nanomaterials

(Some figures in this article are in colour only in the electronic version)

7 These authors contributed equally to this work8 Address for correspondence State Key Laboratory of Biotherapyand Cancer Center West China Hospital West China Medical School

Sichuan University 1 Keyuan Road 4 Gaopeng Street High TechnologicalDevelopment Zone Chengdu 610041 Peoplersquos Republic of China

0957-448410125105+12$3000 copy 2010 IOP Publishing Ltd Printed in the UK1


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NMR-based metabonomic study of the sub-acute toxicity of titanium dioxide nanoparticles in rats...

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Transcript of NMR-based metabonomic study of the sub-acute toxicity of titanium dioxide nanoparticles in rats...