Metabolic Profiling Reveals the Protective Effect of Diammon

ORIGINAL ARTICLE

Metabolic pro?ling reveals the protective effect of diammonium glycyrrhizinate on acute hepatic injury induced by carbon tetrachloride

Xiaoyan Wang?Jingchao Lin?Tianlu Chen?

Mingmei Zhou?Mingming Su?Wei Jia

Received:15July2010/Accepted:8September2010

óSpringer Science+Business Media,LLC2010

Abstract Diammonium glycyrrhizinate(DG),a constitu-tent of Glycyrrhiza uralensis,has a protective effect on hepatic injury,hepatisis and cirrhosis.To date,the mecha-nism has been poorly understood,especially at the metabolic level.A metabolomic pro?ling study was performed to characterize the carbon tetrachloride(CCl4)induced global metabolic alteration and the protective effects of DG in Sprague-Dawley rats.Urinary and hepatic tissue metabolic pro?ling revealed that CCl4perturbed the amino acid metabolism(alanine,glycine,leucine),tricarboxylic acid cycle(citrate),lipid metabolism(unsaturated fatty acids)and gut microbiota related metabolites.Our results also indicated that DG was able to attenuate CCl4perturbed metabolic pathways and ameliorated biochemical markers of alanine aminotransferase(ALT),aspartate aminotransferase(AST), and Total cholesterol(TCHO).This global metabolomic approach also revealed full metabolic recovery takes longer than apparent and conventional histological and biochemical markers.

Keywords MetabolomicsáAcute liver injureáDiammonium glycyrrhizinateáCarbon tetrachloride Abbreviations

DG Diammonium glycyrrhizinate

NC Normal control

TCHO Total cholesterol

AST Aspartate aminotransferase

ALT Alanine aminotransferase

MDA Malondiadehyde

SOD Superoxide dismutase

GSH-px Glutathion peroxidase

ECF Ethylchloroformate

PCA Principal component analysis

PLS-DA Partial least squares discriminant analysis

PC Principal component

GSH Reduced glutathione

GSSG Oxidized glutathione

DOPAC3,4-Dihydroxy-phenylacetate

1Introduction

Diammonium glycyrrhizinate(DG),a puri?ed effective constituent of the traditional Chinese medicinal herb Glycyrrhiza uralensis(liquorices or Gan-Cao)is clinically used in the treatment of hepatic injury,hepatisis and cirrhosis(Feng et al.2007).DG possesses a high anti-in?ammatory effect,which protects the hepatic cell membrane,and ameliorates liver function(Xu et al.2009

; X.WangáJ.LináT.Chen

Ministry of Education Key Laboratory of Systems Biomedicine,

Shanghai Center for Systems Biomedicine,

Shanghai Jiao Tong University,Shanghai200240,China

M.Zhou

Shanghai University of Traditional Chinese Medicine,

Shanghai201203,China

M.SuáW.Jia

David H.Murdock Research Institute,University of North

Carolina at Greensboro,North Carolina Research Campus,

Kannapolis,NC28081,USA

W.Jia(&)

Department of Nutrition,University of North Carolina

at Greensboro,North Carolina Research Campus,

500Laureate Way,Kannapolis,NC28081,USA

e-mail:w_jia@9fa8d17e5acfa1c7aa00cced

123 Metabolomics

DOI10.1007/s11306-010-0244-5

Yuan et al.2006).It would undoubtedly be bene?cial to understand the mechanisms of the hepatoprotective effect of this herbal ingredient in metabolic regulation.Carbon tetrachloride(CCl4)is a widely used hepatotropic poison which induces experimental liver damage histologically as hepatic steatosis,cellular necrosis,?brosis,hepatocellular death and carcinogenicity(Weber et al.2003).Because the CCl4induced hepatic injury model can clearly re?ect the function,metabolism and morphological variations of hepatic cells with high reproducibility,the model is com-monly used to simulate acute/chronic hepatitis(Okamoto and Okabe2000;Cherkashina and Petrenko2006)and hepatic?brosis(Abdel-Salam et al.2007).The metabolic impact of CCl4has been evaluated by pro?ling differential expressed metabolites of plasma,tissue and urine during poisoning process(Robertson et al.2000;Pan et al.2010; Lin et al.2009).In this study,we set up an acute hepatic injury animal model with CCl4to test the biochemical and metabolic mechanism of the anti-hepatotoxicity of DG.

Metabolic pro?ling of biological samples using GC/MS has been extensively used to evaluate the toxic/disease status and test the ef?cacy of drug treatment(Pan et al. 2010;Qiu et al.2007;Wang et al.2007).This technology provides quantitative information on metabolite levels increase or decrease in response to xenobiotic interven-tions,especially in hepatotoxicity research(Chen et al. 2009;Beger et al.2010;Sun et al.2009).This information complements organ-speci?c biochemical and histological variations and can reveal a complex interplay among bio-chemical regulatory pathways and xenobiotics agents in a given biological system.This study aimed to characterize metabolic variations and thus,understand the dynamic pathophysiological process associated with the CCl4 induced acute hepatic toxicity and the protective effect of DG pre-ingestion in Sprague-Dawley(SD)rats upon treatment of a diammonium glycyrrhizinate intervention. 2Materials and methods

2.1Animals and treatments

A total of72Male Sprague-Dawley(SD)rats weighing 200–250g were commercially obtained from Shanghai Laboratory Animal Co.,Ltd.(SLAC,Shanghai,China).All animals were kept in a barrier system with regulated temperature(23–24°C)and humidity(60±10%)and on a 12/12-h light–dark cycle with lights on at08:00a.m.The rats were fed certi?ed standard rat chow and tap water ad libitum for2week acclimation.Rats were randomly pi-ded into three groups of24:(A)normal control(NC),(B) CCl4model(CCl4),(C)DG?CCl4(DG/CCl4).Each animal in the DG/CCl4group was intragastrically administered46.88mg kg-1of DG(dissolved in saline), while rats in the NC and CCl4groups received the same volume of saline,once a day for14days.On the14th day, each animal in the CCl4and DG/CCl4group received an intraperitoneal injection of CCl4in olive oil(25%v/v)at 1.5ml kg-1to induce the acute injury model.Normal control rats received the same volume of olive oil.Eight rats in each group were randomly killed at24and96h after CCl4administration and at the end of the study.The right lobes of the livers(0.5g)were?xed in a10% formaldehyde solution,embedded in paraf?n and then processed for light microscopy.Another part of each liver was washed with saline,wiped dry,and then homogenized to10%homogenate with cold saline for further biochem-ical measurement and metabolic pro?ling analysis.

Twenty-four hour urine samples of each animal were collected from inpidually housed rats in metabolism cages at the initial day(I1),the7th day(I7),and the1st day (P1),the4th day(P4),the7th day(P7)and the10th day (P10)post CCl4administration(Table1).At the end of the study,after the last time point(P10),urine samples were collected,all the rats were sacri?ced and the serum and livers were collected for biochemical measurement.The urine samples were centrifuged at10,000rpm for10min to remove suspended debris and stored at-80°C.The animal experiment was carried out under the Guidelines for Animal Experiment of Shanghai University of Traditional Chinese Medicine(Shanghai,China),and the protocol was approved by the Animal Ethics Committee of the Shanghai University of Traditional Chinese Medicine.

2.2Biochemistry and histopathology study

Blood was collected at the1st(P1),4th(P4)day after CCl4 administration and at the end of the experiment(P10). Serum samples were then removed from the coagulated blood after centrifugation(10,0009g,10min,4°C)and biochemical analysis was performed.Total cholesterol (TCHO)and serum enzymes,including aspartate amino-transferase(AST)and alanine aminotransferase(ALT), were detected using an automatic biochemical analyzer (BST-370)to evaluate the severity of hepatic injury.

Liver tissue was collected at the1st(P1),4th(P4)day after CCl4administration and at the end of the experiment (P10).The liver malondiadehyde(MDA),superoxide dis-mutase(SOD)and glutathion peroxidase(GSH-px)content was detected with commercial Malondiadehyde Assay Kit, Superoxide Dismutase,and glutathion peroxidase Assay Kit(Jiancheng Bioengineer Institute,Nanjing,China), respectively.Brie?y,liver tissues were homogenized with a T10basic homogenizer(IKA,Staufen,Germany)for30s at0°C before the procedures were conducted in strict accordance with instruction of the manufacturers.Values

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of MDA,SOD and GSH-px were obtained from the measurement and the standard curves.

The same part of each liver sample was?xed in10% (v/v)formalin for at least12h,and then processed into wax sections.Tissue sections were subsequently stained with haematoxylin and eosin(H–E),examined under a light microscope(OLYMPUS Co.,Ltd,Japan)for the hepatic cell morphology evaluation,and captured by a digital camera.

Data from the serum and tissue biochemistry determi-nation were expressed as mean±SD.Differences between the means of the treatment and control groups were ana-lyzed using one-way analysis of variance(ANOVA).The critical P value was set at0.05.

2.3Metabolic pro?ling of urine and hepatic tissue

samples

GC/MS-based metabolic pro?ling was performed on the urine samples following our established methods(Qiu et al. 2007).The urine samples were derivatized with ethyl-chloroformate(ECF).A600l l of diluted urine sample (urine/water)1:1,v/v)was added with100l l of L-2-chlorophenylalanine(0.10mg/ml,internal standard for batch quality control),400l l of anhydrous ethanol,100l l of pyridine and derivatized with50l l of ECF at room temperature,and then ultrasonicated at100kHz for60s. The derivatives were extracted with300l l of chloroform, and the pH was adjusted with100l l of NaOH(7mol/l). The derivatization process was repeated by adding an additional50l l of ECF.The resulting mixtures were centrifuged at3000rpm for3min.Then,the aqueous layer was removed and the chloroform layer containing deriva-tives was dehydrated with anhydrous sodium sulfate for subsequent GC/MS analysis.A1l l aliquot of the deriva-tized extract was injected in splitless mode into an Agilent 6890N GC/5975B inert MSD(Agilent Technologies,Santa Clara,CA,USA).Separation was achieved on a DB-5MS capillary column.

Each100mg liver tissue was extracted following the two-step extraction procedure described in our previous report(Pan et al.2010).Brie?y,for each100-mg liver tissue sample,500l l of each of the two solvents(the mixture of chloroform,methanol and water(1:2:1,v/v/v)and methanol alone)were used as the two extraction solvents.After homogenization with the?rst solvent and centrifugation,a 150-l l aliquot of supernatant was transferred to a separate vial and the deposit was re-homogenized with the second solvent before a second centrifugation.Another150-l l aliquot of supernatant was drawn out and mixed with the ?rst150-l l aliquot of supernatant.Then the300l l super-natant from the two extraction step was diluted with300l l of water.The600l l solution was then derivatized with ECF using the aforementioned method.

Either the injection temperature or the interface tem-perature was set to260°C;and the ion source temperature was adjusted to200°C.Initial GC oven temperature was 80°C;2min after injection,the GC oven temperature was raised to140°C with10°C min-1,to240°C at a rate of 4°C min-1,to280°C with10°C min-1again,and?nally held at280°C for3min.Helium was the carrier gas with a ?ow rate set at1ml min-1.The measurements were taken with electron impact ionization(70eV)in the full scan mode(m/z30–550).

2.4GC-MS Data Analysis

The analysis of GC-MS data was performed with a minor modi?cation to our established methods(Wang et al. 2009).Brie?y,unprocessed GC/MS?les were converted into NetCDF format via DataBridge(Perkin-Elmer Inc., U.S.A.)and directly processed by our custom scripts in MATLAB(The MathWorks,Inc.,U.S.A.).This process performed data smoothing,?ltering,de-noising,baseline correction,peak discrimination and alignment(for identi-?cation and extraction of the peaks of the chromatogram indicating the existence and intensities of potential metabolites),internal standard exclusion,and normaliza-tion to the total sum of the chromatogram(Bao et al.2009; Wang et al.2009;Pan et al.2010).The resulting three-dimensional(retention time,M/Z and Intensity of peaks) matrix was introduced into the SIMCA-P12.0Software package(Umetrics,Umea?,Sweden)for multivariate statistical analysis.To ensure the consistency in spectral data transformation and avoid errors introduced to the data

Table1Experimental design

and sampling schedule

Time(day)Abbreviations Experiment content Sampling

1I1Initial time(pretreatment of DG)Urine

7I7Initial time(pretreatment of DG)Urine

14CCl4toxication

15P1Post-toxication Urine,liver tissue,serum

18P4Post-toxication Urine,liver tissue,serum

21P7Post-toxication Urine

24P10Endpoint Urine,liver tissue,serum Metabolic effect of DG on CCl4induced liver injury

123

processing,we examined the peak areas with relatively high intensities and found no drastic?uctuations among those peaks.The data were mean-centered and then pareto-scaled.The mean-centering procedure subtracts the mean of the data and results in a shift of the data towards the mean.The pareto-scaling technique gives the weight of each variable by the square root of its standard deviation, which ampli?es the contribution of lower concentration metabolites but not to such an extent where noise produces a large contribution,this process enhances the identi?ca-tion of metabolites consistently present in the biological samples.The normalized data was analyzed by principal component analysis(PCA)to visualize general clustering, trends,or outliers among the observations.Then,partial least-squares-discriminant analysis(PLS-DA)was con-ducted to identify the metabolites differentially produced by CCl4or DG.R2X and R2Y of the model represent the fraction of the variance,while Q2Y suggests the predictive accuracy of the model.The cumulative values of R2X,R2Y and Q2Y(range of0to1)close to1indicate a satisfactory model.To avoid model over-?tting,the PLSDA model was carefully validated by an iterative7-round cross-validation with1/7of the samples being excluded from the model in each round and random permutation tests(1,000times).

A VIP parameter(denoting the variable importance)of greater than1,combined with a correlation coef?cient pCorr(indicating the reliability of the loading and VIP value)of±0.7was adopted as the cutoff value for selecting the most important variables in terms of the PLSDA model, based on the integrated MS data(Wiklund et al.2008). Differentially expressed variables were identi?ed by chromatogram-MS data and labeled using thresholds of their fold change and P-values of their Kruskal–Wallis test. In addition to the nonparametric Kruskal–Wallis test, classical one-way analysis of variance(ANOVA)was also carried out to validate the statistical signi?cance of these variables.The critical P value of Kruskal–Wallis and ANOVA was set at0.05for this 9fa8d17e5acfa1c7aa00ccedpound identi-?cation of metabolites was performed by comparing the mass fragments of the signi?cant variables with those present in commercially available mass spectral databases such as NIST,Wiley,NBS,and the library we established with a similarity threshed of70%.Finally,about half of them were veri?ed by reference compounds.

3Results and discussion

3.1Biochemistry and histopathology results

As alanine aminotranferease(ALT)and aspartate amino-transferase(AST)are enzymes located in liver cells that are readily released into the general circulation when liver cells are injured,the two enzymes were measured to monitor the hepatocellular damage.At the24th hour post CCl4 administration,the activity of serum ALT and AST increased signi?cantly,while the TCHO level decreased,as shown in Fig.1.Pretreatment with DG signi?cantly attenuated the alterations of several metabolite levels. At96h,the ALT,AST and TCHO activity of every animal in the study recovered to normal status.The value of MDA and SOD in liver changed signi?cantly,and DG was able to attenuate the variation of MDA as well.The activity of GSH-px was altered by CCl4and DG,but not signi?cantly. At the end point of the study,hepatic MDA,SOD and GSH-px levels returned to normal.

The liver sections stained with H–E were examined for the histopathological assessment of CCl4induced liver injury and DG’s protection(Fig.2).At the?rst day post injection of CCl4,signi?cant damage to the hepatic his-tology structure,including cytoplasmic vacuolization,cell swelling,variations in cellular size and morphology and in?ammatory cell in?ltrations,was observed in most CCl4 challenged samples(Fig.2b),compared with the normal liver morphologies in the NC group(Fig.2a).Samples from the DG/CCl4group illustrate reduced liver damage, e.g.intact liver structure and inconspicuous in?ammatory cell in?ltration(Fig.2c).At96h post-dose of CCl4,all the damage was repaired and the histology showed no difference from the normal tissue(Fig.2d,e).We ruled out the possible therapeutic effect due to the use of olive oil as a vehicle in our short-term(1–4day)study,since the anti-in?ammatory effects were generally observed in the subjects with relatively long-term(several weeks) dietary supplementation with olive oil(Beauchamp et al. 2005).

3.2Metabolite Variation Induced by CCl4

The PCA scores plot derived from the GC/MS data shows the clustering of NC and CCl4groups on the?rst two principal components(PC1and PC2),as depicted in Fig.3a.The metabolomic result of this liver injury model displays stable cumulative modeled variation and good prediction capabil-ity with the?rst two components(Component Number=3, R2Xcum=0.626and Q2Ycum=0.864).The3-dimen-sional scores plot derived from the GC/MS data of the CCl4, DG/CCl4and NC group is depicted in Fig.3b(Component Number=3,R2Xcum=0.635,Q2Ycum=0.408).

The trajectories of PCA scores derived from the CCl4 (Component Number=2,R2Xcum=0.296,Q2Ycum= 0.170)and DG/CCl4group(Component Number=2, R2Xcum=0.293,Q2Ycum=0.109)were illustrated in Fig.4.Transient shifts in the trajectory plot revealed the dynamic progress of the metabolic variation induced by CCl4alone or in combination with DG treatment.In the

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CCl4group,the metabolic pro?le on the?rst day post-CCl4 injection(P1)was distinct from those of the other time-points.The trajectory demonstrates that the metabolic regulatory network underwent the most signi?cant meta-bolic?uctuations on the?rst day of exposure to CCl4,and that the perturbed network underwent a recovery process during the following time points,returning to a stable pro?le close to the pre-dose state.This is consistent with the biochemical markers results.The time-dependent tra-jectory of metabolic alteration in the DG/CCl4group appears similar to that in the CCl4group.However,the metabolic perturbation in the DG/CCl4group at the1st and 4th day post-dose of CCl4appeared less signi?cant than those in the CCl4group.

We selected the different expressed metabolites in the rats of CCl4group relative to NC group at the1st day(P1) post CCl4exposure,a key time point of liver injury study, and evaluated their variances at different time points of the DG/CCl4group(Grizzi et al.2003).Sixteen out of21 differentially expressed peaks,including amino acids such as alanine,glycine,proline,and glutamine,and organic acids such as citrate and hexanoate,were identi?ed from spectral dataset,veri?ed by reference standards(Fig.4; Table2).Univariate statistical methods,including one-way ANOVA and the nonparametric Kruskal–Wallis test, were utilized to verify the signi?cance of multivariate statistical method.The two heat-maps generated using differentially expressed metabolites in rats also indicate less signi?cant?uctuation of metabolite levels(in fold change,relative to NC)in the DG/CCl4group,suggest-ing that DG could attenuate the metabolic perturbation in the rats exposed to CCl4.These results support the clinical?ndings that DG has a protective effect on liver injuries.

Signi?cantly expressed metabolites in liver tissues were also analyzed by the Kruskal–Wallis test and classical one-way analysis of variance(ANOVA).P-values were set at 0.05.Figure5is the heat-map of the fold changes of dif-ferentially expressed metabolites in CCl4and DG/CCl4 groups,which consistently shows alleviative effect in

Metabolic effect of DG on CCl4induced liver injury

123

DG/CCl 4group.Decreased levels of alanine,glycine,valine,leucine,arachidonate and eicosapentaenoate,increased levels of aspartate and oleate were observed in CCl 4group at the 1st (P1)and 4th day (P4).Except for arachidonate,the alteration of these metabolites was attenuated in DG/CCl 4group at the 4th day.There was no signi?cant variation in the metabolites of liver tissues in the two groups at the last time

point.Fig.2Liver histology (*200)

of chemical liver injury’s rats

induced by CCl 4.a NC group;

(b )the 1st day post dose in CCl 4

group;(c ):the 1st day post dose

in group;(d )the 4th day post

dose in CCl 4group;(e )the 4th

day post dose in DG/CCl 4group

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3.3Potential pathways associated with CCl 4

and the DG treatment

Our metabolic pro?ling strategy of urine and liver tissues is able to reveal the multi-pathway metabolic perturbation associated with CCl 4and the DG treatment,as summarized in Fig.6.Since the liver is the hub of amino acids metabolism,any hepatic injury might induce amino acids metabolic disturbances.The metabolomic results showed that most of the amino acids that increased in urine and decreased in liver tissue,such as alanine,proline,glutamine,phenylalanine and isoleucine.Generally,the impact of acute liver injury on amino acids concentration results from three factors:protein synthesis and catabolism,BCAAs (branched chain amino acids)metabolism,and hepatic amino acid clearance.When damage occurs,catabolism promoting hormones,such as glucocorticoids and catecholamines,increase in secretion and decrease in deactivation,resulting in increased protein catabolism and therefore,increased levels of a number of amino acids in systemic circulation.Many amino acids are metabolized by liver enzymes and hence directly associated with

the

Fig.3Metabolic pro?les depicted by 2D PCA scores plot of GC/MS spectral data from the urine of CCl 4and NC group (a :Component Number =3,R 2Xcum =0.626and Q 2Ycum =0.864),and 3D PCA scores plot of GC/MS spectral data from the urine of CCl 4,DG/CCl 4and NC group (b :Component Number =3,R 2Xcum =0.635,Q 2Ycum =0.408)1day post injection of CCl

4

Fig.4PCA scores plots of GC/MS spectral data from model CCl 4group (a :Component Number =2,R 2Xcum =0.296,Q 2Ycum =0.170)and CCl 4/DG group (C)visualized using a metabolic pro?ling approach,with the heatmap plot of differential expressed

metabolites(b :Component Number =2,R 2Xcum =0.293,Q 2Y-cum =0.109)(?lled square I1,open square I7,open diamond P1,?lled triangle P4,?lled diamond P7,open circle P10)

Metabolic effect of DG on CCl 4induced liver injury

123

activity of hepatic enzymes.ALT and AST,two important enzymes in liver,were detected as well.Glutamine is an important amino acid for maintaining nitrogen balance (Brosnan 2003).A decrease in liver and an increase in urinary excretion of glutamine may be associated with an out?ow of AST from hepatocellular mitochondrion and an indication of impaired hepatic regulating function.Branched chain amino acids (BCAAs),such as valine,isoleucine and leucine,are essential amino acids typically involved in stress,energy and muscle metabolism (Chou-dry et al.2006).In our study,an increased level of iso-leucine and leucine in urine,while decreased levels of hepatic valine,leucine and isoleucine were observed in CCl 4group at the 1st and 4th day post CCl 4injection (P1,P4).When protein catabolism is being observed,as in some cases of severe toxicity,the BCAAs are used in muscle to create alanine,which is then shuttled to the liver (Holecek et al.1996).Normally blood alanine is transported to liver via glucose-alanine cycle to generate pyruvate which becomes a source of carbon atoms for gluconeogenesis.Alanine was found largely excreted in urine and decreased in liver tissue,suggesting an impaired glucose-alanine cycle due to the CCl 4exposure.DG attenuated the altered levels of alanine and BCAAs but didn’t affect citrate level,suggesting that DG’s interaction is involved in the glucose-alanine cycle and its closely associated amino acids metabolism.It was not able to ameliorate the citrate expression level as part of the impaired TCA cycle,whose metabolic enzymes located in mitochondria (Fig.6).

Aspartic acid is vitally important to the metabolism and construction of many amino acids and intermediates in the tricarboxylic acid (TCA)cycle.It has also shown a protec-tive action on the liver by its capacity to reestablish the cellular de?cit of pyridine nucleotides and thus improve the synthesis of nucleic acids,glycoprotein and glycolipids and/or by its participation in various metabolic pathways (Fodor et al.1976).In this study,aspartate remained a signi?cant higher concentration in the liver at the 1st and 4th day post CCl 4injection,while its decrease in urine excretion could be resisted by either recovery time or DG treatment.

Urinary and hepatic glycine levels signi?cantly decreased after CCl 4exposure.The large amounts of oxygen free radicals generated from CCl 4dose altered hepatic levels of MDA,SOD and GSH-px.As a result,the anti-oxidants involving reduced glutathione hormone (GSH)were presumably over-consumed.As glutathione is synthesized from the amino acids L -cysteine,L -glutamate and glycine,it is understandable that glycine was found signi?cantly depleted soon after CCl 4exposure.However,we were surprised to detect a drastically increased level of glycine in urine (Fig.4a)while decreased in the liver (Fig.5,P4)4days after toxication.Recent research has indicated that glycine signi?cantly decreases liver injury

Table 2List of identi?ed differential metabolites in the urine sam-ples of CCl 4and DG/CCl 4at 1day after CCl 4toxication,P values in CCl 4and DG/CCl 4Metabolites

RT a

VIP b

P c CCl 4

DG/CCl 4Hexanoate d 6.06 1.450.03060.6770Phenylacetate d 7.13 1.190.00030.0332Alanine d 7.51 1.060.03590.1750Glycine

d 7.62 1.190.00490.0331Leucine

d 10.42 1.520.00490.0574Isoleucin

e d

10.70 1.530.04270.3771Proline d 11.12 1.360.02060.1937Unknown 12.84 1.670.03820.3171Aspartate

d 14.03

1.290.00260.0526Glutamine d

14.42 1.670.00040.00733,4-dihydroxy-phenylacetate d

14.87 1.090.04190.0203Citrate d 15.05 1.210.00020.0031Unknown 15.28 1.330.02390.0552Unknown 16.01 1.070.02980.2346Unknown

17.42 1.020.06580.2600Phenylalanine d

18.28

1.590.02730.22074-hydroxy-phenylacetate d

18.46 1.490.04670.0574Indol-3-acetate d

21.28 1.190.03310.0426Tyramine

d

26.16 2.070.00020.01944-ehtoxyphenyl-propenoate d 29.55 1.690.00710.1296Unknown

33.46

1.81

0.0046

0.0172

a Retention time of the metabolites in GC/MS

b

Variable importance in the projection (VIP)was obtained from PLS-DA

c P values were calculate

d from Kruskal–Wallis test vs.NC group d

Metabolites veri?ed by reference compounds,others were directly obtained from library

searching

Fig.5Differential expressed metabolites in liver tissue the 1st and 4th day post intoxication

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via a direct effect on hepatocytes (Froh et al.2008).As the decreased hepatic glycine level was normalized by DG at both the 1st and 4th day after injury,we presumed that the elevation of glycine in the urine may be bene?cial to the recovery of hepatic cells.The urinary glycine level also displayed a rebound in DG/CCl 4group at P4.

Furthermore,several compounds containing benzene ring,such as phenylacetate,phenylalanine,4-hydroxy-phenylacetate,indol-3-acetate,tyramine,3,4-dihydroxy-phenylacetate and 4-ethoxyphenyl-propenoate were discovered signi?cantly altered in the urine of CCl 4treated rats,as compared to the normal group.These metabolites are mainly produced from aromatic compounds,especially aromatic amino acids.Indol-3-acetate,namely indoleacetic acid (IAA)is produced in tryptophan metabolism often with involvement of bacteria in the mammalian gut (Maillet et al.2009).It is believed to be a product of the decarboxylation of tryptamine or the oxidative deamination of tryptophan.Tyramine is formed by decarboxylation of tyrosine in tissues as well as in the gut (Asatoor 1968).Phenylacetate can be produced by the transamination and then decarboxylation of excess phenylalanine,an important precursor of tyrosine,which decreased in liver and increased in urine at the 1st day post dose.4-hydroxy-phenylacetate is an oxidative deaminated metabolite of tyramine and also a metabolite of tyrosine from enteric bacteria (Rechner et al.2004,Nowak and Libudzisz 2006).Another phenolic acid,3,4-dihydroxy-phenylacetate,commonly called DOPAC,is the product of oxidation of the aldehyde produced by deamination metabolite from dopamine,one of the catecholamines derived from tyrosine (Goldstein et al.2003).DOPAC is also one of the major phenolic acids formed during gut microbial fermentation of diets.The variation of these metabolites may re?ect either an alteration in a disturbed symbiotic gut microbiota and/or

the catecholamine metabolic pathway.Furthermore,the aromatic metabolites still ?uctuated at the P4and P7,and ?nally returned to the normal level,suggesting that the impact of CCl 4on the metabolism of aromatic compounds lasted longer than its impact on other metabolic pathways.The normalized expression level of these metabolites in the DG/CCl 4group suggests a protective effect of DG on gut microbiota and/or catecholamine pathways.

It has been reported that CCl 4derived free radicals may attack polyunsaturated fatty acids (PUFA)in cell mem-branes,forming fatty acid free radicals,which initiate an autocatalytic lipid peroxidation process and generate more lipid hydroperoxides and reactive hydroxyalkenals during membrane disruption (Vulimiri et al.2010;Catala 2009).The toxin,CCl 4,can also promote the production of fatty acids and triglyceride inside the liver,accelerate the lipid esteri?cation and cholesterols synthesis,and thus,lower the content of partially unsaturated fatty acids (Boll et al.2001).In our results,octadecanoate,arachidonate and eicosapentaenoate decreased in tissue samples at the 1st or 4th day.We found that the hepatic content of oleate increased at both the 1st and 4th day.DG signi?cantly inhibited the alteration of these fatty acids except arachidonate,suggesting that the anti-in?ammatory effect of DG is not directly associated with arachidonate metabolism.

4Concluding remarks

Our metabolic pro?ling has revealed the CCl 4induced alterations in amino acid,TCA,lipid and gut microbiota metabolism.Most of these metabolic alterations could be attenuated by Diammonium glycyrrhizinate.This global metabolomic approach also revealed that the

experimental

Fig.6Potential metabolic mechanisms of CCl 4induced toxicity and the protection of Diammonium glycyrrhizinate.All the colored character styles were connected with CCl 4induced toxicity.Green :DG effective metabolites;Red :DG ineffective metabolites;Orange :biochemical indices

Metabolic effect of DG on CCl 4induced liver injury 123

rats required a prolonged recovery in metabolic pro?le, although the histological results and biochemical markers indicated a rapid recuperation from CCl4induced liver injury.

Acknowledgments This work was?nancially supported by the National Basic Research Program of China(2007CB914700),the National Science and Technology Major Project(2009ZX10005-020) and the National Natural Science Foundation of China Grant 30901997,20775048and the International Collaborative Project of Chinese Ministry of Science and Technology(2006DFA02700). References

Abdel-Salam,O.M.,Sleem,A.A.,&Morsy,F.A.(2007).Effects of biphenyldimethyl-dicarboxylate administration alone or com-bined with silymarin in the CCL4model of liver?brosis in rats.

Scienti?cWorldJournal,7,1242–1255.

Asatoor,A.(1968).The origin of urinary tyramine.Formation in tissues and by intestinal microorganisms.Clinica Chimica Acta, 22,223–229.

Bao,Y.,Zhao,T.,Wang,X.,Qiu,Y.,Su,M.,Jia,W.,et al.(2009).

Metabonomic variations in the drug-treated type2diabetes mellitus patients and healthy volunteers.Journal of Proteome Research,8,1623–1630.

Beauchamp,G.K.,Keast,R.S.,Morel,D.,Lin,J.,Pika,J.,Han,Q., et al.(2005).Phytochemistry:ibuprofen-like activity in extra-virgin olive oil.Nature,437,45–46.

Beger,R.D.,Sun,J.,&Schnackenberg,L.K.(2010).Metabolomics approaches for discovering biomarkers of drug-induced hepato-toxicity and nephrotoxicity.Toxicology and Applied Pharma-cology,243,154–166.

Boll,M.,Weber,L.W.,Becker, E.,&Stamp?, A.(2001).

Pathogenesis of carbon tetrachloride-induced hepatocyte injury bioactivation of CCI4by cytochrome P450and effects on lipid homeostasis.Z Naturforsch C,56,111–121.

Brosnan,J.T.(2003).Interorgan amino acid transport and its regulation.Journal of Nutrition,133,2068S–2072S.

Catala,A.(2009).Lipid peroxidation of membrane phospholipids generates hydroxy-alkenals and oxidized phospholipids active in physiological and/or pathological conditions.Chemistry and Physics of Lipids,157,1–11.

Chen,C.,Krausz,K.W.,Shah,Y.M.,Idle,J.R.,&Gonzalez,F.J.

(2009).Serum metabolomics reveals irreversible inhibition of fatty acid beta-oxidation through the suppression of PPARalpha activation as a contributing mechanism of acetaminophen-induced hepatotoxicity.Chemical Research in Toxicology,22, 699–707.

Cherkashina, D.V.,&Petrenko, A.Y.(2006).Hepatoprotective effect of fetal tissue cytosol and its thermostable fraction in rats with carbon tetrachloride-induced hepatitis.Bulletin of Experi-mental Biology and Medicine,141,544–547.

Choudry,H.A.,PAN,M.,Karinch,A.M.,&Souba,W.W.(2006).

Branched-chain amino acid-enriched nutritional support in surgical and cancer patients.Journal of Nutrition,136, 314S–318S.

Feng, C.,Wang,H.,Yao, C.,Zhang,J.,&Tian,Z.(2007).

Diammonium glycyrrhizinate,a component of traditional Chinese medicine Gan-Cao,prevents murine T-cell-mediated fulminant hepatitis in IL-10-and IL-6-dependent manners.

International Immunopharmacology,7,1292–1298.

Fodor,O.,Schvartz,M.,Suciu, A.,Barbarino, F.,Duca,S.,& Lezeu,R.(1976).Protecting action of aspartate on the hepatic

changes induced by D-galactosamine.Arzneimittelforschung,26, 855–858.

Froh,M.,Zhong,Z.,Walbrun,P.,Lehnert,M.,Netter,S.,Wiest,R., et al.(2008).Dietary glycine blunts liver injury after bile duct ligation in rats.World Journal of Gastroenterology,14, 5996–6003.

Goldstein,D.S.,Eisenhofer,G.,&Kopin,I.J.(2003).Sources and signi?cance of plasma levels of catechols and their metabolites in humans.Journal of Pharmacology and Experimental Thera-peutics,305,800–811.

Grizzi,F.,Franceschini,B.,Gagliano,N.,Moscheni,C.,Annoni,G., Vergani,C.,et al.(2003).Mast cell density,hepatic stellate cell activation and TGF-beta1transcripts in the aging Sprague-Dawley rat during early acute liver injury.Toxicologic Pathol-ogy,31,173–178.

Holecek,M.,Tilser,I.,Skopec,F.,&Sprongl,L.(1996).Leucine metabolism in rats with cirrhosis.Journal of Hepatology,24, 209–216.

Lin,Y.,SI, D.,Zhang,Z.,&Liu, C.(2009).An integrated metabonomic method for pro?ling of metabolic changes in carbon tetrachloride induced rat urine.Toxicology,256, 191–200.

Maillet,E.L.,Margolskee,R.F.,&Mosinger,B.(2009).Phenoxy herbicides and?brates potently inhibit the human chemosensory receptor subunit T1R3.Journal of Medicinal Chemistry,52, 6931–6935.

Nowak,A.,&Libudzisz,Z.(2006).In?uence of phenol,p-cresol and indole on growth and survival of intestinal lactic acid bacteria.

Anaerobe,12,80–84.

Okamoto,T.,&Okabe,S.(2000).Carbon tetrachloride treatment induces anorexia independently of hepatitis in rats.International Journal of Molecular Medicine,6,181–183.

Pan,L.,Qiu,Y.,Chen,T.,Lin,J.,Chi,Y.,Su,M.,et al.(2010).An optimized procedure for metabonomic analysis of rat liver tissue using gas chromatography/time-of-?ight mass spectrometry.

Journal of Pharmaceutical and Biomedical Analysis,52, 589–596.

Qiu,Y.,Su,M.,Liu,Y.,Chen,M.,GU,J.,Zhang,J.,et al.(2007).

Application of ethyl chloroformate derivatization for gas chro-matography-mass spectrometry based metabonomic pro?ling.

Analytica Chimica Acta,583,277–283.

Rechner,A.R.,Smith,M.A.,Kuhnle,G.,Gibson,G.R.,Debnam,

E.S.,Srai,S.K.,et al.(2004).Colonic metabolism of dietary

polyphenols:in?uence of structure on microbial fermentation products.Free Radical Biology and Medicine,36,212–225. Robertson,D.G.,Reily,M.D.,Sigler,R.E.,Wells,D.F.,Paterson,

D.A.,&Braden,T.K.(2000).Metabonomics:evaluation of

nuclear magnetic resonance(NMR)and pattern recognition technology for rapid in vivo screening of liver and kidney toxicants.Toxicological Sciences,57,326–337.

Sun,J.,Schnackenberg,L.K.,&Beger,R.D.(2009).Studies of acetaminophen and metabolites in urine and their correlations with toxicity using metabolomics.Drug Metabolism Letters,3, 130–136.

Vulimiri,S.V.,Berger,A.&Sonawane,B.(2010).The potential of metabolomic approaches for investigating mode(s)of action of xenobiotics:Case study with carbon tetrachloride.Mutation Research,Accession number:20188855,epub ahead of print. Wang,X.,Su,M.,Qiu,Y.,NI,Y.,Zhao,T.,Zhou,M.,et al.(2007).

Metabolic regulatory network alterations in response to acute cold stress and ginsenoside intervention.Journal of Proteome Research,6,3449–3455.

Wang,X.,Zhao,T.,Qiu,Y.,Su,M.,Jiang,T.,Zhou,M.,et al.(2009).

Metabonomics approach to understanding acute and chronic stress in rat models.Journal of Proteome Research,8, 2511–2518.

X.Wang et al.

123

Weber,L.W.,Boll,M.,&Stamp?,A.(2003).Hepatotoxicity and mechanism of action of haloalkanes:Carbon tetrachloride as a toxicological model.Critical Reviews in Toxicology,33, 105–136.

Wiklund,S.,Johansson,E.,Sjostrom,L.,Mellerowicz,E.J.,Edlund, U.,Shockcor,J.P.,et al.(2008).Visualization of GC/TOF-MS-based metabolomics data for identi?cation of biochemically interesting compounds using OPLS class models.Analytical Chemistry,80,115–122.Xu,X.,Ling,Q.,Gao,F.,He,Z.L.,Xie,H.Y.,&Zheng,S.S.

(2009).Hepatoprotective effects of marine and kuhuang in liver transplant recipients.American Journal of Chinese Medicine,37, 27–34.

Yuan,H.,Ji,W.S.,Wu,K.X.,Jiao,J.X.,Sun,L.H.,&Feng,Y.T.

(2006).Anti-in?ammatory effect of Diammonium Glycyrrhizi-nate in a rat model of ulcerative colitis.World Journal of Gastroenterology,12,4578–4581.

Metabolic effect of DG on CCl4induced liver injury

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