Protective effect of tea polyphenols against paracetamol-induced hepatotoxicity in mice is significanly correlated with cytochrome P450 suppression

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1 Online Submissions: wjg.wjgnet.com World J Gstroenterol 2009 pril 21; 15(15): wjg@wjgnet.com World Journl of Gstroenterology ISSN doi: /wjg The WJG Press nd Bishideng. ll rights reserved. ORIGINL RTICLES Protective effect of te polyphenols ginst prcetmol-induced heptotoxicity in mice is significnly correlted with cytochrome P450 suppression Xi Chen, Chng-Ki Sun, Guo-Zhu Hn, Jin-Yong Peng, Ying Li, Yn-Xi Liu, Yun-Yun Lv, Ke-Xin Liu, Qin Zhou, Hui-Jun Sun Xi Chen, Chng-Ki Sun, Guo-Zhu Hn, Jin-Yong Peng, Ying Li, Yn-Xi Liu, Yun-Yun Lv, Ke-Xin Liu, Qin Zhou, Hui-Jun Sun, Deprtment of Phrmcology, Dlin Medicl University, Dlin , Lioning Province, Chin uthor contributions: Chen X, Sun CK contributed eqully to this work; Hn GZ, Sun HJ designed the reserch; Chen X, Liu YX, Lv YY performed the reserch; Peng JY, Li Y nlyzed the dt; Sun HJ, Liu KX wrote the pper; Zhou Q provided the vitl regents nd nlyticl tools. Supported by Grnt from the Science Foundtion of Eductionl Deprtment of Lioning Province, 05L117 nd Dlin Science & Technology Bureu, 2007J22JH012 Correspondence to: Hui-Jun Sun, Professor, Deprtment of Clinicl Phrmcology, College of Phrmcy, Dlin Medicl University, 9 west section, Lvshun South Rod, Lvshunkou District, Dlin , Lioning Province, Chin. sunhuijun@hotmil.com Telephone: Fx: Received: December 31, 2008 Revised: Mrch 3, 2009 ccepted: Mrch 10, 2009 Published online: pril 21, 2009 bstrct IM: To investigte the heptoprotective ctivity of te polyphenols (TP) nd its reltion with cytochrome P450 (CYP450) expression in mice. METHODS: Heptic CYP450 nd CYPb5 levels were mesured by UV-spectrophotometry in mice 2 d fter intrperitonel TP (25, 50 nd 100 mg/kg per dy). Then the mice were intrgstricly pre-treted with TP (100, 200 nd 400 mg/kg per dy) for six dys before prcetmol (1000 mg/kg) ws given. Their cute mortlity ws compred with tht of control mice. The mice were pre-treted with TP (100, 200, nd 400 mg/kg per dy) for five dys before prcetmol (500 mg/kg) ws given. Heptic CYP2E1 nd CYP12 protein nd mrn expression levels were evluted by Western blotting, immunohistochemicl stining nd trnscriptse-polymerse chin rection. RESULTS: The heptic CYP450 nd CYPb5 levels in mice of TP-treted groups (100, 200 nd 400 mg/kg per dy) were decresed in dose-dependent mnner compred with those in the negtive control mice. TP significntly ttenuted the prcetmol-induced heptic injury nd drmticlly reduced the mortlity of prcetmol-treted mice. Furthermore, TP reduced CYP2E1 nd CYP12 expression t both protein nd mrn levels in dose-dependent mnner. CONCLUSION: TP possess potentil heptoprotective properties nd cn suppress CYP450 expression The WJG Press nd Bishideng. ll rights reserved. Key words: Te polyphenols; Cytochrome P450; Prcetmol-induced heptotoxicity Peer reviewer: Vincent Li, PhD, Derby NHS Foundtion Trust, Utooxeter Rod, Derby DE22 3NE, United Kingdom Chen X, Sun CK, Hn GZ, Peng JY, Li Y, Liu YX, Lv YY, Liu KX, Zhou Q, Sun HJ. Protective effect of te polyphenols ginst prcetmol-induced heptotoxicity in mice is significnly correlted with cytochrome P450 suppression. World J Gstroenterol 2009; 15(15): vilble from: URL: DOI: INTRODUCTION Te hs been consumed in Chin to promote helth nd longevity since 3000 B.C. It is now populr beverge ll over the world. Te polyphenols (TP) re lrge nd diverse clss of compounds extrcted from te. The mjor compounds of TP re pictechin (EC), epiglloctechin (EGC), epictechin gllte (ECG) nd epiglloctechin-3-gllte (EGCG). Recent studies indicte tht TP cn prevent oxidtive stressrelted diseses, including cncer, crdiovsculr nd degenertive diseses nd hve other bioctive properties [1-4]. In recent yers, the interest in understnding the metbolic benefits of TP hs been incresing. Liver is the min orgn responsible for the metbolism of TP. Liu et l [5] reported tht TP cn mrkedly increse cytochrome P450 (CYP450) ctivity in rts. However, the effect of TP on CYP450 ctivity remins controversil [6,7].

2 1830 ISSN CN /R World J Gstroenterol pril 21, 2009 Volume 15 Number 15 Until now, no one could give cler explntion of the different results. CYP450 enzymes ply pivotl role not only in the metbolism of xenobiotics, but lso in the biosynthesis nd ctbolism of endogenous substrtes, such s vitmins, ftty cids, hormones nd prostglndins [8]. ltertion in heptic CYP450 enzyme expression would ffect the phrmcokinetic profiles of cliniclly used drugs. Furthermore, both induction nd suppression of severl CYP450s my led to cellulr oxidtive stress nd tissue injury in response to xenobiotics [9]. Prcetmol, one of the most widely used heptotoxic drugs, is sfe t therpeutic doses, but cuses liver filure in overdoses [10,11]. When norml dose is used, prcetmol is extensively metbolized by conjugtion with sulphte nd glucuronic cid. smll frction of the drug is subjected to oxidtion rections ctlyzed by CYP450 enzymes in the liver, resulting in genertion of N-cetyl-p-benzo-quinoneimine (NPQI), highly electrophilic metbolite tht triggers ensuing liver dmge. Exposure to high doses of prcetmol increses the NPQI level. Normlly, toxic oxidtion metbolites generted in the liver re converted into non-toxic metbolites excreted in urine vi conjugtion with glutthione (GSH) contining sulphydryl groups. However, high doses of prcetmol limit the bility of GSH to detoxify NPQI, nd result in the consumption of liver GSH stores [12,13]. It ws reported tht oxidtive stress constitutes mjor mechnism underlying the pthogenesis of prcetmol-induced liver dmge [14,15]. There re three fmilies of CYP450 isoforms, CYP1, CYP2, nd CYP3, which re minly involved in the biotrnsformtion of xenobiotics [16]. It hs been shown tht CYP2E1, CYP12, nd intrcellulr GSH ply n importnt role in the heptotoxicity induced by prcetmol [17-19]. The present study ws to investigte the heptopro-tective ctivity of TP nd its reltion with CYP450 expression in mice. MTERILS ND METHODS niml tretment TP obtined from Wuyun Te Plnttion (Jingxi Province, Chin) with purity of > 98% contining % o f E G C G n d % o f E C G we r e decffeinized. Kunming mle mice, weighing g, provided by Experimentl niml Center of Dlin Medicl University, were used in the study. The mice were rndomly divided into high, medium, low TP dose groups, positive nd negtive control groups (n = 10). The mice in low, medium nd high dose TP groups were intrperitonelly injected with 25, 50 nd 100 mg/kg of TP per dy for two dys. The mice in positive control group were given 50 mg/kg chlormphenicol one hour before they were killed. The mice in the negtive control group were given the sme volume of sline solution for two dys. On dy 3, ll the mice were killed by decpittion, with their livers removed immeditely nd clened with cold sline solution. Specimens were stored t -80 for preprtion of liver microsomes. Mle mice in ech group received intrgstric TP t the dose of 100, 200, nd 400 mg/kg, once dy for six dys. The mice in positive control group were given 900 mg/kg N-cetylcysteine, once dy. The mice in negtive control group were given the sme volume of sline solution. ll the mice were given 1000 mg/kg prcetmol one hour lter nd the cute mortlity of mice in both groups ws recorded 72 h fter prcetmol ws given. Mle mice in ech group received intrgstric TP t the dose of 100, 200, nd 400 mg/kg, once dy for five dys. The mice in model nd negtive control groups were given the sme volume of sline solution. The mice were given 500 mg/kg prcetmol one hour lter, except for the mice in negtive control group. ll the mice were killed by decpittion 24 h lter with their livers removed immeditely nd clened with cold sline solution. Specimens were fixed in formlin for histologicl exmintion or snp-frozen in liquid nitrogen nd stored t -80 for protein nd RN extrction. Preprtion of liver microsomes Liver microsomes extrcted from frozen smples were prepred s previously described [20]. Microsoml protein level ws mesured s previously described [21] with bovine serum lbumin s stndrd. Mesurement of microsome enzyme levels The levels of CYP450 nd CYPb5 were mesured s previously described [22]. Western blot ssy liquots (80 µg) of liver microsomes from ech smple were loded onto ech lne of 10% SDS-PGE gel electrophoresis nd electroblotted onto nitrocellulose membrnes (Millipore, Bedford, M). The membrnes were probed with rbbit ntibody ginst mice CYP2E1 (Boster Biologicl Technology Co., Ltd, Wuhn, Chin) t dilution of 1:400 or CYP12 (Snt Cruz Biotechnology, Inc., Cliforni, US) t dilution of 1:500 nd peroxidse-conjugted ffinipure got ntirbbit IgG (Zhongshn Golden Bridge Biotechnology Co., Ltd, Beijing, Chin) ccording to the mnufcturer s constructions. The signls were visulized with DB ssy kit (Zhongshn Golden Bridge Biotechnology Co., Ltd, Beijing, Chin) nd nlyzed with Quntitiy One softwre. Histology nd immunohistochemicl ssy ll specimens embedded in prffin were cut into 4-µm thick seril sections. The specimens were mounted on slides nd deprffinized with grded concentrtions of xylene nd ethnol, nd stined with hemtoxylin nd eosin (HE). For immunohistochemicl stining, ll sections were immersed in 3% H2O2 for 15 min t room temperture to block the endogenous peroxidse ctivity. To stin with CYP2E1 nd CYP12, the sections were predigested with 0.4% proteinse K for 5 min t 37. The sections were then incubted with 10% norml got serum for 15 min to reduce the nonspecific bckground

3 Chen X et l. Effects of TPs on CYP Tble 1 Sequences of oligonucleotide primers Primer Sequence CYP2E1 F 5 -CCCGTTGCCTTGCTTGTCTG-3 CYP2E1 R 5 -CTCTGGCTCCGCCTTC-3 CYP12 F 5 -GTGTTCTGGTGGTCGGC-3 CYP12 R 5 -GCGGGTGCTGCGTCG-3 GPDH F 5 -TGGTGGGTCGGTGTGC-3 GPGH R 5 -GTCTTCTGGGTGGCGTGTG-3 Tble 3 Effect of TP on mortlity induced by prcetmol in mice Group (n = 10) Dose (mg/kg) Deth (n ) Deth rte (%) Protection rte (%) Negtive control Positive control TP Low Medium High Tble 2 Effects of TP on CYP450 nd CYPb5 levels (men ± SD) Group (n = 10) CYP450 (nmol/g) CYPb5 (nmol/g) Negtive control ± b ± b Low dose ± 0.014,b ± 0.004,b Medium dose ± 0.016,b ± High dose ± ± Positive control ± ± P < 0.01 vs negtive control group (NS group); b P < 0.01 vs positive control group (chlormphenicol group). stining nd rected with polyclonl rbbit nti-mouse ntibody (b) t 4 overnight. The working dilution of bs used for exmining mouse CYP2E1 nd CYP12 in the liver ws diluted t 1:100 nd 1:50. fter rinsing with phosphte-buffered sline (ph 7.4), the sections were incubted with biotinylted nti-rbbit immunoglobulin secondry bs t room temperture for 30 min. Horserdish peroxidse lbeled streptomycin-vidin complex ws used to detect the second ntibody. Finlly, the sections were counterstined with hemtoxylin before exmintion under light microscope. The brown or drk brown stined cells were considered positive cells. Reverse trnscription-polymerse chin rection (RT- PCR) ssy Totl RN ws extrcted from liver tissue with the TRIquick regent (Solrbio Science & Technology CO., Ltd, Beijing, Chin). RN purity ws ssessed by the opticl densities t 260 nm nd 280 nm, nd the integrity ws verified by electrophoresis on 1% grose gel contining 0.5 µg/ml of ethidium bromide. Reverse trnscription ws conducted for 30 min t 42 from 500 ng of purified RN in 10 µl of reser ve trnscriptions system rection mixture using MV reverse trnscriptse (TKR, Jpn), followed by 30 cycles of PCR (denturtion t 94 for 30 s, nneling t 56 for 30 s, nd extension t 72 for 1 min). The mplified products were electrophoresed on 1.5% grose gel using 100 bp DN ldder mrkers (TKR, Jpn) s stndrd to determine the moleculr size nd visulized with ethidium bromide stining. Imges of the ethidium bromide-stined grose gel were cquired with Gel Doc EQ system nd quntified using Quntity One softwre. The sequences of oligonucleotide primers re provided in Tble 1. Sttisticl nlysis Dt were clculted seprtely nd expressed s men B CYP2E1/b-ctin 64 kd 43 kd ± SD. Sttisticl significnces were nlyzed by onewy NOV followed by Student Newmn-Keuls test using SPSS Version The difference ws considered significnt t two-tiled. P < 0.05 ws considered sttisticlly significnt. RESULTS CYP2E1 b-ctin Figure 1 Western blot nlysis of protein for CYP2E1 in liver tissue of mice showing representtive bnds of ech group () nd normlized densitometic rtios of CYP2E to β-ctin (B). b P < 0.01 vs control group; d P < 0.01 vs model group. Effect of TP on CYP450 nd CYPb5 levels Two dys fter peritonel injection of TP, the CYP450 nd CYPb5 levels in livers of mice were mesured. The CYP450 nd CYPb5 levels were significntly lower in the high, medium nd low dose TP groups thn in the negtive control group (P < 0.01), indicting tht TP cn reduce the CYP450 nd CYPb5 levels in liver of mice. The CYP450 level ws higher in the medium nd low dose groups thn in the positive control group (P < 0.01); but, it ws not significntly different between the high dose nd positive control groups (P > 0.05). The CYPb5 level ws mrkedly different in the low dose group (P < 0.01); but it, ws not significntly different in the high nd medium dose groups (P > 0.05) compred the positive control group, indicting tht TP cn suppress both CYP450 nd CYPb5 in mice in dose-dependent mnner (Tble 2).

4 1832 ISSN CN /R World J Gstroenterol pril 21, 2009 Volume 15 Number 15 B C D E Figure 2 Chnges of HE stining in liver tissue of mice 24 h fter prcetmol dministrtion ( 100) in control group with norml centrl lobulr region (), model group with some centrl lobulr heptocyte necrosis nd microvesiculr ftty chnge (B), low TP dose group (C), medium TP dose group (D) nd high TP dose group (E). The pthologicl chnge of liver ws much milder in different TP dose groups thn in model group. Effect of TP on mortlity of prcetmol-treted mice We exmined the effect of TP on mortlity of prcetmol-treted mice. The mortlity rte of mice in the negtive control group, low, medium nd high dose TP groups ws 90%, 70%, 40% nd 20%, respectively, indicting tht TP cn significntly reduce the mortlity of prcetmol-treted mice. The effect of high dose TP on mortlity of prcetmol-treted mice ws not different from tht of mice in the positive control group (Tble 3). Western blot nlysis Western blot showed strong positive CYP2E1 signls in the control nd model groups. The expression of CYP2E1 in the low dose group ws not significntly different from tht in the control nd model groups. However, TP significntly decresed the CYP2E1 expression level in the medium nd high dose groups in dose-dependent mnner (Figure 1). The expression of CYP12 nd CYP2E1 protein ws similr in the liver microsomes (Dt not shown). Histology nlysis Liver tissues from the control group showed norml lobulr rchitecture with centrl veins nd rditing heptic cords (Figure 2). However, liver tissues from the model group showed formtion of more fibrous tissues extending into the heptic lobules, leding to their complete seprtion. lrge number of inflmmtory cells infiltrted the intrlobulr nd interlobulr regions. The liver structure ws in disorder nd there were more necrotic nd ftty degenerted liver cells thn in the controls (Figure 2B). In the three TP tretment groups, however, heptocyte degenertion, necrosis nd infiltrtion of inflmmtory cells were ll pprently meliorted (Figure 2C-E). Compred with the model group, the liver condition in TP tretment groups ws significntly improved in dose-dependent mnner. Immunohistochemicl nlysis of liver CYP2E1 nd CYP12 The expression of CYP2E1 showed deep brown immunostining. No difference ws found in the expression of CYP2E1 between the control nd model groups. However, in medium nd high dose groups, TP significntly suppressed the CYP2E1 expression in dose-dependent mnner (Figure 3). The expression level of CYP12 (showing deep brown immunostining) ws mrkedly lower in the low, medium nd high dose TP groups thn in the control nd model groups (Figure 3B). Expression levels of CYP2E1 nd CYP12 mrn The expression levels of CYP2E1, CYP12 nd GPDH cdns in the mouse liver were ssyed by RT-PCR (Figure 4), nd the rtio of CYP2E1 nd CYP12/ GPDH cdns ws clculted (Figure 4B). TP significntly inhibited the CYP2E1 mrn expression in medium nd high dose groups (P < 0.01), compred to the control nd model groups (P > 0.05). The expression of CYP12 mrn ws reduced in ll TP groups compred with the control nd model groups (P < 0.05), indicting tht TP cn suppress the expression of CYP2E1 nd CYP12 mrn in dosedependent mnner. DISCUSSION Heptic drug metbolizing enzyme is clled mixedfunction oxidse or monooxygense contining mny enzymes including phse Ⅰ enzymes such s

5 Chen X et l. Effects of TPs on CYP b c d e B b c d e Figure 3 Immunohistochemicl stining showing expression of CYP2E1 () nd CYP12 (B) in liver tissue of mice 24 h fter prcetmol dministrtion ( 400) in control group (), model group (b), low TP dose group (c), medium TP dose group (d) nd high TP dose group (e). The brown or drk brown stined cells were considered positive. CYP450 nd CYPb5, etc, nd plys prominent role in the metbolism of mny phrmceuticl gents nd ctivtion or dectivtion of potentil crcinogens. cquiring metbolic informtion nd determining the effect of chemicls on heptic drug metbolizing enzymes re importnt in developing cliniclly sfe nd efficient medictions [23]. TP hve vrious phrmcologicl effects on cncer, crdiovsculr nd degenertive diseses nd hve other bioctive properties [1-4]. In this experiment, TP significntly suppressed the CYP450 nd CYPb5 levels in liver of mice, suggesting tht TP cn restrin the ctivity of drug metbolizing enzymes. In the present study, 100, 200 nd 400 mg/kg TP could reduce the mortlity of mice fter tretment with prcetmol (Tble 2). The immunohistochemicl study showed tht TP mrkedly llevited prcetmol- induced heptotoxicity, thus improving ltertions in liver tissue pthology. Especilly mrked heptoprotective effects of TP were observed in the high dose TP group with its diffuse hemorrhge nd necrosis chnged into focl hemorrhge nd necrosis (Figure 2) compred to the model group tht received single dose of 500 mg/kg prcetmol, indicting tht TP cn protect liver of mice ginst prcetmol injury, which is consistent with the reported dt [24]. It ws reported tht mny mechnisms re involved in prcetmol heptotoxicity [25], showing tht the toxicity is medited by CYP450 metbolism of prcetmol to NPQI which covlently binds to criticl proteins leding to inctivtion of these proteins, especilly fter GSH depletion. CYP2E1 is usully ssumed to be the

6 1834 ISSN CN /R World J Gstroenterol pril 21, 2009 Volume 15 Number 15 CYP2E1 effects of TP re ssocited with the expression of CYP2E1 nd CYP12 gene nd protein. B CYP/GPDH CYP2E1 Mrker GPDH CYP12 GPDH,b CYP12 Figure 4 RT-PCR nlysis of mrn for CYP2E1 nd CYP12 expression in liver tissue of mice showing representtive bnds of ech group () nd normlized densitometic rtio of CYP2E nd CYP12 to GPDH (B). P < 0.05 vs model group; b P < 0.01 vs control group; d P < 0.01 vs model group. most ctive CYP450 in ctlyzing the metbolism of prcetmol to heptotoxic NPQI [15,18]. Studies with knockout mice showed tht CYP2E1 nd CYP12 ply n importnt role in the metbolism of prcetmol [26,27]. To confirm whether the protective effects of TP on liver re correlted with the inhibition of CYP2E1 nd CYP12, we investigted the effect of TP on the expression of CYP2E1 nd CYP12 mrn nd protein. In the present study, 200 mg/kg nd 400 mg/kg TP significntly down-regulted the CYP2E1 expression either t gene level (Figure 4) or t protein level (Figures 1 nd 3) s shown by RT-PCR, Western blot nd immunohistochemicstry. The effects of TP on the expression of CYP12 nd CYP2E1 gene nd protein were similr. It ws reported tht the CYP12 level in incresed in rts fter drinking 2% solution of green or blck te [28]. Cffeine, component of te, is responsible for the induction of CYP12 [29]. In our study, however, different doses of TP (100, 200 nd 400 mg/kg) significntly suppressed the expression of CYP12 protein nd mrn, prtly becuse TP used in our experiment did not contin cffeine. The bove findings indicte tht the dose-dependent liver-protective effects of TP re likely relted to the suppression of CYP2E1 nd CYP12 expression, which reduces NPQI ssocited with the prcetmol heptotoxicity, nd effectively protects liver ginst prcetmol heptotoxicity. In conclusion, TP restrin the ctivity of drug metbolizing enzymes, CYP450 nd CYPb5, nd protect liver ginst prcetmol-induced injury. The protective CKNOWLEDGMENTS The uthors thnk Professor Zhong-Gun Yn (Center for Moleculr Medicine, Krolinsk Institute, Stockholm, Sweden) nd Jing-Lin Fn (Deprtment of Pthology, University of Ymnshi, Jpn) for their technicl ssistnce. COMMENTS Bckground Prcetmol is one of the most widely used heptotoxic drugs, which is sfe t therpeutic doses, but cuses liver filure t overdoses. High doses of prcetmol limit the bility of glutthione (GSH) to detoxify N-cetyl-pbenzoquinonimine (NPQI), nd results in consumption of liver GSH stores. There re three fmilies of cytochrome P450 (CYP450) isoforms, CYP1, CYP2, nd CYP3, which re minly involved in the biotrnsformtion of xenobiotics. It hs been shown tht CYP2E1, CYP12 nd intrcellulr GSH ply n importnt role in prcetmol-induced heptotoxicity. Te polyphenols (TP) re lrge nd diverse clss of compounds extrcted from te. Liver is the min orgn responsible for the metbolism of TP. Some work hs been done in the field of TP modultion or interction with drug metbolizing enzymes. However, until now, no one could give cler explntion for this. Reserch frontiers TP re lrge nd diverse clss of compounds extrcted from te. Liver is the min orgn responsible for the metbolism of TP. Some work hs been done in the field of TP modultion or interction with drug metbolizing enzymes. However, until now, no one could give cler explntion for this. The reserch hotspot is to investigte the effects of TP on CYP450 nd CYPb5 expression in livers of mle mice. Whether the protective effect of TP on prcetmol-induced heptotoxicity in mice is relted with their effect on CYP450 ws studied. Innovtions nd brekthroughs The protective effect of TP on prcetmol-induced heptotoxicity ws found to be relted with their effect on CYP450. pplictions TP possess potentil heptoprotective properties, which re closely relted with their suppressive effect on CYP450 expression. Terminology Prcetmol-induced heptotoxicity: When norml dose is used, prcetmol is metbolized extensively by conjugtion with sulphte nd glucuronic cid. smll frction of prcetmol is subjected to oxidtion rections ctlyzed by CYP450 enzymes in the liver, resulting in genertion of NPQI, highly electrophilic metbolite tht triggers the ensuing liver dmge. Peer review This is good descriptive study in which the uthors gve cler explntion of the effect of TP on the expression of CYP450, CYP2E1 nd CYP12 in liver of mice t both protein nd mrn levels. The interesting results suggest tht TP cn significntly ttenute prcetmol-induced heptic injury, which is closely relted with their suppressive effect on CYP450 expression. 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