Effect of Conjugated Linoleic Acid (CLA) in Rats Subjected to Damage Liver Induced by Carbon Tetrachloride

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1 32 Journl of Modern Medicinl Chemistry, 2014, 2, Effect of Conjugted Linoleic Acid () in Rts Sujected to Dmge Liver Induced y Cron Tetrchloride Eline Bonifácio Teixeir de Crvlho 1,*, Illn Louise Pereir de Melo 1, An Mr de Oliveir e Silv 2, Dlv Assunção Portri Mncini 3 nd Jorge Mncini-Filho 1 1 Lortory of Lipids, Deprtment of Food nd Experimentl Nutrition, Fculty of Phrmceuticl Sciences, University of São Pulo; 2 Center for Nutrition, University of Sergipe; 3 Butntn Institute, São Pulo, Brzil Astrct: The conjugted linoleic cid () consists in group of positionl nd geometricl isomers of octdecdienoic cid (18:2-6) in which the two doule onds re conjugted nd my hve cis or trns configurtions. s hve een the suject of extensive investigtions for their vrious iologicl ctivities, including ntitherosclerotic, nticrcinogenic nd ntioxidtive ctivities. The im of this study ws to evlute the ntioxidnt ctivity nd heptoprotective properties of in rts sujected to liver injury induced y cron tetrchloride (CCl 4). Eighteen mle Wistr rts were divided into three groups: control; CCl 4 nd. The ws dministrted dily y gvge for 21 dys, nd then sujected to liver injury induced y CCl 4. The serum nd liver were collected nd nlyzed. s supplementtion tke to ttenution of the liver dmge in treted nimls, since the reduction in the levels of thiorituric cid rective sustnces (TBARS) in the liver suggests heptoprotective ctivity of these ftty cids. The levels of ctlse (CAT) nd glutthione reductse (GR) nd reduced glutthione (GSH) levels in liver tissue s result of reduced oxidtive dmge induced y CCl 4, nd incresed fter tretment with, suggesting ntioxidnt cpcity of s. Moreover the nlysis of reverse trnscription / polymerse chin rection (RT / PCR) showed incresed expression of the CAT gene, suggesting inducing effects on this enzyme. These results suggest tht s cn e used s djuncts to ttenute heptic dmge. Keywords: Conjugted linoleic cid, heptoprotective, cron tetrchloride, ntioxidnt ctivity, rts. INTRODUCTION Liver injury often leds to poptosis nd necrosis of heptocytes, regrdless of its cuse. They my e usully cused y virl infections, utoimmune diseses, ischemi, nd xenoiotics, including: drugs, toxins or lcohol. The model of cute liver injury induced y cron tetrchloride (CCl 4 ) is widely used to investigte the mechnisms of liver injury nd regenertion [1]. Liver nd lipid ccumultion in the liver occur within 24 hours of orl dministrtion only. The heptocyte necrosis, dmge to lysosomes nd mitochondri of liver cells re induced y dministrtion of CCl 4 [2]. Even during the cute phse of liver dmge, firosis does not occur, there is the ctivtion of heptic stellte cells which re the key firogenic cells of the liver injury [3]. Epidemiologicl studies hve consistently shown tht nutrition is strongly ssocited with reduced risk of developing chronic diseses such s cncer nd crdiovsculr disese, nd lso in the pthogenesis of cute nd chronic liver disese [3]. In this context ftty cids my hve protective effects on the liver injury induced y endotoxin. Reserch reported protective *Address correspondence to this uthor t the Lortory of Lipids, Deprtment of Food nd Experimentl Nutrition, Fculty of Phrmceuticl Sciences, University of São Pulo, Av.Prof.Lineu Prestes, Bloco 14, Butntn, cep: So Pulo, SP, Brzil; Tel: ; Fx: (11) ; E-mil: elineonifcio@yhoo.com.r effects of olive oil on liver injury cused y CCl 4 dministrtion [4]. Olive oil prevents liver injury induced y CCl 4 nd liver firosis, since the oleic cid inhiited the ctivtion of heptic stellte cells. Thus, the ftty cids my ply n importnt role in liver firosis [3]. Polyunsturted ftty cids (PUFAs) such s linoleic cid, -linolenic cid nd rchidonic cid re importnt for the mintennce of iologicl functions in mmmls. In clinicl studies with humns nd nimls is oserved tht other polyunsturted ftty cids omeg fmily, such s eicospentenoic cid nd docoshexenoic cid re correlted with the reduction in the risk of developing cncer nd crdiovsculr disese [5]. Some studies hve shown tht oils rich in polyunsturted ftty cids hve heptoprotective effect in rts nd mice sujected to liver dmge y CCl 4, y elevting the ctivity of endogenous ntioxidnt enzymes superoxide dismutse, ctlse nd glutthione peroxidse (SOD, CAT nd GPx, respectively) nd reduce lipid peroxidtion [6, 7]. Recently, studies re focused on conjugted linoleic cid (), which is the generl term used to descrie group of positionl nd geometric isomers of linoleic ftty cid with conjugted doule onds [2, 8]. The interest in the possile helth enefits is incresing, since they hve een shown to hve ntitumor ctivity, immunomodultory ctivity nd lipid lowering of E-ISSN: / Synergy Pulishers

2 Effect of Conjugted Linoleic Acid () in Rts Sujected Journl of Modern Medicinl Chemistry, 2014 Vol. 2, No serum [9]. The ftty cid conjugtes re known to hve phrmcologicl ctivities relevnt for the prevention nd tretment of therosclerosis, oesity, hypertension nd cncer [10]. In vivo study showed tht diet enriched with 1%, significntly inhiited the increse of collgen fiers in the livers of nimls, indicting heptoprotective effects of s on liver firosis induced y CCl 4 [11]. The im of this study ws to evlute the protective effect of the isomers of conjugted linoleic cid (9cis, 11trns nd 10trns, 12cis) in nimls sujected to cute liver disese y the dministrtion of CCl 4. MATERIAL AND METHODS Smple Smples of (Free Ft Acid - Conjugted Linoleic Acid) hve een provided y the compny Cognis, the product ws Tonlin FFA 80, with vlidity of 12 months, consisting of free ftty cids of which pproximtely 80% conjugted linoleic cids. These smples of re derived from sfflower oil nd the product comprises pool (50/50) of the two ctive isomers: 18:2 cis 9, trns-11 nd 18:2 trns-10, cis-12. smples were frctionted in mer ottles previously clened in volumes of 20mL nd 50mL, seled, identified nd stored in freezer t -20 C. Dily ottles with smple mounts to e used were removed in dvnce to thw t room temperture. Identifiction of Isomers For the preprtion of methyl esters of ftty cids present in the smples ws conducted method of esterifiction Alkline s descried y Christie, Séédio nd Junéd [12]. The lipid frction (50 mg oil) ws sujected to esterifiction, the smple ws diluted in toluene, sodium methoxide methnolic 0.5 M (NOCH 3 ) nd heted t 50 C for 10 minutes. After cooling glcil cetic cid wter nd hexne ws dded. After phse seprtion the lower phse ws wshed with hexne, vortexed for 20 seconds nd llowed to stnd for phse seprtion. The upper phse ws recovered nd filtered. The solvent ws evported under N 2, the methyl esters were resuspended with 2 ml of hexne nd the solution ws trnsferred to the vil nd injected into the chromtogrph. Anlysis of Ftty Acid Compositions The ftty cid (FA) compositions of the oil nd heptic tissue, were determined y gs chromtogrphy using Shimdzu GC chromtogrph model 2010 with flme ioniztion detector nd fused silic cpillry column (100 m; 0.25 mm de internl dimeter; 0,2 μm thick film / SP-2560) in column temperture ws: isotherml t 162 C for 32 minutes wrming of 1.4 C/minute up to 195 C, kept t this temperture for 15 minutes. Susequently it ws heted t 2 C/minute up to 235 C nd this temperture ws mintined for 5 minutes. The tempertures of the injector nd detector were 250 C nd 250 C, respectively [13]. Helium (1 ml / min) ws used s the crrier gs nd the split rtio of the smple injector equl to 1/50. The identifiction of ftty cids ws sed on comprison with the retention times for the mixture of methyl esters of stndrds C4- C24 (Sigm 18919). Animls This study ws pproved y the Ethics Committee on Animl Experiments of the Fculty of Phrmceuticl Sciences, University of São Pulo, Brzil. To evlute the effects of FFA- in vivo were used 18 rts, mle linege "Wistr", weighing etween 50 nd 70g, rered in Production nd Experimenttion of the Fculty of Phrmceuticl Sciences nd the Institute Chemistry / USP. The nimls hd free ccess to wter nd using lnced commercil diet (Nuvil CR-1) decontminted y irrdition t 12KGy offered d liitum. Supplementl FFA- were performed y gvge for 21 dys. The nimls were housed in polypropylene cges (six rts ech) in n environment with controlled lighting (12 h light / drk) t 25 C nd 60% humidity throughout the experimentl period. We dopted n djustment period of seven dys, nd fter this period, the nimls were divided into three groups. Tle 1: Ftty Acid Composition of Oil FAT Ftty Acid Composition (%) 18:1 18:2 c9, t11 C18:2 t10,c ± ± ± 0.89

3 34 Journl of Modern Medicinl Chemistry, 2014 Vol. 2, No. 1 Crvlho et l. Scheme of distriution of nimls: : treted dily with wter, orlly (po). CCl 4 : : dily treted with wter (po) followed y tretment with CCl 4. treted dily with 2% (compred to the verge dily consumption of diet, po), followed y tretment with CCl 4. The heptic dmge ws induced in the nimls on dy 21 of the experiment, y dministrtion of single dose of 3 ml of CCl 4 (1:1 dilution in olive oil) sucutneously in groups CCl 4 nd. After 24 hours of induction, the nimls were nesthetized with n intrperitonel injection of mixture of ketmine (90 mg / kg) nd xylzine (10 mg / kg), the recommended dose Vivrium Production nd Experimenttion of the Fculty of Phrmceuticl Sciences nd the Institute Chemistry / USP, nd euthnized. Blood smples were collected y dominl rtery for determintion of liver function enzymes - lnine (ALT) nd sprtte (AST) minotrnsferses nd nlysis of iochemicl prmeters - triglycerides, totl cholesterol nd HDL cholesterol using commercil kit Ltest nd to nlyze the levels of thiorituric cid rective sustnces (TBARS). The liver ws perfused through the injection of 0.9% NCl into the portl vein, collected nd homogenized in potssium phosphte uffer 0.1M (ph 7.0) nd the homogente ws centrifuged. The superntnt portion of the homogente ws intended for the testing of TBARS nd ntioxidnt enzymes. The feed intke nd niml weight were monitored throughout the test, in order to check the weight gin nd feed efficiency rtio (CEA). Anlysis of TBARS TBARS were mesured in serum nd liver homogentes the method ws descried y Ohkw et l. [14]. The TBARS concentrtions were clculted using stndrd curve for 1,1,3,3 -(TEP) tetrethoxypropne (10-4 mol/l) nd were expressed s mol of mlondildehyde (MDA) per milligrm of protein. Extrction nd Esterifiction of Heptic Lipids Lipids from the heptic tissue were otined y the Folch method [15], using the originl extrtion rtio of 20 prts 2:1 chloroform/metnol to 1 prt tissue. A wek slt solution (0,88% KCL) is then dded to chieve finl rtion of 8:4:3 chloroform/metnol/wter fter including the wter contined in the tissue. The ftty cid methyl ester content nd nlysis of ftty cid compositions ws determined ccording to the methods lredy descried previously. Determining Superoxide Dismutse (SOD) The cytoplsmic SOD ctivity ws determined ccording method of Mccord nd Fridovich [16] using rection contining cytochrome C (100 mm), xnthine (500 mm), ethylenediminetetrcetic cid (1 mm) nd KCN (200 mm) nd potssium phosphte uffer (0.05 M - ph 7.8). The results were expressed s units per milligrm of protein. One unit of SOD ctivity ws defined s the mount of enzyme required to inhiit the rection rte y 50% t 25 C nd ph 7.8. Determintion of Activity Ctlse (CAT) The ctlse (CAT) ctivity in the liver homogente ws mesured spectrophotometriclly y clculting the rte of degrdtion of H 2 O 2, the sustrte of the enzyme t 37 C nd ph 8.0. The results were expressed s mmol of hydrogen peroxide decomposed per minute per milligrm of protein, ccording to the method of Beutler [17]. Reduced Glutthione (GSH) Assy The intrcelulr GSH contentes in the liver homogente ws quntifiied spectrophotometriclly ccording to the method of Tietze [18]. The sme is sed on the rection of reduced glutthione (GSH) present in homogentes with sustnce dithiois nitroenzoic cid (DTNB) to produce colored compound tht sors t 412 nm spectrophotometriclly. The concentrtion of GSH ws quntified using stndrd curve of GSH (50 to 800 mm). Determintion of Glutthione Peroxidse (GPX) The ctivity GPx in the liver homogente ws determined s descried y Sies et l. [19]. The results were expressed s units per milligrm of protein. The unit of enzyme ctivity ws defined s the mount of enzyme required to oxidize one mol of NADPH per minute t 30 C, ph 7.0. Determintion of Glutthione Reductse (GR) The ctivity GR in the liver homogente ws determined spectrophotometriclly ccording to the method of Sies et l. [19]. We dded 50 μl of smple to solution of 50 mm GSSG, 0.5 M EDTA, in 0.1 M

4 Effect of Conjugted Linoleic Acid () in Rts Sujected Journl of Modern Medicinl Chemistry, 2014 Vol. 2, No phosphte uffer (ph 7.0) nd 4mM NADPH ws dded just efore the enzymtic determintion s the strting regent. The ssy ws 6.22 nm run t 340 nm t 37 C. GR ctivity ws estimted using NADPH extinction coefficient of 6.2 mm -1.cm -1 nd expressed s unit/mg of protein. Anlysis of Protein The determintion of the content of proteins in tissues ws performed ccording to the colorimetric method [20]. The mount of protein in the smple ws clculted from stndrd curve of ovine serum lumin nd results expressed in mg of protein per ml. Reverse Trnscription / Polymerse Chin Rection (RT / PCR) RNA Extrction RNA extrction ws crried out with mixture of 100 mg of rt liver nd 1000 μl of Trizol regent (Invitrogen, New York, New York). Following y ddition of 200μL of chloroform (Merck, Drmstdt, Hessen, Germny) y vortexing for 15 seconds, incuted t room temperture for 5 minutes nd centrifugtion t 12,000 x g nd 4 C for 15 minutes. The superntnt (400 μl) ws collected, voiding the interphse, nd mixed with 500 μl of isopropnol y vortexing for 5 seconds. Then it ws centrifuged t 12,000 x g t 4 C for 5 minutes nd the superntnt ws discrded. The resulting pellet ws wshed with 1 ml of ethnol (75%), vortex gently shken nd centrifuged t 7,500 x g nd 4 C for 10 minutes. The superntnt ws gin discrded. The pellet ws resuspended in 20 μl of RNse-free distilled wter, incuted t 50 C for 10 minutes, nd stored t - 70 C. Reverse Trnscription Five μl of RNA ws dded to 1.0 μlof primer (Cu / Zn SOD or CAT) 1.0 μldntp (10 mm), nd 4,0 μlof sterile distilled wter. The rection ws initited y heting step t 65 C for 5 minutes nd then quickly cooled on ice. After ddition of 4.0 μlof 5X uffer First- Strnd (Invitrogen), 2.0 μlof DTT (0.1 M, Invitrogen), nd 1.0 μlrnaseoutrionuclese inhiitor (Invitrogen), the mixture ws incuted t 37 C for 2 minutes. Therefter, 1.0 μlm-mlv reverse trnscriptse (200 U / μl, Invitrogen) ws dded nd the mixture ws incuted t 37 C for 50 minutes. The rection ws stopped y heting step t 70 C for 15 minutes. The PCR product (cdna) ws stored t -70 C. PCR Amplifiction The mplifiction rection ws performed with five microliters of mixture contining cdna 5.0 μltris (hydroxymethylminomethn) t 20 mm, ph 8.4, 1.5 μl MgCl 2 (50 mm), 1.0 μl dntp (10 mm), 1.0 μl of primer (SOD or CAT) nd 0.4 μl to TqDNApolimerse (5 U/uL). After initil denturtion t 94 C for 3 minutes in therml cycler (Bio-Rd, Hercules, Cliforni, USA), 35 cycles (94 C for 45 seconds, 55 C for 30 seconds, 72 C for 1.3 minutes nd 72 C for 10 minutes) were conducted. Finlly, the mix ws cooled to 4 C. The PCR mplifiction products were nlyzed y electrophoresis on n grose gel 2,0% (Sigm, St. Louis, Missouri, USA) t 60 V. The gel ws stined with 0.1 μl / ml Syr sfe (Invitrogen), visulized on tle fluorescence (Viler-Lourmt, Mrne-l-Vllée, Frnce), nd photogrphed with digitl cmer. The primers CAT-262p (C to T) nd SOD-242 p (C to T) were genotyped using the following sequences (Promeg, Mdison, AL, USA): CAT 1-5'-GCG AAT GGA GTG GAG GCA TAC - 3 ' CAT 2-5'-GAG TGA CGT TGT CTT CAC CAT TAG TG - 3 ' Cu / Zn SOD 1-5'-TCT AAG CAT GGC AAA GGT CC - 3 ' Cu / Zn SOD 2-5 '- CAG GGC TTAGCA CAGCAG AT - 3' Sttisticl Anlysis The experiments were performed in triplicte nd results were expressed s men nd stndrd devition, p vlue <0.05 ws considered sttisticlly significnt. It ws used nlysis of vrince (ANOVA) followed y Tukey's comprison test using Prism 5.0 softwre (GrphPd). RESULTS DISCUSSION The results of consumption, food intke, weight gin nd CEA re shown in Tle 2. The supplementtion does not significntly lter food intke, weight gin nd niml CEA. This pttern ws oserved [21] which investigted the effect of supplementtion nd phytosterols lone or in comintion, on the process of uto-oxidtion of lipids nd ctivities of ntioxidnt enzymes in helthy Sprgue-Dwley rts for nine weeks. Concerning the feed intke nd weight gin of the nimls during the period of supplementtion, it cn e sid tht, lthough

5 36 Journl of Modern Medicinl Chemistry, 2014 Vol. 2, No. 1 Crvlho et l. Tle 2: Food Intke, Weight Gin nd Feed Efficiency Rtio (CEA) in Animls Supplemented with for 21 Dys nd Undergoing Liver Injury y CCl 4 Groups Smple (ml/d) Food Intke (g/ d) Weight Gin (g) CEA ± ± CCl ± ± ± ± *Dt re presented s men nd stndrd devition (n = 6). there were slight differences mong the groups, the development of the nimls were norml. The levels of totl cholesterol nd HDL-cholesterol in the present study, it ws oserved tht there ws no sttisticl difference etween groups (Figures 1 nd 1). As for the levels of triglycerides, there ws n increse in levels for the group supplemented with (Figure 1c). Other study [22] lso oserved n increse in triglyceride levels fter supplementtion with s, esides the increse in dominl circumference of nimls nd induced crdic oxidtive stress. However, other reserch hs shown tht supplementtion with s significntly reduces ody weight gin [23], plsm levels of free ftty cids nd triglycerides [24], nd totl cholesterol [24, 25]. The results otined in this study regrding serum levels of AST (Figure 2) nd ALT (Figure 2), did not differ significntly (p> 0.05) of the results found in controls. In contrst, other study [2] demonstrted tht the levels of AST nd ALT were significntly higher fter dministrtion of CCl 4, nd supplementtion ws not effective in reducing these levels of AST nd ALT induced y CCl 4. It is possile in this study were lredy in the liver regenertion process nd the levels of mrkers of liver dmge, such s ALT nd AST detectle in the loodstrem were lredy normlized. Reserch demonstrted tht the cute liver injury induced y CCl 4 reversile nd is temporrily ccompnied y significnt inflmmtion nd necrosis of heptocytes, followed y complete restortion with heptocyte regenertion in the delyed phse of injury [26]. These uthors report tht the genes responsile for representtive liver function such s ctlse, lumin, nd PPAR- were significntly dysregulted in 24 h nd 48 h were restored fter dministrtion of CCl 4. The proposed mechnism of heptotoxicity of CCl 4 involves the ioctivtion of CCl 4 to give rective free rdicls. Rdicls originting cuse peroxidtion of ftty cids in memrne phospholipids, resulting in lipid peroxide rdicls, lipid hydroperoxides nd other products, cting s ctive oxidizing gents. When the liver is the toxic condition with excess trichloromethyl rdicl, the ctivity of the enzyme xnthine oxidse, which produces superoxide nion nd hydrogen peroxide, increses with the progression of cell injury. Therefore, lipid peroxidtion plys key role in liver cused y CCl 4 [27, 28]. The iochemicl mechnisms involved in the development of heptotoxicity hve een investigted mg cholesterol/dl mg cholesterol HDL/dL mg triglycerides/dl () () (c) Figure 1: Levels of totl cholesterol (), HDL-cholesterol (), nd triglycerides (c) in the serum of rts. Groups (n = 6): control, CCl 4 nd. Different letters indicte significnt differences (p <0,05).

6 Effect of Conjugted Linoleic Acid () in Rts Sujected Journl of Modern Medicinl Chemistry, 2014 Vol. 2, No ALT (U/mL) AST (U/mL) () () Figure 2: Plsm levels of lnine minotrnsferse () nd sprtte minotrnsferse () in rts. Groups (n = 6) control, CCl 4 nd. nd mlondildehyde (MDA) is widely used s mrker of lipid peroxidtion [6]. Liver injury induced y CCl 4, comes significnt increse of pproximtely 270% nd 188% in the liver content of mlondildehyde nd hydroxyproline, respectively, nd supplementtion with olive oil, significntly reduced heptic levels of mlondildehyde nd hydroxyproline to 48 nd 74% respectively [7]. Another study lso showed tht lmond oil significntly inhiited the formtion of MDA in the liver during the cute dmge cused y dministrtion of CCl 4 [6]. The TBARS re produced s result of lipid peroxidtion. In this study we found tht serum showed no sttisticl difference (p> 0.05) etween groups. As for liver tissue, there ws reduction in the rte of lipid peroxidtion in nimls treted with 2% s, when compred to CCl 4, illustrted in Figure 3. This result indictes tht s hve the ility to protect heptic tissue dmge cused y ltered redox stte y the dministrtion of CCl 4. In this study the incorportion of isomers on heptic tissue ws evluted. The results re shown in Tle 3. The isomers 18:2 cis-9, trns-11 nd 18:2 trns-10, cis-12, were incorported in the liver of nimls supplemented with, the incorportion ws 2.7%. The rective oxygen species (ROS), such s nionic superoxide nd hydrogen peroxide (H 2 O 2 ), re produced during norml cellulr eroic metolism. The min components of the ntioxidnt system in mmmlin cells is composed of three enzymes, Serum TBARS (μmol/mg protein) () () Figure 3: TBARS levels in serum () nd liver tissue () in rts Groups (n = 6). control, CCl 4 nd. Different letters indicte significnt differences (p <0,05).

7 38 Journl of Modern Medicinl Chemistry, 2014 Vol. 2, No. 1 Crvlho et l. Tle 3: Ftty Acid Composition in Liver Tissue FA Composition (%) CCl 4 C 13:0 4.5 ± ± ± 0,1 C 16: ± ± ± 0,4 C 18: ± ± ± 0,9 C 18:1t 6.4 ± ± ± 1,4 C 18:1c 2.5 ± ± ± 0,1 C 18:2c 24.3 ± ± ± 1,0 9c11t 1.7 ± t12c 1.0 ± 0.2 C 20:4 n ± ± ± 2.0 C 22:6 n3 3.5 ± ± ± 0.4 Sturted 33.9 ± ± ± 0.6 Monounsturted 2.5 ± ± ± 0.1 Polyunsturted 52.6 ± ± ± 0.8 Trns 6.4 ± ± ± 1.4 Different letters indicte significnt differences (p <0,05). FA Ftty Acid. nmely superoxide dismutse (SOD), ctlse (CAT) nd glutthione peroxidse (GPx). These enzymes constitute mutully supportive tem of defense ginst ROS. The decrese in SOD ctivity in the liver in rts treted with CCl 4 my e due to incresed lipid peroxidtion or inctivtion of ntioxidnt enzymes. The decrese in GPx ctivity cused y CCl 4 toxicity my e due to decresed vilility of GSH, which results in incresed lipid peroxidtion. The reduction in the level of SOD, CAT nd GPx re consequences of liver dmge in rts following dministrtion of CCl 4 [6]. The ctivities of enzymtic ntioxidnts, ssessed in this study, re presented in Figures 4, 5 nd 6. In liver tissue, there ws significnt reduction in ctlse ctivity (p <0.05), CCl 4 group (Figure 4) compred to the control group. Supplementtion with incresed the ctivity of CAT ctivity vlues close to the control group. There ws no significnt difference in SOD ctivity etween the groups (Figure 4). The evlution y RT / PCR shows the chnges in gene expression of ctlse nd superoxide dismutse etween groups evluted (Figure 5). The levels of CAT incresed in the liver tissues fter repeted dministrtion of, this figure shows chnges in the expression of ctlse evluted y RT / PCR gene. The nds representing the expression of the SOD gene show no chnge, y reproducing the results of Unit.(mg/protein) Unit.(mg/protein) () () Figure 4: Activity of ctlse (CAT - ) nd superoxide dismutse (SOD - ) in rt liver. Groups (n = 6) control, CCl 4 nd. Different letters indicte significnt differences (p <0,05).

8 Effect of Conjugted Linoleic Acid () in Rts Sujected Journl of Modern Medicinl Chemistry, 2014 Vol. 2, No CAT SOD Ldder Figure 5: Gene expression (RT-PCR) of the ctivity of ctlse (CAT) nd superoxide dismutse (SOD) in liver tissue of rts. Groups (n = 6): control (1), CCl 4(2), (3). ntioxidnt ctivity for this enzyme. It is possile tht for the CAT, the CCl4 increse the expression of this enzyme nd tht there is physicl rrier, or my e eing used s sustrte for the tetrchloride or cting s n ntioxidnt nd thus would remin ville for more CAT enzyme ctivity s comproved in the Figure 4. Regrding the group of glutthione, one cn oserve significnt increse (p <0.05) reduced glutthione (GSH), the -supplemented group, reching levels similr to the control group (Figure 6). It is cler, lso slight increse in the ctivities of GR to the group treted with (Figure 6) confirming the effectiveness of the ntioxidnt cpcity of the. The dministrtion of CCl 4 did not interfere in the ctivity of GPx in the control group, on the other hnd, the ctivity of GPx in the group supplemented with ws lower thn the control group, lthough not presenting difference from CCl 4 group, results shown in Figure 6c. Reserch shows tht dministrtion of oils with clims ntioxidnt properties reduce the levels of mrkers of lipid peroxidtion, nd provide incresed ntioxidnt ctivity, ut ecuse they re olive oil [7], lmond oil [6], nd pumpkin seed oil [29], the effects cn e ttriuted to the presence of unsturted ftty cids, phenolic compounds nd tocopherols. Unit.(mg/protein) () Unit.(mg/protein) Unit.(mg/protein) () (c) Figure 6: Vlues of reduced glutthione (GSH - ), nd the ctivity of glutthione reductse (GR - ) nd glutthione peroxidse (GPx - c) in rt liver. Groups (n = 6): control, CCl 4 nd. Different letters indicte significnt differences (p <0,05).

9 40 Journl of Modern Medicinl Chemistry, 2014 Vol. 2, No. 1 Crvlho et l. Oxidtive stress plys criticl role in the pthophysiology of severl liver diseses, nd mny complictions of these diseses re medited y oxidtive stress, oxidtive stress relted meditors, nd inflmmtion. Oxidtive stress in the liver is induced y numerous systemic diseses such s hypertension, dietes mellitus, nd hypercholesterolemi; y gents such s ntiiotics, chemotherpeutics, nd rdio contrsts; nd environmentl toxins, occuptionl chemicls, smoking, s well s lcohol consumption [29]. hs different isomers in respect to position nd configurtion of doule onds. Different isomers hve different in vitro nd in vivo nti-oxidtive nd ntiinflmmtory effects tht lredy hve een studied extensively [26, 30-32]. Some reserchers report the ntioxidnt effect of the AGC s possile explntion of the eneficil helth effects [30, 33], However few studies hve evluted the ntioxidnt ctivity of these components in vitro. The conjugted ftty cids re lipidic components, which mke the ppliction of very limited testing due to its lipossolule. Fced with this feture, ssessment is confined to test scn DPPH rdicl (2,2-diphenyl picrylhydrzyl) in determining the ntioxidnt cpcity of conjugted linoleic cid (s) isomers [31]. These uthors [30-33], found tht s hve high scnning ctivity of free rdicl y DPPH, which my contriute to its iologicl ctivity. Another study lso demonstrted the ility of s isomers in reducing free rdicls y the methods photoemission (using tert-utyl hidroxiperóxide to induce peroxidtion of PUFAs nd mesuring the reduction of the chemiluminescence rection y dding the s isomers) nd spectrophotometric evlution y mesuring the decrese the sornce DPPH solution with the ddition of s isomers [32]. The s ctivity s ntioxidnts hs een studied in recent yers, ut the results re contrdictory nd do not llow cler conclusions on the ntioxidnt ctivity of these compounds. Reserch evluted the supplementtion effect of high ft diet plus 3% of s (cis-9, trns-11 nd trns-10, cis-12) for 30 dys in helthy niml model (CF1 mice). The results showed tht the s fed group showed n increse in the content of reduced glutthione (GSH) levels in liver tissue. The ctivity of glutthione peroxidse (GPx) nd ctlse (CAT) did not increse with supplementtion of s [34]. In nother study [21], reported tht supplementtion with s in helthy Sprgue-Dwley provides reduction in MDA levels in plsm nd liver tissue when compred to the control group supplemented with soyen oil. s supplementtion significntly incresed y out 3.7 times, the CAT ctivity in plsm, since the ctivity of SOD, GPx, GR nd GSH plsm levels did not differ significntly in the control group. Studies on the effects of s on models with ltered redox stte re scrce. Reserch exmined the iologicl effects of commercil mixture of isomers of s in model of firosis induced y cron tetrchloride (CCl 4 ). The s diet significntly inhiited the increse of collgen fiers in the liver of nimls in the control group ws oserved n increse in the numer of collgen fiers nd firosis round the centrl vein. Although the control group ws oserved n increse in the numer of cells -SMA ( -smooth muscle ctin) round the portl vein. However, in the group fed the cell numer s -SMA ws similr to tht oserved round the vein of norml liver [11]. These results indicte heptoprotective effects of s on liver firosis induced y CCl 4. In the present study, the results lso suggest tht supplementtion with s hs heptoprotective effect in model rts y CCl 4 oxidtive stress y reducing lipid peroxidtion nd incresing the ctivity of CAT nd GSH content.some reserchers ttempted to explin the ppernce of stetosis nd stetoheptitis y the determintion of the expression of nd peroxisome prolifertor ctivted receptors (PPAR nd PPAR ). There is hypothesis tht cts s lignd of PPAR, especilly PPAR nd PPAR [23, 35, 36]. If this is indeed the cse, supplementtion my e efficient in heptic lipid disese prevention, nd my ct s n djuvnt in its tretment. CONCLUSIONS s supplementtion led to ttenution of the liver dmge induced y CCl 4 in rts, s shown y reduction in the levels of thiorituric cid rective sustnces nd n increse in the content of reduced glutthione, s well s the ctivity of ctlse nd glutthione reductse enzymes in the liver. Although oils nd fts re considered risk fctors for liver dmge due to susceptiility to free rdicl ttck, the dt presented here suggest heptoprotective ctivity of s, due to its ility to improve the oxidtive stress cused y CCl 4. ACKNOWLEDGEMENTS The uthors would like to thnk São Pulo Stte Reserch Foundtion (FAPESP), which supports our

10 Effect of Conjugted Linoleic Acid () in Rts Sujected Journl of Modern Medicinl Chemistry, 2014 Vol. 2, No reserch (Grnts 09/ nd 09/ ) ndntionl Council of Technologicl nd Scientific Development (CNPq) which supports (Grnts /2010-9). REFERENCES [1] Aleksunes LM, Slitt AM, Cherrington NJ, Thiodeu MS, Assen CD, Mnutou JE. Differentil expression of mouse heptic trnsporter genes in response to cetminophen nd cron tetrchloride. Toxicol Sci 2005; 83: [2 ] Oikw D, Akimoto Y, Mizoe Y, Tsuym S, Furuse M. Functions of nd ARA for prevention of induced ftty liver in mice. J Anim Vet Adv 2010; 9: [3] Tnk N, Kono H, Ishii K, Hosomur N, Fuji H. Dietry olive oil prevents cron tetrchloride-induced heptic firosis in mice. J Gstroenterol 2009; 44: [4] Szende B, Timár F, Hrgiti B. Olive oil decreses liver dmge in rts cused y cron tetrchloride (CCl 4 ). Exp Toxicol Pthol 1994; 46: [5] Crvlho EBT, Melo ILP, Mncini-Filho J. Chemicl nd physiologicl spects of isomers of conjugted linoleic ftty cids. Brz Food Sci Technol 2010; 30: [6] Ji X, Zhng Q, Zhng Z, et l. Heptoprotective effects of lmond oil ginst cron tetrchloride induced liver injury in rts. Food Chem 2011; 125: [7] Fng H, Li J, Lin W. Inhiitory effect of olive oil on firosis induced y cron tetrchloride in rt liver. Clin Nutr 2008; 27: [8] Yun GF, Sinclir AJ, Sun HY, Li D. Ftty cid composition in tissues of mice fed diets contining conjugted linolenic cid nd conjugted linoleic cid. J Food Lipids 2009; 16: [9] Co Y, Chen J, Yng L, Chen ZY. Differentil incorportion of dietry conjugted linolenic nd linoleic cids into milk lipids nd liver phospholipids in lctting nd suckling rts. J Nutr Biochem 2009; 20: [10] Trn HN, Be SY, Song BH, et l. Pomegrnte (punic grntum) seed linolenic cid isomers: concentrtiondependent modultion of estrogen receptor ctivity. Endocr Res 2010; 35: [11] Yun HS, Do SH, Jeong WI, et l. Cytotoxic effects of the conjugted linoleic cid isomers t10c12, c9t11- nd mixed form on rt heptic stellte cells nd CCl4-induced heptic firosis. J Nutr Biochem 2008; 19: [12] Christie WW, Séédio JL, Junéd P. A prcticl guide to the nlysis of conjugted linoleic cid (). Inform 2001; 12: [13] Bulits RT, Pohlmn FW, Brown Jr AH, et l. Injection of conjugted linoleic cid into eef strip loins. Met Sci 2007; 75: [14] Ohkw H, Ohishi N, Ygi K. Assy of lipid peroxides in niml tissues y thiorituric cid rection. Anl Biochem 1979; 95: [15] Folch J, Lees M, Stnley GHS. A simple method for the isoltion nd purifiction of totl lipids. J Biol Chem 1957; 226: [16] Mccord JM, Fridovich I. Superoxide dismutse, n enzyme function for erythrhrocuprein (hemocuprein). J Biol Chem 1969; 244: [17] Beutler E. Red cell metolism: mnul of iochemicl methods. 2.ed. New York, London: Grune & Strtton [18] Tietze F. Enzymtic method for quntittive determintion of nnogrm mounts of totl nd oxididsed gluttione: pplictions to mmmlin lood nd other tisues. Anl Biochem 1969; 27: [19] Sies H, Koch OR, Mrtino E, Boveris A. Incresed iliry glutthione disulfide relese in chroniclly ethnol treted rts. FEBS Lett 1979; 103: [20] Lowry OH, Roserough NJ, Frr AL, Rndll RJ. Protein mesurement with the folin phenol regent. J Biol Chem 1951; 193: cittion [21] Mrineli RS, Mrques AC, Furln CPB, Mróstic Jr MR. Antioxidnt effects of the comintion of conjugted linoleic cid nd phytosterol supplementtion in Sprgue Dwley rts. Food Res Int 2012; 49: [22] Diniz YS, Sntos PP, Asslin HB, et l. Conjugted linoleic cid nd crdic helth: oxidtive stress nd energetic metolism in stndrd nd sucrose-rich diets. Eur J Phrmcol 2008; 579: [23] Kennedy A, Mrtinez K, Schmidt S, Mndrup S, Lpoint K, Mcintosh M. Antioesity mechnisms of ction of conjugted linoleic cid. J Nutr Biochem 2010; 21: [24] Zhou X, Sun C, Liu J, Zho D. Dietry conjugted linoleic cid increses PPAR gene expression in dipose tissue of oese rt, nd improves insulin resistnce. Growth Horm IGF Res 2008; 18: [25] Giudetti AM, Beynen AC, Lemmens AG, Gnoni GV, Geelen MJH. Heptic lipid nd cerohydrte metolism in rts fed commercil mexture of conjugted linoleic cids (Clrinol G- 80). Eur J Nutr 2005; 44: [26] Tniguchi M, Tkeuchi T, Nktsuk R, Wtne T, Sto K. Moleculr process in cute liver injury nd regenertion induced y cron tetrchloride. Life Sci 2004; 75: [27] Fng HL, Lin WC. Corn oil enhncing heptic lipid peroxidtion induced y CCl4 does not ggrvte liver firosis in rts. Food Chem Toxicol 2008; 46: [28] Peng WH, Tien YC, Hung CY, et l. Frxinus rhynchophyll ethnol extrct ttenutes cron tetrchloride-induced liver firosis in rts vi down-regulting the expressions of upa, MMP-2, MMP-9 nd TIMP-1. J Ethnophrmcol 2010; 127: [29] Sh SS, Ghosh M. Protective effect of conjugted linolenic cid isomers present in vegetle oils ginst rseniteinduced renl toxicity in rt model. Nutrition 2013; 29: [30] Ali YM, Kdir AA, Ahmd Z, et l. Free rdicl scvenging ctivity of conjugted linoleic cid s single or mixed isomers. Phrm Biol 2012; 50:

11 42 Journl of Modern Medicinl Chemistry, 2014 Vol. 2, No. 1 Crvlho et l. [31] Elflleh W, Ying M, Nsri N, et l. Ftty cids from Tunisin nd Chinese pomegrnte (Punic grntum L.) seeds. Int J Food Sci Nutr 2011; 62: [32] Fgli N, Ctlá A. Antioxidnt ctivity of conjugted linoleic cid isomers, linoleic cid nd its methyl ester determined y photoemission nd DPPH techniques. Biophys Chem 2008; 137: [33] Yu L. Free rdicl scvenging properties of conjugted linoleic cids. J Agric Food Chem 2001; 49: [34] Andreoli MF, Gonzlez MA, Mrtinelli MI, Mocchiutti NO, Bernl CA. Effects of dietry conjugted linoleic cid t highft levels on tricylglycerol regultion in mice. Nutrition 2009; 25: [35] Reynolds CM, Roche HM. Conjugted linoleic cid nd inflmmtory cell signling. Prostglndins Leukot Essent Ftty Acids 2010; 82: [36] Sntos-Zgo LF, Botelho AP, Oliveir AC, Mróstic-Junior MR. Conjugted linoleic cid supplementtion: lipid content nd heptic histology in helthy Wistr rts. Brz Food Sci Technol 2011; 31: Received on Accepted on Pulished on DOI: