Preventive Effects of Lycopene-Enriched Tomato Wine against Oxidative Stress in High Fat Diet-Fed Rats

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1 J Food Sci Nutr Vol 16, p 95~103 (2011) DOI: 1746/jfn Preventive Effects of Lycopene-Enriched Tomto Wine ginst Oxidtive Stress in High Ft Diet-Fed Rts A-Young Kim 1,2, Seon-Min Jeon 1,2, Yong-Jin Jeong 3, Yong Bok Prk 4, Un Ju Jung 1,2, nd Myung-Sook Choi 1,2 1 Deprtment of Food Science nd Nutrition, Kyungpook Ntionl University, Degu , Kore 2 Center for Food nd Nutritionl Genomics Reserch, Kyungpook Ntionl University, Degu , Kore 3 Deprtment of Food Science nd Technology, Keimyung University, Degu , Kore 4 School of Life Sciences nd Biotechnology, Kyungpook Ntionl University, Degu , Kore Astrct This study ws performed to investigte the ntioxidnt mechnism of tomto wine with vrying lycopene content in rts fed high ft diet (HFD). Mle Sprgue-Dwley rts were rndomly divided into five groups (n=10 per group) nd fed n HFD (35% of totl energy from ft) plus ethnol (7.2% of totl energy from lcohol), tomto wine with vrying lycopene content (0.425 mg%, mg% or mg% lycopene) or n isocloric control diet for 6 weeks. Mice fed HFD plus ethnol significntly incresed erythrocyte hydrogen peroxide nd thiorituric cid rective sustnces (TBARS) levels with increses in ctivities of erythrocyte ntioxidnt enzymes such s superoxide dismutse (SOD), ctlse (CAT), glutthione peroxidse (GSH-Px) nd glutthione reductse (GR) compred to pir-fed rts. Supplementtion of tomto wine with vrying lycopene content decresed ethnol-medited increses of erythrocyte lipid peroxidtion nd ntioxidnt enzyme ctivities in HFD-fed rts, nd tomto wine with higher lycopene ppered to e more effective. Tomto wine lso dose-dependently lowered TBARS levels with decresed pro-oxidnt enzyme, xnthine oxidse (XOD) ctivity in plsm of HFD-fed rts. In contrst to erythrocytes, the inhiitory effects of tomto wine on heptic lipid peroxidtion were linked to incresed heptic ntioxidnt enzymes (SOD nd CAT) nd lcohol metolizing enzyme (lcohol dehydrogense nd ldehyde dehydrogense) ctivities. There were no significnt differences in heptic XOD nd cytochrome P450-2E1 ctivities mong the groups. Together, our dt suggest tht tomto wine fortified with lycopene hs the potentil to protect ginst ethnol-induced oxidtive stress vi regultion of ntioxidnt or pro-oxidnt enzymes nd lcohol metolizing enzyme ctivities in plsm, erythrocyte nd liver. Key words: ntioxidnt effects, high ft liquid diet, lipid peroxidtion, lycopene, tomto wine INTRODUCTION Chronic consumption of lcoholic everges is mjor cuse of liver injury such s serious ftty liver, cirrhosis nd cncer (1). Ethnol-induced oxidtive stress is known to ply mjor role in the mechnisms y which ethnol produces liver injury (2,3). In prticulr, the comintion of high ft diet (HFD) nd ethnol cuses n increse in lipid peroxidtion, most likely s consequence of decrese in ntioxidnt defense induced y n HFD nd n increse in rective oxygen species (ROS) production induced y ethnol (4). However, epidemiologicl evidence suggests tht some lcoholic everges, such s red wine, reduce the risk of coronry hert disese nd my hve protective effect on the liver (5,6). It hs een shown tht dietry intke of tomtoes nd tomto-sed products such s tomto juice nd tomto pste cn decrese risk of chronic diseses such s crdiovsculr diseses nd cncer (7), ut there re no studies on the potentil effects of tomto wine. The eneficil properties of tomto pper to e relted to the ntioxidnt content, prticulrly lycopene nd β-crotene, which my ply role in inhiiting rections medited y ROS (8). Lycopene, red crotenoid pigment found in tomtoes nd other fruits or vegetles, is n isomer of β-crotene ut does not hve provitmin A ctivity. It is n cyclic, highly unsturted, stright chin hydrocron with thirteen doule onds (11 conjugted, 2 nonconjugted) (9,10) (Fig. 1). Becuse of its high numer of conjugted doule onds, it is one of the most potent Fig. 1. Structure nd numering of lycopene. Corresponding uthor. E-mil: mschoi@knu.c.kr Phone: , Fx:

2 96 A-Young Kim et l. ntioxidnts mong dietry crotenoids. It is reported tht lycopene exhiits higher singlet oxygen quenching ility thn β-crotene or α-tocopherol (11) nd it my reduce the risk of therosclerosis nd cncer y preventing the oxidtive dmge of iomolecules including lipids, proteins nd DNA (12-16). Furthermore, severl studies suggest tht lycopene cn decrese lcohol-induced oxidtive stress (17,18). Lycopene decreses lcohol-induced hydrogen peroxide nd glutthione levels in HepG2 cells overexpressing cytochrome P450-2E1 (CYP2E1) (17). An epidemiologicl study lso reported n inverse ssocition etween lycopene nd lcohol-induced liver dmge in moderte nd hevy drinkers (25 g of ethnol/dy) (18). However, severl studies showed the ntioxidnt nd pro-oxidnt properties of lycopene my e dose-dependent (19,20). Veermchneni (21) reported tht supplementtion of lycopene t high dose (3.3 mg/kg BW/dy), ut not t lower dose (1.1 mg/kg BW/dy), induced heptic oxidtive stress nd inflmmtion in lcohol-fed rts. In light of these collective dt, we hypothesized tht tomto wine contining vrying lycopene my protect or excerte oxidtive stress induced y high ft nd lcohol intke. Thus, we evluted the effects of tomto wine contining low, medium nd high lycopene content on oxidtive stress nd ethnol-induced heptic iomrkers in HFD-fed rts. MATERIALS AND METHODS Mterils Tomtoes were purchsed from locl mrket (Degu, Kore). D,L-cystein, D,L-methionine, cholin itrte, nd xnthn gum were purchsed from Sigm Chemicl Co. (St. Louis, MI, USA). Supplement preprtion Full-mture rsped tomto (1 kg) without stem ws fortified with sugr to e 22 o Brix, initil cidity ws revised to 0.45% y using trtric cid, S. ynus Llvin EC-1118 (5%) ws inoculted s strter nd then sttic culture ws cultivted in n incutor t 25 o C for 3 dys. After lcohol fermenttion, the solution ws filtered through non-woven fric nd centrifuged t 13,000 rpm for 5 min, which resulted in superntnt of tomto wine with low lycopene content (4.25 mg%). Tomto wine with medium nd high lycopene contents (11.40 mg% nd mg%, respectively) ws mde y dding either 10% (w/v) tomto puree for medium lycopene levels or 20% (w/v) tomto puree for high lycopene levels. The three types of tomto wine with vrying lycopene content were used for the dietry intervention. Experimentl nimls Fifty mle Sprgue-Dwley rts (4-week-old) weighing etween 80 nd 100 g were purchsed from the Centrl L. Animl Inc. (Seoul, Kore). The nimls were ll individully housed in stinless steel cges in n ir-conditioned room with controlled temperture (20 ~23 o C) nd utomtic lighting (lternting 12 hr period of light nd drk). All of the rts were treted in strict ccordnce with the Kyungpook Ntionl University guidelines for the cre nd use of lortory nimls. Experimentl design All rts were fed pellets of commercil chow for 1 week fter rrivl. The rts were rndomly divided into five groups (n=10 per group), which were ssigned to one of five dietry ctegories; high ft liquid diet (35% of totl energy) plus ethnol (7.2% of totl energy) (ethnol control, EC), norml diet tht includes the sme mount of ft (35% of totl energy) nd energy s ech ethnol group consumed in the previous dy (pir-fed control, PC), high ft liquid diet (35% of totl energy) plus low lycopene (0.425 mg%) tomto wine (TW-LL, 7.2% of totl energy), high ft diet (35% of totl energy) plus medium lycopene (1.140 mg%) tomto wine (TW-ML, 7.2% of totl energy), high ft diet (35% of totl energy) plus high lycopene (2.045 mg%) tomto wine (TW-HL, 7.2% of totl energy) for 6 wk. The composition of the experimentl diet ws sed on the modified Lier-DeCrli liquid diet (22) s shown in Tle 1. Ech group ws given the modified liquid diet (~100 ml/dy). Ethnol ws introduced grdully into the diet strting from 5% of energy during the first nd second dy, incresing to 6% of energy during the third nd fourth dy nd ws finlly served to 7.2% (w/v) from the fifth dy. The rts in the PC group received n ethnol-free norml liquid diet contining dextrin-mltose insted of ethnol. Smple preprtion At the end of the experimentl period, the rts were nesthetized with ketmin-hcl following 12 hr fst. Blood ws drwn from the inferior ven cv into heprin-coted tues to determine plsm iomrkers. The liver ws removed, rinsed with physiologicl sline, nd weighed immeditely. All smples were stored t -70 o C until iomrkers nlysis. The preprtion of the enzyme source in the erythrocytes ws s follows: fter the heprin treted lood smples were centrifuged t 1,000 g for 15 min t 4 o C, the plsm nd uffy cot were crefully removed. The seprted cells were then wshed three times y resuspension in 0.9% NCl solution nd recentrifuged. The wshed cells were lysed in n equl volume of wter

3 Lycopene-Enriched Tomto Wine Protects ginst Ethnol-Induced Oxidtive Stress 97 Tle 1. Composition of experimentl nd control diets 1) (g/l/1,000 cl) Groups Csein L-Cystein D,L-Methionine Corn oil Olive oil Dextrin mltose 2) Cholin itrtrte Fier Xnthn gum Vitmin mixture 3) Minerl mixture 4) Ethnol TW-LL TW-ML TW-HL ) The liquid diet ws mixed in 1 L of distilled wter. 2) The ethnol in the ethnol diet ws replced with dditionl dextrin mltose in the pir-fed group. 3) AIN-76 vitmin mixture. 4) AIN-76 minerl mixture. EC, ethnol control; PC, pir-fed control; TW-LL, tomto wine with low lycopene content; TW-ML, tomto wine with medium lycopene content; TW-HL, tomto wine with high lycopene content. nd mixed thoroughly. The hemogloin concentrtion ws estimted in n liquot of the hemolyste, using commercil ssy kit (No. 525-A, Sigm Chemicl Co.). An pproprite dilution of the hemolyste ws then prepred from the erythrocytes suspension y the ddition of distilled wter to estimte ctlse (CAT) nd glutthione peroxidse (GSH-Px) ctivities. Hemogloin ws removed y precipittion with chloroform : ethnol s follows (23): 0.2 ml of n ethnol : choloroform (3:5, v/v) mixture ws dded to n liquot of the hemolyste cooled in ice. This mixture ws stirred constntly for 15 min nd then diluted with wter. After centrifugtion for 10 min t 1,600 g, the ple yellow superntnt ws seprted from the protein precipitte nd used to ssy superoxide dismutse (SOD). The enzyme source frction in the liver ws prepred ccording to the method developed y Hulcher nd Oleson (24), with slight modifiction. A 20% (w/v) homogente ws prepred in uffer contining 0.1% M triethnolmine, 0.02 M EDTA nd 2 mm dithiothreitol (ph 7.0). This homogente ws centrifuged t 600 g for 10 min to discrd ny cellulr deris. The superntnt ws then centrifuged t 10,000 g for 20 min to isolte the mitochondril pellet. Susequently, the superntnt ws ultrcentrifuged twice t 100,000 g for 60 min t 4 o C to otin the cytosolic superntnt. The mitochondril nd microsoml pellets were then redissolved in 800 µl of homogeniztion uffer nd their protein content ws determined y the method of Brdford (25) using ovine serum lumin s the stndrd. Lipid peroxidtion nd hydrogen peroxide ssy Lipid peroxidtion levels were mesured using plsm, erythrocyte (26) nd heptic thiorituric cid sustnces (TBARS) (27). Plsm nd erythrocyte were mixed with 5% trichlorocetic cid (TCA) nd 60 mmol/l thiorituric cid (TBA). After incution t 80 o C for 90 min, the superntnts were centrifuged t 1000 g for 15 min t 4 o C. The sornce of the superntnt ws determined t 535 nm using tetrmethoxypropne (Sigm Chemicl Co.) s the stndrd. Heptic homogentes contining 8.1% sodium dodecyl sulfte were mixed with 20% (w/v) cetic cid (ph 3.5), distilled wter nd 0.8% (w/v) TBA. The rection mixture ws heted t 95 o C for 60 min. After cooling the mixture, n-utnol:pyridine (15:1, v/v) ws dded nd centrifuged t 800 g for 15 min. The resulting colored lyer ws mesured t 535 nm. The hydrogen peroxide levels in erythrocyte nd liver were mesured y Wolff s method (28). FOX 1 (ferrous oxidtion) regent ws prepred from 0.25 M H 2 SO 4, 1 M soritol, 2.5 mm mmonium iron (II), 1 mm xylenol ornge, nd hydrogen peroxide levels were determined t 560 nm sornce. Enzyme ssys The SOD ctivity ws mesured ccording to the method of Mrklund nd Mrklund (29) with slight modifiction. One unit ws determined s the mount of enzyme tht inhiited the oxidtion of pyrogllol y 50%. The CAT ctivity ws mesured using Aei s (30) method, in which the disppernce of hydrogen peroxide ws monitored spectrophotometriclly t 240 nm for 5 min using spectrophotometer. The GSH-Px ctivity ws mesured using the spectrophotometric ssy, s descried previously y Pgli nd Vlentine s (31) methods with slight modifiction. The rection mixture included 30 mm glutthione, 6 mm NADPH nd H 2 O 2 in 0.1 M Tris-HCl (ph 7.2) uffer. The rection ws initited y dding the enzyme source nd the sornce ws mesured t 340 nm for 5 min. The glutthione reductse (GR) ctivity ws mesured y monitoring the oxidtion of NADPH t 340 nm (32). Plsm proxonse (PON) ctivity ws ssyed using the methods descried y Mckness et l. (33), which mesured the increse in sornce for 60 sec t 405 nm nd 25 o C. The lcohol dehydrogense (ADH) ctivity ws mesured y monitoring the formtion of NADH t 340 nm in the heptic cytosol (34). The ldehyde dehydrogense (ALDH) ctivity in the mitochondri of liver ws ssyed using Koivul nd Koivuslo s method (35). The ctivity of CYP2E1 ws mesured in microsome frction of liver

4 98 A-Young Kim et l. y the formtion of 4-nitroctechol detected spectrophotometiclly s previously descried (36). The xnthine oxidse (XOD) ctivity ws mesured y monitoring the production of uric cid t 290 nm in plsm nd liver (37). Plsm ethnol concentrtion Plsm ethnol concentrtion ws determined using commercil kit (ECET-100, BioAssy Systems, Hywrd, CA, USA). Sttisticl nlysis All dt were presented s the men±se. Sttisticl nlysis ws crried out using SPSS sttisticl softwre (version 11.0, SPSS Inc., Chicgo, IL, USA). Significnt differences mong the groups were determined y onewy ANOVA. Post-hoc Duncn s multiple-rnge tests were performed when differences were identified etween the groups. Significnt differences were ccepted t p0.05. RESULTS Effects of tomto wine with lycopene on plsm, erythrocyte nd heptic hydrogen peroxide nd lipid peroxidtion levels In rts fed HFD plus vrying lycopene content, erythrocyte nd heptic hydrogen peroxide level tended to e dose-dependently decresed compred to ethnol/ HFD-fed rts (Fig. 2). Plsm TBARS level ws significntly decresed y tomto wine with high lycopene content compred to ethnol plus HFD diet. Supplementtion of tomto wine over 6 weeks lso decresed eryth- () () c c c (c) c (d) c c c (e) 0 NS Fig. 2. Effects of tomto wine contining low, medium nd high lycopene content on TBARS nd hydrogen peroxide levels in plsm, erythrocyte nd liver of HFD-fed rts. Vlues re men±se. -c Mens not shring common letter re significntly different mong groups t p0.05. EC, ethnol control; PC, pir-fed control; TW-LL, tomto wine with low lycopene content; TW-ML, tomto wine with medium lycopene content; TW-HL, tomto wine with high lycopene content; H 2O 2, hydrogen peroxide; TBARS, thiorituric cid rective sustnce.

5 Lycopene-Enriched Tomto Wine Protects ginst Ethnol-Induced Oxidtive Stress 99 Tle 2. Effects of tomto wine contining low, medium nd high lycopene content on ntioxidnt enzyme ctivities in plsm, erythrocyte nd liver of HFD-fed rts Groups Liver SOD (unit/mg protein) CAT (μmol/min/mg protein) GSH-Px (nmol/min/mg protein) GR (nmol/min/mg protein) Erythrocyte SOD (unit/mg H) CAT (μmol/min/mg H) GSH-Px (nmol/min/mg H) GR (nmol/min/mg H) Plsm PON (μmol/min/ml) 24.73± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±0.014 c 32.13± ± ± ± ± ± ± ± ±0.010 c 51.29± ± ± ± ± ± ± ± ±0.006 c Vlues re men±se. -c Mens not shring common letter in row re significntly different mong groups t p0.05. EC, ethnol control; PC, pir-fed control; TW-LL, tomto wine with low lycopene content; TW-ML, tomto wine with medium lycopene content; TW-HL, tomto wine with high lycopene content; SOD, superoxide dismutse; CAT, ctlse; GSH-Px, glutthion peroxidse; GR, glutthione reductse; PON, proxonse. rocyte TBARS levels in HFD-fed rts compred to ethnol/hfd-fed rts, ut not pir-fed rts. Notly, tomto wine with high lycopene content ppered to e more effective t suppression of plsm nd erythrocyte TBARS thn tomto wine with lower lycopene content. However, there ws no dose dependent effect of lycopene content on heptic TBARS. Heptic TBARS level ws significntly lowered in tomto wine with low lycopene content thn in oth ethnol/hfd-fed rts nd pir-fed rts. Effects of tomto wine with lycopene on ntioxidnt nd pro-oxidnt enzyme ctivities Next, we compred how the ethnol/hfd diet vs. the isocloric control diet influences plsm, erythrocyte nd heptic ntioxidnt enzyme ctivities, nd determined whether tomto wine with vrying lycopene cn regulte these ntioxidnt enzyme ctivities in plsm, erythrocyte nd liver of HFD-fed rts (Tle 2). Supplementtion of tomto wine in HFD-fed rts over 6 weeks effectively incresed heptic SOD nd CAT ctivities compred to the ethnol/hfd control group. Among the tomto wines tested, high lycopene induced the strongest response y incresing heptic SOD nd CAT ctivities. There were no differences in the heptic GSH-Px nd GR ctivities mong groups. In contrst to heptic ntioxidnt ctivities, ethnol/ HFD-fed rts showed higher ctivities of erythrocyte SOD, CAT, GSH-Px nd GR compred to the pir-fed control group nd tomto wine contining lycopene dosedependently decresed these erythrocyte ntioxidnt enzyme ctivities compred to ethnol/hfd control group (Tle 2). Plsm PON ctivity ws lso significntly lowered in rts fed tomto wine with medium or high lycopene content. To further exmine whether supplementtion of tomto wine with lycopene inhiits pro-oxidnt enzymes, we determined plsm nd heptic XOD ctivities (Fig. 3). The plsm XOD ctivity ws incresed in ethnol/ HFD-fed rts compred to pir-fed rts, wheres supplementtion of tomto wine with medium or high lycopene content ppered to significntly inhiit plsm XOD ctivity compred to ethnol/hfd-fed rts. Heptic XOD ctivity ws not significntly different etween pir-fed controls nd HFD-fed rts regrdless of the lycopene content of tomto-wine. Effects of tomto wine with lycopene on plsm ethnol concentrtion nd heptic lcohol-metolizing enzyme ctivities We next investigted the potentil effects of tomto wine with lycopene on plsm ethnol concentrtion nd heptic lcohol-metolizing enzyme ctivities in HFDfed rts. Tomto wine with vrying lycopene content dose-dependently lowered ethnol concentrtion in the plsm when compred mong the ethnol-treted groups (Tle 3). No ethnol ws detected in the plsm from the pir-fed rts (Tle 3). Heptic ADH ctivity ws not significntly different etween ethnol/hfd rts nd pir-fed controls, wheres heptic ALDH ctivity ws significntly higher in the ethnol/hfd-fed rts compred to pir-fed controls (Tle 3). Supplementtion of tomto wine with vrying doses of lycopene resulted in increses of heptic ADH nd ALDH ctivities compred to ethnol/hfd-fed rts (Tle 3). The ctivity of heptic CYP2E1, nother enzyme involved in ethnol metolism, ws not significntly different mong groups (Fig. 3).

6 100 A-Young Kim et l. () () (c) Fig. 3. Effects of tomto wine contining low, medium nd high lycopene content on XOD nd CYP2E1 ctivities in plsm nd liver of HFD-fed rts. Vlues re men±se. -c Mens not shring common letter re significntly different mong groups t p0.05. EC, ethnol control; PC, pir-fed control; TW-LL, tomto wine with low lycopene content; TW-ML, tomto wine with medium lycopene content; TW-HL, tomto wine with high lycopene content; XOD, xnthine oxidse; CYP2E1, cytochrome P450-2E1. c DISCUSSION Oxidtive stress is chrcterized y n imlnce etween ROS genertion nd ntioxidnt cpcity. ROS re nturlly generted in smll mounts through norml metolic rections nd re constntly produced nd eliminted, since cells hve endogenous ntioxidnt cpcity. However, overproduction of ROS cn cuse dmge to lipids, proteins nd DNA, which cn then induce pthologicl events leding to heptic stetosis, crdiovsculr diseses nd cncer. It is well known tht chronic nd excessive lcohol consumption cn promote the genertion of ROS through the vrious pthwys relted to ethnol metolism. Ethnol is extensively oxidized into cetldehyde y the ADH nd then into cette y ALDH in the liver. Acetldehyde is highly toxic metolite of ethnol nd the ccumultion of cetldehyde in the liver fter chronic lcohol consumption hs een implicted in the pthogenesis of lcoholic liver disese (3). In the current study, lcohol feeding led to inhiition of heptic ALDH, ut not ADH, compred to pir-fed rts. This result indictes tht moderte lcohol intke in comintion with n HFD over 6 weeks cn e efficiently converted into cetldehyde y norml ADH ctivity ut the susequent conversion of cetldehyde into cette cn e delyed due to low ALDH ctivity. Interestingly, supplementtion of tomto wine with lycopene effectively ctivted oth ADH nd ALDH ctivities in HFD-fed rts, therey enling the conversion of ethnol into cette vi cetldehyde nd contriuting to lower plsm ethnol concentrtion. Acetldehyde is lso oxidized into cette y XOD in liver, producing superoxide nd hydrogen peroxide (38,39). However, others hve previously demonstrted tht XOD does not ply mjor role in oxygen rdicl production in the liver during cute lcohol intoxiction (40). Our present study lso showed tht heptic XOD ctivity ws not significntly different etween pir-fed controls nd lcohol-fed rts regrdless of lycopene content of tomto-wine. Furthermore, we oserved no effect of moderte lcohol consumption in HFD-fed rts on Tle 3. Effects of tomto wine contining low, medium nd high lycopene content on plsm ethnol concentrtion nd heptic lcohol metolizing enzyme ctivities in HFD-fed rts Ethnol (mg/dl) Alcohol dehydrogense (nmol/min/mg protein) Aldehyde dehydrogense (nmol/min/mg protein) Groups 6.59± ±0.11 c 1.16±6 6.25±9 c 5.21± ±4 7.83±1 5.42±0 3.35± ±0.60 c 6.31±1.93 0± ± ±0.97 Vlues re men±se. -c Mens not shring common letter in row re significntly different mong groups t p0.05. EC, ethnol control; PC, pir-fed control; TW-LL, tomto wine with low lycopene content; TW-ML, tomto wine with medium lycopene content; TW-HL, tomto wine with high lycopene content; ADH, lcohol dehydrogense; ALDH, ldehyde dehydrogense.

7 Lycopene-Enriched Tomto Wine Protects ginst Ethnol-Induced Oxidtive Stress 101 heptic CYP2E1 ctivity, which lso helps oxidize ethnol prticulrly following chronic lcohol intke (41,42), compred to pir-fed controls nd tomto wine with lycopene-supplemented groups. An in vitro study showed tht lycopene ttenuted lcoholic induced-oxidtive stress nd poptosis in HepG2 cells overexpressing CYP2E1 (17). In contrst, in vivo supplementtion of lycopene t high dose (3.3 mg/kg BW/dy), ut not lower dose (1.1 mg/kg BW/dy) is reported to significntly induce heptic CYP2E1 levels nd inflmmtion in lcohol-fed rts (21), suggesting need for cution mong individuls consuming high mounts of oth lcohol nd lycopene. Some vrition mong pulished reports regrding the potency of lycopene on CYP2E1- relted oxidtive stress my e due to mny fctors such s species, dietry intervention period, lycopene source nd mount of lcohol. On the other hnd, lcohol feeding over 6 weeks resulted in significntly lowered ctivity of heptic CAT, ntioxidnt enzyme, compred to pir-fed group in the present study s previously oserved y others (40). In contrst, supplementtion of tomto wine contining lycopene ppered to effectively ctivte heptic SOD nd CAT ctivities compred to ethnol plus HFD group. Furthermore, we oserved trend of decresed heptic lipid peroxidtion with tomto wine contining lycopene in HFD-fed rts. Antioxidnt enzymes such s SOD, CAT nd GSH-Px re importnt in protecting cells from oxidtive dmge. SOD converts superoxide rdicls into hydrogen peroxide, which is then further metolized y CAT, which ctlyzes the conversion of hydrogen peroxide to wter nd oxygen. If CAT ctivity is not sufficiently enhnced to metolize hydrogen peroxide, this cn led to incresed hydrogen peroxide nd TBARS levels (43). Our results re consistent with the study y Hsu et l. (44), who demonstrted tht supplementtion with tomto pste contining lycopene for 8 weeks incresed heptic SOD, CAT nd GSH-Px ctivities nd decresed lipid peroxidtion in cholesterol-fed hmsters. However, in the current study, there were no dose-dependent effects of lycopene content on TBARS levels in liver, suggesting tht the low dose of lycopene contined in tomto wine ws sufficient to suppress heptic lipid TBARS levels. Tken together, our findings indicte tht tomto wine nd lycopene supplementtion my suppress heptic lipid peroxidtion y ctivting ntioxidnt enzymes s well s y suppressing pro-oxidnt enzymes such s ADH nd ALDH in liver of HFD-fed rts. Erythrocytes re lso susceptile to lipid peroxidtion due to lcohol consumption (45), nd we found tht lcohol feeding significntly incresed erythrocyte TBARS nd hydrogen peroxide levels concomitnt with incresed ctivities of heptic ntioxidnt enzymes like SOD, CAT, GSH-Px nd GR compred to pir-fed rts. Accordingly, our study suggests tht the comintion of lcohol nd n HFD my result in overproduction of ROS-stimulting ntioxidnt enzyme ctivity to cope with the incresed lipid peroxidtion in erythrocytes. Interestingly, the erythrocyte lipid peroxidtion nd ntioxidnt enzyme ctivities were recovered to the norml level in ll mice on tomto wine with lycopene. Notly, tomto wine with high lycopene content ppered to e the most effective t suppressing the increse in erythrocyte lipid peroxidtion nd ntioxidnt enzyme ctivities in HFD fed rts, suggesting tht the lycopene content, rther thn other functionl components in tomto wine, ws primrily responsile for suppression of oxidtive stress induced y ethnol. Bose nd Agrwl (46-48) lso showed tht tomto or lycopene decresed erythrocyte lipid peroxidtion levels in ptients with crdiovsculr disese nd type 2 dietes. Zim et l. (49) reported tht ethnol significntly increses plsm XOD ctivity in oth rts nd hmsters, suggesting tht plsm XOD my e sensitive mrker of liver dmge. In our study, we found tht plsm XOD ctivity ws higher in lcohol-fed group compred to pir-fed group, wheres ll mice on tomto wine with vrying lycopene content mrkedly decresed the ctivity of plsm XOD. Furthermore, tomto wine with lycopene effectively suppressed plsm lipid peroxidtion levels, indicted y significntly lower plsm TBARS in these rts. Previous studies lso supported role for tomto products in the prevention of plsm lipid peroxidtion (50,51). Plsm PON, n HDL-ssocited ntioxidnt enzyme (52), protects ginst plsm lipid oxidtion (53). In generl, PON ctivity ws suggested to e inversely ssocited with oxidtive stress in plsm (54) nd ntioxidnts cn increse PON ctivity (55). However, two previous studies reported tht high intke of vegetles rich in vitmins E nd C ws negtively correlted with serum PON ctivity in Finnish popultions (56,57). We lso showed tht tomto wine nd lycopene supplementtion in HFD-fed rts resulted in lower plsm PON ctivity compred to ethnol/ HFD-fed rts. There ws no effect of ethnol on plsm PON ctivity compred to the pir-fed group. This finding is in ccordnce with previous experimentl dt showing no ssocition etween lcohol consumption nd serum PON ctivity in helthy men (58). In conclusion, the present study suggests tht supplementtion of tomto wine with vrying lycopene content protects ginst lcohol-induced oxidtive stress in HFD-fed rts. Tomto wine with high lycopene content

8 102 A-Young Kim et l. ppered to e more effective thn tomto wine with lower lycopene content for suppressing lipid peroxidtion. The eneficil effects of tomto wine pper to e prtly medited through regultion of pro-oxidnt nd ntioxidnt enzyme ctivity in plsm, erythrocyte nd liver. Thus, supplementtion of tomto wine fortified with lycopene could prove promising in decresing lcohol-induced heptic stetosis nd crdiovsculr disese. ABBREVIATIONS ADH, lcohol dehydrogense; ALDH, ldehyde dehydrogense; CAT, ctlse; CYP2E1, cytochrome P450-2E1; GSH-Px, glutthion peroxidse; GR, glutthione reductse; HFD, high ft diet; H 2O 2, hydrogen peroxide; PON, proxonse; ROS, rective oxygen species; SOD, superoxide dismutse; TBA, thiorituric cid; TBARS, thiorituric cid-rective sustnce; XOD, xnthine oxidse ACKNOWLEDGEMENTS This work ws supported y the Ntionl Reserch Foundtion of Kore (NRF) grnt funded y the Kore government (No ) nd Kyungpook Ntionl University Reserch Fund None of the uthors hd ny conflicts of interest. REFERENCES 1. An JH Proteomic nlysis of the protective effects of Pltycodi Rdix in liver of chroniclly lcoholic rts. J Med Food 12: Lieer CS Heptic metolic nd toxic effect of ethnol. Alcohol Clin Exp Res 5: Lier CS Alcohol nd the liver. Heptology 106: Demori I, Voci A, Fugss E, Burlndo B Comined effects of high-ft diet nd ethnol induce oxidtive stress in rt liver. Alcohol 40: Renud S Wine, lcohol, pltelets, nd the French prdox for coronry hert disese. Lncet 339: Lieer CS Alcoholic ftty liver: its pthogenesis nd mechnism of progression to inflmmtion nd firosis. Alcohol 34: Alshtwi AA Tomto powder is more protective thn lycopene supplement ginst lipid peroxidtion in rts. Nutr Res 30: Oshim S, Ojim F, Skmoto H, Ishiguro Y, Tero J Supplementtion with crotenoids inhiits singletoxygen-medited oxidtion of humn plsm low-density lipoprotein. J Agric Food Chem 44: Nguyen ML, Schwrtz SJ Lycopene: chemicl nd iologicl properties. Food Technol 53: Suhsh C, Bose K, Agrwl BK Effect of lycopene from cooked tomtoes on serum ntioxidnt enzymes, lipid peroxidtion rte, lipid profile nd glycted hemogloin in type 2 dietes. Indin J Nutr Diet 43: Di MP, Devsgym TP, Kiser S, Sies H Crotenoids, tocopherols nd thiols s iologicl singlet moleculr oxygen quenchers. Biochem Soc Trns 18: Agrwl S, Ro AV Tomto lycopene nd low density lipoprotein oxidtion: humn dietry intervention study. Lipids 33: Ro AV, Agrwl S Role of lycopene s ntioxidnt crotenoids in the prevention of chronic diseses. Nutr Res 19: Khchik F, Crvlho L, Bernstein PS Chemistry, distriution, nd metolism of tomto crotenoids nd their impct on humn helth. Exp Biol Med 227: Upritchrd JE, Sutherlnd WH, Mnn JI Effect of supplementtion with tomto juice, vitmin E, nd vitmin C on LDL oxidtion nd products of inflmmtory ctivity in type 2 dietes. Dietes Cre 23: Kiokis S, Gordon MH Dietry supplementtion with nturl crotenoid mixture. Eur J Clin Nutr 57: Xu Y, Leo MA, Lieer CS Lycopene ttenutes lcoholic poptosis in HepG2 cells expressing CYP2E1. Biochem Biophys Res Commun 308: Sugiur M, Nkmur M, Ikom Y, Yno M, Ogw K, Mtsumoto H, Kto M, Ohshim M, Ngo A High serum crotenoids re inversely ssocited with serum gmm-glutmyltrnsferse in lcohol drinkers within norml liver function. J Epidemiol 15: Lowe GM, Booth LA, Young AJ, Bilton RF Lycopene nd et-crotene protect ginst oxidtive dmge in HT29 cells t low concentrtions ut rpidly lose this cpcity t higher doses. Free Rdic Res 30: Yeh S, Hu M Antioxidnt nd pro-oxidnt effects of lycopene in comprison with et-crotene on oxidnt-induced dmge in Hs68 cells. J Nutr Biochem 11: Veermchneni S High dose lycopene supplementtion increses heptic cytochrome P4502E1 protein nd inflmmtion in lcohol-fed rts. J Nutr 138: Lieer CS, DeCrli LM The feeding of ethnol in liquid diets. Alcohol Clin Exp Res 10: McCord JM, Fridovich I Superoxide dismutse. An enzymic function for erythrocuprein (hemocuprein). J Biol Chem 244: Hulcher FH, Oleson WH Simplified spectrophotometric ssy for microsoml 3-hydroxy-3-methylglutryl CoA reductse y mesurement of coenzyme A. J Lipid Res 14: Brdford MM A rpid nd sensitive method for the quntittion of microgrm quntities of protein utilizing the principle of protein-dye inding. Anl Biochem 72: Bsil G, Trldgis AM, Person LR, Dugn J Chemistry of the 2-thiorituric cid test for etermintion of oxidtive rncidity in foods. II. formtion of the t-mlonldehyde complex without cid-het tretment. J Sci Food Agric 15: Ohkw H, Ohishi N, Ygi K Assy for lipid peroxides in niml tissues y thiorituric cid rection. Anl Biochem 95: Wolff SP Ferrous ion oxidtion in presence of ferric ion indictor xylenol ornge for mesurement of hydroperoxides. Methods Enzymol 233:

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