effects of swim training on liver carcinogenesis in male wistar rats fed a low-fat or high-fat diet

Effects of swim training on liver carcinogenesis inmale Wistar rats fed a low-fat or high-fat diet

Marco Aurélio Aguiar e Silva, Ivan José Vechetti-Junior,André Ferreira do Nascimento, Kelly Silva Furtado, Luciana Azevedo,Daniel Araki Ribeiro, and Luis Fernando Barbisan

Abstract: The present study aimed to investigate the beneficial effects of swim training on the promotion–progression stagesof rat liver carcinogenesis. Male Wistar rats were submitted to chemically induced liver carcinogenesis and allocated into4 major groups, according their dietary regimen (16 weeks) and swim training of 5 days per week (8 weeks): 2 groups werefed low-fat diet (LFD, 6% fat) and trained or not trained and 2 groups were fed high-fat diet (HFD, 21% fat) and trained ornot trained. At week 20, the animals were killed and liver samples were processed for histological analyses; immunohisto-chemical detection of persistent or remodeling preneoplastic lesions (pPNL and rPNL) expressing placental glutathione S-transferase (GST-P) enzyme; or proliferating cell nuclear antigen (PCNA), cleaved caspase-3, and bcl-2 protein levels byWestern blotting or malonaldehyde (MDA) and total glutathione detection by HPLC. Overall analysis indicated that swimtraining reduced the body weight and body fat in both LFD and HFD groups, normalized total cholesterol levels in theHFD group while decreased the MDA levels, increased glutathione levels and both number of GST-P-positive pPNL andhepatocellular adenomas in LFD group. Also, a favorable balance in PCNA, cleaved caspase-3, and bcl-2 levels was de-tected in the liver from the LFD-trained group in relation to LFD-untrained group. The findings of this study indicate thatthe swim training protocol as a result of exercise postconditioning may attenuate liver carcinogenesis under an adequate diet-ary regimen with lowered fat intake.

Key words: swim training, rat liver carcinogenesis, high-fat diet, preneoplastic–neoplastic lesions.

Résumé : Cette étude se propose d’analyser les effets bénéfiques de l’entraînement à la nage sur la promotion–progressiondes stades de la carcinogenèse du foie de rat. On induit chimiquement une carcinogenèse du foie à des rats Wistar mâles di-visés en quatre groupes principaux selon leur régime alimentaire (16 semaines) et leur entraînement à la nage à raison de5 jours par semaine (8 semaines) : deux groupes nourris avec un régime alimentaire faible en gras (LFD, 6 % de gras) etsoumis ou non à l’entraînement et deux groupes nourris avec un régime alimentaire riche en gras (HFD, 21 % de gras) etsoumis ou non à l’entraînement. À la 20e semaine, on sacrifie les animaux et on prélève des échantillons du foie pour desanalyses histologiques et la détection immunohistochimique de lésions prénéoplasiques persistantes ou en remodelage(pPNL et rPNL) exprimant la glutathion S-transférase placentaire (GST-P) ou de l’antigène nucléaire de prolifération cellu-laire (PCNA), de la caspase 3 clivée, des protéines de la famille bcl-2 par buvardage de western ou la malonaldéhyde(MDA) et la détection du glutathion total par chromatographie liquide à haute performance (HPLC). D’après l’analyse glo-bale, l’entraînement à la nage suscite une diminution de la masse corporelle et du gras corporel dans les groupes LFD etHFD, normalise la concentration de cholestérol total dans le groupe HFD et diminue la concentration de MDA, augmente laconcentration de GSH et le nombre de pPNL positives à la GST-P et d’adénomes hépatocellulaires dans le groupe LFD. Enoutre, on détecte dans le foie du groupe LFD entraîné un équilibre favorable de PCNA et des concentrations de caspase 3clivée et de bcl-2 comparativement au groupe LFD non entraîné. Selon les observations de cette étude, le protocole d’entraî-nement à la nage, résultat du conditionnement subséquent de l’exercice, semble atténuer la carcinogenèse du foie en pré-sence d’un régime alimentaire faible en gras.

Mots‐clés : entraînement à la nage, carcinogenèse du foie de rat, régime riche en gras, lésions prénéoplasiques–néoplasiques.

[Traduit par la Rédaction]

Received 8 May 2012. Accepted 27 July 2012. Published at www.nrcresearchpress.com/apnm on 10 September 2012.

M.A. Aguiar e Silva and I.J. Vechetti-Junior. Post-Graduation Program in General and Applied Biology, Institute of Biosciences,UNESP, Sao Paulo State University, Botucatu 18618-970, SP, Brazil.A.F. Nascimento. Department of Clinical Medicine, School of Medicine, UNESP, Sao Paulo State University, 18618-970 Botucatu, SP,Brazil.K.S. Furtado. School of Medicine, Department of Pathology, UNESP, Sao Paulo State University, 18618-970 Botucatu, SP, Brazil.L. Azevedo. Faculty of Nutrition, UNIFAL, Federal University of Alfenas, 37130-000, Alfenas, MG, Brazil.D.A. Ribeiro. Department of Biosciences, UNIFESP, Federal University of Sao Paulo, 11060-001, Santos, SP, Brazil.L.F. Barbisan. Department of Morphology, Institute of Biosciences, UNESP, Sao Paulo State University, 18618-970 Botucatu, SP, Brazil.

Corresponding author: Luis Fernando Barbisan (e-mail: [email protected]).

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Appl. Physiol. Nutr. Metab. 37: 1101–1109 (2012) doi:10.1139/H2012-129 Published by NRC Research Press

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Introduction

Hepatocellular carcinoma (HCC) is the fifth most frequentcancer worldwide, ranking third among all cancer-relatedmortalities in the world (El-Serag and Rudolph 2007; Jemalet al. 2010). HCC arises from hepatocytes undergoing malig-nant transformation in response to various noxious chronicstimuli, such as hepatitis B or C virus, aflatoxin B1 contami-nation, chronic alcohol abuse, and nonalcocholic steatothpati-tis (NASH) (Farazi and Depinho 2006; Siegel and Zhu 2009;McGlynn and London 2011). Survival remains poor in pa-tients presenting intermediate and advanced stage HCC be-cause of the aggressiveness of the lesions at the time ofdiagnosis, lack of curative therapy, and high recurrence rate(Farazi and Depinho 2006; El-Serag and Rudolph 2007; Je-mal et al. 2010). Thus, it is important to develop more effec-tive strategies for the prevention and quality of life for peopleaffected with this malignance (El-Serag and Rudolph 2007;Jemal et al. 2010; Farazi and Depinho 2006; McGlynn andLondon 2011).Several rodent models have been used in the study of de-

velopment and pathogenesis of HCC, contributing to the cur-rent knowledge of this liver disease as well as its preventionand treatment (Heindryckx et al. 2009; Newell et al. 2008).The resistant hepatocyte (RH) model is a well-defined livercancer model for reproducing human HCC developmentsince RH induces a rapid growth of the initiated hepatocytessubpopulations into larger preneoplastic lesions (PNL)(French 2010; Andersen et al. 2010). Some PNL eventuallyprogress into highly visible nodules and during a period ofsome months, HCC arises (French 2010; Andersen et al.2010). The evolution of PNL to HCC is a long-term processand requires chronic exposure to a tumor-promoting environ-ment, such as oxidative stress, inflammation, and (or) nutri-tional imbalances (e.g., increase in reactive oxygen species(ROS) by high fat consumption and low antioxidant intake)(Farazi and Depinho 2006; Siegel and Zhu 2009; McGlynnand London 2011). Specifically, recent studies have revealedthat a high-fat diet may promote NASH and carcinogenesisin rodent and human liver in contrast to the low-fat diet(Hill-Baskin et al. 2009; Wang et al. 2009; Siegel and Zhu2009).Regular aerobic exercise has been suggested as an appro-

priate means for the activation of antioxidant and immuno-logical defenses, reduction of inflammatory processes, andimprovement in quality of life, mainly in metabolic syndromediseases (Kuo et al. 2007; Kelley et al. 2011). Also, the exer-cise combined with healthy eating, including low fat intake,is highly efficacious for improving serum lipid profiles, qual-ity-of-life, and complications of obesity, metabolic syndrome,and NASH (Zivkovic et al. 2007; Nobili et al. 2011). Inchemically induced rodent carcinogenesis models, moderateexercise (running and swimming) reduces the developmentof preneoplastic and neoplastic lesions in the colon andmammary gland (Hoffman-Goetz 2003; Na and Oliynyk2011). In contrast, a high fat ingestion may compromise thebeneficial effects of moderate exercise against rodent colonand mammary carcinogenesis (Cohen et al. 1992; Baltgalviset al. 2009; Perše et al. 2012). On the other hand, no reportsexist on the possible beneficial effects of regular exercise on

the promotion–progression stages of human or rodent livercarcinogenesis.Considering the beneficial effects of regular exercise to

health and quality of life, the present study aimed to investi-gate the beneficial effects of swim training on the promotion–progression stages of liver carcinogenesis in male Wistar ratssubmitted to the RH model. This study also analyzed themodifying effects of exercise intervention combined with alow-fat or high-fat dietary regimen, which mimics food habitsof humans living in Western countries.

Materials and methods

Animal and experimental designThis study was approved by the Institute of Biosciences of

Sao Paulo State University (UNESP) Ethics Committee forAnimal Research (Protocol no. 242). Four-week-old maleWistar rats were obtained from Multidisciplinary Center forBiological Investigation (CEMIB, UNICAMP Campinas-SP,Brazil). The animals were housed in polypropylene cages(4 animals per cage) covered with metallic grids in a roommaintained at 22 ± 2 °C, 55% ± 10% humidity and with a12-h light/12-h dark cycle, during a 2-week acclimatizationperiod. Then, all animals were submitted to the RH modelof liver carcinogenesis adapted for male Wistar rats (Mazzan-tini et al. 2008). At the beginning of the experiment, animalsreceived a single i.p. dose of 200 mg·kg–1 body weight of di-ethylnitrosamine (DEN, Sigma–Aldrich Co., St. Louis, Mo.,USA) to initiate liver carcinogenesis. After a recovery periodof 2 weeks, the initiated hepatocytes were selected–promotedby 5 i.g. doses of 2-acetylaminofluorene (2-AAF, Sigma–Aldrich Co., St. Louis, Mo., USA). The first 4 doses(200 mg·kg–1 body weight) were administered on 4 consec-utive days before 70% partial hepatectomy (HP) surgery.The remaining doses of 2-AAF (75 mg·kg–1 body weight)were administered on days 2 and 4 after the HP. Threeweeks after HP, the animals were randomly allocated into 4groups, consisting of 10 animals in each group (n = 10).Then, groups were fed low-fat diet (LFD; 6% fat, 51% car-bohydrate 26% protein, and 3.64 kcal·g–1 with calories fromsaturated fat (2.5%) and unsaturated fat (9.5%) Agroceres(Rio Claro-SP, Brazil)) or high-fat diet (HFD; 21% fat;42% carbohydrate, 24% protein and 4.54 kcal·g–1 with calo-ries from saturated fat (9.95%) and unsaturated fat (39.1%),Agroceres) for 16 weeks. Fatty acid composition in LFDand HFD were 16.56% and 15.09% (palmitic (C16:0));3.90% and 4.39% (stearic (C18:0)); 27.96% and 37.94%(oleic (C18:1n9c)); 47.10% and 40.83% (linoleic(C18:2n6)); and 4.48% and 1.75% (others, lauric (C12:0),miristic (C14:0), palmitoleic (C16:1), and linoleic(C:18:3n3)), respectively (Nascimento et al. 2011).After 8 weeks of feeding LFD or HFD, groups were un-

trained or trained (swimming during 5 days per week) for anadditional 8-week period, consisting of 10 animals in eachgroup (n = 10). The trained groups performed individually aprogressive aerobic individual training program in an aquar-ium of 110 × 70 × 22 cm, subdivided into 5 compartments,filled with 50 cm of the water at 30 ± 2 °C. Before the initialtraining program, all the animals were adapted to the water.The adaptation consisted of keeping the animals in shallowwater at 30 ± 2 °C for 5 days during 1 week. The animals

1102 Appl. Physiol. Nutr. Metab. Vol. 37, 2012

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swam using washers as overload attached by a rubber bandon their tails. Training sessions and intensity were progres-sive: 10 min without overloading (first week); 20 min, 1%overload (second week); 25, 30, 35, and 40 min, 3% overload(third week); 45, 50, 55, and 60 min, 3%–5% overload(fourth week); and 60 min, 3%–5% overload (fifth to eighthweeks) (Gobatto et al. 2001). According to Gobatto et al.(2001), 5% overload represents the maximal lactate steadystate in rats. The overload training was calculated based onbody weight data to ensure the same training intensitythroughout the experiment; the animals were weighted daily.Untrained groups fed LFD or HFD were kept in shallowwater throughout the training period to mimic the stress lev-els in these animals. The adaptation phase and all trainingsessions were performed from 1400 to 1600 h in a darkroom.

Tissue processing, histology, and immunohistochemicalproceduresImmediately before necropsy, blood samples were col-

lected and the serum levels of glucose, total cholesterol, andtriglycerides were measured spectrometrically. During nec-ropsy, the liver was removed and weighed, and representativesamples were either fixed in 4% phosphate buffered formalinduring 24 h for paraffin embedding or frozen in liquid nitro-gen for Western blotting analysis. The paraffin blocks werecut into 5-µm-thick sections and stained with hematoxylin–eosin (HE) for histological evaluation and immunohistochem-ical reaction for glutathione S-transferase placental form(GST-P) enzyme (Furtado et al. 2009).PNL and tumors were classified as altered foci of hepato-

cytes (AFH) showing clear–eosinophilic or basophilic celltypes or hepatocellular adenomas (HA) or hepatocellular cellcarcinomas (HCC), respectively, according to previously pub-lished criteria (Bannasch et al. 2003).GST-P expression in the liver sections was immunohisto-

chemically detected using a polymer system (NovoLink MaxPolymer Detection System, Leica/Novocastra, Newcastle,UK). Briefly, histological liver sections were put on silanizedcoated slides, deparaffinized, and rehydrated with graded al-cohol. Then, the slides were treated with 3% H2O2 in phos-phate-buffered saline for 20 min, nonfat milk for 60 min,rabbit polyclonal anti-GST-P (Medical and Biological Labo-ratories Co., Tokyo, Japan, clone 311, 1:1000 dilution) anti-body for 16 h at 4 °C, post-primary blocked for 30 min, andpolymer for 30 min. Chromogen generation was achievedwith 3,3-diaminobenzidine tetrahydrochroride (DAB, Sigma–Aldrich Co., St. Louis, Mo., USA) as the substrate to demon-strate the sites of peroxidase binding. The slides were coun-terstained with Harris’s hematoxylin.PNL expressing uniform (distinct borders) and nonuniform

(indistinct borders) staining for GST-P were classified as per-sistent and remodeling phenotypes, respectively, according topreviously published criteria (Imai et al. 1997; Mazzantini etal. 2008).

Protein expression analysisProtein levels of PCNA, cleaved caspase-3, and bcl-2 from

liver samples were determined by Western blot technique, us-ing b-actin protein as a normalizer. Liver samples were ho-mogenized in lysis buffer (1% Triton X-100, 10 mmol·L–1

sodium pyrophosphate, 100 mmol·L–1 NaF, 10 µg·mL–1 aproti-nin, 1 mmol·L–1 phenylmethylsulfonylfluoride, 0.25 mmol·L–1

Na3VO4, NaCl 150 mmol·L–1, and Tris-HCl 50 mmol·L–1

pH 7.5). The samples were centrifuged at 11 000 r·min–1for 20 min, and 50 mL of homogenate fraction was re-sus-pended in 25 mL of Laemmli loading buffer (2% SDS, 20%glycerol, 0.04 mg·mL–1 bromophenol blue, 0.12 mol·L–1

Tris-HCl, pH 6.8, and 0.28 mol·L–1 b-mercaptoethanol). Anamount of 70 mg of total protein was fractioned by SDS–PAGE gel (12%) and stained with Coomassie blue to con-firm equal loading of each sample. Proteins were transferredfrom gel to a nitrocellulose membrane (Bio-Rad Laborato-ries, Hercules, Calf., USA). Nonspecific binding sites wereblocked using 3% bovine serum albumin solution (BSA) inphosphate–saline buffer (PBS-T: NaH2PO4·H2O 0.1 mol·L–1,Na2HPO4·7H2O 0.1 mol·L–1, NaCl 0.15 mol·L–1, Tween-200.1% pH 7.4) for 10 min. Membranes were incubated withspecific primary antibodies for PCNA (1:200), bcl-2(1:200), cleaved caspase-3 (1:500), and b-actin (1:100) di-luted in BSA 1% solution for 10 min. PCNA, bcl-2, andb-actin antibodies were purchased from Santa Cruz Biotech-nology (Calif., USA) and cleaved caspase-3 from and CellSignaling Technology Inc. (Calif., USA). After 4 wash stepswith PBS-T, membranes were incubated with specific horse-radish peroxidase-conjugated secondary antibodies, accord-ing to the primary antibodies used (1:2000). Finally, after 4wash steps, the membranes were submitted to immunoreac-tive protein signals detected using a SuperSignal West PicoChemiluminescent Substrate Kit (Thermo Fisher Scientific,Rockford, Ill., USA), according to manufacturer’s instruc-tions. Signals were captured in an immunoblotting film pa-per and the band intensities were quantified usingdensitometry analysis software (Image J software for win-dows, version 1.71, 2006, Austria).

Lipid peroxidation and total glutathione analysis

Malonaldehyde (MDA) analysisLipid peroxidation was assessed through the levels of

MDA, measured by HPLC. Approximately 1 g of liver washomogenized in phosphate buffer pH = 6.5, following a ratioof 1:5, and was then centrifuged for 5 min at 3500 r·min–1. In50 mL of each sample was added 250 mL of phosphoric acid(1.22 mol· L–1), 450 mL of deionized water, and 250 mL ofthiobarbituric acid (TBA 0.67% — prepared in equal propor-tions in acetic acid and water). The reaction was then incu-bated for 1 h in a water bath at 95 °C and then cooled in anice bath at 4 °C (Young and Trimble 1991). To each 200 mLof sample was added 360 mL of methanol and 40 mL of so-dium hydroxide (1 mol·L–1), after being filtered throughMillipore coupled to the syringe, straight to vial. The sampleswere chromatographed on ODS RP18 column VC-250 mm ×4.6 mm with 25 mmol·L–1 phosphate buffer 50:50 (v/v) andpH 6.5. The methanol was the mobile phase at a flow rateof 0.8 mL·min–1. The fluorimetric detection was performedwith lexc 532 nm and lemm 553 nm and using a detectormodel RF-10AXL (Shimadzu, Tokyo, Japan). The resultswere expressed as mmol·mL–1 and the peak of the MDA–TBA adduct was calibrated with a standard solution proc-essed 1,1,3,3 tetraethoxypropane equimolar amounts ofMDA.

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Glutathione analysisLiver samples were thawed, minced, and homogenized to a

concentration of 1:10 (w/v) in 1.15% KCl. Then, liver homo-genates (600 µL) were added to ice-cold 5% trichloroaceticacid and centrifuged at 300 r·min–1 (7000g) for 15 min at4 °C, and the supernatants were used to quantify glutathionecontent in spectrophotometer at 412 nm (Biochrom, Libra522, Berlin, Germany). The results were expressed asmmol·mL–1 and compared with a standard curve using cys-teine (2 mmol·L–1) (Sedlak and Lindsay 1968).

Statistical analysisBody weight, food intake, relative liver weight, body fat,

biochemical markers, GSTP-positive lesions and multiplicityof neoplastic lesion, PCNA, cleaved caspase-3, and bcl-2protein levels were analyzed by ANOVA test or the Kruskal–Wallis test. The incidences of different types of PNL andneoplastic lesions were examined using c2 test or the Fish-er’s exact test. Significance was set at p < 0.05. Statisticalanalysis was performed using the Jandel Sigma Stat Soft-ware (Jandel Corp., San Rafael, Calif., USA).

Results

During the stages of promotion–progression of liver carci-nogenesis in the RH model, groups fed HFD presented a re-duction in body weight when compared with the respectivecontrol groups fed LFD (Table 1). At the end of the experi-mental period, the mean values of body weight from groupssubmitted to swim training were significantly lower (p <0.05) than respective untrained groups fed LFD or HFD (Ta-ble 1). Due to higher fat content, groups receiving HFD pre-sented a significant reduction (p < 0.001) in food intakewhen compared with the groups receiving LFD (Table 1).The mean values of relative liver weights were similar amonggroups but body fat mass and total cholesterol were signifi-cantly higher in the untrained group fed HFD than respectiveuntrained group fed LFD (p < 0.05). A significant reduction

(p < 0.05) in body weight, body fat mass, and total choles-terol was detected in groups fed HFD and submitted to swimtraining when compared with their respective untrainedgroup.Oxidative stress in the liver was evaluated by measuring

the concentration of MDA, as a lipid peroxidation marker,and reduced glutathione (GSH) (Fig. 1). MDA and totalGSH concentrations did not differ between untrained groupsfed LFD or HFD. However, a significant reduction (p < 0.05)in MDA but an increase in reduced GSH levels (p < 0.05)was observed in group fed LFD and swim-trained when com-pared with their respective untrained group.PCNA, cleaved caspase-3, and bcl-2 protein levels were

analyzed in the liver through Western blotting technique(Fig. 2). There was a significant reduction (p < 0.05) inPCNA protein levels in the group fed LFD and swim-trainedcompared with the respective untrained control group. Also,a significant reduction (p < 0.001) in bcl-2 protein levels as-sociated to a significant increase (p < 0.05) in cleaved caspase-3 protein levels were observed in groups submitted to swimtraining in comparison to the respective untrained groups.After the histopathological analyses (Table 2), the main

PNL observed in carcinogens-treated groups was character-ized by AFH, showing different types of clear–eosinophiliccell (most frequent PNL, Fig. 3C) or basophilic cell pheno-types. Neoplastic lesions included HA, showing eosinophiliccell phenotype (Fig. 3D). The incidence of different types ofPNL and HA did not differ among groups, but the meannumber of large tPNL, pPNL (Figs. 3A–3B) and HA perliver area was significantly reduced (p < 0.02, p < 0.05, andp < 0.03, respectively) in the group fed LFD and submittedto swim training when compared with the respective controluntrained group.

Discussion

The results indicated that promotion–progression stages ofliver carcinogenesis were successively obtained herein because

Table 1. Effects of swim training on general and biochemical parameters evaluated in male Wistar rats fed a low-fat diet (LFD) or high-fat diet (HFD).

DEN/2-AAF

Parameters LFD–untrained LFD–trainedHFD–untrained(n = 10)

HFD–trained(n = 10)

GeneralBody weight a at week 5(g) 380.4±9.33 382.0±8.58 380.1±5.57 380.4±9.04Body weight at week 13 (g) 447.7±11.84 448.1±12.57 429.2±10.13 430.6±14.88Body weight at week 22 (g) 460.3±15.3 411.1±15.4* 424.4±10.5* 391.7±22.5*,**Food consumption (g·rat–1·d–1)b 24.59±3.10 23.25±5.71 16.86±5.26* 18.89±5.03*Fat body (%)c 4.91±0.26 3.96±0.36* 5.91±0.14* 4.30±0.38**Relative liver weight (%) 2.81±0.23 2.85±0.21 2.90±0.13 2.91±0.21Biochemical dataGlucose (mg·dL–1) 147.32±6.75 138.28±10.43 153.53±6.33 156.01±9.35Triglycerides (mg·dL–1) 48.41±4.26 47.40±9.53 59.65±8.01 51.12±5.67Cholesterol (mg·dL–1) 72.70±5.16 65.89±5.49 93.53±7.34* 72.18±5.01**

Note: Data are means ± SE. DEN/2AAF, diethylnitrosamine and 2-acetaminofluorene hepatocarcinogens. *, **, Differentfrom LFD–untrained or HFD–untrained groups, respectively, 0.05 < p < 0.001.

aWeeks 5, 13, and 22 represent HFD introduction, exercise introduction, and sacrifice, respectively.bAfter HFD introduction.cAbdominal, retroperitoneal, and epididymal white adipose tissues.



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of rapid and synchronized development of preneoplastic andneoplastic lesions in RH model. The beneficial action ofswim training was well characterized by body weight andbody fat loss in the LFD and HFD groups. Also, swimtraining normalized total cholesterol levels in the HFDgroup, and it reduced the MDA/total GSH levels, numberof GST-P-positive pPNL, and adenomas in the LFD group.Also in groups fed LFD, a favorable balance in PCNA,cleaved caspase-3, and bcl-2 levels was detected in the liverfrom the trained group in relation to untrained group.Some studies have shown that high-fat feeding regimens

induce obesity, insulin resistance, dyslipidemia, cardiovascu-lar changes, and nonalcoholic fatty liver disease (Young andKirkland 2007; Ahmed et al. 2009; Wu et al. 2011). More-over, the most hepatic deleterious effects of the high-fat diet-ary regimen have been assessed using NASH liquid diets orlipogenic methionine choline-deficient diet (Wang et al.2009; Ahmed et al. 2009; Wu et al. 2011). These dietary reg-imens induce steatohepatitis without a significant incrementin body mass or an elevation of serum levels in total choles-terol or triglycerides (de Lima et al. 2008; Wang et al. 2008,2009; Ahmed et al. 2009; Wu et al. 2011). The same high-fatdiet used in the present study was effective in promoting obe-sity in noncancerous male Wistar rats, which was demon-strated by an increasing adiposity index in association with ahigher body weight (Nascimento et al. 2011). In contrast,male Wistar rats initiated–promoted for liver carcinogenesisand after being fed 16-week HFD presented lowered bodyweights in relation to the LFD group, although cholesteroland body fat mass was increased. During progression ofHCC transformation in the RH model, a reduction in total

biliary bile acid output and bile flow and an increase in en-ergy carbohydrate metabolism have been described (Monte etal. 2000; Bannasch et al. 2003). Thus, the findings on thebody weight gain in the HFD groups could be associatedwith differences in physiology, metabolism, and liver diseasestatus induced by carcinogens treatment and the balance be-tween higher fat and lower carbohydrate ingestion in relationto the LFD group (Covasa 2010).Chemically induced liver carcinogenesis is a multi-step

process that has been well documented by morphological, bi-ochemical, and genetic changes in the liver (De Miglio et al.2003; Pitot 2007; Mazzantini et al. 2008; French 2010). Afterthe DNA-damaging event that initiates the carcinogenesisprocess, surrogate PNL appear and depending on the treat-ment or time, may be followed by benign tumors (adenomas)and eventually by HCC (Bannasch et al. 2003; Pitot 2007). Atypical feature of PNL in the RH model is their capability ofexpressing 1 of 2 options under genetic control: spontane-ously remodeling to a normal-appearing liver by the majority(95%–98%) or persistence with cell proliferation and evolu-tion to cancer by a small minority (2%–5%) (Tatematsu et al.1983; Farber and Rubin 1991). Persistence of PNLs could in-dicate a block in remodeling with increases in cell prolifera-tion, by loss of differentiation or reduction of apoptosis,which appears to be genetically linked to enhanced evolutionof hepatocellular carcinoma (Imai et al. 1997; De Miglio etal. 2003; Mazzantini et al. 2008). The present study demon-strated that swim training reduced the number of larger PNL(persistent and remodeling) but mainly the number of persis-tent GST-P positive PNL (pPNL) in the group fed on a LFD.Taken together, these data demonstrates that swim training asa result of exercise postconditioning has a protective effect onthe expansion of persistent PNL in male Wistar rats exposedto a dietary regimen with a lowered fat intake.Cell proliferation and apoptosis balance play an important

role during the progression of rat liver carcinogenesis (Pitot2007). Proliferating cell nuclear antigen, a co-factor forDNA-polymerase-d that leads to DNA replication and DNAdamage repair, has been considered a feasible marker for cellproliferation (Iatropoulos and Williams 1996). Apoptosis isan event that results in cellular death, which occurs underboth physiological and pathological conditions, and is regu-lated by numerous modulators, including the Bcl-2 familyand caspases (Kuwana and Newmeyer 2003; Brunelle andLetai 2009; Ulukaya et al. 2011). The Bcl-2 family membersare pivotal in the regulatory processes that either repress (e.g.,bcl-2 and bcl-x1) or induce (e.g., bak, bax, and bad) apop-tosis or programmed cellular death (Kuwana and Newmeyer2003; Brunelle and Letai 2009; Ulukaya et al. 2011). Thecaspases are a family of cysteine proteases that function ascentral regulators of cell death (Ulukaya et al. 2011). Somecaspases are known as initiator caspases (caspase 2, 8, 9,and 10), whereas the others are effector caspases (caspase 3,6, and 7) (Ulukaya et al. 2011). The results of Westernblotting suggest that regular and moderate swim training re-duced the bcl-2 and PCNA levels but it increased cleavedcaspase-3 in the liver from the group feeding on a LFD. Incontrast, bcl-2 and cleaved caspase-3 protein levels werealso changed by exercise in the liver from the group trainedand fed on a HFD. Therefore, the protective effect of swimtraining in the group feeding on a LFD could be due to a

Fig. 1. (A) Malonaldehyde (MDA, mmol·mL–1) and (B) reducedglutathione (GSH, mmol·mL–1) levels in the liver from differentgroups. LFD, low-fat diet; HFD, high-fat diet; UT, untrained ani-mals; T, trained animals. Values are means ± SE. *, Different fromLFD–UT group, p < 0.05.



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favorable balance in PCNA, cleaved caspase-3, and bcl-2protein levels. Thus, the cell proliferation levels rather thanapoptosis constitute the probably major determinant factorfor progression of liver carcinogenesis.Oxidative stress is potentially deleterious to cells and tis-

sues and associated with the progression of many diseases,including experimental and human liver carcinogenesis in ro-dents and humans (Sánchez-Pérez et al. 2005; Sasaki 2006).In general, the eukaryotic cells are continuously attacked byreactive oxygen species (ROS), which arise through endoge-nous pathways (i.e., mitochondrial and P450 metabolism, de-generative or inflammatory diseases, etc.) as well as byexogenous pathways (i.e., exhaustive exercise, radiation,ozone, pollutants, etc.) (Ziech et al. 2011). The oxidativeDNA damage induced by ROS may induce DNA base modi-

fications, single- and double-strand breaks, and the formationof apurinic–apyrimidinic lesions, resulting in mutations andgenomic instability when they are not accurately repaired(Ziech et al. 2011). Physical exercise and training can havepositive or negative effects on oxidative stress depending onthe training load, training specificity, and the basal level oftraining (Finaud et al. 2006; Packer et al. 2008; da Silva etal. 2009). A recent study conducted by our research grouphas revealed that swimming administrated as exercise precon-ditioning was able to protect various rat tissues against ge-netic damage induced by doxorubicin, an anti-tumor drugthat also induces free radical formation and lipid peroxidation(Martins et al. 2012). The results of the MDA and reducedGSH analyses, which reflect lipid peroxidation status, indi-cate that moderate swim training reduced lipid peroxidation

Fig. 2. (A) Representative immunoblots of PCNA (34 kDa), cleaved caspase-3 (17/19 kDa), bcl-2 (28 kDa), and b-actin (43 kDa) assayed byWestern blotting. (B–D) Data from (mean ± SE) IOD calculated from the ratio of PCNA, cleaved caspase-3, and bcl-2 to b-actin levels forthe different groups. LFD, low-fat diet; HFD, high-fat diet; UT, untrained animals; T, trained animals. Values are means ± SE. *, Differentfrom LFD–UT or HFD–UT groups, < 0.05 < p < 0.001.



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and increased GSH in the liver from the group fed LFD, in-dicating attenuation in hepatic oxidative stress in a dietaryregimen with a lowered fat intake.

In summary, the present study demonstrated that the swimtraining protocol may attenuate liver carcinogenesis under anadequate dietary regimen with lowered fat intake. Also, the

Table 2. Effects of swim training on histopathology and development of GST-P-positive PNL evaluated in male Wistarrats fed a low-fat diet (LFD) or high-fat diet (HFD).

DEN/2-AAF

ParametersLFD–untrained(n = 10)

LFD–trained(n = 10)

HFD–untrained(n = 10)

HFD–trained(n = 10)

HE analysisPNL (incidence) 100% 100% 100% 100%HA (incidence) (multiplicity) 70% (1.2±0.3) 40% (0.3±0.2)* 80% (1.6±0.3) 70% (0.9±0.2)GST-P analysis (number/cm2)pPNL 12.46±1.50 4.84±1.08* 13.40±2.41 10.96±1.90rPNL 18.13±3.14 17.21±8.18 19.73±3.57 17.69±2.74tPNL (diameter 0.2 to <1.1 mm) 24.01±3.64 19.30±4.94 31.71±5.37 26.02±4.83tPNL (diameter 1.2 to 2.1 mm) 7.91±1.13 3.45±0.95* 8.89±1.10 6.25±1.30

Note: Data are in percentages or means ± SE. DEN/2AAF, diethylnitrosamine and 2-acetaminofluorene hepatocarcinogens; PNL,preneoplastic lesion; HA, hepatocellular adenoma; multiplicity, mean number of HA; pPNL, persistent preneoplastic lesion;rPNL, remodeling preneoplastic lesion; tPNL, total pPNL + rPNL. *, Different from LFD–untrained group, 0.05 < p < 0.02.

Fig. 3. (A–B) Representative persistent and remodeling glutathione S-transferase-positive preneoplastic lesions (20× objective) in a immuno-histochemically-stained liver section, respectively; (C–D) representative preneoplastic lesion (arrows, 20× objective ) and hepatocellular ade-noma (arrows, 4× objective) in a HE-stained liver section, respectively.



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exercise regimen leads to body weight and adiposity massloss, and improved serum cholesterol in high-fat feeding reg-imen.

AcknowledgementsThe study was supported by Fundação para o Desenvolvi-

mento da UNESP (FUNDUNESP DFP- 0028610). Marco A.Aguiar e Silva and Luis F Barbisan were recipients of fellow-ships from FAPESP (2010/03056-9) and CNPq (301585/2009-1), respectively.

ReferencesAhmed, U., Redgrave, T.G., and Oates, P.S. 2009. Effect of dietary fat

to produce non-alcoholic fatty liver in the rat. J. Gastroenterol.Hepatol. 24(8): 1463–1471. doi:10.1111/j.1440-1746.2009.05870.x. PMID:19702912.

Andersen, J.B., Loi, R., Perra, A., Factor, V.M., Ledda-Columbano,G.M., Columbano, A., and Thorgeirsson, S.S. 2010. Progenitor-derived hepatocelular carcinoma model in the rat. Hepatology, 51(4):1401–1409. doi:10.1002/hep.23488. PMID:20054870.

Baltgalvis, K.A., Berger, F.G., Peña, M.M., Davis, J.M., and Carson,J.A. 2009. The interaction of a high-fat diet and regular moderateintensity exercise on intestinal polyp development in Apc Min/+mice. Cancer Prev. Res. (Phila.), 2(7): 641–649. doi:10.1158/1940-6207.CAPR-09-0017. PMID:19549797.

Bannasch, P., Haertel, T., and Su, Q. 2003. Significance of hepaticpreneoplasia in risk identification and early detection of neoplasia.Toxicol. Pathol. 31(1): 134–139. doi:10.1080/01926230390173923.PMID:12597458.

Brunelle, J.K., and Letai, A. 2009. Control of mitochondrialapoptosis by the Bcl-2 family. J. Cell Sci. 122(4): 437–441.doi:10.1242/jcs.031682. PMID:19193868.

Cohen, L.A., Boylan, E., Epstein, M., and Zang, E. 1992. Voluntaryexercise and experimental mammary cancer. Adv. Exp. Med. Biol.322: 41–59. doi:10.1007/978-1-4684-7953-9_5. PMID:1442300.

Covasa, M. 2010. Deficits in gastrointestinal responses controllingfood intake and body weight. Am. J. Physiol. Regul. Integr. Comp.Physiol. 299(6): R1423–R1439. doi:10.1152/ajpregu.00126.2010.PMID:20861277.

da Silva, L.A., Pinho, C.A., Rocha, L.G., Tuon, T., Silveira, P.C., andPinho, R.A. 2009. Effect of different models of physical exerciseon oxidative stress markers in mouse liver. Appl. Physiol. Nutr.Metab. 34(1): 60–65. doi:10.1139/H08-132. PMID:19234586.

de Lima, V.M.R., Oliveira, C.P.M.S., Alves, V.A.F., Chammas, M.C.,Oliveira, E.P., Stefano, J.T., et al. 2008. A rodent model of NASHwith cirrhosis, oval cell proliferation and hepatocellular carcinoma.J. Hepatol. 49(6): 1055–1061. doi:10.1016/j.jhep.2008.07.024.PMID:18929425.

De Miglio, M.R., Simile, M.M., Muroni, M.R., Calvisi, D.F., Virdis,P., Asara, G., et al. 2003. Phenotypic reversion of rat neoplasticliver nodules is under genetic control. Int. J. Cancer, 105(1): 70–75. doi:10.1002/ijc.11044. PMID:12672032.

El-Serag, H.B., and Rudolph, K.L. 2007. Hepatocellular carcinoma:epidemiology and molecularcarcinogenesis. Gastroenterology,132(7): 2557–2576. doi:10.1053/j.gastro.2007.04.061. PMID:17570226.

Farazi, P., and Depinho, R.A. 2006. The genetic and environmentalbasis of hepatocelular carcinoma. Discov. Med. 6(35): 182–186.PMID:17234139.

Farber, E., and Rubin, H. 1991. Cellular adaptation in the origin anddevelopment of cancer. Cancer Res. 51(11): 2751–2761. PMID:2032214.

Finaud, J., Lac, G., and Filaire, E. 2006. Oxidative Stress: Relation-

ship with Exercise and training. Sports Med. 36(4): 327–358.doi:10.2165/00007256-200636040-00004. PMID:16573358.

French, S.W. 2010. Molecular events in hepatic preneoplasia: areview. Exp. Mol. Pathol. 88(2): 219–224. doi:10.1016/j.yexmp.2010.01.005. PMID:20097196.

Furtado, K.S., Pires, P.W., Justulin, L.A., Jr, Rodrigues, M.A.,Felisbino, S.L., and Barbisan, L.F. 2009. Metalloproteinases 2 and9 activity during promotion and progression stages of rat livercarcinogenesis. J. Mol. Histol. 40(1): 1–11. doi:10.1007/s10735-008-9206-x. PMID:19052882.

Gobatto, C.A., de Mello, M.A., Sibuya, C.Y., de Azevedo, J.R., dosSantos, L.A., and Kokubun, E. 2001. Maximal lactate steady statein rats submitted to swimming exercise. Comp. Biochem. Physiol.A Mol. Integr. Physiol. 130(1): 21–27. doi:10.1016/S1095-6433(01)00362-2.

Heindryckx, F., Colle, I., and Van Vlierberghe, H. 2009. Experi-mental mouse models for hepatocellular carcinoma research. Int. J.Exp. Pathol. 90(4): 367–386. doi:10.1111/j.1365-2613.2009.00656.x. PMID:19659896.

Hill-Baskin, A.E., Markiewski, M.M., Buchner, D.A., Shao, H.,DeSantis, D., Hsiao, G., et al. 2009. Diet-induced hepatocellularcarcinoma in genetically predisposed mice. Hum. Mol. Genet.18(16): 2975–2988. doi:10.1093/hmg/ddp236. PMID:19454484.

Hoffman-Goetz, L. 2003. Physical activity and cancer prevention:animal tumor models. Med. Sci. Sports Exerc. 35(11): 1828–1833.doi:10.1249/01.MSS.0000093621.09328.70. PMID:14600546.

Iatropoulos, M.J., and Williams, G.M. 1996. Proliferation markers.Exp. Toxicol. Pathol. 48(2–3): 175–181. doi:10.1016/S0940-2993(96)80039-X. PMID:8672872.

Imai, T., Masui, T., Ichinose, M., Nakanishi, H., Yanai, T., Masegi,T., et al. 1997. Reduction of glutathione S-transferase P formmRNA expression in remodeling nodules in rat liver revealed by insitu hybridization. Carcinogenesis, 18(3): 545–551. doi:10.1093/carcin/18.3.545. PMID:9067555.

Jemal, A., Siegel, R., Xu, J., and Ward, E. 2010. Cancer statistics,2010. CA Cancer J. Clin. 60(5): 277–300. doi:10.3322/caac.20073. PMID:20610543.

Kelley, G.A., Kelley, K.S., Roberts, S., and Haskell, W. 2011.Efficacy of aerobic exercise and a prudent diet for improvingselected lipids and lipoproteins in adults: a meta-analysis ofrandomized controlled trials. BMC Med. 9(1): 74. doi:10.1186/1741-7015-9-74. PMID:21676220.

Kuo, H.K., Yen, C.J., Chen, J.H., Yu, Y.H., and Bean, J.F. 2007.Association of cardiorespiratory fitness and levels of C-reactiveprotein: Data from the National Health and Nutrition ExaminationSurvey 1999–2002. J. Cardiol. 114(1): 28–33. doi:10.1016/j.ijcard.2005.11.110. PMID:16678922.

Kuwana, T., and Newmeyer, D.D. 2003. Bcl-2-family proteins andthe role of mitochondria in apoptosis. Curr. Opin. Cell Biol. 15(6):691–699. doi:10.1016/j.ceb.2003.10.004. PMID:14644193.

Martins, R.A., Minari, A.L., Chaves, M.D., dos Santos, R.W.,Barbisan, L.F., and Ribeiro, D.A. 2012. Exercise preconditioningmodulates genotoxicity induced by doxorubicin in multiple organsof rats. Cell Biochem. Funct. 30(4): 293–296. doi:10.1002/cbf.2799. PMID:22287211.

Mazzantini, R.P., de Conti, A., and Moreno, F.S. 2008. Persistent andremodeling hepatic preneoplastic lesions present differences in cellproliferation and apoptosis, as well as in p53, Bcl-2 and NF-kBPathways. J. Cell. Biochem. 103(2): 538–546. doi:10.1002/jcb.21420. PMID:17546582.

McGlynn, K.A., and London, W.T. 2011. The global epidemiology ofhepatocellular carcinoma: present and future. Clin. Liver Dis.15(2): 223–243, vii–x. doi:10.1016/j.cld.2011.03.006. PMID:21689610.



App

l. Ph

ysio

l. N

utr.

Met

ab. D

ownl

oade

d fr

om w

ww

.nrc

rese

arch

pres

s.co

m b

y G

eorg

e M

ason

Uni

vers

ity o

n 05

/02/

13Fo

r pe

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al u

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nly.

Monte, M.J., Palomero, F., Sainz, G.R., Domínguez, M., Díez, M.,Toraño, A., and Marin, J.J. 2000. Bile acid secretion during ratliver carcinogenesis. Life Sci. 66(12): 1085–1095. doi:10.1016/S0024-3205(00)00412-4. PMID:10737359.

Na, H.K., and Oliynyk, S. 2011. Effects of physical activity on cancerprevention. Ann. N. Y. Acad. Sci. 1229(1): 176–183. doi:10.1111/j.1749-6632.2011.06105.x. PMID:21793853.

Nascimento, A.F., Luvizotto, R.A., Leopoldo, A.S., Lima-Leopoldo,A.P., Seiva, F.R., Justulin, L.A., Jr, et al. 2011. Long-term high-fatdiet-induced obesity decreases the cardiac leptina receptor withoutapparent lipotoxicity. Life Sci. 88(23–24): 1031–1038. doi:10.1016/j.lfs.2011.03.015. PMID:21457721.

Newell, P., Villanueva, A., Friedman, S.L., Koike, K., and Llovet, J.M.2008. Experimental models of hepatocellular carcinoma. J.Hepatol. 48(5): 858–879. doi:10.1016/j.jhep.2008.01.008.PMID:18314222.

Nobili, V., Carter-Kent, C., and Feldstein, A.E. 2011. The role oflifestyle changes in the management of chronic liver disease. BMCMed. 9(1): 70. doi:10.1186/1741-7015-9-70. PMID:21645344.

Packer, L., Cadenas, E., and Davies, K.J. 2008. Free radicals andexercise: an introduction. Free Radic. Biol. Med. 44(2): 123–125.doi:10.1016/j.freeradbiomed.2007.05.031. PMID:18191747.

Perše, M., Injac, R., Štrukelj, B., and Cerar, A. 2012. High fat mixedlipid diet modifies protective effects of exercise on 1,2dimethylhydrazine induced colon cancer in rats. Technol. CancerRes. Treat. 11(3): 289–299. doi:10.7785/tcrt.2012.500297. PMID:22417061.

Pitot, H.C. 2007. Adventures in hepatocarcinogenesis. Annu. Rev.Pathol. 2(1): 1–29. doi:10.1146/annurev.pathol.2.010506.092027.PMID:18039091.

Sánchez-Pérez, Y., Carrasco-Legleu, C., García-Cuellar, C., Pérez-Carreón, J., and Hernández-García, S. 2005. Oxidative stress incarcinogenesis. Correlation between lipid peroxidation and induc-tion of preneoplastic lesions in rat hepatocarcinogenesis. CancerLett. 217(1): 25–32. doi:10.1016/j.canlet.2004.07.019. PMID:15596293.

Sasaki, Y. 2006. Does oxidative stress participate in the developmentof hepatocellular carcinoma? J. Gastroenterol. 41(12): 1135–1148.doi:10.1007/s00535-006-1982-z. PMID:17287893.

Schmitz, K.H., Holtzman, J., Courneya, K.S., Mâsse, L.C., Duval, S.,and Kane, R. 2005. Controlled physical activity trials in cancersurvivors: a systematic review and meta-analysis. Cancer Epide-miol. Biomarkers Prev. 14(7): 1588–1595. doi:10.1158/1055-9965.EPI-04-0703. PMID:16030088.

Sedlak, J., and Lindsay, R.H. 1968. Estimation of total, protein-bound

and non-protein sulfhydryl groups in tissue with Ellman’s reagent.Anal. Biochem. 25(1): 192–205. doi:10.1016/0003-2697(68)90092-4. PMID:4973948.

Siegel, A.B., and Zhu, A.X. 2009. Metabolic syndrome andhepatocellular carcinoma: two growing epidemics with a potentiallink. Cancer, 115(24): 5651–5661. doi:10.1002/cncr.24687.PMID:19834957.

Tatematsu, M., Nagamine, Y., and Farber, E. 1983. Redifferentiationas a basis for remodeling of carcinogen-induced hepatocytenodules to normal appearing liver. Cancer Res. 43(11): 5049–5058. doi:10.1002/cbf.1774. PMID:6137276.

Ulukaya, E., Acilan, C., and Yilmaz, Y. 2011. Apoptosis: why andhow does it occur in biology? Cell Biochem. Funct. 29(6): 468–480. doi:10.1002/cbf.1774. PMID:21773978.

Wang, Y., Ausman, L.M., Russell, R.M., Greenberg, A.S., and Wang,X.D. 2008. Increased Apoptosis in high-fat diet-induced nonalco-cholic steatothpatitis in rats is associated with c-jun NH2- terminalkinase activation and elevated proapoptotic bax. J. Nutr. 138(10):1866–1871. PMID:18806094.

Wang, Y., Ausman, L.M., Greenberg, A.S., Russell, R.M., and Wang,X.-D. 2009. Nonalcoholic steatohepatitis induced by a high-fatdiet promotes diethylnitrosamine-initiated early hepatocarcinogen-esis in rats. Int. J. Cancer, 124(3): 540–546. doi:10.1002/ijc.23995.PMID:19004024.

Wu, T.C., Chen, L.K., Tsai, S.H., Liaw, Y.H., and Hwang, B. 2011.Hepatic steatosis: an experimental model for quantification. Arch.Gerontol. Geriatr. 52(2): 164–166. doi:10.1016/j.archger.2010.03.009. PMID:20398952.

Young, G.S., and Kirkland, J.B. 2007. Rat models of caloric intakeand activity: relationships to animal physiology and human health.Appl. Physiol. Nutr. Metab. 32(2): 161–176. doi:10.1139/h06-082.PMID:17486157.

Young, I.S., and Trimble, E.R. 1991. Measurement of malondialde-hyde in plasma by high performance liquid chromatography withfluorimetric detection. Ann. Clin. Biochem. 28(Pt. 5): 504–508.PMID:1958055.

Ziech, D., Franco, R., Pappa, A., and Panayiotidis, M.I. 2011.Reactive oxygen species (ROS) induced genetic and epigeneticalterations in human carcinogenesis. Mutat. Res. 711(1–2): 167–173. doi:10.1016/j.mrfmmm.2011.02.015. PMID:21419141.

Zivkovic, A.M., German, J.B., and Sanyal, A.J. 2007. Comparativereview of diets for the metabolic syndrome: implications fornonalcoholic fatty liver disease. Am. J. Clin. Nutr. 86(2): 285–300.PMID:17684197.



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effects of swim training on liver carcinogenesis in male wistar rats fed a low-fat or high-fat diet

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