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Nutrition 28 (2012) 81–91

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Nutrition

journal homepage: www.nutr i t ionjrnl .com

Basic nutritional investigation

Hypolipidimic and antioxidant activities of virgin olive oil and its fractions in2,4-diclorophenoxyacetic acid–treated rats

Amel Nakbi Ph.D. a,*, Wafa Tayeb Ph.D. a, Samia Dabbou Ph.D. a, Issam Chargui Ph.D. b,Manel Issaoui Ph.D. a, Abdelfattah Zakhama Dr. c, Abdelhedi Miled Ph.D. d,Mohamed Hammami Ph.D. a,e

aBiochemistry Laboratory, UR03/ES08, Human Nutrition and Metabolic Disorders, USCR Mass Spectrometry, Faculty of Medicine of Monastir, Monastir, Tunisiab Laboratory of Histology and Cytogenetic, Faculty of Medicine of Monastir, Monastir, Tunisiac Laboratory of Anatomopathology, Faculty of Medicine of Monastir, Monastir, TunisiadDepartment of Biochemistry CHU Hached, Sousse, TunisiaeKing Saud University, Riyadh, Saudi Arabia

a r t i c l e i n f o

Article history:Received 22 September 2010Accepted 15 February 2011

Keywords:Virgin olive oilHydrophilic fractionLipophilic fraction2,4-DHypolipidemicAntioxidant activitiesLiver damage

This research was supported by a grant from the MiSup�erieur et de la Recherche Scientifique (UR03ESD�esordres M�etaboliques et USCR-SM).* Corresponding author. Tel.: þ216-73-462-200; fax

E-mail address: nakbia@yahoo.fr (A. Nakbi).

0899-9007/$ - see front matter � 2012 Elsevier Inc. Adoi:10.1016/j.nut.2011.02.009

a b s t r a c t

Objective: We examined the effects of extra virgin olive oil (EVOO) and its hydrophilic and lipo-philic fractions on serum lipids, oxidative stress, and morphologic and functional liver damagesinduced by 2,4-diclorophenoxyacetic acid (2,4-D).Methods: Male Wistar rats were divided randomly into eight groups: control; 2,4-D at a dose of5 mg/kg of body weight (2,4-D); 2,4-D plus EVOO (2,4-D/EVOO); 2,4-D plus the hydrophilic fraction(2,4-D/OOHF); 2,4-D plus the lipophilic fraction (2,4-D/OOLF); only EVOO (EVOO); only thehydrophilic fraction (OOHF); and only the lipophilic fraction (OOLF). These components wereadministered daily by gavage for 4 wk.Results: A hepatic architecture aberration, increased activities of aspartate and alanine amino-transferase enzymes, total and low-density lipoprotein cholesterol, and malondialdehyde (MDA)level, and a decreased antioxidant defense system were observed in the 2,4-D group. Theadministration of EVOO restored the damage caused by 2,4-D by a significant decrease of plasmatotal and low-density lipoprotein levels and a moderate increase of high-density lipoproteincholesterol. The 2,4-D/OOHF group exhibited a pronounced enhancement of the antioxidantdefense system by an increase of superoxide dismutase, catalase, and glutathione peroxidase levelsand a decrease of plasma and liver MDA levels. However, less improvement in the liver histo-architecture and antioxidant status was observed in rats supplemented with OOLF diet, despite itsrichness in a-tocopherol.Conclusion: Extra virgin olive oil may be a potential functional food source of antioxidants than candecrease the frequency of cardiovascular diseases and liver damage.

� 2012 Elsevier Inc. All rights reserved.

Introduction

Oxidative stress produced by reactive oxygen species hasbeen linked to the development of cardiovascular and neuro-degenerative diseases and cancer [1,2]. However, reactive oxygenspecies are constantly formed as a byproduct of normal

nist�ere de l’Enseignement08 Nutrition Humaine et

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metabolic reactions and their formation is accelerated by acci-dental exposure to occupational chemicals such as pesticides.

In Tunisia, 2,4-diclorophenoxyacetic acid (2,4-D), which ischemically derived from phenoxyacetic acid, is a commonly usedpesticide in the north, the most important agricultural region inTunisia. Its herbicidal activity is mediated by an auxin-likecapacity to alter normal protein synthesis and cell division inplant meristems and leaves [3]. Furthermore, it has recently beensuggested that its herbicidal activity may be due to an increase inthe production of reactive oxygen species leading to the gener-ation of oxidative stress in the weed [4]. Several reports haveshown that 2,4-D produces oxidative stress and/or depletes

Table 1Composition of standard diet fed to rats

Ingredients Quantities (g/kg)

DL-Methionine 3Corn starch 393Corn oil 155Sucrose 154Cellulose 50Mineral mix* 35Vitamin mixy 10Caseinz 200

A. Nakbi et al. / Nutrition 28 (2012) 81–9182

antioxidants in vitro and in vivo [5–7]. The liver is a primarytarget of adverse pesticide reactions because it is the predomi-nant site for the biotransformation of pesticides. However, 2,4-Dproduces hepatotoxicity and initiates the process of cell death bydecreasing cellular reduced glutathione, adenosine triphosphate,and reduced nicotinamide adenine dinucleotide [8].

Since antiquity, virgin olive oil has been known for itsmedicinal properties, and for many years, olive oil consumptionwas claimed as the basic food supply responsible for the lowincidence of cardiovascular diseases in some societies [9]. Oliveoil, a rich source of monounsaturated fatty acid (MUFA), is a maincomponent of the Mediterranean Diet. Virgin olive oil retains allthe lipophilic components of the fruit, small amounts ofa-tocopherol, and sizeable amounts of phenolic compoundswithstrong antioxidant properties, whereas refined olive oil losesmost of its antioxidants during the refining process [10].

Moreover, the beneficial effects of olive oil on coronary heartdisease risk factors are now recognized but often only attributedto the high MUFA content of the olive oil. It is known that anincreased consumption of MUFA instead of polyunsaturated fattyacid decreases the risk of atherosclerosis because it makes thecirculating lipoprotein less sensitive to peroxidation [11].Furthermore, the health effects of dietary MUFA have beenattributed to endothelial activation [12] and susceptibility oflow-density lipoprotein (LDL) to oxidation [13,14].

Besides containing large quantities of oleic acid, virgin oliveoil is rich in phenolic compounds. Previous research has sug-gested that the minor constituents of olive oil might have moreeffects on health than once believed [15,16]. The biologicalactivities of olive oil phenolic compounds have promptedseveral studies on their potential activity in the prevention ofcardiovascular diseases and cancer. However, the increasedhigh-density lipoprotein cholesterol (HDL-C) and decreasedoxidative damage to lipids were related to the phenolic contentof extra virgin olive oil (EVOO) in a dose-dependent manner [17].In recent years, the anti-inflammatory and vascular protectiveproperties of EVOO polyphenols have been extensively investi-gated [18,19]. Many studies have shown the powerful antioxi-dant properties of olive oil in vitro and in vivo [20–22], butwhether the effects really occur and the mechanisms respon-sible for these beneficial effects are not presently known [23].

Regarding the great importance in the protective effect ofvirgin olive oil against cardiovascular disease, studying differentfractions separately may be of great interest. The aim of thisstudy was to determine the effects of olive oil and its lipophilicand hydrophilic components on serum lipids and oxidative stressand morphologic and functional liver damages induced by 2,4-Din rats.

Materials and methods

Materials

The commercial formulation of 2,4-D (D�esormone Lourd; Nufarm Company,Wyke, Bradford, West Yorkshire, England), consisting of 600 g/L of 2,4-D esterbutyl glycol (register number H.96064, SEPCM), available in Tunisia was used in

Fig. 1. Chemical structure of 2,4-diclorophenoxyacetic acid.

the experiments (Fig. 1). 2-Thiobarbituric acid (TBA) was obtained from SigmaChemical Company (Taufkirchen, Germany); 1,1,3,3-tetramethoxypropane waspurchased from Sigma Chemical Company (St. Louis, MO, USA), and the Folin-Ciocalteu phenol reagent was purchased from Fluka Biochemika (Buchs,Switzerland). All other chemicals used were of analytical grade and wereobtained from Sigma Chemical Company or Merck (Darmstadt, Germany).

Oil sample analysis

The EVOO used in this experiment was obtained from a Ch�etoui cultivargrown in the north of Tunisia. The hydrophilic fraction (OOHF) was extractedfrom EVOO by the method of Montedoro et al. [24] using water instead ofmethanol to avoid its toxic effect in rats. Briefly, 10 g of EVOO was homogenizedwith 10 mL of water by a mixer (Ultra-Turrax T25 [IKA Labortechnik, Janke &Kunkel, Staufen, Germany]; 15 000 g/min) and centrifuged at 5000 � g for 10min. The extraction was performed two times.

The lipophilic fraction (OOLF) was obtained from EVOO as follows. The EVOOwas homogenized for 1 min with water (1:1, v/v), and the oil was separated bycentrifugation; this procedure was repeated six times. Then, the OOLF wasfiltered through a cellulose acetate membrane.

Fattyacidswere converted into fatty acidmethyl esters preparedbydissolving0.1 gof EVOOorOOLF in2mLof heptaneand0.2mLofKOH(0.2N) inmethanol andincubated for 1 h. Individual fatty acid methyl esters were separated and quanti-fied by a gas chromatograph (model 5890 Series II; Hewlett-Packard, Palo Alto, CA,USA) equippedwith aflame ionization detector and a fused silica capillary column(HP-INNOWAX [J & W Scientific, Agilent , Palo Alto, CA, USA]; 30 m length �0.25-mm inner diameter and 0.25 mm film thickness). The temperature wasprogrammed to increase from 170�C to 270�C at a rate of 5�C/min. Nitrogen ultrawas used as a carrier gas. Results were expressed as relative area percentage oftotal fatty acid methyl esters [25].

The oxidative stability index was evaluated by the Rancimat apparatus(model 743; Metrohm Schweiz AG, Zofingen, Switzerland) using 3 g of oil sampleheated to 120�C with 20l/h air flow [26]. The oxidative stability index wasexpressed as oxidation induction time (hours).

Carotenoids and chlorophylls (milligrams per kilogram of oil) were deter-mined at 470 and 670 nm, respectively, in cyclohexane using the specificextinction values according to the method of Minguez-Mosquera et al. [27].

Phenolic compounds were extracted, estimated colorimetrically at 765 nmusing the Folin-Ciocalteau reagent, and expressed as hydroxy tyrosyl equivalentsas reported by Montedoro et al. [24].

For the tocopherol analysis, the sample was diluted with n-hexane (1:10),the mixture was Vortex-mixed, and 200 mL was transferred to a test tube con-taining 600 mL of methanol and 200 mL of internal standard (300 mg/mL).High-performance liquid chromatographic separation was carried out ona Hewlett-Packard system (Waldbronn, Germany) comprising an HP-1100pump, a Rheodyne model 7725 injector (Cotati, CA, USA; loop volume 20 mL),an HP-1200 M multi-array detector, and a Supelcosil ODS-2 column (150 �4.5-mm inner diameter, 5 mm film thickness) [28].

Animal treatment

Male adult Wistar rats (Central Pharmacy, Tunis, Tunisia), weighing about200 to 230 g, were housed at 22 � 3�C, with 12-h light–dark periods, 40%

Total energy (kJ/100 g) 1631.2

* Mineral mixture (milligrams per kilogram of diet): CaHPO4, 17 200; KCl,4000; NaCl, 4000; MgO, 420; MgSO4, 2000; Fe2O3, 120; FeSO4$7H2O, 200; traceelements, 400 (MnSO4$H2O, 98; CuSO4$5H2O, 20; ZnSO4$7H2O, 80; CoSO4$7H2O,0.16; KI, 0.32; sufficient starch to 40 g/kg of diet).

y Vitamin mixture (milligrams per kilogram of diet): retinol, 12; cholecalcif-erol, 0.125; thiamine, 40; riboflavin, 30; pantothenic acid, 140; pyridoxine, 20;inositol, 300; cyanocobalamin, 0.1; menadione, 80; nicotinic acid, 200; choline,2720; folic acid, 10; p-amino benzoic acid, 100; biotin, 0.6; sufficient starch to20 g/kg of diet.

z Casein represents 86.3% protein.

Table 2Mean values of analytical parameters, fatty acid composition, oxidative stabilityand antioxidant content of EVOO, OOHF, and OOLF*

EVOO OOHF OOLF

Fatty acid (%)Palmitic acid (C16:0) 10.28 � 0.04 d 10.40 � 0.01Palmitoleic acid (C16:1u�7) 0.77 � 0.30 d 0.79 � 0.60Stearic acid (C18:0) 3.39 � 0.14 d 3.56 � 0.37Oleic acid (C18:1u�9) 64.80 � 1.99 d 62.58 � 3.71Linoleic acid (C18:2u�6) 14.34 � 0.90 d 15.04 � 0.54Linolenic acid (C18:3u�3) 2.90 � 1.99 d 1.66 � 0.82Arachidic acid (C20:0) 1.12 � 0.59 d 0.98 � 0.31Gadoleic acid (C20:1u-9) 0.62 � 0.03 d 0.57 � 0.03Behenic acid (C22:0) 2.84 � 0.37 d 2.87 � 0.80SFA 17.63 � 0.31 d 17.81 � 0.13MUFA 66.19 � 2.34 d 63.94 � 3.07PUFA 17.25 � 1.09 d 16.70 � 0.27MUFA/PUFA 3.83 � 0.1 d 3.82 � 0.24OSI (h) 9.6 d 4.7Chlorophylls (mg/kg) 12.65 � 0.29 d 5.4 � 0.76b-Carotene (mg/kg) 7.15 � 0.20 d 5.43 � 0.08Total polyphenols (mg/kg)y 579.17 � 71.40 384 � 23.18 d

a-tocopherol (mg/kg) 484.56 � 11.11 d 491.64 � 10.36

EVOO, extra virgin olive oil; MUFA, monounsaturated fatty acid; OOHF, hydro-philic fraction of olive oil; OOLF, lipophilic fraction of olive oil; OSI, oxidativestability index; PUFA, polyunsaturated fatty acid; SFA, saturated fatty acids

* Data are expressed as mean � SD of three independent experiments.y As hydroxy tyrosyl equivalents.

A. Nakbi et al. / Nutrition 28 (2012) 81–91 83

minimum relative humidity, and free access to water and a standard diet (SICO,Sfax, Tunisia). The dietary composition of the animals’ diet is presented in Table 1.All breeding phases and experiments were conformable to the rules of theTunisian Society for the Care and Use of Laboratory Animals. All experimentswere conducted at the animal facilities of the Faculty of Medicine of Monastirwith the approval of the Faculty of Medicine ethics committee.

After acclimatization to the laboratory conditions for 1 wk, the animals weredivided into eight groups of 10 animals each. Group C included the controlanimals and received 1 mL of distilled water by gavage daily and a standard diet.Group D received a daily dose of 2,4-D by gavage at a concentration of 5 mg/kg ofbody weight and fed with the standard diet. Group D/EVOO was treated simul-taneously with 2,4-D at a dose of 5 mg/kg of body weight and EVOO (300 mL)daily by gavage. Groups D/OOHF and D/OOLF received a daily dose of 5 mg/kg ofbody weight of 2,4-D followed by supplementation of hydrophilic fraction (1 mL)and lipophilic fraction (300 mL) by gavage, respectively. Animals in groups EVOO,OOHF, and OOLF were given EVOO (300 mL), OOHF (1 mL; the same amount ofphenols that we found in 300 mL of oil ¼ 0.16 mg), and OOLF (300 mL),respectively.

Each group was kept on treatment for 4 wk. Water and food consumptionand individual animal body weights were recorded daily throughout the exper-iment. At the end of the experimental period, rats were kept fasting overnightand were sacrificed under diethyl ether anesthesia.

Blood collection

The blood samples were obtained by a cardiac puncture using a syringe forthe determination of serum enzyme levels. Blood samples were put immediatelyinto ice-chilled silicon disposable glass tubes. Then, serum samples wereobtained by centrifuging blood samples at 3500 rpm for 15 min at 4�C and storedat �80�C in aliquots until analysis. The protein content of serumwas determinedaccording to the method of Bradford [29].

Serum lipids

The determination of serum total cholesterol (TC), LDL cholesterol (LDL-C),HDL-C, and triacylglycerol (TG) concentrations were assessed with commerciallyavailable diagnostic kits supplied by Randox Laboratories (Ardmore, NorthernIreland, UK).

Antioxidant enzyme activities

Antioxidant enzyme activities were analyzed using a BioRad UV-Visiblespectrophotometer with a “kinetics” program (BioRad, Mares la Coquette,France). The measurements of superoxide dismutase and glutathione peroxidaseactivities in serumwere performed by the commercially available diagnostic kitssupplied by Randox Laboratories. Catalase activity was measured at 25�Caccording to the method of Aebi [30] by measuring the H2O2 concentrationdecrease at 240 nm.

TBA-reactive substances assay

The level of lipid peroxidation products was measured as TBA-reactivemetabolites according to the method of Yagi [31]. One hundred twenty-fivemicroliters of serum was homogenized by sonication with 50 mL of Tris buff-ered saline,125 ml of trichloroacetic acid- butylated hydroxytoluene to precipitateproteins, and then centrifuged (1000 � g, 10 min, 4�C). Two hundred microlitersof the supernatant was mixed with 40 mL of HCl (0.6 M) and 160 mL of TBA dis-solved in Tris and the mixture was heated at 80�C for 10 min. The absorbance ofthe resultant supernatant was read at 530 nm. The TBA-reactive metabolitesamount was calculated using a 156 mmol/ L � cm extinction coefficient.

Histopathologic studies

Fresh tissue pieces of liver were fixed in Bouin liquid. After 2 d of fixation, thespecimens were washed and dehydrated through a graded series of ethanol.Then, they were embedded in paraffin wax. Blocks were made and sectioned at5 mm thickness using a rotary microtome. Sections were rehydrated in distilledwater, stained with hematoxylin and eosin, and then examined under lightmicroscopy.

Statistical analyses

The data were analyzed using SPSS 11.0 for Windows (SPSS, Inc., Chicago, IL,USA). In each assay, the experimental data represent the mean of 10 independentassays � standard deviations. The results were analyzed using the Student t testfor comparison between the different treatment groups. The Tukey test was usedto determine any significant differences between means. Statistical significancewas set at P < 0.05.

Results

Analytical parameters of EVOO and its compounds

The EVOO analytical parameters (fatty acid, oxidativestability, and antioxidant composition) are presented in Table 2.It can be seen that the phenolic compounds were efficientlyremoved from the EVOO, being much lower in the lipophilicfraction. The procedure employed to produce the OOLF, at vari-ance with a washing process, was developed to eliminateselectively hydrophilic substances, such as phenolic compounds,leaving unmodified the other olive oil components such astocopherols and fatty acids (Table 2).

Body weight and weight gain

All the results from various treatment groups were comparedwith group C. However, results from the D/EVOO, D/OOHF, andD/OOLF groups were also compared with the data of group D.During this study, death was not observed during the experi-mental period. Rats exhibited normal status compared withgroup C. There were no significant differences between thetreated and control rats in body weight gain and food and waterintake during the experiment (Table 3).

Biochemical indicators of liver function

No significant differences were observed for serum albumin,total protein, and glycemia levels in control and treated rats(Table 4). However, a significant increase in total bilirubin andthe ratio of aspartate to alanine aminotransferase was found inthe 2,4-D–treated rats compared to controls, which indicatedliver function damage induced by 2,4-D. The supplementation ofEVOO and its fractions cancelled the effect of 2,4-D on thetransaminase (alanine and aspartate) activities and total bili-rubin level and returned these to normal levels consistent withthe controls. Nevertheless, no significant differences were

Table 3Body weight, weight gain, water and food intake of control and experimental rats*

Initial body weight (g) Final body weight (g) Weight gain (%) Food intake (g/day/rat) Water intake (ml/day/rat)

C 216.80 � 18.10a 273.70 � 27.10a 26.16 � 4.05a 17.18 � 1.10a 20.06 � 3.51a

2,4-D 220.00 � 11.11a 276.70 � 15.23a 25.62 � 5.66a 18.42 � 0.92a 18.15 � 1.68a

2,4-D/EVOO 222.80 � 19.03a 280.60 � 19.77a 26.21 � 6.15a 18.02 � 1.05a 19.40 � 2.08a

2,4-D/OOHF 222.60 � 25.29a 279.10 � 34.74a 25.32 � 5.67a 18.82 � 2.43a 18.74 � 4.20a

2,4-D/OOLF 219.10 � 16.17a 277.40 � 21.66a 26.69 � 5.90a 17.94 � 1.45a 17.71 � 1.90a

EVOO 221.30 � 22.84a 271.10 � 22.96a 22.81 � 6.10a 17.55 � 1.07a 20.56 � 4.10a

OOHF 225.50 � 23.10a 286.10 � 25.86a 27.08 � 4.33a 18.97 � 2.05a 21.20 � 4.86a

OOLF 221.50 � 30.64a 284.50 � 29.53a 29.82 � 6.85a 17.47 � 2.04a 19.26 � 4.44a

2,4-D, 2,4-diclorophenoxyacetic acid–treated group at 5 mg/kg of body weight; 2,4-D/EVOO, 2,4-diclorophenoxyacetic acid plus extra virgin olive oil; 2,4-D/OOHF,2,4-diclorophenoxyacetic acid plus hydrophilic fraction of olive oil; 2,4-D/OOLF, 2,4-diclorophenoxyacetic acid plus lipophilic fraction of olive oil; C, control group;EVOO, group treated with only extra virgin olive oil; OOHF, group treated with only hydrophilic fraction of olive oil; OOLF, group treated with only lipophilic fraction ofolive oil

* Data are expressed asmean� SD (n¼ 10 rats per group). Comparison between groups was performed using one-way analysis of variance followed by the Tukey test(P < 0.05). Values followed by the same letters are not significantly different.

A. Nakbi et al. / Nutrition 28 (2012) 81–9184

observed between groups of rats supplemented with EVOO andits fractions without 2,4-D treatment (Table 4).

Serum lipids

At the end of the fourth week, when the 2,4-D–treated groupwas compared with the control group, there was a significantincrease in TC and LDL-C levels (þ33% and þ25%, respectively)against decreases in TG and HDL levels (�24% and �19%,respectively) in 2,4-D–treated rats (P < 0.05; Fig. 2). However,the supplementation of EVOO and its fractions to rats treatedwith 2,4-D involved a significant decrease in concentrations ofTC and LDL-C (P < 0.05), with a moderate increase in the level ofHDL-C observed only in the D/EVOO rats (P ¼ 0.058). Asa consequence, the incidences measured with a cardiovasculardisease risk index were significantly lowered mainly by oraladministration of EVOO. In fact, TC/HDL-C and LDL-C/HDL-Cratios were decreased by 39% and 44%, respectively. Neverthe-less, a significant decrease of TG level was found after EVOOsupplementation compared with the control group (Fig. 2).

Antioxidant enzyme activities

Results listed in Table 5 show plasma biochemical parameters,which indicated oxidative damage. Exposure of rats to 2,4-Dproduced a significant decrease in plasma superoxide dismutase,glutathione peroxidase, and catalase enzyme activities comparedwith those of the control group. In contrast, the oral administra-tion of EVOO and its fractions improved the enzyme activities ofrats treatedwith 2,4-D. The decreasewas significantly restored inthe presence of the hydrophilic fraction (Table 5).

Table 4Effect of 2,4-D, extra virgin olive oil and its extracts on serum albumin, total protein,

Albumin (g/dL) Total Protein (g/dL)

C 4.44 � 0.44a 8.62 � 1.09a

2,4-D 3.56 � 0.51a 7.57 � 0.94a

2,4-D/EVOO 3.55 � 0.87a 7.55 � 0.37a

2,4-D/OOHF 3.90 � 0.76a 7.38 � 0.19a

2,4-D/OOLF 3.84 � 1.03a 7.53 � 0.22a

EVOO 4.11 � 0.42a 7.68 � 0.41a

OOHF 4.03 � 0.61a 8.15 � 0.75a

OOLF 4.22 � 1.21a 8.41 � 1.89a

2,4-D, 2,4-diclorophenoxyacetic acid–treated group; ALT, alanine aminotransferase; Aacetic acid plus extra virgin olive oil; D/OOHF, 2,4-diclorophenoxyacetic acid plus hydrfraction of olive oil; EVOO, group treated with only extra virgin olive oil; OOHF, grouplipophilic fraction of olive oil; TB, total bilirubin

* Data are expressed asmean� SD (n¼ 10 rats per group). Comparison between gro(P < 0.05). Values followed by the same letters are not significantly different.

TBA-reactive substances levels

Compared with the controls, liver and plasma malondialde-hyde (MDA) levels were determined to have increased signifi-cantly in the 2,4-D–treated group (Fig. 3). Furthermore,a significant decrease of the MDA rate was observed afteradministration of EVOO and OOHF. In fact, the liver MDA levelwas decreased by 82% and 64%, respectively, whereas plasmaMDA level was decreased by 38% and 52%, respectively. Incontrast, the administration of the lipophilic fraction to 2,4-D–treated animals showed no significant differences in liver andplasma MDA levels compared with the 2,4-D–treated group.These findings indicated that free radicals released in the liverwere effectively scavenged when treated with OOHF.

Hepatic histoarchitecture

The histologic findings from the liver in various treatmentgroups are presented in Figures 4–7. The liver of control ratsshowed a normal structure (Fig. 4). The liver histologic sectionrevealed clear-cut hepatic lobules, separatedby interlobular septaand traversed by portal veins. The hepatocytes were polyhedral.The nuclei were round and the size was roughly uniform.

However, subacute 2,4-D exposure at 5 mg/kg for 4 wkresulted in severe pathologic liver injury marked by necrosis,dilatation of sinusoids, inflammation, and strong vacuolization ofhepatocytes (Fig. 5). In contrast, coadministration of EVOO and itsfractions to 2,4-D–treated rats resulted in marked improvementin the structure of hepatocytes (Fig. 6). The livers of rats coad-ministrated EVOO or OOHF with 2,4-D presented a normalappearance in the arrangement of hepatocytes and the aspect of

glycemia, TB profiles, and AST/ALT ratio of experimental rats*

Glycemia (g/L) TB (U/L) AST/ALT

1.18 � 0.42a 5.66 � 1.18a 2.79 � 0.31a

1.52 � 0.33a 12.05 � 0.81b 4.19 � 0.66b

1.27 � 0.30a 7.00 � 0.91a 2.84 � 0.51a

1.57 � 0.12a 7.56 � 1.70a 2.95 � 0.65a

1.79 � 0.59a 7.42 � 0.61a 2.87 � 0.93a

1.32 � 0.39a 6.14 � 1.09a 2.97 � 0.36a

1.61 � 0.43a 6.86 � 1.33a 3.54 � 0.41ab

1.42 � 0.38a 5.58 � 1.68a 3.09 � 0.62a

ST, aspartate aminotransferase; C, control group; D/EVOO, 2,4-diclorophenoxy-ophilic fraction of olive oil; D/OOLF, 2,4-diclorophenoxyacetic acid plus lipophilictreated with only hydrophilic fraction of olive oil; OOLF, group treated with only

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Fig. 2. Effect of 2,4-D, EVOO, OOHF, and OOLF on rat TC (A), TG (B), HDL-c (C), LDL-c (D) levels and atherogenic index, i.e., TC/HDL-c (E) and LDL-c/HDL-c (F). Each barrepresents mean � SE of 10 rats. Bars with different letters differ significantly (P � 0.05). 2,4-D, 2,4-diclorophenoxyacetic acid–treated group at 5 mg/kg of body weight; 2,4-D/EVOO, 2,4-diclorophenoxyacetic acid plus extra virgin olive oil; 2,4-D/OOHF, 2,4-diclorophenoxyacetic acid plus hydrophilic fraction of olive oil; 2,4-D/OOLF, 2,4-diclor-ophenoxyacetic acid plus lipophilic fraction of olive oil; C, control group; EVOO, group treated with only extra virgin olive oil; HDL-c, high-density lipoprotein cholesterol;LDL-c, low-density lipoprotein cholesterol; OOHF, group treated with only hydrophilic fraction of olive oil; OOLF, group treated with only lipophilic fraction of olive oil; TC,total cholesterol; TG, triacylglycerol.

A. Nakbi et al. / Nutrition 28 (2012) 81–91 85

the sinusoidal spaces,with total disappearance of vacuolization ofcells (Fig. 6A, C). A similar morphology of the hepatocyte nucleiwas observed compared with controls (Fig. 6B, D), althoughvascular congestion, smaller proportion of vacuolized degen-erated hepatocytes, some regions of necrosis, and an abundanceof Kupffer cells were still present in livers of rats treated with D/OOLF (Fig. 6E, F).

Treatment with EVOO and its fractions alone did not result inany alteration in hepatic histoarchitecture (Fig. 7).

Discussion

Recent lines of evidence have suggested that the beneficialeffects of olive oil are related not only to its high content of oleicacid but also to the antioxidant potential of its polyphenols. Inthis study, olive oil and its hydrophilic and lipophilic fractionswere tested for their lipid-lowering, antioxidative, and hep-atoprotective effects against oxidative damage induced by 2,4-D.Our results revealed that the hypolipidemic effect was strongly

Table 5Plasma antioxidant enzymes activities (SOD, CAT, and GPx) in control andexperimental rats*

SOD(U/g protein)

CAT(mmol/min g protein)

GPx(U/g protein)

C 12.92 � 1.98b 711.45 � 175.15c 176.63 � 38.68ab

2,4-D 7.86 � 1.30a 411.88 � 137.42a 116.14 � 47.33a

2,4-D/EVOO 9.08 � 1.41ab 573.44 � 147.32abc 164.42 � 68.38ab

2,4-D/OOHF 12.52 � 2.43b 581.01 � 115.03abc 256.87 � 70.76b

2,4-D/OOLF 12.73 � 2.81b 568.12 � 153.02abc 129.08 � 56.89a

EVOO 11.56 � 1.22ab 697.35 � 117.36c 183.98 � 47.31ab

OOHF 12.96 � 0.59b 645.94 � 206.46bc 186.53 � 17.94ab

OOLF 9.21 � 2.96ab 460.48 � 155.67ab 185.72 � 76.77ab

2,4-D, 2,4-diclorophenoxyacetic acid–treated group; 2,4-D/EVOO, 2,4-diclor-ophenoxyacetic acid plus extra virgin olive oil; 2,4-D/OOHF, 2,4-diclor-ophenoxyacetic acid plus hydrophilic fraction of olive oil; 2,4-D/OOLF,2,4-diclorophenoxyacetic acid plus lipophilic fraction of olive oil; C, controlgroup; CAT, catalase; EVOO, group treated with only extra virgin olive oil; GPx ,glutathione peroxidase; OOHF, group treated with only hydrophilic fraction ofolive oil; OOLF, group treated with only lipophilic fraction of olive oil; SOD,superoxide dismutase

* Data are expressed as mean � SD (n ¼ 10 rats per group). Comparisonbetween groups was performed using one-way analysis of variance followed bythe Tukey test (P < 0.05).Values followed by the same letters are not significantly different.

a

b

aa

ab

aa

a

0

10

20

30

40

50

60

70

80

C

24,

D- OO

VE/

D-4,

2

FH

OO/

D-4,

2

FL

OO/

D-4,

2

OO

VE O

O

FH

OO

FL

)ni

et

or

pa

ms

alp

g/M

m(

AD

M

Plasma

b

ab

a

ab

a

abb

a

0

0,2

0,4

0,6

0,8

1

1,2

1,4

1,6

C

,2

4-D

24,

D-

E/V

OO

24,-

OO/

D

FH

24,

O/D-

O

FL

OO

VE

FH

OO

FL

OO

)ni

et

or

pr

evil

g/M

µ(

AD

M

Liver

A

B

Fig. 3. Plasma (A) and liver (B) MDA levels of treatment groups over 4 wk. Data areexpressed as mean � SD (n ¼ 10 rats per group). Comparison between groups wasperformed using one-factor analysis of variance. Values followed by the sameletters are not significantly different (P < 0.05, Tukey test). 2,4-D, 2,4-diclor-ophenoxyacetic acid–treated group at 5 mg/kg of body weight; 2,4-D/EVOO,2,4-diclorophenoxyacetic acid plus extra virgin olive oil; 2,4-D/OOHF, 2,4-diclor-ophenoxyacetic acid plus hydrophilic fraction of olive oil; 2,4-D/OOLF, 2,4-diclor-ophenoxyacetic acid plus lipophilic fraction of olive oil; C, control group; EVOO,group treated with only extra virgin olive oil; MDA, malondialdehyde; OOHF, grouptreated with only hydrophilic fraction of olive oil; OOLF, group treated with onlylipophilic fraction of olive oil.

A. Nakbi et al. / Nutrition 28 (2012) 81–9186

visualized in the 2,4-D–treated rats with supplementation ofEVOO, whereas the antioxidant activity was well observed in the2,4-D–treated rats with supplementation of OOHF. In addition,supplementationwith EVOO and its fractions resulted in markedimprovement in hepatic histoarchitecture, emphasizing theirprotective potential.

In Tunisia, 2,4-D is a commonly used pesticide in the north,the most important agricultural region in Tunisia. In this study,we used 2,4-D at 5 mg/kg to determine its disordering lipidicprofile and possible oxidative damage in rats. However, it isconceivable that the 2,4-D herbicide, as a toxicologic agent likeother pesticides, might be interacting primarily with the liverand muscle tissue cell membrane. Consequently, structural liverdamage and changes in enzyme leakage were observed. Ourresults showed that 2,4-D induced an increase in total bilirubinand alterations in serum enzymatic activities such as alanine andaspartate aminotransferases. These changes pointed to thefunctional disorder of the liver [32,33], which was proved by theliver histologic study. In fact, rats treated with 2,4-D exhibitedhistopathologic changes such as vacuolization, necrosis, markedleukocytic infiltration, and widened sinusoidal spaces. Thesedata are in agreement with our previous study that reported that2,4-D, at doses of 15, 75, and 150 mg/kg of body weight for 28 d,contributes to liver cellular injury in rats [34]. Similar findingsconcerning histologic damagewere observed byMountassif et al.[35], such as manifold centrilobular necrotic areas in the liverjerboa exposed to 2,4-D at 3 mg/kg of body weight for 4 wk.However, such anomalies were significantly decreased withEVOO and its fractions supplemented to 2,4-D–treated rats. Themechanism underlying the hepatoprotection of EVOO and itsfractions might be related to its radical scavenging proprietiesand its indirect effect as a regulator of antioxidative systems.

In addition to the fluctuating levels of serummarker enzymesand morphologic liver alterations, the 2,4-D–treated groupshowed amarked perturbation of serum lipidmetabolism. In fact,the 2,4-D treatment caused an important increase in plasma TCand LDL-C levels and a decrease inTG andHDL-C,which indicateda change in the permeability of hepatic cells [36]. These resultsconfirmed the findings of Mountassif et al. [35]. Moreover, ourdata clearly demonstrated that subacute exposure to 2,4-Dsignificantly increased the LDL/HDL and TC/HDL ratios, which arepertinent indices of the incidence of cardiovascular risk [37].

In this investigation, simultaneous administration of EVOO,OOHF, and OOLF with 2,4-D resulted in a decrease in serum TC,TG, and LDL-C levels compared with the 2,4-D–treated rats,whereas the HDL-C level was significantly increased only in 2,4-D–treated rats supplemented with EVOO. Lowering levels of TC,TG, and LDL-C and improving the level of HDL-C have beenlinked to a lower risk of cardiovascular disease [38]. In thiscontext, a marked improvement in TC/HDL-C and LDL-C/HDL-Cratios (atherosclerotic index) was mainly observed in 2,4-D–treated rats administered EVOO. Recent studies have reinforcedthe proposed mechanisms by which EVOO can exert its benefi-cial effects on cardiovascular risk [39,40]. EVOO has beenreported to improve the lipid profile through a decrease in TCand LDL-C and an increase of the HDL-C/TC ratio [11,41]. Inaddition, it can decrease LDL susceptibility to oxidation [11,42]and improve endothelial function [12,42]. Further findingshave shown that EVOO improves blood pressure control [43,44].

However, for years, the beneficial effects of olive oil oncardiovascular disease have been attributed exclusively to its highMUFA content, mostly in the form of oleic acid (18:1u-9) becausethis effect on TG, LDL-C, and HDL-C has been demonstrated inevery diet with a high content of MUFA [45,46]. Furthermore, the

Fig. 4. Photomicrographs of histologic section from a control animal showing a hepatic lobule with the uniform pattern of a polyhedral hepatocyte radiating from the centralvein and presenting a normal aspect of the sinusoidal spaces. Sections (n ¼ 6) were stained with hematoxylin and eosin (magnifications 320� in A, 1000� in B). cv, centralvein; Hp, polyhedral hepatocyte; SC, sinusoidal spaces.

A. Nakbi et al. / Nutrition 28 (2012) 81–91 87

Effect of Olive Oil on Oxidative Damage in European Populations(EUROLIVE) Study, a controlled trial of different types of olive oil[17], showed a decrease in serumTG thatwas related to theMUFAcontent of the test oils. Other human studies have suggested thatMUFAs are as effective as polyunsaturated fats in loweringplasma cholesterol concentration [47].

However, the beneficial effects of EVOO intake on theprevention of cardiovascular diseases cannot be attributed onlyto the content of oleic acid. In the present study, although TC, TG,and LDL-C decreased, no improvement in HDL-C was detected ingroups administered theOOLF. Ruiz-Gutierrez et al. [48] observedthat, in contrast to virgin olive oil, high-oleic sunflower oil did notdecrease blood pressure in hypertensive patients. Moreover, byshowing that phenolic compounds in EVOO can decreasecardiovascular risk factor levels, the EUROLIVE study providedclear evidence that EVOO is more than just a MUFA. In that study,olive oil with different phenolic contents was given, and all formsdecreased serum TG and increased HDL-C and the reduced-to-oxidized glutathione ratio [20]. However, the increased HDL-Cwas related to the phenolic content of EVOO in a dose-dependent manner [17]. Therefore, our data revealed that onlyan EVOO-supplemented diet to 2,4-D–treated rats induced animprovement inHDL-C level and the atherosclerotic index,which

Fig. 5. Liver sections of animals treated with 2,4-diclorophenoxyacetic acid at 5 mg/kg reaspect and hepatocyte vacuolization. Sections (n ¼ 6) were stained with hematoxylin andSC, sinusoidal spaces; V, vacuolization.

might have resulted from the synergistic action of the two frac-tions (hydrophilic and lipophilic).

Therefore, a decrease of TC and LDL-C is a primary step in theprevention of vascular disease. Furthermore, preventing oxida-tive stress could delay the development of atherosclerosis. Thereis evidence that high antioxidant levels in the plasma are asso-ciated with a lower frequency of coronary disease [49], and thatdecreased antioxidant levels in the plasma can promote theformation of free radicals and consequently LDL peroxidation[50,51].

In the present study, a significant decrease in antioxidantpotential in 2,4-D–treated rats was observed. The lower antioxi-dant status of this group was augmented by a decrease in antiox-idant enzyme activities such as superoxide dismutase, glutathioneperoxidase, and catalase and an increase in liver and plasma MDAlevels as a marker of lipid peroxidation. The obtained resultsconfirmed the findings of Celik et al. [7] who studied the effects of2,4-D on serum marker enzymes, antioxidant defense systems,and lipidperoxidationcontent invarious tissues of rats. They foundthat the administration of 1.5 and 3mg/d of 2,4-D for 25 d inducedin vivo oxidation. Furthermore, previous studies have shown that2,4-D produces oxidative stress and/or depletes antioxidants invitro and in vivo [7,35,52,53].

vealed (A) marked vascular congestion and wide sinusoidal spaces and (B) a necroticeosin (magnifications 320� in A, 1000� in B). cv*, vascular congestion; Nc, necrotic;

Fig. 6. Effect of extra virgin olive oil and its fractions on liver damage induced by 2,4-diclorophenoxyacetic acid. (A, B) Liver from a rat treated with extra virgin olive oil plus2,4-diclorophenoxyacetic acid; (C, D) liver from a rat treated with the hydrophilic fraction of olive oil plus 2,4-diclorophenoxyacetic acid; (E, F) liver from a rat treated withthe lipophilic faction of olive oil plus 2,4-diclorophenoxyacetic acid. Sections (n ¼ 6) were stained with hematoxylin and eosin (magnifications 320� in A, C, E, 1000� in B, D,F). *, vascular congestion; **, degenerated hepatocytes; cv, central vein; Hp, normal aspect of hepatocytes; Kc, Kupffer cells; Nc, hepatocyte nuclei; Nr, necrosis; sc, sinusoidalspaces; V, vacuolization.

A. Nakbi et al. / Nutrition 28 (2012) 81–9188

Furthermore, improved antioxidant status by increasedantioxidant enzyme activity and decreased lipid peroxidationhas been reported after the intake of EVOO compared with 2,4-Dtreatment. These beneficial effects of EVOO were then related toits phenolic content. However, the OOLF-supplemented dietgenerally did not show any improvement in antioxidant status ofrats, although it is rich in a-tocopherol. In this context, mostrandomized intervention trials completed thus far have shownno significant decrease in cardiovascular disease with vitamin Esupplementation [54,55]. In addition, the amount (1200 mg/kg)

of vitamin E in sunflower oil is not enough to protect the highlyunsaturated LDL against oxidation [14]. In our previous study,we found that EVOO with a higher phenolic content seemed tohave beneficial effects on LDL particle oxidization [56]. Otherstudies have shown that EVOO increases resistance to lipidperoxidation and the antioxidant defense system more thanrefined olive oil [57].

Moreover, in our experiment, rats fed theOOHFdiet presentedthe highest levels of antioxidant activity enzymes and lower liverand plasma MDA levels, providing an appropriate model for

Fig. 7. Photomicrograph of stained liver sections from rats supplemented with extra virgin olive oil (A, B), the hydrophilic fraction of olive oil (C, D), and the lipophilic fractionof olive oil (E, F). Sections (n ¼ 6) were stained with hematoxylin and eosin (magnifications 320� in A, C, E, 1000� in B, D, F). cv, central vein; Hp, normal aspect ofhepatocytes; Nc, hepatocyte nuclei; SC, sinusoidal spaces; V, vacuolization.

A. Nakbi et al. / Nutrition 28 (2012) 81–91 89

dietary oxidative damage defense. In this regard, this observationcan be explained by the direct contact of phenolic compounds tofree radicals and their ability to decompose them by quenchingreactive oxygen species and by trapping radicals before reachingtheir cellular targets [58]. This assumption has reinforced theproposed suggestions on phenolic compounds, which are able tointeractwith biological systems and act as bioactivemolecules; inparticular, they are important inhibitors of lipid peroxidation [59]and are believed to be effective through their free radical-scavenging and metal-chelating properties [60].

Conclusion

As expected, our results indicated that 2,4-D causedmorphologic and functional liver damage, an increased athero-sclerosis index, and oxidative stress in rats. A diet enriched withthe test EVOO exhibited a pronounced hypolipidemic effect andlowered the atherosclerosis index in 2,4-D–treated rats moreeffectively than a diet rich in hydrophilic or lipophilic fractions.However, a marked enhancement of the antioxidant defensesystem was found in rats supplemented with OOHF diet. This

A. Nakbi et al. / Nutrition 28 (2012) 81–9190

finding suggests that including olive oil in the diet may offerbenefits in decreasing liver damage and the atheroscleroticprocess during 2,4-D exposure in rats.

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