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Contents lists available at SciVerse ScienceDirect

The International Journal of Biochemistry& Cell Biology

journa l h o me page: www.elsev ier .com/ locate /b ioce l

mega-3 fatty acids enhance mitochondrial superoxide dismutase activity in ratrgans during post-natal development

atherine Garrela, Jean-Marc Alessandrib, Philippe Guesnetb, Kaïs H. Al-Guboryc,∗

Unité de Biochimie Hormonale et Nutritionnelle, Département de Biologie – Toxicologie – pharmacologie, Centre Hospitalier Universitaire de Grenoble,8043 Grenoble Cedex 9, FranceUnité de Nutrition et Régulation Lipidique des Fonctions Cérébrales (UR 909), Département Alimentation Humaine, Institut National de la Recherche Agronomique,-78352 Jouy-en-Josas, FranceUnité de Biologie du Développement et de la Reproduction (UMR 1198), Département Physiologie Animale et Systèmes d’Elevage, Institut National de la Recherche Agronomique,-78352 Jouy-en-Josas, France

r t i c l e i n f o

rticle history:eceived 23 May 2011eceived in revised form 5 October 2011ccepted 16 October 2011vailable online xxx

eywords:ntioxidant enzymesxidative stressolyunsaturated fatty acidsipid peroxidationat

a b s t r a c t

The protection of the developing organism from oxidative damage is ensured by antioxidant defensesystems to cope with reactive oxygen species (ROS), which in turn can be influenced by dietarypolyunsaturated fatty acids (PUFAs). PUFAs in membrane phospholipids are substrates for ROS-inducedperoxidation reactions. We investigated the effects of dietary supplementation with omega-3 PUFAs onlipid peroxidation and antioxidant enzyme activities in rat cerebrum, liver and uterus. Pups born fromdams fed a diet low in omega-3 PUFAs were fed at weaning a diet supplying low �-linolenic acid (ALA),adequate ALA or enriched with eicosapentaenoic acid (EPA) plus docosahexaenoic acid (DHA). Malondi-aldehyde (MDA), a biomarker of lipid peroxidation, and the activities of superoxide dismutase 1 (SOD1),SOD2, catalase (CAT) and glutathione peroxidase (GPX) were determined in the three target organs.Compared to low ALA feeding, supplementation with adequate ALA or with EPA + DHA did not affect thecerebrum MDA content but increased MDA content in liver. Uterine MDA was increased by the EPA + DHA

ost-natal development diet. Supplementation with adequate ALA or EPA + DHA increased SOD2 activity in the liver and uterus,while only the DHA diet increased SOD2 activity in the cerebrum. SOD1, CAT and GPX activities were notaltered by ALA or EPA + DHA supplementation. Our data suggest that increased SOD2 activity in organs ofthe growing female rats is a critical determinant in the tolerance to oxidative stress induced by feedinga diet supplemented with omega-3 PUFAs. This is may be a specific cellular antioxidant response to ROSproduction within the mitochondria.

. Introduction

Reactive oxygen species (ROS) are generated as by-productsf cellular aerobic respiration and metabolism. Physiological lev-ls of ROS are essential for cellular signaling and gene expressionAllen and Tresini, 2000), but are harmful at higher levels andontribute to the development and pathology of several humaniseases (Halliwell, 2011). Growing body of literature indicateshat ROS-induced oxidative stress plays a role in prenatal defects,regnancy-related disorders and post-natal developmental com-lications (Agarwal et al., 2008; Al-Gubory et al., 2010).

The key intracellular antioxidant enzymes, copper–zinc

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ontaining SOD (SOD1), manganese containing SOD (SOD2),lutathione peroxidases (GPXs) and catalase (CAT), repre-ent interdependently operating network of defenses against

∗ Corresponding author. Tel.: +33 1 34 65 23 62; fax: +33 1 34 65 23 64.E-mail address: kais.algubory@jouy.inra.fr (K.H. Al-Gubory).

357-2725/$ – see front matter © 2011 Elsevier Ltd. All rights reserved.oi:10.1016/j.biocel.2011.10.007

© 2011 Elsevier Ltd. All rights reserved.

ROS-induced oxidative stress and lipid peroxidation by limitingthe generation and propagation of highly reactive and toxicROS (Jezek and Hlavatá, 2005; Valko et al., 2007; Halliwell,2009). SOD1 is a dimeric protein, predominantly localized inthe cytosol, but it is also found in mitochondria. SOD2 is ahomotetrameric protein, located in the mitochondria. GPXspresent in the cytoplasm and the mitochondria, and CAT foundwithin peroxisomes, both convert H2O2 to water and oxygen.There are five members of the selenium-containing GPX fam-ily (GPX1–GPX5) and GPX4 is a unique antioxidant (Imai andNakagawa, 2003) that specifically acts in membranes on hydroper-oxy groups of peroxidized phospholipid side chains and oncholesterol hydroperoxides (Jezek and Hlavatá, 2005). In thepresence of free iron ions, ROS such as H2O2 and •O2

− interactin a Haber–Weiss reaction to generate the highly reactive and

ce mitochondrial superoxide dismutase activity in rat organs duringl.2011.10.007

oxidative •OH (Jezek and Hlavatá, 2005). Nitric oxide (•NO) actsas a regulator of many cellular events (Moncada et al., 1991).However, •NO-derived potent oxidants, mainly the reactive per-oxynitrite (ONOO−), can mediate macromolecules damage (Jezek

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Table 1Lipid composition of experimental diets.

Low ALA(20 mg/100 gdiet)

adequate ALA(300 mg/100 gdiet)

Enriched DHA(500 mg/100 gdiet)

Oil (g/100 g of diet)Sunflower 6.3 3.0 4.6Rapeseed 0.2 3.5 0.0Tuna 0.0 0.0 2.2

Fatty acid (FA) (% by wt of total FA)Saturated

16:0 4.2 4.518:0 2.7 2.120:0 0.3 0.322:0 0.8 0.524:0 0.3 0.3

Monounsaturated16:1 (n-7) 0.1 0.218:1 (n-9) 70.9 66.220:1 (n-9) 0.3 0.7

Polyunsaturated18:2 (n-6) 19.9 20.5 14.218:3 (n-3) 0.3 4.6 0.318:4 (n-3) 0.320:4 (n-3) 0.2

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nd Hlavatá, 2005). •NO reacts with •O2− in a reaction controlled

y the bioavailability of both radicals to form ONOO− (Garrel andontecave, 1995; Packer et al., 1996). ROS, mainly •OH and ONOO−,nduce lipid peroxidation and thereby cause malfunctioning ofiological membranes by altering their fluidity and membrane-ound proteins synthesis (Jezek and Hlavatá, 2005; Valko et al.,007).

Long chain polyunsaturated fatty acids (LC-PUFAs) are impor-ant constituents of biological membranes where they contributeo maintain the structural and functional integrity of cells and cel-ular components (Salem et al., 1985; Stillwell et al., 1993, 1997).hey are supplied by dietary fats, either from vegetable fats underhe form of their essential precursor linoleic acid [LA, 18:2(n-6)]f the omega-6 series and �-linolenic acid [ALA, 18:3(n-3)] of themega-3 series, or from fatty fishes, particularly eicosapentaenoiccid [EPA, 20:5(n-3)] and docosahexaenoic acid [DHA, 22:6(n-)]. Omega-3 PUFAs are crucial for the biogenesis of membranehospholipids and are sources of energy during development andunction of organs and tissues (Hamosh and Salem, 1998; Xiang andetterström, 1999; Herrera, 2002), especially the brain (Alessandrit al., 2004; Mccann and Ames, 2005; Innis, 2007). PUFAs are alsorecursor of prostaglandin synthesis and thus have indirect roles

n the control of many functions, especially uterine function andemale fertility (Wathes et al., 2007; Coyne et al., 2008). On thether hand, high content of PUFAs in phospholipids of biologicalembranes are potential substrates for ROS-induced peroxidation

eactions (Piché et al., 1988; Meydani et al., 1991; Halliwell andhirico, 1993; Ando et al., 2000; Song et al., 2000). Dietary intake ofUFAs may be disadvantageous because the oxidative peroxidationf PUFAs can lead to the formation of highly toxic products, mainlyalondialdehyde, MDA (Piché et al., 1988; Draper et al., 1986)hich has been referred to as potentially mutagenic and athero-

enic factor (Del et al., 2005). PUFAs, particularly DHA, have beenhown to be rapidly incorporated into phospholipids of the plasmaembrane (Zerouga et al., 1996) and mitochondria (Stillwell et al.,

997) where they may affect cell functions by enhancing suscep-ibility of biological membranes to lipid peroxidation (Song et al.,000). The susceptibility to peroxidation of biomembrane PUFAsy ROS raises the question of oxidizing properties exerted by theseatty acids, especially if their content is substantially augmented iniet.

Our hypothesis is that an increase of omega-3 PUFAs cellularncorporation which can be provoked by nutritional handling dur-ng the post-weaning period could modify the organ and tissuentioxidant status as a consequence of lipid peroxidation (Sawadat al., 1992). We aimed therefore to investigate the effects ofupplementing young rats with omega-3 PUFAs on lipid peroxi-ation and activities of the key antioxidant enzymes, SOD1, SOD2,AT and GPX, in three target organs, cerebrum, liver and uterus.

n the present study, diets were designed to cover the range ofmega-3 PUFA supplies which can be found in human foods, i.e.nadequate or adequate amounts of PUFAs, and under the formf the metabolic precursor ALA or of LC-PUFAs (EPA + DHA). Tohis end, rats born with a low omega-3 PUFA status receivedt weaning a diet containing low or adequate amounts of ALArom vegetable oil or a diet enriched in EPA + DHA from fishil. Under these conditions, the repletion in omega-3 PUFAs isapid and the LC-PUFA status in tissues covers a broad spec-rum of omega-3 PUFAs and omega-6 PUFAs levels. The contentf LC-PUFA of the omega-3 and omega-6 docosapentaenoic acidDPA, C22:5(n-6)] in the major classes of biological membranehospholipids, namely phosphatidylcholine (PC) and ethanolamine

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hosphoglycerides (EPG) were determined in cerebrum whereC-PUFAs are intensively incorporated throughout development,nd in liver which is the main organ responsible for LC-PUFAsynthesis.

20:5 (n-3) 2.722:6 (n-3) 8.2

2. Materials and methods

2.1. Experimental animals and diets

Animals were treated in accordance with the European Com-munity Council Directive of 24 November 1986 (86/609/EEC). Allprocedures were approved by the institutional animal care and usecommittee according to the French regulation for animal exper-imentation (authorization no. 78-34). Wistar rats were fed frommating to pregnancy and lactation a diet low in omega-3 PUFAs.This pre-experimental semi-synthetic diet contained 1.2 g LA/100 gand only 5 mg ALA/100 g from sunflower oil (6.2% by weight ofdry matter) as the sole lipid source. We used a variety of sun-flower oil containing 20% by weight of total fatty acids as 18:2(n-6)and less than 0.1% as 18:3(n-3). There was no other omega-3PUFA in the diet. At birth, 18 females were chosen on weighthomogeneity among 8 litters (of males and females) born from 8pre-experimental dams fed a diet low in omega-3 PUFAs, and wererandomly redistributed under 3 pre-experimental lactating damsto reconstitute 3 new litters of 6 females each. Eighteen pups (3-week-old) were separated from the dams and were fed for 5 weeksa replenishing diet supplying low ALA (20 mg/100 g diet, group 1,G1), adequate ALA (300 mg/100 g diet, group 2, G2) or enriched withEPA + DHA (500 mg/100 g diet, group 3, G3). The experimental dietswere based on a blend of sunflower and rapeseed oils for G1 andG2, and of sunflower and tuna oils for G3 (Table 1).

2.2. Tissue collection

At 8-weeks of age, animals were weighed and killed by decap-itation in accordance with protocols approved by the institutionalethical committee. Cerebrums, livers and uteri were removed andimmediately snap-frozen in liquid nitrogen and then stored at−80 ◦C until processed for MDA content, SOD1, SOD2, CAT and GPXactivities, isolation of phospholipid classes PC and EPG and analysisof fatty acids composition.

ce mitochondrial superoxide dismutase activity in rat organs duringl.2011.10.007

2.3. Antioxidant enzyme activity assays

Cerebrum, liver and uterine tissues were homogenizedin cold Tris–HCl buffer (10 mM, pH 7.4) containing 1 mM

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iethylenetriaminepentaacetic acid and 1 mM phenylmethanesul-onylfluoride. The homogenates were centrifuged at 4000 × g for0 min at 4 ◦C and the supernatants were used for determination ofrotein concentration (Lowry et al., 1951), MDA content and enzy-atic activities (Garrel et al., 2007; Al-Gubory et al., 2008). Total

OD activity was measured using the pyrogallol assay based onhe competition between pyrogallol oxidation by superoxide radi-als and superoxide dismutation by SOD (Marklund and Marklund,974). Enzymatic activity of SOD2 was determined by assaying forOD activity in the presence of sodium cyanide, which selectivelynhibits SOD1 but not SOD2 (Jin et al., 2005). SOD1 activity was cal-ulated by subtracting SOD2 activity from total SOD activity. Theate of auto-oxidation is taken from the increase in the absorbancet 420 nm. One unit of SOD activity is defined as the amount of thenzyme required to inhibit the rate of pyrogallol auto-oxidation by0%. CAT activity was determined as described previously (Nzenguet al., 2008). Activity was assayed by determining the rate of decom-osition of H2O2 by CAT in 10 mM of potassium phosphate bufferpH 7). The reaction rate was related to the amount of CAT presentn the mixture. The rate of H2O2 decomposition by CAT was fol-owed at 240 nm. One unit was defined as the decomposition of

mmol hydrogen peroxide per min per mg protein. GPX activ-ty was measured using the glutathione reductase-NADPH methodTappel, 1978). Activity was determined by a coupled assay systemn which oxidation of glutathione was coupled to NADPH oxidationatalyzed by glutathione reductase. The rate of glutathione oxidizedy tertiary butyl hydroperoxide was evaluated by the decreasef NADPH in the presence of EDTA, excess reduced glutathionend glutathione reductase. The rate of decrease in concentrationf NADPH was recorded at 340 nm. GPX activity was expressed inerms of nM of NADPH oxidized per min per mg of protein.

.4. Malondialdehyde (MDA) measurement

The most widely used method for MDA determination, the endroducts of lipid peroxidation (Hartley et al., 1999), is based on

ts reaction with thiobarbituric acid (TBA). Reversed-phase higherformance liquid chromatography (HPLC) technique in whichhe MDA–TBA adducts are separated from interfering substancesLondero and Lo Greco, 1996) was used for determining MDA inrain, liver and uterus tissue homogenates. The breakdown prod-ct of 1,1,3,3-tetraethoxypropane (TEP) was used as standard. TEPndergoes hydrolysis to liberate stoichiometric amounts of MDA.tandard solution (480 �l of TEP in 100 ml ethanol) was preparednd this primary solution was diluted to the concentrations of 0,, 2, 3, 4, 5 and 6 �M. Tissue extract aliquots or standards (100 �l)ere mixed with 750 �l of 0.8% TBA. The tubes were placed in aater bath at 95 ◦C for 1 h, and then they were cooled. Samples wereeutralized with methanol-NaOH mixture (pH 6). After centrifu-ation, 50 �l of protein-free supernatant was chromatographed inhe HPLC system. The column used for the separation was Adsor-osphere C18 (5 �m particle diameter, 250 mm × 4.6 mm ID). TheDA–TBA adduct is eluted from the column with potassium dihy-

rogen phosphate buffer (10 mM, pH 6.0)-acetonitrile (17%). Theuantification of MDA derivative was established by comparing thebsorption to the standard curve of MDA equivalents generated bycid-catalyzed hydrolysis of TEP as �moles per gram tissue protein.

.5. Fatty acid analysis

Total lipids were extracted from cerebrum and liver with ml of chloroform–methanol (2:1, v/v) in the presence of 0.02%

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w/v) butylated hydroxytoluene. The phospholipid classes, PCnd EPG, were isolated by solid-phase extraction on a 500-mgminopropyl-bonded silica column, as described (Alessandri et al.,008). Phospholipids were then derivatized to fatty acid methyl

PRESSemistry & Cell Biology xxx (2011) xxx– xxx 3

esters (FAMEs) at 90 ◦C for 20 min with 10% boron trifluoride inmethanol (Fluka, Socolab, Paris, France). FAMEs were separated bygas liquid chromatography using a 50 m × 0.20 mm capillary col-umn (CP-Wax 52 CB; Varian, Les Ulis, France) and a one-stagetemperature program (from 54 ◦C to 212 ◦C at 3 ◦C/min). They weredetected through a flame ionization detector. The resulting peakswere automatically identified by comparison to standards andexpressed as percentage by weight of total fatty acids on the basisof peak areas.

2.6. Statistical analysis

Phospholipids DHA and omega-6 DPA proportion, tissue MDAcontent and SOD1, SOD2, CAT and GPX enzymatic activities wereanalyzed by one-way ANOVA and the Newman–Keuls multiplecomparison test (PRISM Graph Pad version 2; Graph Pad Soft-ware, San Diego, CA). The acceptable level of significance was setat P < 0.05. Data are presented as the mean ± SEM.

3. Results

3.1. Body weight

Body weights (mean ± SEM) of the rats were 193 ± 2.2, 196 ± 3.7and 207 ± 2.9 for the low ALA, adequate ALA and EPA + DHA groups,respectively. There were no differences in the body weight betweenthe low and adequate dietary groups. The rats fed the EPA + DHAdiet had significantly higher body weight than that of rats fed thelow ALA diet (P < 0.01) or the adequate ALA diet (P < 0.05).

3.2. Fatty acid compositions

The cumulated proportion of DHA and omega-6 DPA was higherin the EPG fraction than that in the PC fraction of cerebrum (Fig. 1A)and liver (Fig. 1B) of the three groups. Compared to low ALA feeding,there was no change in the sum of DHA and omega-6 DPA in thebrain EPG and PC of rats fed the adequate ALA diet or the enrichedEPA + DHA diet (Fig. 1A). There was a significant increase in the sumof DHA and omega-6 DPA in the liver PC (Fig. 1B) of rats fed theenriched EPA + DHA diet (P < 0.001, G3 vs. G1, G3 vs. G2), and in theliver EPG (Fig. 1B) of rats fed the adequate ALA diet (P < 0.001, G2vs. G1) or the enriched EPA + DHA diet (P < 0.001, G3 vs. G1, G3 vs.G2). The individual proportions of DHA and of omega-6 DPA in boththe brain and liver tissues responded to diets as follows: DHA washigher (P < 0.001), whereas omega-6 DPA was lower (P < 0.001), inPC and in EPG of cerebrum (Fig. 2A and B) and liver (Fig. 3A and B)of rats fed the adequate ALA diet (G2) or the enriched EPA + DHAdiet in comparison with those fed the low ALA diet.

3.3. MDA content

The content of MDA in the cerebrum was unaffected by neitherthe adequate ALA diet nor the enriched EPA + DHA diet (Fig. 4A). TheMDA content was higher in the liver of the rats received the ade-quate ALA diet or the enriched EPA + DHA diet compared to thosefed the low ALA diet (P < 0.05, G2 vs. G1; P < 0.01, G3 vs. G1, Fig. 4B).Uterine MDA content was increased only by the enriched EPA + DHAdiet (Fig. 4C) in comparison with the adequate ALA diet (P < 0.05,G3 vs. G2, Fig. 4C).

3.4. Antioxidant enzyme activities

ce mitochondrial superoxide dismutase activity in rat organs duringl.2011.10.007

The effects of feeding omega-3 PUFAs on antioxidant enzymeactivities in cerebrum, liver and uterus tissues are given in Figs. 5–7,respectively. Feeding the enriched EPA + DHA diet increased SOD2activity (P < 0.05) in the cerebrum (Fig. 5B). Feeding the adequate

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Fig. 1. Total omega-3 docosahexaenoic acid [DHA, C22:6(n-3)] and omega-6docosapentaenoic acid [DPA, C22:5(n-6)] content in phosphatidylcholine (PC) andethanolamine phosphoglycerides (EPG) phospholipids of cerebrum (A) and liver (B)tissues of female rats born from dams fed a diet low in omega-3 PUFA and fedfor 5 weeks a replenishing diet supplying low �-linolenic acid (ALA) (20 mg/100 gdiet, group 1, G1), adequate ALA (300 mg/100 g diet, group 2, G2) or enriched withD*

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Fig. 2. Omega-3 docosahexaenoic acid [DHA, C22:6(n-3)] and omega-6 docos-apentaenoic acid [DPA, C22:5(n-6)] content in phosphatidylcholine (PC) andethanolamine phosphoglycerides (EPG) phospholipids of cerebrum of female ratsborn from dams fed a diet low in omega-3 PUFA and fed for 5 weeks a replen-ishing diet supplying low �-linolenic acid (ALA) (20 mg/100 g diet, group 1, G1),adequate ALA (300 mg/100 g diet, group 2, G2) or enriched with DHA (500 mg/100 g

HA (500 mg/100 g diet, group 3, G3). Values are means ± SEM for 6 rats per group.**P < 0.001.

LA diet or the enriched EPA + DHA diet increased SOD2 activityP < 0.05) in the liver (Fig. 6B) and uterus (Fig. 7B). The activitiesOD1, CAT and GPX in cerebrum, liver and uterus were unaffectedy neither the adequate ALA diet nor the enriched EPA + DHA dietFigs. 5–7A, C, and D).

. Discussion

Dietary omega-3 PUFAs are of major importance for humanealth, particularly for the optimum development of brain func-ions and the prevention of neurodevelopmental and neurologicalisorders (Guesnet and Alessandri, 2010; Schuchardt et al., 2010).aily intakes of ALA or DHA have been recommended by several

nternational committees of human nutrition, importantly dur-ng pre- and post-natal development. However, the relationshipetween the antioxidant enzyme systems and the fatty acid com-osition of the cell membranes of organs and tissues under in vivo

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onditions of omega-3 PUFA supplementation has not been inves-igated. Our hypothesis is that accumulation and incorporation ofC-PUFAs in cell membrane phospholipids may alter the activity of

diet, group 3, G3). Values are means ± SEM for 6 rats per group. *P < 0.05, **P < 0.01,and ***P < 0.001.

the key ROS-scavenging antioxidant enzymes in response to lipidperoxidation, if any, exerted by omega-3 dietary supplementation.

Our data showed that rats born with a low DHA status andfed adequate ALA or enriched EPA + DHA diet exhibit a significantincrease of DHA and a decrease of omega-6 DPA in cerebrum andliver phospholipids. The ratio of omega-6 DPA to DHA was invertedin the cerebrum and liver of rats feeding the adequate ALA or theenriched EPA + DHA diet compared to those fed the low ALA diet.This inversion from the omega-6 to omega-3 series in the cerebrumdid not modify the total contents of these fatty acids, as the rise inDHA being compensated for by the collapse of omega-6 DPA. Thus,there was a strict homeostasis of the sum of LC-PUFAs from the

ce mitochondrial superoxide dismutase activity in rat organs duringl.2011.10.007

omega-3 and omega-6 series in the brain phospholipids, while theliver was highly sensitive to diets, especially in the EPG fraction.This data confirms that the level in total LC-PUFAs is thoroughly

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Fig. 3. Omega-3 docosahexaenoic acid [DHA, C22:6(n-3)] and omega-6 docos-apentaenoic acid [DPA, C22:5(n-6)] content in phosphatidylcholine (PC) andethanolamine phosphoglycerides (EPG) phospholipids of liver tissues of female ratsborn from dams fed a diet low in omega-3 PUFA and fed for 5 weeks a replenishingdiet supplying low �-linolenic acid (ALA) (20 mg/100 g diet, group 1, G1), adequateA3

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blaaeaNan

Fig. 4. Malondialdehyde (MDA) contents in brain (A), liver (B) and uterus (C) offemale rats born from dams fed a diet low in omega-3 PUFA and fed for 5 weeksa replenishing diet supplying low �-linolenic acid (ALA) (20 mg/100 g diet, group1, G1), adequate ALA (300 mg/100 g diet, group 2, G2) or enriched with DHA

LA (300 mg/100 g diet, group 2, G2) or enriched with DHA (500 mg/100 g diet, group, G3). Values are means ± SEM for 6 rats per group. ***P < 0.001.

egulated to maintain a high level of total polyunsaturation in brainnder condition of inadequate dietary supplies or in relative excess.he metabolic activity of liver contributes to this regulation in pro-iding the brain the neo-formed LC-PUFAs from both omega-3 andmega-6 series, when omega-3 PUFAs are inadequate in diet orhen ALA is provided in adequate amount.

In the present study, increased lipid peroxidation, as evidencedy a significant increase in MDA content, was observed in the

iver of rats supplemented with adequate ALA, and in the livernd uterus of rats supplemented with EPA + DHA. The increasedctivity of SOD2 in the liver and uterus, particularly of rats fednriched EPA + DHA diet, may be a compensatory defense mech-nism in response to increased lipid peroxidation in these organs.

Please cite this article in press as: Garrel C, et al. Omega-3 fatty acids enhanpost-natal development. Int J Biochem Cell Biol (2011), doi:10.1016/j.bioce

evertheless, this hypothesis cannot be validated for the cerebrums there was no change in the MDA content in spite of the sig-ificantly increased incorporation of DHA in phospholipids after

(500 mg/100 g diet, group 3, G3). Values are means ± SEM for 6 rats per group.*P < 0.05, **P < 0.01.

feeding of rats with adequate ALA or enriched EPA + DHA diet. Thisis due to the specific maintaining of the omega-3 + omega-6 LC-PUFA content in phospholipids membranes of the cerebrum. It isimportant to note that the MDA content in cerebrum of rats fed thelow ALA, adequate ALA or enriched EPA + DHA diet was higher thanthat in the liver and uterus (present study). High lipid peroxida-tion already observed in cerebrum of rats fed the low ALA diet maybe related to the high omega-3 + omega-6 polyunsaturation level inthis tissue. Moreover, this may be also explained by the fact that thecerebrum contains low activity of SOD1, GPX and CAT as comparedto the liver or uterus, irrespective of the replenishing diet. The low

ce mitochondrial superoxide dismutase activity in rat organs duringl.2011.10.007

activity of antioxidant enzymes and the elevated content of perox-idizable LC-PUFAs in phospholipids of the cerebrum in our animalmodels feeding adequate ALA or enriched EPA + DHA diet prompted

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Fig. 5. Activities of SOD1, SOD2, CAT and GPX in brain of female rats born from dams fed a diet low in omega-3 PUFA and fed for 5 weeks a replenishing diet supplyinglow �-linolenic acid (ALA) (20 mg/100 g diet, group 1, G1), adequate ALA (300 mg/100 g diet, group 2, G2) or enriched with DHA (500 mg/100 g diet, group 3, G3). Values aremeans ± SEM for 6 rats per group. *P < 0.05.

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Fig. 6. Activities of SOD1, SOD2, CAT and GPX in liver of female rats born from dams fed a diet low in omega-3 PUFA and fed for 5 weeks a replenishing diet supplying low�-linolenic acid (ALA) (20 mg/100 g diet, group 1, G1), adequate ALA (300 mg/100 g diet, group 2, G2) or enriched with DHA (500 mg/100 g diet, group 3, G3). Values aremeans ± SEM for 6 rats per group. *P < 0.05.

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ig. 7. Activities of SOD1, SOD2, CAT and GPX in uterus of female rats born from dow �-linolenic acid (ALA) (20 mg/100 g diet, group 1, G1), adequate ALA (300 mg/1

eans ± SEM for 6 rats per group. *P < 0.05.

s to suggest that the cerebrum, although highly susceptible toOS-induced oxidative damage (Wang and Michaelis, 2010), has

specific protective mechanism against lipid peroxidation whichurrently remains unknown.

A decrease in activities of antioxidant enzymes is one of theain alterations that promote oxidative damage to proteins and

ipids (Slater et al., 1987). Under the feeding experimental condi-ions of the present study, the activities of SOD1, CAT and GPX in theerebrum, liver and uterus were unaffected by feeding the grow-ng female rats with the adequate ALA or enriched EPA + DHA diet.n contrast, a significant increase in SOD2 activity was observed inerebrum collected from rats fed on the diet supplemented withnriched EPA + DHA, and in liver and uterus of rats fed on theiet supplemented with adequate ALA or enriched EPA + DHA. Pre-ious animal studies have reported interesting but contradictoryndings. Activity of liver SOD was not different between rats fed

or 4 weeks coconut or fish oil (D’Aquino et al., 1991) or rats fedor 8.5 weeks groundnut or fish oil (Ramaprasad et al., 2005). Inhese studies only total SOD activity was measured which is notnformative of the changes in activity of cytosolic SOD1 and/or

itochondrial SOD2. Activity of SOD2 was significantly reduced inearts of rats born from dams fed a fish-oil diet during the last twoeeks of pregnancy and then were fed this diet from 3 through

weeks of age as compared to that in hearts of rats fed an oillend (Chapman et al., 2000). The differences between the data ofhese studies and that of the present study could be explained, ateast in part, by experimental conditions. In particular, we used

sequence of pre-natal deficiency and post-weaning replenish-

Please cite this article in press as: Garrel C, et al. Omega-3 fatty acids enhanpost-natal development. Int J Biochem Cell Biol (2011), doi:10.1016/j.bioce

ent which may differently impact on SOD2 activity compared toermanent feeding with a fish oil-based diet.

The generation of •O2− by a single electron donation to molec-

lar oxygen is the first step in the formation and propagation

fed a diet low in omega-3 PUFA and fed for 5 weeks a replenishing diet supplyingiet, group 2, G2) or enriched with DHA (500 mg/100 g diet, group 3, G3). Values are

of H2O2, •OH and ONOO− within mitochondria (Adam-Vizi andChinopoulos, 2006). These mitochondrial ROS can be releasedinto the cytosol and could function as a second messenger toactivate ROS-induced ROS release in neighboring mitochondria.Such mitochondrion-to-mitochondrion ROS-signaling constitutesa positive feedback mechanism for ROS production leading to mito-chondrial injury (Zorov et al., 2006). Mitochondria are endowedwith NOS and thus •NO is produced by these organelles (Ghafourifarand Richter, 1997). The interaction of accumulated mitochondrial•O2

− and •NO yields ONOO− and alters levels of •NO, which in turnaffect cellular functions (Garrel and Fontecave, 1995; Packer et al.,1996). Furthermore, cellular •O2

− accumulation can increase lipidperoxidation (Slater et al., 1987) by affecting the redox cycling ofiron (Minotti and Aust, 1992). Indeed, •O2

− enhances cell concen-trations of Fe2+, which is a key reactive element in the formation of•OH from H2O2 via the Fenton reaction (Kehrer, 2000). Since there isno known direct enzymatic pathway to scavenge •OH and ONOO−,these highly reactive and toxic free radicals are considered as themain ROS that damage DNA, proteins and lipids in biological sys-tems (Beckman and Koppenol, 1996). It is of interest to highlightthat supplementation of 5-week-old male rats with fish oil rich inomega-3 PUFA for 6 weeks offer protection against ROS-inducedoxidative stress and DNA damage in liver as compared with ratssupplemented with safflower oil rich in omega-6 PUFA (Kikugawaet al., 2003). Reduced expression and production of SOD2 and theconsequently accumulated •O2

− leading to cardiac hypertrophy,dilated cardiomyopathy and premature death (Ding et al., 2007)and render the peripheral nervous system more susceptible to

ce mitochondrial superoxide dismutase activity in rat organs duringl.2011.10.007

hyperglycemia-induced oxidative stress (Vincent et al., 2007). Wesuggest that the enhanced activity of SOD2 in cerebrum, liver anduterus of female rats born from omega-3 PUFA-deficient damsand fed a replenishing diet supplying adequate ALA or enriched

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8 C. Garrel et al. / The International Journal of Biochemistry & Cell Biology xxx (2011) xxx– xxx

Fig. 8. A schematic overview of the potential protective role of omega-3 polyunsaturated fatty acid (PUFA) against reactive oxygen species (ROS)-induced oxidative cellulardamage and death. The production of superoxide radical (•O2

−) by a single electron donation to molecular oxygen (O2) is the initial step in the formation and propagation ofROS within and out of the mitochondria. Imbalance between ROS production and the enzymatic antioxidant systems increases tissues and organs vulnerability to oxidativedamage. The •O2

− radicals react with nitric oxide (•NO) in a reaction controlled by the production and bioavailability of both radicals to form peroxynitrite (ONOO−). Inthe presence of transition iron, •O2

− and hydrogen peroxide (H2O2) interact in a Haber–Weiss reaction to generate hydroxyl radical (•OH). The highly reactive ONOO− and•OH both can damage cellular nucleic acids, proteins or lipids with the ensuing deleterious consequences for the organism. Decreased •O2

− bioavailability by increasingm − anda emova

Epqapcpo

5

tSdicfpordo

A

M

itochondrial superoxide dismutase (SOD2) activity subsequently deceases ONOOctivity, and consequently reduces ONOO− and •OH formation. The H2O2 could be rre unchanged by omega-3 PUFA.

PA + DHA (present study) may act as protective mechanism torevent accumulation and bioavailability of •O2

− and the subse-uent production and propagation of harmful ROS, such as •OHnd ONOO−, within and out of the mitochondria. Such mechanismrevents alteration in mitochondrial DNA, membranes damage andell death. Based on our findings, a schematic overview of theotential protective role of omega-3 PUFAs against ROS-inducedxidative damage is presented in Fig. 8.

. Conclusions

The present in vivo study demonstrates that dietary supplemen-ation with omega-3 PUFAs enhances the activity of mitochondrialOD2 in the growing cerebrum, liver and uterus of rats born fromams fed a diet low in omega-3 PUFAs. Our data suggest that

ncreased SOD2 activity in organs of the growing female rats is aritical determinant in the tolerance to oxidative stress induced byeeding a diet supplemented with omega-3 PUFAs. As mitochondriaroduce a large flux of ROS, omega-3 PUFAs-induced enhancementf SOD2 activity is believed to be a specific cellular antioxidantesponse to prevent •O2

− accumulation and the subsequent pro-uction and propagation of potentially harmful ROS within and outf the mitochondria during post-natal development.

Please cite this article in press as: Garrel C, et al. Omega-3 fatty acids enhanpost-natal development. Int J Biochem Cell Biol (2011), doi:10.1016/j.bioce

cknowledgements

The authors would like to thank Krawiec Angele, Catherineangournet, Sandra Grange, Christine Tozzoli, Cécile Mounioz,

•OH formation. Omega-3 PUFA decreases •O2− bioavailability by increasing SOD2

ed by glutathione peroxidase (GPX) and catalase (CAT) whose enzymatic activities

Laurence Puillet-Anselme (CHU Grenoble), and Alain Linard, Marie-Sylvie Lallemand, Philippe Bolifraud (INRA, Jouy-en-Josas) for theirexcellent technical assistance. We are very grateful to PatriceDahirel and Claire Maudet (INRA, Jouy-en-Josas) for outstandingrodents’ management and care.

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