larvi 2013 Oxidative stress in sea bass (Dicentrarchus labrax) larvae interaction of high dietary DHA contents and several antioxidant nutrients

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1 lrvi th fish & shellfish lrviculture symposium Oxidtive stress in se ss (Dicentrrchus lrx) lrve interction of high dietry DHA contents nd severl ntioxidnt nutrients Monic Betncor ghent university, elgium, 2-5 septemer 2013

2 Oxidtive stress in se ss (Dicentrrchus lrx) lrve fed on high DHA microdiets. Involvement of severl ntioxidnt nutrients University of Ls Plms de GC, Cnry Islnds, Spin Mónic B Betncor Mª José Cllero Author present ddress Mrisol Izquierdo

3 Energy Lipids 1-20% PLs Protein 15-20% Crohydrtes <2% Crriers Hormones LC-PUFA DHA 22:6n-3 Memrne function Izquierdo & Koven, 2010 EPA 20:5n-3 Growth & stress resistnce Wtne & Kiron, 1994 ARA 20:4n-6 Selectively retined Rinuzzo et l., 1993

4 Mourente et l., 2003; Benítez-Sntn et l., 2007 Srgent et l., 1995 Izquierdo, 2005 Incresed susceptiility to Ngok et l., 1990 ROS ROS ROS Species Sprus urt Scophtlmus mximus Lrve DHA Lrve DHA content Decrese in DHA 10 dph Authors (Requirements) requirements (%) (% totl ftty cid) Wtne, 1993 Izquierdo, 2005 >3 30.1± 0.4 Le Milinire, ± Pgrus pgrus Hernández et l., 1999 Dentex dentex Mourente et l., 1999 Seriol dumerilii Lurel et l., 2010 Fernández-Plcios et l., 2011 Izquierdo, 2005

5 Wht is the effect of high DHA dietry levels on se ss lrve?

6 Experimentl conditions: -Se ss lrve L grey cylinder fire glss tnks -Temperture C -Oxygen 5-8 g L-1 -Slinity 34 g L-1 -Photoperiod 12:12 -Mnully clened -Wter flow L min-1

7 Diets nd Experimentl Design -Five experiments -Two, three or five weeks DHA (g/100g) α-toh (mg/100g) /150 5/ /300 3/300 5/300

8 Betncor et l., Aqucult Nutr Finl survivl Totl length 15,0 100 c 14,5 95 cd mm d % c 14, , , ,5 75 3/150 Dietry DHA/vitmin E 5/150 c 3/150 Dietry DHA/vitmin E High DHA Reduced growth nd survivl 5/150

9 Fire swelling Eosinophilic nd ple cytoplsm Grnulr cytoplsm Fire rekge Monocyte infiltrte Musculr deris+infiltrte Musculr dystrophy Lovell et l., 1984; Gtlin et l., 1986; Frischknecth et l., 1994; Bowter & Burren, 2007; Leold et l., 2013

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11 1,20E+04 MyHC LESIONS 1,00E+04 8,00E+03 6,00E+03 4,00E+03 2,00E MyHC mrna copy nº/ng totl RNA IGF-I mrna copy nº/ng totl RNA IGF-I 4,00E+07 3,50E+07 3,00E+07 2,50E+07 2,00E+07 1,50E+07 1,00E+07 5,00E+06 5/ / Regenerting fires Rowlerson et l., 1997 Stellite cell pool control Grounds,1999; Sele & Rudnicki, 2000 Muscle regenertion process Betncor et l., CBPA

12 α-ctin ROS Memrne lesion 1,20E+08 1,10E+08 Permeility Severl α-ctin isoforms H2O C2+ NWte, 1,00E+08 9,00E+07 8,00E+07 7,00E+07 SR oedem 6,00E+07 Clpins 5,00E Cellulr oedem Mitochondri oedem Proteolysis 34 5/150 MPT Cytoskeleton lesion Cytochrome c Cpn1 3,00E+05 Necrosis 2,80E+05 Apoptosis 2,60E+05 2,40E+05 2,20E+05 2,00E+05 1,80E+05 1,60E+05 1,40E /150 Betncor et l., CBPA

13 THIN SECTIONS N+, C2+ nd wter influx Filure of ion pumps Hydropic degenertion Cotrn et l., 2004 Dilttion Vcuoles SR

14 1. High DHA (5%) microdiets led to reduced growth nd survivl nd fvoured the ppernce of musculr lesions in se ss lrve 2. One of the first ltertions tkes plce t the cell memrne, proly due to the direct ttck of ROS to phospholipids, cusing ltertion of itsispermeility nd leding to cell nd orgnelles Wht the effect of high DHA dietry swelling. Mssive influx of clcium occurs nd consequent levels onlysis se ss lrve? ctivtion of µ-clpins protein Is there n in cuses vivo oxidtion?? Could it e prevented withmuscle incresed levels ofshown vitmin 3. A compenstory response ws found, s y thee? increse in insulin-like growth fctors nd myosin hevy chin gene expression, s well s the undnt ctivted stellite cells

15 Totl length Incidence of musculr lesions , ,5 Totl length mm % 25 16, ,5 c cd d 14,0 13,5 13,0 12,5 12,0 0 1/300 3/150 3/300 Dietry DHA/vitminE 5/150 1/300 3/150 3/300 5/150 Dietry DHA/vitmin E Vitmin E increse cn protect se ss lrve from incresed DHA contents Betncor et l., Aqucult Nutr

16 TBARS content 700 Incidence of musculr lesions t 35 dph % nm MDA/tissues Initil 5/150 High DHA+High VitE Incresed oxidtion 5/300 5/150 5/300 Betncor et l., J Fish Dis

17 = ROS O2 Cell Interior = AOE O2 - SOD H2O2 Fe+3 α-toh O2- Fenton CAT H2O 2GSH OH + α-toh +OH- GR Fe+2 GPX GSSG H2O α-toh α-toh Cell Exterior

18 Which other nutrients could protect se ss lrve when high levels of DHA re used in their diets?

19 Active sites of the GPX Felton et l., 1996 Synergistic ction etween α-toh nd Se Poston et l., 1976; Bell & Cowey, 1985 Spring effect etween α-toh nd Se Lin & Shiu, 2009 Involved in thyroid hormone nd insulin function, mintennce of fertility nd regultion of cell growth Lll, 2002; Kohlmeier, 2003

20 Vitmin E content TBARS µg/g dry mss nmol/g dry mss c Initil / /300+Se Initil 5/300 5/300+Se Diet Lrve No dose dependnt effect Kiron et l., 2004 Antioxidnt protection Betncor et l., Br J Nutr Antioxidnt Tissue vitmin E Pungkew et l., 2005

21 Incidence of musculr lesions % /300 5/300+Se Se ccumultion in muscle Monteiro et l., 2009; Eli et l., 2011

22 Selenium content 7 Tissue concentrtion µg/mg dry mss % 83.46% % 3 2 Antioxidnt c 1 Low retention rte GPX?? 0 Initil Diet 5/300 Betncor et l., Br J Nutr Lrve 5/300+Se

23 GPX GPX mrna copy nº/ng totl RNA 6,50E+05 6,00E+05 Antioxidnt defence 5,50E+05 (Burk et l., 1997) 5,00E+05 4,50E+05 4,00E+05 3,50E+05 3,00E+05 2,50E selenocysteine residues (Tujev et l., 2000) Lrgely used s n ntioxidnt 2,00E Dys post htch 5/ /300+Se Sturted t norml nutritionl intkes Betncor et l., Br J Nutr

24 RO RO+Se DHA DHA+Se DHA (% totl ftty cids) Se (ppm) months tril Two ges 7 months 20 months Glutthione peroxidses ID2 SEPW 15 KD GPX4 Thyroid Hormone Deiodinses Thioredoxin reductses SEPP1 GPX3 TR1 Selenoproteins Selenocysteine iosynthesis SPS2 SEC43

25 Young fish more sensitive to oxidtive stress Selenium content 0,7 0, nmol MDA/g lipid 2500 mg/kg 0,4 0,3 0,2 c 0,1 RO+Se Old DHA Young RO ,5 TBARS DHA+Se 0 RO RO+Se Old DHA Young DHA+Se

26 Young Old Reduced mrna copy no. TR1 1,6 1,2 1 0,8 0,6 0,4 0,2 0 RO DI2 1,4 Hierrchichl regultion of selenoprotein expression (Schomurg nd 1,2 Schweizer, 2009) 1,4 1,6 RO+Se Old DHA Young DHA+Se Reltive expression 1,8 Reltive expression Thioredoxin reductses 2 Thyroid Hormone Deiodinses (Benner et l., 2010) 1 0,8 0,6 0,4 0,2 0 RO RO+Se Old DHA Young DHA+Se

27 Young Old Selenide+ATP SPS2 Selenophosphte Selenoproteins 1,2 1 Sep W 0,8 0,6 0,4 0,2 0 RO RO+Se Old DHA Young DHA+Se Selenophosphte synthetse Low levels WMD 1,4 1,2 SPS2 1 0,8 0,6 0,4 0,2 0 RO RO+Se Old DHA Young DHA+Se

28 Protects low density LP Levine, 1986 Recycling of α-toh Hmre et l., 1997; Seley & Gtlin, 2002 Spring effect etween α-toh nd AA Shiu & Shu, 2002; Yildirim-Aksoy et l., 2008

29 TBARS Tppel, 1962 Adpted from Hmre, NADH 2500 NAD Asc-H 1000 c 0 µg/g dry mss nmol/g dry mss Vitmin E content NADP NADPH Asc O O O Wter phse Lipid phse Diet Initil Hmre & Lie, 1995; Phospholipid Kolkovski with PUFAs et l., 2000 with PUFAs Tocopheroxyl rdicl Asc-H+ DHA 2 Asc O H0 α-tocopherol Phospholipid depletion GSSG GSH 5/300 Lrve 5/300+AA Vitmin E spring/recycling Phospholipid Seley with PUFAs& Gtlin, α-tocopherol 2002; Shiu & Shu, 2002

30 Incidence of musculr lesions % /300 5/300+AA SR

31 Inclusion of oth orgnic selenium (5 mg kg-1) nd scoric cid (180 mg 100 g-1 ) to diets with 5% of DHA nd 300 mg 100 g-1 of Which other nutrients could protect se vitmin E controlled in vivo lipid peroxidtion nd decresed the incidence of musculr lesions compred to the ss lrve when highwhen levels of DHA re unsupplemented diets used in their diets? In zerfish muscle TR1, ID2, SEPW nd SPS2 expression is ffected y the ddition of 7 ppm of Se when high DHA diets re used, indicting tht these could e good iomrkers of the oxidtive sttus in teleost fish

32 Thnks for your ttention University of Ls Plms de GC, Cnry Islnds, Spin Mónic B Betncor Mª José Cllero Mrisol Izquierdo