diapositiva 1 - ugent 4 - wednesday... · benítez-santana et al., 2007 sargent et al., 1995...
TRANSCRIPT
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larvi 2013
ghent university, belgium, 2-5 september 2013
6th fish & shellfish larviculture symposium
Oxidative stress in sea bass(Dicentrarchus labrax) larvae
interaction of high dietary DHA contentsand several antioxidant nutrients
Monica Betancor
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Oxidative stress in sea bass (Dicentrarchus labrax)larvae fed on high DHA microdiets. Involvement of
several antioxidant nutrients
Mónica B Betancor*Mª José CaballeroMarisol IzquierdoUniversity of Las Palmas de GC,
Canary Islands, Spain*Author present address
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Carbohydrates<2%
Protein15-20%
Lipids1-20%
Carriers
PLs
Energy
Hormones
EPA20:5n-3
ARA20:4n-6
DHA22:6n-3
Growth & stressresistance
Watanabe & Kiron, 1994
Membrane functionIzquierdo & Koven, 2010
Selectively retainedRainuzzo et al., 1993
LC-PUFA
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Fernández-Palacios et al., 2011
Decrease in DHA 10 dphWatanabe, 1993
Species Larvae DHArequirements (%)
Larvae DHA content(% total fatty acid)
Authors (Requirements)
Sparus aurata >3 30.1± 0.4 Izquierdo, 2005
Scophtalmusmaximus
3.2 27.0 ± 0.3 Le Milinaire, 1984
Seriola dumerilii 4 - Izquierdo, 2005
Pagrus pagrus 3.4 26.9 Hernández et al., 1999
Dentex dentex 4 27.6 Mourente et al., 1999
Mourente et al., 2003;Benítez-Santana et al., 2007
Sargent et al., 1995 Izquierdo, 2005
Laurel et al., 2010
ROS
ROS
ROS
Increased susceptibility to
Nagaoka et al., 1990
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What is the effect of high DHA dietarylevels on sea bass larvae?
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Experimental conditions:
- 170 L grey cylinder fibre glass tanks
-Photoperiod 12:12
-Sea bass larvae
-Temperature 19.5-20°C
-Oxygen 5-8 g L-1
-Salinity 34 g L-1
-Manually cleaned-Water flow 1.0-1.5 L min-1
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Diets and Experimental Design
DHA (g/100g)
α-TO
H(m
g/10
0g)
1 3 5
150
300
1/150
1/300 3/300 5/300
3/150 5/150
-Five experiments-Two, three or five weeks
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Betancor et al., 2011. Aquacult NutrBetancor et al., 2011. Aquacult Nutr
12,5
13,0
13,5
14,0
14,5
15,0
1/150 3/150 5/150
mm
Dietary DHA/vitamin E
d
bc
cd
Total length
High DHA
Reduced growth and survival
75
80
85
90
95
100
1/150 3/150 5/150
%
Dietary DHA/vitamin E
c
bc
ab
Final survival
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Muscular dystrophyLovell et al., 1984; Gatlin et al., 1986;Frischknecth et al., 1994; Bowater &
Burren, 2007; Lebold et al., 2013
Fibre swelling Eosinophilic and pale cytoplasm
Muscular debris+InfiltrateFibre breakage Monocyte infiltrate
Granular cytoplasm
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2,00E+03
4,00E+03
6,00E+03
8,00E+03
1,00E+04
1,20E+04
14 26 35
IGF-
I mRN
A co
py n
º/ng
tota
l RN
A
1/150 5/150
b
a
b
a
IGF-I
Regenerating fibresRowlerson et al., 1997Regenerating fibresRowlerson et al., 1997Regenerating fibresRowlerson et al., 1997
Satellite cell pool controlGrounds ,1999; Seale & Rudnicki, 2000
Satellite cell pool controlGrounds ,1999; Seale & Rudnicki, 2000
Satellite cell pool controlGrounds ,1999; Seale & Rudnicki, 2000
Muscle regeneration processMuscle regeneration processMuscle regeneration process
5,00E+06
1,00E+07
1,50E+07
2,00E+07
2,50E+07
3,00E+07
3,50E+07
4,00E+07
14 26 35MyH
C m
RNA
copy
nº/
ng to
tal R
NA
1/150 5/150
b
a
b
a
MyHC
↑ LESIONS
Betancor et al., 2013. CBPABetancor et al., 2013. CBPA
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α-actin
5,00E+07
6,00E+07
7,00E+07
8,00E+07
9,00E+07
1,00E+08
1,10E+08
1,20E+08
14 19 24 29 34
1/150 5/150
1,40E+05
1,60E+05
1,80E+05
2,00E+05
2,20E+05
2,40E+05
2,60E+05
2,80E+05
3,00E+05
14 19 24 29 34
1/150 5/150
ba
Capn1
↑ ROS
Membrane lesion
↑ Permeability
↑ Ca2+ ↑ Na+↑ H2O
Cellularoedema
Mitochondriaoedema
MPT
Cytochrome c
ApoptosisNecrosis
↑ Calpains
↑ Proteolysis
Cytoskeleton lesion
SR oedema
Several α-actin isoformsWatabe, 2001
Betancor et al., 2013. CBPABetancor et al., 2013. CBPA
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THIN
SEC
TIO
NS
*
*
***
VacuolesDilatation SR
*
Hydropic degenerationCotran et al., 2004
Failure of ion pumps
Na+, Ca2+ and water influx
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What is the effect of high DHA dietarylevels on sea bass larvae?
1. High DHA (5%) microdiets lead to reduced growth and survival andfavoured the appearance of muscular lesions in sea bass larvae
2. One of the first alterations takes place at the cell membrane,probably due to the direct attack of ROS to phospholipids, causingalteration of its permeability and leading to cell and organellesswelling. Massive influx of calcium occurs and a consequentactivation of µ-calpains causes protein lysis
3. A compensatory muscle response was found, as shown by theincrease in insulin-like growth factors and myosin heavy chain geneexpression, as well as the abundant activated satellite cells
Is there an in vivo oxidation?? Could it beprevented with increased levels of vitamin E?
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0
5
10
15
20
25
30
35
1/150 1/300 3/150 3/300 5/150
%
Dietary DHA/vitaminE
Incidence of muscular lesions
ab
b
ab
ab
a
Vitamin E increase can protect seabass larvae from increased DHAcontents
Betancor et al., 2011. Aquacult NutrBetancor et al., 2011. Aquacult Nutr
12,0
12,5
13,0
13,5
14,0
14,5
15,0
15,5
16,0
1/150 1/300 3/150 3/300 5/150
Tota
llen
gth
mm
Dietary DHA/vitamin E
a
d
bcb
cd
Total length
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0
5
10
15
20
25
30
1/150 5/150 5/300
%
TBARS content Incidence of muscular lesions at 35 dph
0
100
200
300
400
500
600
700
Initial 1/150 5/150 5/300
nMM
DA/t
issu
es
Betancor et al., 2012. J Fish DisBetancor et al., 2012. J Fish Dis
High DHA+High VitE↓
Increased oxidation
b
a a
aa
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Cell ExteriorCell Exterior
Cell InteriorCell InteriorO2
H2O
H2O
+OH-Fenton
Fe+2
Fe+3
GSSG
2GSH
O2-· H2O2
O2-·
OH·+
CATSOD
GPXGR
= ROS
= AOE
α-TOH α-TOHα-TOH α-TOH
α-TOH α-TOH α-TOH α-TOH
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Which other nutrients could protectsea bass larvae when high levels of DHA
are used in their diets?
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Active sites of the GPX
Felton et al., 1996
Sparing effect between α-TOH and Se
Lin & Shiau, 2009
Synergistic action between α-TOH and Se
Poston et al., 1976; Bell & Cowey, 1985
Involved in thyroid hormone and insulinfunction, maintenance of fertility andregulation of cell growth
Lall, 2002; Kohlmeier, 2003
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0
500
1000
1500
2000
2500
3000
Initial 1/150 5/300 5/300+Se
nMol
/gdr
ym
ass
TBARS
a
cb
0
500
1000
1500
2000
2500
3000
3500
Diet Larvae
µg/g
dry
mas
s
Initial1/1505/3005/300+Se
Vitamin E content
AntioxidantAntioxidant protection
No dose dependant effectKiron et al., 2004
↓ Tissue vitamin EPuangkaew et al., 2005
Betancor et al., 2012. Br J NutrBetancor et al., 2012. Br J Nutr
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0
10
20
30
40
50
60
70
1/150 5/300 5/300+Se
%
Incidence of muscular lesions
**
*
* *
*
ab
a
b
**
↓ Se accumulation in muscleMonteiro et al., 2009; Elia et al., 2011
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0
1
2
3
4
5
6
7
Diet Larvae
µg/m
gdr
ym
ass
Initial 1/150 5/300 5/300+Se
Selenium content
a
cb
35.88%
83.46%
100%
Antioxidant
↓ Tissue concentration
Low retention rate – GPX??
Betancor et al., 2012. Br J NutrBetancor et al., 2012. Br J Nutr
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2,00E+05
2,50E+05
3,00E+05
3,50E+05
4,00E+05
4,50E+05
5,00E+05
5,50E+05
6,00E+05
6,50E+05
14 26 35
GPX
mRN
A co
py n
º/ng
tota
l RN
A
Days post hatch
1/150 5/300 5/300+Se
ab
a
b
a
b
GPX
Antioxidant defence(Burk et al., 1997)
17 selenocysteine residues(Tubajeva et al., 2000)
Largely used as an antioxidant
Saturated at normal nutritionalintakes
Betancor et al., 2012. Br J NutrBetancor et al., 2012. Br J Nutr
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RO RO+Se DHA DHA+Se
DHA (% total fatty acids) 0.7 0.2 19.0 18.5
Se (ppm) 1.7 7.0 1.3 6.3
Two ages7 months 20 months
2 months trial
Glutathione peroxidases
Thyroid Hormone Deiodinases
Thioredoxin reductases
Selenoproteins
Selenocysteine biosynthesis
GPX3 GPX4b
ID2
TR1
SEPP1 SEPW 15 KDa
SPS2 SEC43
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0
0,1
0,2
0,3
0,4
0,5
0,6
0,7
RO RO+Se DHA DHA+Se
mg/
kg
Selenium content
Old Young
a a
a
b
b
bb
c
0
500
1000
1500
2000
2500
3000
3500
RO RO+Se DHA DHA+Se
nMol
MDA
/g li
pid
TBARS
Old Young
a
b
b
b
Young fish more sensitive to oxidative stress
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Old Young
Thyr
oid
Horm
one
Deio
dina
ses
0
0,2
0,4
0,6
0,8
1
1,2
1,4
1,6
1,8
2
RO RO+Se DHA DHA+Se
Rela
tive
expr
essi
on
TR1
Old Young
*
*
aaab
b
0
0,2
0,4
0,6
0,8
1
1,2
1,4
1,6
RO RO+Se DHA DHA+Se
Rela
tive
expr
essi
on
DI2
Old Young
*
a
bab
b
Thio
redo
xin
redu
ctas
es Hierarchichal regulation of selenoprotein expression(Schomburg and Schweizer, 2009)
Reduced mRNA copy no.(Benner et al., 2010)
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Sele
nopr
otei
ns
Old Young
0
0,2
0,4
0,6
0,8
1
1,2
RO RO+Se DHA DHA+Se
Sep W
Old Young
a a
b b
0
0,2
0,4
0,6
0,8
1
1,2
1,4
RO RO+Se DHA DHA+Se
SPS2
Old Young
Sele
noph
osph
ate
synt
heta
se aab ab
b
Low levels→WMDSelenide+ATP
↓SPS2Selenophosphate
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Protects low density LP
Levine, 1986
Recycling of α-TOH
Hamre et al., 1997; Sealey & Gatlin, 2002
Sparing effect between α-TOH and AA
Shiau & Shu, 2002; Yildirim-Aksoy et al., 2008
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0
500
1000
1500
2000
2500
3000nM
ol/g
dry
mas
sTBARS
0
500
1000
1500
2000
2500
3000
3500
Diet Larvae
µg/g
dry
mas
sInitial 1/150 5/300 5/300+AA
c
a
b
ab ab
Vitamin E content
Vitamin E sparing/recyclingSealey & Gatlin, 2002; Shiau & Shu, 2002
α-tocopherol depletionHamre & Lie, 1995; Kolkovski et al., 2000
NADP NADPH
O
O·
O
OH
Asc-H
NADHNAD
Asc· Asc-H+DHA
2 Asc·
GSSGGSH
Water phase
Lipid phase
Phospholipidwith PUFAs
Phospholipidwith PUFAs
Phospholipidwith PUFAs
Tocopheroxylradical
α-tocopherol
Tappel, 1962Adapted from Hamre, 2011
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0
10
20
30
40
50
60
70
1/150 5/300 5/300+AA
%
Incidence of muscular lesions
b
a
b
SR
*
*
*
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Inclusion of both organic selenium (5 mg kg-1) and ascorbic acid(180 mg 100 g-1 ) to diets with a 5% of DHA and 300 mg 100 g-1 ofvitamin E controlled in vivo lipid peroxidation and decreased theincidence of muscular lesions when compared to theunsupplemented diets
Which other nutrients could protect seabass larvae when high levels of DHA are
used in their diets?In zebrafish muscle TR1, ID2, SEPW and SPS2 expression is affectedby the addition of 7 ppm of Se when high DHA diets are used,indicating that these could be good biomarkers of the oxidativestatus in teleost fish
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Thanks for your attention
Mónica B BetancorMª José CaballeroMarisol IzquierdoUniversity of Las Palmas de GC,
Canary Islands, Spain