Ecotossicologia delle ossilipine da Diatomee · - Sphaerechinus granularis - Paracentrotus lividus....
Transcript of Ecotossicologia delle ossilipine da Diatomee · - Sphaerechinus granularis - Paracentrotus lividus....
Giornate di studio - Livorno ottobre 2010
Ecotossicologia delle ossilipine da Diatomee..
Giovanna RomanoGiovanna RomanoStazione Zoologica Anton Stazione Zoologica Anton DohrnDohrn
There are more than 200 genera of living diatoms, and it is estimated that there are approximately 100,000 speciesDiatoms can be found in the oceans and in freshwater. Most live pelagically in open water, although some live as surface films at the
water-sediment interface (benthic), or even under damp atmospheric conditions. They are especially important in oceans, where they are estimated
to contribute up to 45% of the total oceanic primary production.
Ecotossicologia delle ossilipine da Diatomee Introduction
Ecotossicologia delle ossilipine da Diatomee Introduction
High diatomconcentration
Low viability
February 1997 February 1998June 1997Egg production Egg production Egg production
Ecotossicologia delle ossilipine da Diatomee Introduction
Miralto Miralto etet al. Nature 1999al. Nature 1999
Ecotossicologia delle ossilipine da Diatomee Introduction
Diatom densityDiatom density--dependent effect on dependent effect on CalanusCalanus hatching successhatching success
104cells ml-1 P.minimum
P.tricornutum104
105
106
(Cells ml-1)
1020304050 60708090
100
10 20 30 40
% e
gg v
iabi
l ity
ChaudronChaudron et al. et al. Mar.Ecol.Prog.SerMar.Ecol.Prog.Ser. 1996. 1996
Days
Introduction
ab
%
0 2 4 6 8 10
0
20
40
60
80
100
abno
rmal
nau
plii
Days
0 2 4 6 8 100
20
40
60
80
100
% h
atch
ed n
aupl
ii
Days
Effect of Skeletonema marinoi diet on Calanus helgolandicus
CHO
CHO
CHO
A1
A2
A3
0.0 0.5 1.0 1.5 2.00
20
40
60
80
100
% E
gg h
atch
ing
µg ml-1
IanoraIanora etet al. Nature 2004al. Nature 2004
IntroductionOxylipins in diatoms
CO2H
OOHCO2H
OHCO2H
CO2HOOH
CO2HO
CO2HOH
CO2H
OOHCO2H
OHCO2H
CO2HOOH
CO2HO
CO2HOH
HTA
6-LOXHTrA
6-LOX 9-LOX
9-LOX
O
OCO2H
CHO
CO2H
HOO
CO2H
HOCO2H
OOH
CO2H
O
CO2HCHO
OOH
OH CO2H
CO2H
CO2HOOH
OHC
CO2H
HO
EPA11R-LOX
15S-LOX
OH
9S-LOX
5R-LOX
14-LOX
CO2HOH
CO2H
HOO
CO2H
OH
decatrienal
octatrienal
octadienal
C16-PUFAs
C20-PUFA
eptadienal
Fontana et al 2007
Polyunsaturated C16 or C 20 fatty acids
Lipoxygenases
Hydroxides
Hydroperoxides
Aldehydes and oxoacids Epoxy alcohols
O2
R R'
OH
RR
OR
R R'
OOH
ROH
OR
Polyunsaturated C18 fatty acids
Lipoxygenases
Hydroxides
Hydroperoxides
Aldehydes and
oxoacids
Epoxy alcohols
O2
R R'
OOH
R R'
OH
RR
R
OH
O
RR O
Cyclizedcompounds
Epoxides
Divinylethers
R R'
O
R RO
OCOOH
Bleè, TRENDS in Plant Science 2002
Glycolipids Phospholipids
Lipolytic Activity
Glycolipids Phospholipids
Lipolytic Activity
Fontana et al., ChemBioChem 2007
Oxylipin metabolism Introduction
IntroductionWound-activated defence
CHO
Introduction
0
25
50
75
100
0 10 20 30 40min.
alde
hyde
(fg/
cell)
AldehydeAldehyde synthesis insynthesis in S. S. marinoimarinoi
0.0 2.5 5.0 7.5 10.0 12.5 15.00.00
0.05
0.10
0.15
0.20
0.25
time (days)
Abs
orba
nce
AldehydeAldehyde detection in detection in sea watersea water
Fontana et al., 2007 Romano et al. 2010
Effect of diatom aldehydes on marine organisms
Polychaeta (Anellida) - Arenicola marina- Nereis virens
- Cleavage inhibition- Hatching reduction - Larval toxicity- Inhibition of sperm motility
Echinodermata- Asterias rubens- Psammechinus miliaris- Sphaerechinus granularis- Paracentrotus lividus
Crustacea- Tisbe holothuriae (copepod)
- Artemia salina- various species of
Copepods
Results
Caldwell 2009; Ianora and Miralto 2009
ResultsEffect of hydroperoxydes, aldehydes and oxylipins on egg viability and naupliar abnormalities
Fontana Fontana etet al. 2007al. 2007
ResultsApoptosis Induction
Copepods
Agarose gel analysis of PCR amplified DNA isolated from copepod embryos.(C: control; 3h and 1h: time of incubation in 5 µg/ml DD; M: markers)
Calanus helgolandicus embryos observed by confocal laser scanning microscopy in fluorescent (A, C, E) and in trasmitted(B, D, F) light. In (A) and (C) nuclei are positively stained (green) by TUNEL. Bar, 40µm. (E) Three-dimensional image of control embryo. Nuclei are not stained in green and appear as black shadows.
A B
C D
E F
C 3h 1h M
contr
olde
cadie
nal in
vivo
deca
diena
l in vi
tro
0
25
50
75
100
% o
f apo
ptot
ic e
mbr
yos
Romano Romano etet al, J al, J ExpExp BiolBiol 20032003
ResultsEffect of aldehydes on sea urchin embryos
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.50
25
50
75
100
(a)
eptadienaleoctadienalmix HD+OD+DD
decadienal
µg ml-1
% c
leav
age CHO
2,4,7 OT
2,4 HD
2,4 ODCHO
CHO
2,4,7 DTCHO
tridecanal
octatrienal
CHO
ResultsApoptosis Induction
0 30 60 90 1200
25
50
75 Control 5 µg/ml DD
+ inhibitor 10 µg/ml DD
Time (min)
nmol
AFC
/min
/mg
Prot
.
0 30 60 90 1200
20
40
60
80
100 DDcontrol
methanoldecanal
Incubation time
% tu
nel p
ositi
ve e
mbr
yos
Romano Romano etet al, J al, J ExpExp BiolBiol 20032003
ResultsApoptosis Induction
Mitochondrial integrity assessment
Control Decadienal
30 min
130 min
Romano, unpublished
ResultsDisruption of Disruption of TubulinTubulin polymerization in sea urchins polymerization in sea urchins
Buttino et al. 1999
ResultsInterference on Actin microfilament reorganization
Control 10 min Control 90min
DD5ug 10min DD5ug 90min
Control 120min
DD5ug 120min
Romano Romano unpublishedunpublished
0.00 0.2 0.4 0.6 0.8 1.00
50
100
µg ml-1
% a
bnor
mal
ities
/mor
talit
y
Results
ControlControl DD 0.2DD 0.2
DD 0.6DD 0.6DD 0.4DD 0.4 DDDD 0.80.8
Impairment of larval fitnessImpairment of larval fitness
Long term exposure to DD subLong term exposure to DD sub--lethal concentrationslethal concentrations
Romano Romano etet al. 2010al. 2010
ResultsLong term exposure to Long term exposure to subletalsubletal concentrattionconcentrattion
Induction of apoptosis at larval stageInduction of apoptosis at larval stage
Romano Romano etet al. 2010al. 2010
Fig. 1 TUNEL assay showing DNA fragmentation in P. lividusembryos, at pluteus stage. Equatorial optical sections of embryosobserved under Confocal Laser Scanning Microscopy. DNA fragmentation (green channel) (a); totality of nuclei by propidiumiodide (red channel) (b); merging of a and b (c). Control, 36 h ofdevelopment (1); TPA ? 31C treated (2); CdCl2 1 mM treated (3
ResultsEffect of aldehydes on sea urchin embryos
Tosti Tosti etet al. 2003al. 2003
ResultsGenes expression in response to subGenes expression in response to sub--lethal lethal aldehydealdehyde concentrationconcentration
Romano et al, in preparation
Nitric oxyde increase after treatment with 1, 2.5 and 5 µg/ml decadienal, revealed with the fluorescent specific dye DAF-FM
Results
Expression of stress-related genes in C. helgolandicusExpression of stressExpression of stress--related genes in C. related genes in C. helgolandicushelgolandicusApproaches:• Induce stress (feeding) and quantify gene expression with
Quantitative Real-Time PCR (qPCR)• cDNA libraries of C. helgolandicus after diatom feeding
“Suppression Subtractive Hybridization”
Quantitative expression of stress-related genes:C. helgolandicus
Genes of interestGenes of interest FunctionFunction
AldehydeAldehyde dehydrogenasesdehydrogenases (ALDH) (ALDH) AldehydeAldehyde detoxificationdetoxification
CytochromeCytochrome P450 (CYP) P450 (CYP) BiotransformationBiotransformation
Glutathione SGlutathione S--transferasetransferase (GST) (GST) BiotransformationBiotransformation
HeatHeat--shock proteins (HSP) shock proteins (HSP) General stress responseGeneral stress response
Superoxide dismutase (SOD) Superoxide dismutase (SOD) Radical detoxificationRadical detoxification
CatalaseCatalase (CAT) (CAT) Radical detoxificationRadical detoxification
SummaryMechanism of action of diatom aldehydes
Effects: cleavage inhibitionteratogenesisdevelopmental delay
► Induction of Apoptosis
► Disruption of Tubulin polymerization
► Interference on Actin microfilament reorganization
► Influence on stress-related gene expression
Giovanna RomanoGiovanna RomanoStazione Zoologica Anton Stazione Zoologica Anton DohrnDohrn