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