EFECTOS DE LOS CONTAMINANTES

EFECTOS “GRANDES”INTOXICACION, ENVENENAMIENTO, MUERTE

EFECTOS “PEQUEÑOS”SISTEMA ENDOCRINODNAEXTRÉS OXIDATIVOPAUTAS DE COMPORTAMIENTODINÁMICA DE LAS POBLACIONES

NIVELES DE LOS EFECTOS

MOLECULAR, CELULAR, BIOQUÍMICO

MORFOLOGÍA Y COMPORTAMIENTO DEL INDIVIDUO

CAPACIDAD REPRODUCTIVA DEL INDIVIDUO

POBLACIÓN

ECOSISTEMA

Endocrine Disrupting Chemicals (EDCs)

The Endocrine System produces hormones that guide the development, growth, reproduction, and behavior of humans & animals

EDCs are synthetic or naturally occurring chemicals that interfere with endocrine (hormone) system function

EFECTOS ENDOCRINOS TRANSTORNOS REPRODUCTIVOS MASCULINIZACIÓN/FEMINIZACIÓNMENOR FERTILIDADMAYOR MORTALIDAD DE LA PROGENIE    FEMINIZACIÓN O MASCULINIZACIÓN DE PECES QUE VIVEN EN AGUAS QUE RECIBEN DESCARGAS DE EFLUENTES INDUSTRIALES Y MUNICIPALES    CIRCULACIÓN DE NIVELES ANORMALES DE HORMONAS EN PECES EXPUESTOS A EFLUENTES DE PLANTAS DE FÁBRICAS DE PULPA DE PAPEL    DIFERENCIACIÓN Y DESARROLLO SEXUAL ANORMAL EN CAIMANES DEL LAGO APOPKA (FLORIDA CENTRAL)    IMPOSEX: DESARROLLO DE LAS CARACTERÍSTICAS SEXUALES MASCULINAS (PENE Y VASOS) EN GASTERÓPODOS HEMBRA POR EXPOSICIÓN A TRIBUTILESTAÑO    RESPUESTAS FEMENINAS EN EMBRIONES DE GAVIOTA POR EXPOSICIÓN A DDT

EN HUMANOS

(EFECTOS OBSERVADOS QUE PUEDEN ESTAR RELACIONADOS CON LOS CONTAMINANTES)

 

   DESCENSO DE LA CALIDAD Y CANTIDAD DEL ESPERMA HUMANO ENTRE 1938-1990

   AUMENTO DE CANCER DE TESTÍCULO, PECHO Y PÁNCREAS EN MUCHOS PAISES

   BAJA CAPACIDAD REPRODUCTORA EN LOS HOMBRES

   TESTÍCULOS QUE NO DESCIENDEN (RECIÉN NACIDOS)

   MALFORMACIONES CONGÉNITAS

   FUNCIÓN TIROIDEA ANORMAL

MECANISMOS DE DISRUPCIÓN DE FUNCIONES VITALES DEL SISTEMA ENDOCRINO 1.   Some of the endocrine-modulators are similar enough in structure to hormones that they are able to bind to cellular receptors designed to be the target for natural hormones. This causes unpredictable and abnormal cell activity.2.   Some appear to block the binding sites, so that natural hormones are unable to bind to them, thus impairing normal cell activity.3.   Other endocrine-modulators induce the creation of extra receptor sites in the cell, with the consequence of amplifying the impact of hormones on cellular activity.4.   Endocrine-modulators can directly or indirectly interact with natural hormones, changing the hormones´ message and thus altering cell activity.5.   Some endocrine-modulators can also alter the natural pattern of hormone synthesis and metabolism, resulting in improper balance or quantity of circulating hormones.

EN MACHOS, LA GtH I SE INCREMENTA DURANTE LA ESPERMATOGÉNESIS Y DECRECE DURANTE LA FREZA

LA GtH II ESTÁ EN BAJAS CONCENTRACIONES DURANTE TODO EL PROCESO DE CRECIMIENTO Y SE INCREMENTA EN LA FREZA.

LAS GONADOTROPINAS ESTIMULAN LA PROLIFERACIÓN DE ESPERMATOGONIA Y LA SÍNTESIS DE ANDRÓGENOS NECESARIA PARA LA GAMETOGÉNESIS Y EL DESARROLLO DE LOS CARACTERES SEXUALES SECUNDARIOS

Classes of environmental estrogens

Natural products

Environmental pollutants

Industrial chemicals

Pharmaceutical chemicals

Complex mixtures

Genistein

Naringenin

Coumestrol

Zearalenone

Equol

Phloretin

Enterlactone

-sitosterol

Daidzen

Kaempferol

DDT

Dioxins

Kepone

PCBs

PAHs

BFRs

Bisphenol A

Nonionic surfactants

Endosulfan

Phthalate esters

Insecticides

Parabens

Ethinyl estradiol

Diethylstilbestrol

Norgestrel

Gestodene

Contraceptives

Sewage effluents

Industrial effluents

Air particulates

Sediment extracts

Tissue extracts

BFRs: Brominated flame retardants; DDT: dichlorodiphenyltrichloroethane; PCBs: polychlorinated biphenyls; PAHs: polycyclic aromatic hydrocarbons.

EFECTOS SOBRE LA SÍNTESIS DE HORMONAS Y METABOLISMO ALTERACIÓN DE LAS HORMONAS SEXUALES EN PLASMAALTERACIÓN DE LAS ENZIMAS PRODUCTORAS DE ESTEROIDES O DE LOS MECANISMOS DE CONTROL DE LAS MISMASCAMBIOS EN LAS HIDROXILASAS Y REDUCTASAS QUE MODIFICAN LA MOLÉCULA DE COLESTEROL ORIGINAL

Receptor d´estrogens (ELRA)- Muestras de la planta d´AGBAR en Barcelona -

MUESTRAS

1. Agua del río

2. Después precloración

3. Después filtro arena

4. Después de ozonizar

5. Después del filtro de carbón activo

6. Después de la cloración (efluente final)

7. Agua intersticial fango de floculación

8. Extracto del fango de floculación

0

20

40

60

80

100

120

140

160

1 2 3 4 5 6

17-

-est

rad

iol e

qu

ival

ent

(nM

)

1:10

1:100

0

2

4

6

8

10

12

14

1 2 3 4 5 6

g/L

NPEONPEC (nEO=0-1)NPBrNPEO (nEO=2-10)BrNPEC (nEO=0)BrNP

0

200

400

600

800

1000

1200

1400

1600

1800

2000

7 8

1:100

1:1000

0

1000

2000

3000

4000

5000

6000

8g

/kg

0

2

4

6

8

10

12

14

16

18

7

g/L

EFECTOS OXIDATIVOS

The reactivity and properties of the different ROS vary considerably. Neither O2·

- nor H2O2 are considered particularly reactive in aqueous solution,

OH· reacts instantly and indiscriminately with virtually all organic molecules (rate constants of 108 to 1010 mol-1 sec-1). O2·

- (1-electron reduced form of O2) can dismutate to H2O2 (2-electron reduced

form of O2) via

 2 H+ + 2 O2·

- → H2O2 + O2;

 O2·

- and H2O2 can react together to yield OH· (3-electron reduced form of O2)

 O2·

- + Fe3+ → O2 + Fe2+

H2O2 + Fe2+ → OH· + OH- + Fe3+

O2·- + H2O2 → OH· + OH- + O2

 This reaction is dependent on the presence of a suitable redox cycling catalyst, such as an iron-chelate, and therefore the generation of OH· is point-specific to the location of the appropriate catalyst.

Reaction with biomolecules

½ O2 + R2CH → R2COH free energy: ~60 Kcal/mol

Oxidation of biomolecules does not readily occur because the impaired electrons in the O2 molecule are of parallel spin putting O2 in a triplet state (3O2).

For O2 to act as an oxidizing agent, the molecule to be oxidized must donate electrons that are also of parallel spin.

However, most biomolecules are organic non-radicals with covalent bonds and therefore are paired in opposite spin (i.e., they are in a singlet state). The reaction of 3O2 with most biomolecules is quantum mecanically forbidden.

Potentially toxic ROS are continually produced in animals, principally as unwanted bi-products, from various endogenous sources and processes

 

enzymes (e.g. nitric oxide synthase, aldehyde oxidase, tryptophan dioxygenase)

auto-oxidation (e.g. of reduced FAD and FMN, glyceraldehyde)

haem proteins

mitochondrial, endoplasmic reticulum and nuclear membrane electron transport.

Rates or amounts of ROS production can be increased by the presence of a wide range of natural and man-made xenobiotics.

Possible anthropogenic-related sources of enhanced ROS and other pro-oxidant free radical production include organic contaminants:redox cycling compounds (quinones, nitroaromatics, nitroamines, bipyridyl herbicides)

PAHs (benzene, PAH oxidation products)halogenated hydrocarbons (bromobenzene, dibromomethane, PCBs, lindane)DioxinsPentachorophenolMetal contaminants (Al, As, Cd, Cr, Hg, Ni, V)Air contaminants (NO2, O3, SO2)

PeroxidesUV-radiationHypoxiaHyperoxia

Contaminant stimulation of ROS production

 redox cycling catalysed by flavoprotein

reductases (e.g. quinones and others)

redox reactions with O2 and ROS (e.g. Co, Cr, Ni, Va)

Autoxidation (e.g. cytochrome P450s (CYPs) and PCBs)

Enzyme induction (e.g. CYPs, flavoprotein reductases)

Disruption of membrane-bound electron transport (e.g. mitochondrial, microsomal electron transport and lipophilic contaminants)

Depletion of antioxidant defences (e.g. reduced glutathione (GSH) involved in phase II biotransformation of organic contaminants)

Generation of ROS is an inevitable scenario in the toxic mechanisms of many environmental contaminants

Examples of in vitro organic xenobiotic-stimulated NAD(P)H-dependent ROS production by subcellular fractions of tissues of aquatic organisms from invertebrate and vertebrate groups.

Tissues: liver (fish - Vertebrata), digestive gland (bivalves - Mollusca), hepatopancreas (crab - Arthropoda) and pyloric caeca (starfish - Echinodermata). AH, aromatic hydrocarbon

Chemical Species Reference AH-quinones1 Larval turbot (S.

maximus) Peters et al. (1996)

Nitrofurantoin, p- & m-dinitrobenzoic acid

Catfish (I. Punctatus), rainbow trout (O. mykiss), large-mouth bass (M. salmoides)

Washburn & Di Giulio (1988, 1989)

Nitrofurantoin, AH-quinones2, lindane

Flounder (P. flesus), perch (P. fluviatilis)

Lemaire et al. (1994)

9 AH-quinones (1–5 ring)2

Flounder (P. flesus) Lemaire & Livingstone (1997)

Paraquat Ribbed mussel (G. demissa), wedge clam (R. cuneata)

Wenning & Di Giulio (1988), Wenning et al. (1988)

Nitrofurantoin Mussel (M. edulis) Garcia Martinez et al. (1995)

4-nitroquinoline N-oxide

Mussel (M. edulis) Garcia Martinez et al. (1992)

9 AH-quinones (1-5 ring)2

Mussel (M. edulis) Garcia Martinez & Livingstone (1995), Sjölin & Livingstone (1997)

Nitrofurantoin, 1-nitropyrene, p-nitrobenzoic acid

Mussel (M. edulis), shore crab (C. maenas), starfish (A. rubens)

Hetherington et al. (1996)

1tetramethyl-,14-benzoquinone (duroquinone), 1,4-naphthoquinones, 2-methyl-1,4-naphthoquinone (menadione),9,10-phenanthrenequinone; 2as for 1 plus 1,4-benzoquinone, 1,2-naphthoquinone, anthraquinone and 1,6-, 3,6- and 6,12-benzo[a]quinones.

Examples of oxidative damage studies with whole-animal or cell culture exposures of aquatic invertebrates and fish to single contaminants Parameter Chemical Species &

tissue Change Reference

Lipid peroxidation1

Cadmium, copper

Sea bass (D. labrax) kidney

Increase with both metals (Cu > Cd)

Roméo et al. (2000)

Iron African catfish (C. gariepinus) liver & heart

Increase Baker et al. (1997)

Arsenic (As3+& As5+), methyl arsonate

Channel catfish (I. Punctatus) liver

No change Schlenk et al. (1997)

BaP Mussel (M. edulis) digestive gland

Increase Livingstone et al. (1990)

Copper M. edulis digestive gland and gill

Increase Viarengo et al. (1988, 1990)

Paraquat Ribbed mussel (G. demissa) digestive gland

Increase Wenning et al. (1988)

Copper Mediterranean clam (R. decussatus) digestive gland and gill

Increase (digestive gland) and no change (gill)

Roméo & Gnassia-Barelli (1997)

Parameter Chemical Species & tissue

Change Reference

Copper and thiram

Mussel (U. timidus) digestive gland and gill

Increase Doyotte et al. (1997)

Lipofuschin H2O2 Limpet (N. concinna) digestive gland

Increase Abele et al. (1998)

Phenanthrene, fluoranthene, BaP

M. edulis digestive gland

Increase Krishnakumar et al. (1997)

Phenanthrene Periwinkle (L. littorea) digestive gland

Increase Moore et al. (1985)

Oxidised protein (non-peptide carbonyl formation)

Copper Mussel (M. edulis) digestive gland

Increase Kirchin et al. (1992)

8-OH-deoxy-guanosine

Menadione, nitrofurantoin

M. edulis digestive gland

No change Marsh et al. (1993)

BaP Mussel (M. gallo-provincialis) digestive gland & gill

Increase Canova et al. (1998)

Nitrofurantoin Sole (P. vetulus)

Increase Nishimoto et al. (1991)

H2O2 Trout (O. mykiss) liver

Increase Kelly et al. (1992)

Nitrofurantoin S. maximus, dab (L. limanda), sole (S. solea) liver

No change Mitchelmore et al. (1996)

1. Malonaldehyde equivalents

Examples of oxidative damage studies with whole-animal exposures of aquatic invertebrates and fish to mixed-contaminants Parameter Chemical Species Change Reference Lipid peroxidation

Sediment (PAHs, PCBs)

Dab (L. limanda) liver

Increase Livingstone et al. (1993)

Sediment (PAHs, PCBs)

Catfish (I. Punctatus) liver

Increase Di Giulio et al. (1993)

Field (sediment PAHs, PCBs, others)

U. tumidus digestive gland and gill

Increase and no change

Cossu et al. (1997, 2000)

Field (sediment PAHs, metals)

American oyster (Crassostrea virginica)

Increase at many contaminated sites

Ringwood et al (1999)

8-hydroxy-deoxy-guanosine

Field (N. Sea)

L. limanda No site differences

Chipman et al. (1992)

8-hydroxy-guanine

Field (tissue metals)

Rock oyster (Saccostrea commercialis) gill

No site differences

Avery et al. (1996)

2,6-diamino-4-OH-5-formamido-pyrimidine (FapyGua)

Field (Puget Sound, USA - PAHs, PCBs)

Sole (P. vetulus)

Present in pre-malignant and cancerous liver

Malins et al. (1990); Malins & Gunselman (1994)

FapyGua & Fapyadenine

Field (lake with high iron-ore tailings loading)

Trout (S. namaycush)

Elevation at contaminated sites

Payne et al. (1998)

Oxidised protein (non-peptide carbonyl formation

Field (The Netherlands)1

Flounder (P. flesus)

Elevation at contaminated sites

V. Fessard & D.R. Livingstone (unpublished data)

New tools for evaluation of toxicity of environmental samples

Benjamin Piña

Dpt. Química Ambiental, IDAEA

Alle Ding' sind Gift, und nichts ohn' Gift; allein die Dosis macht, daß ein Ding kein Gift ist

Everything is poisonous, nothing is absolutely innocuous; only the dose makes a substance not a poisonParacelsus, s. XVI

Ecotoxicology is to determine:-What is poisonous-To whom is it poisonous (to which species and taxa, at which stage of development)-Why is it poisonous (by which mechanism)-At what dose-Which are the consequences…

-for the ecosystems-for human populations-for economic activities (Ecological services)

BIOASSAYSBIOASSAYS

The determination of the relative strength of a substance (drug or pollutant) by comparing its effect on a test organism with that of a standard preparation

BIOASSAYSBIOASSAYS

The determination of the relative strength of a substance (drug or pollutant) by comparing its effect on a test organism with that of a standard preparation

Animal T

esting: n

ot reco

mmended

ANIMAL-FREE BIOASSAYS

-Assays with microorganisms (bacteria, yeast, algae…)

-Assays in cultured cell lines

-Assays in invertebrates (except cephalopods and large crustaceans

-Animal embryos before independent feeding

-Assays in animals that produce a pain similar to or less than and I.V. injection performed under veterinarian conditions

ANIMAL-FREE BIOASSAYS

-Assays with microorganisms (bacteria, yeast, algae…)

-Assays in cultured cell lines

-Assays in invertebrates (except cephalopods and large crustaceans)

-Animal embryos before independent feeding

-Assays in animals that produce a pain similar to or less than an I.V. injection performed under veterinarian conditionsThe “3

Rs conce

pt”: Reduce

, Replace

, Refin

e

A yeast-based bioassay for dioxin-like compounds

Recombinant Yeast Assay (RYA)AhR-RYA

Air samples (PM10) from Ispra, in the Italian Alps-Summer pollution due to vehicles (minimum)-Winter pollution from wood burning (maximum)

JRC - Ispra

Sample Processing

Extraction by SonicationAir Sample Collection24h

Data Analysis

Direct Sample Introduction TD-GC-MS

0.000

0.200

0.400

0.600

0.800

1.000

1.200

80

320

1280

5120 40

160

640

2560

1024

0 80

320

1280

5120

Sample Dilutions

Rel

ativ

e A

ctiv

ity

ValuesTeorAhR-RYA

Chemical Analysis

Biological Analysis

Fig. 5 Air samples extracts in MeOH

ß-galactosidaseAhR-L-ARNT

DRE mRNALACZ

PAHsTCDDPCBs

Ligand

AhR-L

AhR

+

Fluorogenic assay

Yeast strain YCM4, which contains the human AhR and ARNT genes plus a reporter gene

Yeast assays were performed in parallel with GC-MS chemical analyses

Variation of Atmospheric Pollution according to the period of the year

Concentration (ng/m3) profiles of BaPeq (RYA bioassay), ΣPAH13, Benzo[a]pyrene and BaP toxic equivalents predicted from chemical data using the REP coefficients.

Correlation between RYA bioassay and Chemical data

( , , ) Estimated contributions of wood combustion to the total PAH contents in %.

R2 = 0.8381

1

10

100

1000

0.1 1 10 100 1000

Predicted BaP TEQ (ng·m-3)

BaP

eq (n

g·m

-3)

>50%

30-50%

<30%

R2 = 0.8342

1

10

100

1000

0.1 1 10 100

Sum PAH (ng·m-3)

BaP

eq (n

g·m

-3)

>50%

30-50%

<30%

R2 = 0.5981

1

10

100

1000

10 100

PM10 (µg·m-3)

BaP

eq (n

g·m

-3)

>50%

30-50%

<30%

REP ΣPAHs PM10

Results from the yeast assay correlated best with the predicted toxicity of the samples (TEQ values). However, 70 to 85% of the total activity was not explained by the chemical analysis

Olivares, A., van Drooge, B.L.., Ballesta, P.P., Grimalt, J.O., Piña, B. (2011) Assessment of dioxin-like activity in ambient air particulate matter using the recombinant yeast assay. Atmos. Env. 45, 271-274

•Daphnia magna - Emerging contaminants

Endocrine drisruption

Campos and Barata (unpublished)

Prozac (5-80ug/l)

Tamaño descendencia (mm)

0.93 0.94 0.95 0.96

Núm

ero

de d

esce

ndia

ntes

50

55

60

65

70

Control

1 mm

Molt1st brood

Daphnia on Prozac….

…produces more and larger descendants

Low serotonin secretion

Low synaptic activity

Depression

Blocking serotonine recycling SSRI

Higher synaptic activity

Recovery

Effects of SSRIs in people….

Low serotonin secretion

Low synaptic activity

Depression

Blocking serotonine recycling SSRI

Higher synaptic activity

Recovery

Low food

Delayed reproduction

Smaller clutches

Earlier reproduction

Larger clutches

Effects of SSRIs in people….

…and in Daphnia!!!!

Low serotonin secretion

Low synaptic activity

Depression

Blocking serotonine recycling SSRI

Higher synaptic activity

Recovery

Low food

Delayed reproduction

Smaller clutches

Earlier reproduction

Larger clutches

Effects of SSRIs in people….

…and in Daphnia!!!!The “happy D

aphnia”

model

Analysis of dioxin-like activity in water samples using the zebrafish

scale assay

Zebra fish

Fish scales (gene expression)

qRT –PCR

Advantages It avoids animal killingThe same fish can be monitored during timeRelatively inexpensiveFast

Working principle Pollutan

tChanges in cDNA

3 types of pollutant studied:

Cadmium (heavy metal)

β-estradiol (estrogen)

β-napphthoflavone (dioxin-like)

Changes in mRNA

Quantification (amount of cDNA)

(fluoresence)

Zebra fish

Fish scales (gene expression)

qRT –PCR

Advantages It avoids animal killingThe same fish can be monitored during timeRelatively inexpensiveFast

Working principle Pollutan

tChanges in cDNA

3 types of pollutant studied:

Cadmium (heavy metal)

β-estradiol (estrogen)

β-naphthoflavone (dioxin-like)

Changes in mRNA

Quantification (amount of cDNA)

(fluoresence)

***

***

***

***

******

******

Dioxin-like activity in the Llobregat River by the zebrafish scale assayPelayo, S, López-Roldán, R., González, S., Casado, M., Raldúa, D., Cortina, J.L., Piña, B. Submited

-Treat zebrafish embryos with different compounds during the 48-120 hpf period -Morphological analysis (microscopy, immunochemistry, in situ hybrisisation…)-Microarray analysis-Design and validation of gene expression biomarkers by qRT-PCR48-120 hpf period:

Embryo already developed, but not feeding yet (and still a replacement method)

Development of zebrafish embryo bioassaysDevelopment of zebrafish embryo bioassays

Zebrafish on lipid regulators

CLOFIBRATE

INHIBITION OF LIPOPROTEIN LIPASE FUNCTION

D. Raldúa et al. / Toxicology and Applied Pharmacology 228 (2008) 301–314

Control

MMI

Thyroid disruptors in zebrafish

Raldúa and Babin/ Environ. Sci. Tech. 43, 6844

qRT-PCR analysis of transcription in zebrafish embryos

Incubation

Snap-freezing in liquid N2

RNA extraction

Retrotranscription to DNA

quantitative RT-PCR

Gene expression = (CT ref. gene – CT targ. Gene) + Log2 (1000)

95ºCDNA denaturing

60ºCPrimer

annealing and

extension

95ºC 60ºC

95ºC

60ºC

Fluorescence Fluorescence

Mechanistic comparison between yeast and zebrafish-based bioassays

PAHsTCDDPCBs

Ligand

AhR-L

AhR-L-ARNT

DRE mRNACYP1A1

qRT-PCR

AhR

+

ß-galactosidaseAhR-L-ARNT

DRE mRNALACZ

Fluorogenic assay

PAHsTCDDPCBs

Ligand

AhR-L

AhR

+

0.00

0.20

0.40

0.60

0.80

1.00

0.1 1.0 10.0 100.0 1000.0

g/L

Benzo-a-Pirè Llevat

Benzo-a-Pirè Zebrafish

Dioxin-like activity in air and burnt coal gangue extracts

Dioxin-like activity in air and burnt coal gangue extracts

Zebrafish

Dioxin-like activity in air and burnt coal gangue extracts

Zebrafish

BNF 720 µg/l

DTAR2 1:1500B[a]Pyr 500 µg/L

B[k]Flu 500 µg/L

A

B C

D

Dioxin-like effects in zebra fish embryos

Olivares, A., van Drooge, B., Hamers, T., Grimalt, J.O., Piña, B. et. al., In preparation

Future trends

• Development of genomic tools for invertebrates (mollusks, crustaceans), including microarrays

• Zebrafish-based bioassays for new biological activities: retinoids, PPAR

• Analysis of enviromental impacts for primary producers: Chlorophyta, Cianobacteria, Diatoms

• Toxicity evaluation of particulate samples (including air particles, soil, sediments, and micro and nano-particles)

C3 C2R C2 C1 T2R T2 T1 T3

Pelayo, S, Oliveira, E, Thienpont, B, Babin P.J., Raldúa, D, André, M., Piña, B. Submitted

Dreissena MicroArray Design

eArray de Agilent Array design:

8 x 15,000 spots60 nucleotide- probes

3 best probes3 best position

4,057 different sequences:3,253 from D. rostriformis 750 from D. polymorpha 54 from other bivalves (Veneridae, Unionidae)

Cyclin B D. polymorpha Multixenobiotic Resistance Protein D.polymorpha

Venerupis Catalasa

Verenupis Cu/Zn SOD

Conclusions: •Stronger effects with TBT than with Hg

•Oxidative stress genes and MRPs genes induced by TBT ( confirmed by qRT-PCR)

•Detection of Hg-specific genes, but none of them identified yet.

Navarro, A., Campos, B., Barata, C. Piña, B., in preparation