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