EFECTOS DE LOS CONTAMINANTES
EFECTOS “GRANDES”
INTOXICACION, ENVENENAMIENTO, MUERTE
EFECTOS “PEQUEÑOS”
SISTEMA ENDOCRINO
DNA
EXTRÉS OXIDATIVO
PAUTAS DE COMPORTAMIENTO
DINÁMICA 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/FEMINIZACIÓN
MENOR FERTILIDAD
MAYOR 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
DDT
BFRs
Ethinyl estradiol
Naringenin
Dioxins
Bisphenol A
Diethylstilbestrol
Sewage
effluents
Coumestrol
Kepone
Nonionic
surfactants
Norgestrel
Zearalenone PCBs
Equol
PAHs
Endosulfan
Gestodene
Contraceptives
Industrial
effluents
Air
particulates
Phloretin
Phthalate
esters
Sediment
extracts
Enterlactone
Insecticides
-sitosterol
Parabens
Tissue
extracts
Daidzen
Kaempferol
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 PLASMA
ALTERACIÓN DE LAS ENZIMAS PRODUCTORAS DE ESTEROIDES O DE LOS
MECANISMOS DE CONTROL DE LAS MISMAS
CAMBIOS EN LAS HIDROXILASAS Y REDUCTASAS QUE MODIFICAN LA
MOLÉCULA DE COLESTEROL ORIGINAL
Receptor d´estrogens (ELRA)
- Muestras de la planta d´AGBAR en Barcelona 2000
160
140
1800
1:100
1600
120
MUESTRAS
1:1000
1400
100
1200
80
1000
800
60
600
40
400
20
200
0
0
1
2
3
4
5
7
6
8
18
14
6000
16
NPEO
NPEC (nEO=0-1)
NP
BrNPEO (nEO=2-10)
BrNPEC (nEO=0)
BrNP
10
8
6
4
5000
14
mg/L
12
mg/L
1:100
mg/kg
17--estradiol equivalent (nM)
1:10
12
10
8
6
1000
2
0
0
1
2
3
4
5
6
3000
2000
4
2
4000
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)
0
7
8
7. Agua intersticial
fango de
floculación
8. Extracto del fango
de floculación
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 · - → H2 O2 + 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)
Dioxins
Pentachorophenol
Metal contaminants (Al, As, Cd, Cr, Hg, Ni, V)
Air contaminants (NO2, O3, SO2)
Peroxides
UV-radiation
Hypoxia
Hyperoxia
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)Hdependent 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
1
AH-quinones
Nitrofurantoin, p- &
m-dinitrobenzoic acid
Nitrofurantoin, AHquinones2, lindane
9 AH-quinones (1–5
ring)2
Paraquat
Nitrofurantoin
Species
Reference
Larval turbot (S.
maximus)
Catfish (I. Punctatus),
rainbow trout (O.
mykiss), large-mouth
bass (M. salmoides)
Flounder (P. flesus),
perch (P. fluviatilis)
Flounder (P. flesus)
Peters et al. (1996)
Ribbed mussel (G.
demissa), wedge clam
(R. cuneata)
Mussel (M. edulis)
4-nitroquinoline Noxide
9 AH-quinones (1-5
ring)2
Mussel (M. edulis)
Nitrofurantoin, 1nitropyrene, pnitrobenzoic acid
Mussel (M. edulis),
shore crab (C.
maenas), starfish (A.
rubens)
1
Mussel (M. edulis)
Washburn & Di
Giulio (1988, 1989)
Lemaire et al. (1994)
Lemaire &
Livingstone (1997)
Wenning & Di Giulio
(1988), Wenning et al.
(1988)
Garcia Martinez et al.
(1995)
Garcia Martinez et al.
(1992)
Garcia Martinez &
Livingstone (1995),
Sjölin & Livingstone
(1997)
Hetherington et al.
(1996)
tetramethyl-,14-benzoquinone (duroquinone), 1,4-naphthoquinones, 2-methyl-1,4naphthoquinone (menadione),9,10-phenanthrenequinone; 2as for 1 plus 1,4benzoquinone, 1,2-naphthoquinone, anthraquinone and 1,6-, 3,6- and 6,12benzo[a]quinones.
Examples of oxidative damage studies with whole-animal or cell culture
exposures of aquatic invertebrates and fish to single contaminants
Parameter
Chemical
Lipid
Cadmium,
peroxidation copper
1
Iron
Arsenic
(As3+&
As5+),
methyl
arsonate
BaP
Copper
Paraquat
Copper
Species &
tissue
Sea bass (D.
labrax)
kidney
Change
Increase
Roméo et al.
with both
(2000)
metals (Cu >
Cd)
Increase
Baker et al.
(1997)
African
catfish (C.
gariepinus)
liver & heart
Channel
No change
catfish (I.
Punctatus)
liver
Mussel (M.
edulis)
digestive
gland
M. edulis
digestive
gland and
gill
Ribbed
mussel (G.
demissa)
digestive
gland
Mediterrane
an clam (R.
decussatus)
digestive
gland and
gill
Reference
Schlenk et
al. (1997)
Increase
Livingstone
et al. (1990)
Increase
Viarengo et
al. (1988,
1990)
Increase
Wenning et
al. (1988)
Increase
(digestive
gland) and
no change
(gill)
Roméo &
GnassiaBarelli
(1997)
Parameter Chemical
Species &
tissue
Copper and
thiram
Mussel (U.
timidus)
digestive
gland and
gill
Lipofuschin H2O2
Limpet (N.
concinna)
digestive
gland
Phenanthrene, M. edulis
fluoranthene, digestive
BaP
gland
Phenanthrene Periwinkle
(L. littorea)
digestive
gland
Oxidised
Copper
Mussel (M.
protein
edulis)
(non-peptide
digestive
carbonyl
gland
formation)
8-OHMenadione,
M. edulis
deoxynitrofurantoin digestive
guanosine
gland
BaP
Mussel (M.
galloprovincialis)
digestive
gland & gill
Nitrofurantoin Sole (P.
vetulus)
H2O2
Trout (O.
mykiss) liver
Nitrofurantoin S. maximus,
dab (L.
limanda),
sole (S.
solea) liver
1.
Malonaldehyde equivalents
Change
Reference
Increase
Doyotte et
al. (1997)
Increase
Abele et al.
(1998)
Increase
Krishnakum
ar et al.
(1997)
Moore et al.
(1985)
Increase
Increase
Kirchin et
al. (1992)
No change
Marsh et al.
(1993)
Increase
Canova et
al. (1998)
Increase
Nishimoto et
al. (1991)
Kelly et al.
(1992)
Mitchelmore
et al. (1996)
Increase
No change
Examples of oxidative damage studies with whole-animal exposures of
aquatic invertebrates and fish to mixed-contaminants
Parameter
Chemical
Species
Lipid
Sediment
peroxidation (PAHs,
PCBs)
Sediment
(PAHs,
PCBs)
Field
(sediment
PAHs,
PCBs,
others)
Field
(sediment
PAHs,
metals)
8-hydroxyField (N.
deoxySea)
guanosine
8-hydroxyField (tissue
guanine
metals)
Dab (L.
limanda)
liver
Catfish (I.
Punctatus)
liver
U. tumidus
digestive
gland and
gill
Increase
Livingstone
et al. (1993)
Increase
Di Giulio et
al. (1993)
American
oyster
(Crassostrea
virginica)
L. limanda
Increase at
many
contaminate
d sites
No site
differences
Ringwood et
al (1999)
Rock oyster
(Saccostrea
commercialis
) gill
Sole (P.
vetulus)
No site
differences
Avery et al.
(1996)
Present in
premalignant
and
cancerous
liver
Elevation at
contaminate
d sites
Malins et al.
(1990);
Malins
&
Gunselman
(1994)
2,6-diamino4-OH-5formamidopyrimidine
(FapyGua)
Field (Puget
Sound, USA
- PAHs,
PCBs)
FapyGua & Field (lake
Fapyadenine with
high
iron-ore
tailings
loading)
Oxidised
Field (The
protein
Netherlands
(non-peptide )1
carbonyl
formation
Trout (S.
namaycush)
Change
Reference
Increase and Cossu et al.
no change
(1997, 2000)
Flounder (P. Elevation at
flesus)
contaminate
d sites
Chipman et
al. (1992)
Payne et al.
(1998)
V. Fessard
& D.R.
Livingstone
(unpublishe
d 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 poison
Paracelsus, 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)
BIOASSAYS
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
BIOASSAYS
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-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 conditions
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
Yeast strain YCM4, which contains the human AhR and ARNT genes plus
Ligand
AhR-L-ARNT
PAHs
TCDD
PCBs
+
ß-galactosidase
AhR-L
DRE
AhR
mRNA
LACZ
Fluorogenic
assay
Yeast assays were performed in parallel with GC-MS chemical analyses
Sample Processing
Chemical Analysis
Direct Sample Introduction
TD-GC-MS
Data Analysis
1.200
AhR-RYA
Biological Analysis
Values
Teor
1.000
Relative Activity
Extraction by Sonication
0.800
0.600
0.400
0.200
Sample Dilutions
5120
1280
80
320
10240
640
2560
40
160
320
5120
Fig. 5 Air samples extracts in MeOH
1280
0.000
80
Air Sample
Collection
24h
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
REP
ΣPAHs
1000
>50%
>50%
30-50%
30-50%
30-50%
<30%
<30%
10
<30%
BaP eq (ng·m-3)
100
100
10
R2 = 0.8381
10
100
Predicted BaP TEQ (ng·m-3)
(
,
,
10
R2 = 0.5981
1
1
100
R2 = 0.8342
1
0.1
PM10
1000
>50%
BaP eq (ng·m-3)
BaP eq (ng·m-3)
1000
1000
1
0.1
1
10
100
10
Sum PAH (ng·m-3)
100
PM10 (µg·m-3)
) Estimated contributions of wood combustion to the total PAH contents in %.
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)
Daphnia on Prozac….
1st brood
Molt
1 mm
70
Número de descendiantes
65
Prozac (5-80ug/l)
60
Control
55
50
0.93
0.94
0.95
Tamaño descendencia (mm)
…produces more and larger descendants
0.96
Low serotonin secretion
Effects of SSRIs in people….
Low synaptic activity
Depression
Blocking serotonine
recycling SSRI
Higher synaptic activity
Recovery
Low serotonin secretion
Effects of SSRIs in people….
Low food
Low synaptic activity
Delayed reproducti
Depression
Smaller clutches
Blocking serotonine
recycling SSRI
Earlier reproductio
Higher synaptic activity
…and in Daphnia!!!!
Recovery
Larger clutches
Low serotonin secretion
Effects of SSRIs in people….
Low food
Delayed reproducti
Low synaptic activity
Smaller clutches
Depression
Blocking serotonine
recycling SSRI Earlier reproductio
…and in Daphnia!!!!
Larger clutches
Analysis of dioxin-like activity in
water samples using the zebrafish
scale assay
Fish scales (gene expression)
Zebra
Advantages
fish
It avoids animal killing
The same fish can be monitored during
time
Fast
Relatively inexpensive
qRT –PCR
Working
principle
Changes
Quantification (amount of
Pollutan
Changes
in cDNA
cDNA)
t
in mRNA
(fluoresence)
3 types of pollutant
studied:
β-estradiol (estrogen)
Cadmium (heavy
metal)
β-napphthoflavone (dioxinlike)
Fish scales (gene expression)
Zebra
Advantages
fish
It avoids animal killing
The same fish can be monitored during
time
Fast
Relatively inexpensive
qRT –PCR
Working
principle
Changes
Quantification (amount of
Pollutan
Changes
in cDNA
cDNA)
t
in mRNA
(fluoresence)
3 types of pollutant
studied:
β-estradiol (estrogen)
Cadmium (heavy
metal)
β-naphthoflavone (dioxinlike)
Dioxin-like activity in
the Llobregat River by
the zebrafish scale assay
Pelayo, S, López-Roldán, R., González, S., Casado, M.,
Raldúa, D., Cortina, J.L., Piña, B. Submited
Fall
Spring
1000
1000
100
Site 1
Site 2
***
100
100
Site 3
***
***
***
***
***
**
***
***
10
***
10
**
1
Site 2
Site 3
R² = 0.6251
1
0.1
Site 2
10/4/10
9/29/10
9/27/10
6/18/10
6/16/10
6/14/10
6/11/10
6/9/10
6/7/10
10
0.01
9/23/10
0.1
0.1
9/21/10
Site 1
10/1/10
1
Total micropollutants (µg/L)
***
1
10
100
cyp1a expression (‰ of reference gene)
Development of zebrafish embryo bioassays
-Treat zebrafish embryos with different compounds during the 48120 hpf period
-Morphological analysis (microscopy, immunochemistry, in situ
hybrisisation…)
-Microarray analysis
48-120 hpf
period:
-Design and validation of gene expression biomarkers
by qRT-PCR
Embryo already
developed, but
not feeding yet
(and still a
replacement
method)
Zebrafish on lipid regulators
CLOFIBRATE
INHIBITION OF LIPOPROTEIN LIPASE FUNCTION
D. Raldúa et al. / Toxicology and Applied Pharmacology 228 (2008) 301–314
Thyroid disruptors in zebrafish
Control
MMI
Raldúa and Babin/ Environ. Sci. Tech. 43, 6844
qRT-PCR analysis of transcription in zebrafish embryos
RNA extraction
Retrotranscription to DNA
quantitative RT-PCR
Incubation
95ºC
95ºC
DNA denaturing
60ºC
60ºC
Primer
annealing
and
extension
9
5
º
C
Fluorescence
60ºC
Fluorescence
Snap-freezing in liquid N2
Gene expression = (CT ref. gene – CT
targ. Gene) + Log2 (1000)
Mechanistic comparison between yeast and zebrafish-based bioassays
Ligand
PAHs
TCDD
PCBs
+
AhR-L-ARNT
AhR-L
qRT-PCR
mRNA
CYP1A1
DRE
AhR
Ligand
PAHs
TCDD
PCBs
+
ß-galactosidase
AhR-L-ARNT
AhR-L
DRE
mRNA
LACZ
AhR
Fluorogenic
assay
1.00
Benzo-a-Pirè Llevat
0.80
Benzo-a-Pirè Zebrafish
0.60
0.40
0.20
0.00
0.1
1.0
10.0
mg/L
100.0
1000.0
Dioxin-like activity in air and burnt coal gangue extracts
5
5000
4500
BaP
4
BkF
4000
Other PAHs
3500
AhR-RYA
3
3000
DR-LUC
2500
2
2000
1500
1
1000
500
0
0
A1
A2
A3
A4
DTAR2
DTAR3
Nothern Italy
China
Semirural Area
Coal Mine
BNFeq (mg/L)
PAHs content (g/L)
MolDarT (cyp1a)
Dioxin-like activity in air and burnt coal gangue extracts
5
5000
BaP
Zebrafish
BkF
Other PAHs
4000
MolDarT (cyp1a)
PAHs content (g/L)
AhR-RYA
3500
DR-LUC
3
3000
Recombinant Yeast
2500
2
2000
Cultured mouse cells
1500
1
1000
500
0
0
A1
A2
A3
A4
DTAR2
DTAR3
Nothern Italy
China
Semirural Area
Coal Mine
BNFeq (mg/L)
4
4500
Dioxin-like activity in air and burnt coal gangue extracts
5
5000
BaP
Zebrafish
BkF
Other PAHs
4000
MolDarT (cyp1a)
PAHs content (g/L)
AhR-RYA
3500
DR-LUC
3
3000
Recombinant Yeast
2500
2
2000
Cultured mouse cells
1500
1
1000
500
0
0
A1
A2
A3
A4
DTAR2
DTAR3
Nothern Italy
China
Semirural Area
Coal Mine
BNFeq (mg/L)
4
4500
A
Dioxin-like effects in zebra fish embryos
D
DTAR2 1:1500
B[a]Pyr 500 µg/L
B
C
BNF 720 µg/l
B[k]Flu 500 µg/L
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
Cumulativde Fluorescence (A.U.)
20000
hbbe3
15000
hbbe2
hbbe1
hbae3
10000
hbae1
ba2
hbaa1
5000
alpha globin type-2
si:xx-by187g17.5
0
Cumulative fluorescence (A.U.)
Control
T3-treated
30000
hbbe3
25000
hbbe2
hbae3
20000
hbae1
15000
ba2
hbaa1
10000
5000
0
1
Pelayo, S, Oliveira, E, Thienpont, B, Babin P.J., Raldúa, D, André, M., Piña, B. Submitted
2
4
5
14
Days post-fertilization
30
90
Dreissena MicroArray Design
eArray de Agilent
Array design:
8 x 15,000 spots
60 nucleotide- probes
3 best probes
3 best position
4,057 different sequences:
3,253 from D. rostriformis
750 from D. polymorpha
54 from other bivalves (Veneridae, Unionidae)
Venerupis Catalasa
Verenupis Cu/Zn SOD
Multixenobiotic Resistance Protein D.polymorpha
Cyclin B D. polymorpha
Conclusions:
•
Stronger effects with TBT than with Hg
•
Oxidative stress genes and MRPs genes
induced by TBT ( confirmed by qRTPCR)
•
Detection of Hg-specific genes, but
none of them identified yet.
Navarro, A., Campos, B., Barata, C. Piña, B., in preparation