1 EFECTOS DE LOS CONTAMINANTESEFECTOS “GRANDES” INTOXICACION, ENVENENAMIENTO, MUERTE EFECTOS “PEQUEÑOS” SISTEMA ENDOCRINO DNA EXTRÉS OXIDATIVO PAUTAS DE COMPORTAMIENTO DINÁMICA DE LAS POBLACIONES

2 NIVELES DE LOS EFECTOS MOLECULAR, CELULAR, BIOQUÍMICOMORFOLOGÍA Y COMPORTAMIENTO DEL INDIVIDUO CAPACIDAD REPRODUCTIVA DEL INDIVIDUO POBLACIÓN ECOSISTEMA

3 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

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

5 EN HUMANOS (EFECTOS OBSERVADOS QUE PUEDEN ESTAR RELACIONADOS CON LOS CONTAMINANTES) ü   DESCENSO DE LA CALIDAD Y CANTIDAD DEL ESPERMA HUMANO ENTRE ü   AUMENTO DE CANCER DE TESTÍCULO, PECHO Y PÁNCREAS EN MUCHOS PAISES ü   BAJA CAPACIDAD REPRODUCTORA EN LOS HOMBRES ü   TESTÍCULOS QUE NO DESCIENDEN (RECIÉN NACIDOS) ü   MALFORMACIONES CONGÉNITAS ü   FUNCIÓN TIROIDEA ANORMAL

6 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.

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

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12 EFECTOS SOBRE LA SÍNTESIS DE HORMONAS Y METABOLISMOALTERACIÓN 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

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14 Receptor d´estrogens (ELRA) - Muestras de la planta d´AGBAR en Barcelona - 20 40 60 80 100 120 140 160 1 2 3 4 5 6 17-b-estradiol equivalent (nM) 1:10 1:100 8 10 12 14 mg/L NPEO NPEC (nEO=0-1) NP BrNPEO (nEO=2-10) BrNPEC (nEO=0) BrNP 200 400 600 800 1000 1200 1400 1600 1800 2000 7 1:1000 3000 4000 5000 6000 mg/kg 16 18 MUESTRAS Agua del río Después precloración Después filtro arena Después de ozonizar Después del filtro de carbón activo Después de la cloración (efluente final) Agua intersticial fango de floculación Extracto del fango de floculación

15 EFECTOS OXIDATIVOS

16 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.

17 ½ O2 + R2CH → R2COH free energy: ~60 Kcal/molReaction 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.

18 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.

19 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

20 Contaminant stimulation of ROS productionredox 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

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31 New tools for evaluation of toxicity of environmental samplesBenjamin Piña Dpt. Química Ambiental, IDAEA

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

33 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

34 Animal Testing: not recommendedBIOASSAYS 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 Testing: not recommended

35 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

36 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 The “3Rs concept”: Reduce, Replace, Refine

37 A yeast-based bioassay for dioxin-like compoundsRecombinant Yeast Assay (RYA) AhR-RYA

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

39 Yeast assays were performed in parallel with GC-MS chemical analysesYeast strain YCM4, which contains the human AhR and ARNT genes plus a reporter gene ß-galactosidase AhR-L-ARNT DRE mRNA LACZ PAHs TCDD PCBs Ligand AhR-L AhR + Fluorogenic assay Yeast assays were performed in parallel with GC-MS chemical analyses Sample Processing Extraction by Sonication Air Sample Collection 24h Data Analysis Direct Sample Introduction TD-GC-MS AhR-RYA Chemical Analysis Biological Analysis Fig. 5 Air samples extracts in MeOH

40 Variation of Atmospheric Pollution according to the period of the yearConcentration (ng/m3) profiles of BaPeq (RYA bioassay), ΣPAH13, Benzo[a]pyrene and BaP toxic equivalents predicted from chemical data using the REP coefficients.

41 REP ΣPAHs Correlation between RYA bioassay and Chemical data PM10( , , ) Estimated contributions of wood combustion to the total PAH contents in %. 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,

42 Daphnia magna - Emerging contaminantsEndocrine drisruption Campos and Barata (unpublished)

43 …produces more and larger descendantsMolt 1st brood Daphnia on Prozac…. 1 mm 70 Prozac (5-80ug/l) 65 Número de descendiantes 60 Control 55 50 0.93 0.94 0.95 0.96 Tamaño descendencia (mm) …produces more and larger descendants

44 Low serotonin secretionLow synaptic activity Depression Blocking serotonine recycling SSRI Higher synaptic activity Recovery Effects of SSRIs in people….

45 Low serotonin secretionLow synaptic activity Depression Blocking serotonine recycling SSRI Higher synaptic activity Recovery Low food Effects of SSRIs in people…. Delayed reproduction Smaller clutches Earlier reproduction Larger clutches …and in Daphnia!!!!

46 The “happy Daphnia” modelLow serotonin secretion Low synaptic activity Depression Blocking serotonine recycling SSRI Higher synaptic activity Recovery Low food Effects of SSRIs in people…. Delayed reproduction Smaller clutches The “happy Daphnia” model Earlier reproduction Larger clutches …and in Daphnia!!!!

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

48 Fish scales (gene expression)Advantages Zebra fish It avoids animal killing The same fish can be monitored during time Fast Relatively inexpensive Working principle qRT –PCR Changes in cDNA Quantification (amount of cDNA) (fluoresence) Pollutant Changes in mRNA 3 types of pollutant studied: β-estradiol (estrogen) Cadmium (heavy metal) β-napphthoflavone (dioxin-like)

49 Fish scales (gene expression)Advantages Zebra fish It avoids animal killing The same fish can be monitored during time Fast Relatively inexpensive Working principle qRT –PCR Changes in cDNA Quantification (amount of cDNA) (fluoresence) Pollutant Changes in mRNA 3 types of pollutant studied: β-estradiol (estrogen) Cadmium (heavy metal) β-naphthoflavone (dioxin-like)

50 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 ***

51 Development of zebrafish embryo bioassays-Treat zebrafish embryos with different compounds during the hpf period -Morphological analysis (microscopy, immunochemistry, in situ hybrisisation…) -Microarray analysis -Design and validation of gene expression biomarkers by qRT-PCR hpf period: Embryo already developed, but not feeding yet (and still a replacement method)

52 First Workshop on Advanced Scientific Results from IDAEAZebrafish on lipid regulators CLOFIBRATE INHIBITION OF LIPOPROTEIN LIPASE FUNCTION Here i present some results. We’ve got two diferent exposures here. On the left clofibrate the expression of a Lipoportotein lipase, and on the right the expression of FABP3 fatty acid binding protein. Here a clear dose-response is observed for the expression of LPL And no sifnificative differences are observed for The expression of FABP3 resulting from the Troglitazone D. Raldúa et al. / Toxicology and Applied Pharmacology 228 (2008) 301–314

53 Thyroid disruptors in zebrafishControl MMI Raldúa and Babin/ Environ. Sci. Tech. 43, 6844

54 qRT-PCR analysis of transcription in zebrafish embryosRNA extraction Retrotranscription to DNA quantitative RT-PCR Incubation 95ºC DNA denaturing 60ºC Primer annealing and extension Fluorescence Snap-freezing in liquid N2 Gene expression = (CT ref. gene – CT targ. Gene) + Log2 (1000)

55 Mechanistic comparison between yeast and zebrafish-based bioassaysPAHs TCDD PCBs Ligand AhR-L AhR-L-ARNT DRE mRNA CYP1A1 qRT-PCR AhR + ß-galactosidase AhR-L-ARNT DRE mRNA LACZ Fluorogenic assay PAHs TCDD PCBs Ligand AhR-L AhR +

56 Dioxin-like activity in air and burnt coal gangue extracts

57 Dioxin-like activity in air and burnt coal gangue extractsZebrafish

58 Dioxin-like activity in air and burnt coal gangue extractsZebrafish

59 Dioxin-like effects in zebra fish embryos ADTAR2 1:1500 B[a]Pyr 500 µg/L C B BNF 720 µg/l Olivares, A., van Drooge, B., Hamers, T., Grimalt, J.O., Piña, B. et. al., In preparation B[k]Flu 500 µg/L

60 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)

61 C C2R C2 C T2R T T1 T3 Thyroid ligands Otra de las aplicaciones de la toxicogenómica, además de analizar posibles mecanismos de acción, es en la evaluación de efectos, al permitir seleccionar un grupo de genes específicamente regulados por un compuesto y que puedan ser utilizados para detectar la presencia del compuesto en el medio a través de su efecto sobre la expresión génica (firma genética). Esta es la aproximación que estamos utilizando actualmente para abordar el problema de los disruptores tiroideos. Se ha realizado un microarray con larvas de 5 dpf control y tratadas con hormona tiroidea, encontrado expresión diferencial de uno 700 genes en los animales tratados. Por el momento, se ha confirmando la expresión diferencial de un pequeño grupo de estos genes mediante qPCR. En la imagen podemos ver que el tratamiento con hormona tiroidea durante 3 días induce la expresión de una globina típica de adultos e inhibe la expresión de una globina embrionaria. Una vez seleccionados un pequeño grupo de genes regulados positiva y negativamente, se analizará el efecto de antagonistas del sistema tiroideo, intentando de este modo diseñar un bioensayos de 3 días para detectar disruptores tiroideos. Pelayo, S, Oliveira, E, Thienpont, B, Babin P.J., Raldúa, D, André, M., Piña, B. Submitted

62 Dreissena MicroArray DesigneArray 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)

63 Conclusions: Stronger effects with TBT than with HgCyclin 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