EFECTOS DE LOS CONTAMINANTES EFECTOS “GRANDES” INTOXICACION, ENVENENAMIENTO, MUERTE

1. EFECTOS DE LOS CONTAMINANTES EFECTOS “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, BIOQUÍMICO MORFOLOGÍA 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. EFECTOS ENDOCRINOS TRANSTORNOS REPRODUCTIVOS MASCULINIZACIÓN/FEMINIZACIÓN MENOR FERTILIDAD MAYOR MORTALIDAD DE LA PROGENIE üFEMINIZACIÓN O MASCULINIZACIÓN DE PECES QUE VIVEN EN AGUAS QUE RECIBEN DESCARGAS DE EFLUENTES INDUSTRIALES Y MUNICIPALES üCIRCULACIÓN DE NIVELES ANORMALES DE HORMONAS EN PECES EXPUESTOS A EFLUENTES DE PLANTAS DE FÁBRICAS DE PULPA DE PAPEL üDIFERENCIACIÓN Y DESARROLLO SEXUAL ANORMAL EN CAIMANES DEL LAGO APOPKA (FLORIDA CENTRAL) üIMPOSEX: DESARROLLO DE LAS CARACTERÍSTICAS SEXUALES MASCULINAS (PENE Y VASOS) EN GASTERÓPODOS HEMBRA POR EXPOSICIÓN A TRIBUTILESTAÑO üRESPUESTAS FEMENINAS EN EMBRIONES DE GAVIOTA POR EXPOSICIÓN A DDT

5. EN HUMANOS (EFECTOS OBSERVADOS QUE PUEDEN ESTAR RELACIONADOS CON LOS CONTAMINANTES) üDESCENSO DE LA CALIDAD Y CANTIDAD DEL ESPERMA HUMANO ENTRE 1938-1990 üAUMENTO DE CANCER DE TESTÍCULO, PECHO Y PÁNCREAS EN MUCHOS PAISES üBAJA CAPACIDAD REPRODUCTORA EN LOS HOMBRES üTESTÍCULOS QUE NO DESCIENDEN (RECIÉN NACIDOS) üMALFORMACIONES CONGÉNITAS üFUNCIÓN 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.

10. 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

12. 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

14. 2000 160 1:10 1:100 1800 140 1:100 1:1000 1600 120 1400 100 1200 1000 17-b-estradiol equivalent (nM) 80 800 60 600 40 400 20 200 0 0 7 8 1 2 3 4 5 6 14 18 6000 16 12 5000 NPEO 14 10 NPEC (nEO=0-1) 12 4000 NP mg/kg mg/L 8 mg/L 10 BrNPEO (nEO=2-10) 3000 BrNPEC (nEO=0) 6 8 BrNP 6 2000 4 4 2 1000 2 0 0 0 1 2 3 4 5 6 7 8 Receptor d´estrogens (ELRA) - Muestras de la planta d´AGBAR en Barcelona - 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. 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.

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 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

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

32. 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)

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. 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 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 compounds Recombinant Yeast Assay (RYA)AhR-RYA

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

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

40. 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.

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

42. Endocrine drisruption • Daphnia magna - Emerging contaminants Campos and Barata (unpublished)

43. 1st brood Molt 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 secretion Low synaptic activity Depression Blocking serotonine recycling SSRI Higher synaptic activity Recovery Effects of SSRIs in people….

45. Low 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 Earlier reproduction Larger clutches …and in Daphnia!!!!

46. Low 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) Zebra fish Advantages 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) Zebra fish Advantages 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-likeactivity in the Llobregat Riverbythezebrafishscaleassay Pelayo, S, López-Roldán, R., González, S., Casado, M., Raldúa, D., Cortina, J.L., Piña, B. Submited *** *** *** *** *** *** *** ***