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Steroid Levels, Steroid Metabolic

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Steroid Levels, Steroid Metabolic Universitat Autònoma de Barcelona Programa de Doctorat de Farmacologia, Terapèutica i Toxicologia STEROID LEVELS, STEROID METABOLIC PATHWAYS AND THEIR MODULATION BY ENDOCRINE DISRUPTORS IN INVERTEBRATES Tesi doctoral Gemma Janer Gual 2005 Universitat Autònoma de Barcelona CONSELL SUPERIOR UNIVERSITAT AUTÒNOMA DE D’INVESTIGACIONS CIENTÍFIQUES BARCELONA Institut d’Investigacions Químiques i Programa de Doctorat de Farmacologia, Ambientals de Barcelona Terapèutica i Toxicologia Departament de Química Ambiental Memòria presentada per optar al títol de Doctora en Farmacologia, Terapèutica i Toxicologia per GEMMA JANER GUAL DIRECTORA: TUTOR: Cinta Porte Visa Raimon Guitart Bas Investigadora Titular d’Universitat Dept. Química Ambiental Dept. Farmacologia, Terapèutica i IIQAB-CSIC Toxicologia, UAB Aquest treball, que ha estat realitzat al departament de Química Ambiental del IIQAB- CSIC, ha estat possible gràcies a una beca predoctoral del Ministerio de Educación y Ciencia i als projectes europeus BEEP ( ENVK3-CT-2000-00025) i COMPRENDO (EVK1- CT-2002-00129). AGRAÏMENTS Si aquesta tesi ha estat possible és gràcies a la Cinta Porte, que em va donar l’oportunitat de treballar en el seu laboratori i que m’ha dirigit aquesta tesis amb constància i esperit científic. Gràcies per la confiança dipositada en mi, per haver-me escoltat sempre, per enviar-me a reunions i congressos, pel suport en el meu treball i per tot el que m’ha ensenyat. En Raimon Guitart per la tutoria d’aquest treball i per ajudar-me en els tràmits burocràtics. Als membres del tribunal per haver acceptat a formar-hi part. Als meus companys de laboratori (Rebeca, Ramón, Rémi, Denise, Angeliki, Sabine, Carlos, Itziar, Alice, Arun, i Demetrio), per la seva ajuda a la feina (tinc dos tubs per proteïnes....) i a mantenir el bon humor (happy Friday!), per la seva paciència amb mi (crec que el boli que busques el tinc jo...) i per les bones estones que hem passat dins (food!!) i fora del laboratori (otra vez salsa, no!). A la Dori i la Roser per la seva ajuda amb l’espectrometria de masses. Als altres companys del departament, per les seves lliçons de química i per no oblidar-se (casi) mai de mi a l’hora d’esmorzar. I a l’Anna, perquè ha estat genial poder compartir amb tu les meves històries. To Prof. Kauffman, Prof. LeBlanc, and Prof. Tyler, for welcoming me to their labs, for all the support and confidence they gave me. To my colleagues in those three labs (Tim, Robin, Cynthia, Mu, Heather, Michela and the Exeter team) and especially to Sonia, Ronny and Eduarda for taking care of me in and outside the lab, and for their priceless lessons and discussions. Also, to my friends in US and in England, you made me feel at home. To the COMPRENDO team for all the good moments in our meetings. For welcoming me to the project from the very beginning and making me feel part of it. For all the fantastic discussions we have had (about science and about life!). To all those people I’ve met during these four years who helped me in enjoying life and keeping up the enthusiasm for science. Als amics de sempre, per donar-me suport, per recordar-me la sort que he tingut amb totes les oportunitats que he viscut durant aquests anys de tesi, i perquè sou els millors! I naturalment no puc oblidar-me del Sr. Ernest Michaux, que va inventar la bicicleta amb pedals, o de l’Ino, que potser no va inventar la salsa, però sí que la va introduir a la meva família. Al Roberto, per estar al meu costat en els últims moments de la tesi. I per últim i molt especialment, a la Núria, als meus pares i a la resta de la família, perquè tot i no acabar d’entendre què és això que estic fent, sempre m’heu recolzat en les meves decisions i m’heu animat a tirar endavant. Espero no haver oblidat ningú, però en tot cas, gràcies a tu per llegir aquesta tesi. ACRONIM LIST ACRONIM LIST 11-kT: 11-keto-testosterone ELISA: enzyme-linked immunosorbent 2,4-D: 2,4-dichlorophenoxyacetic acid assay 4-A-diol: 4-androsten-3,17-diol EPA: environmental protection agency NP: nonylphenol ER: estrogen receptor 5-A-diol: 5-androstan-3,17-diol ERE: estrogen response element 5-R: 5-reductase ESI: electrospray ionization AACs: androgenic and antiandrogenic FEN: fenarimol compounds GC: gas chromatography AD: androstenedione HCB: hexachlorobenzene AhR: aryl hydrocarbon receptor HCH: hexachlorocyclohexane (lindane) APCI: atmospheric pressure chemical HPLC: high performance liquid ionization chromatography APE: alkylphenol ethoxylate HPTE: 2,2-bis[p-hydroxyphenyl]-1,1,1- ATAT: acyl-CoA acyl transferase trichloroethane BBP: butylbenzyl phthalate HRE: hormone response element BPA: bisphenol A HSD: hydroxysteroid dehydrogenase BSA: bovine serum albumine IC50: inhibitory concentration 50% BSTFA: JH: juvenile hormone bis(Trimethylsilyl)trifluoroacetamide LC-MS: liquid chromatography-mass cAMP: adenosine 3’,5’-cyclic spectrometry monophosphate LLE: liquid liquid extraction CNS: central nervous system LOEC: lowest observed effect CoA: coenzyme A concentration CYP: cytochrome P450 MAPK: mitogen-activated protein kinase DBP: di-n-butyl-phthalate METI: ministry of energy, trade and DDE: dichlorodiphenyldichloroethylene industry DDT: dichlorodiphenyltrichloroethane MS: mass spectrometry DEHP: di-(2-ethylhexyl)phthalate MT: methyl testosterone DES: diethylstilbestrol NADPH: -nicotinamide adenine DHA: dihydroandrostenedione dinucleotide phosphate DHEA: dehydroepiandrosterone NCI: negative chemical ionization DHT: dihydrotestosterone NGO: non-governamental organization E1: estrone OECD: organization for economic E2: estradiol cooperation and development ED: endocrine disruptor compounds OH-T: hydroxylated testosterone EDSTAC: endocrine disruptor screening metabolites and testing advisory committee P450arom: cytochrome P450 EDTA: ethylenediaminetetraacetic acid aromatase EE2: ethynylestradiol P450c17: cytochrome P450 17- EI: electron impact hydroxylase and C17-20-lyase P450scc: cytochrome P450 side chain SULT: sulfotransferase cleavage T: testosterone PAH: polyaromatic hydrocarbon TBT: tributyltin PAPS: adenosine 3′-phosphate 5′- TCDD: tetrachlorodibenzodioxin phosphosulfate TLC: thin layer chromatography PBB: polybrominated biphenyl TMS: trimethylsilyl PCB: polychlorinated biphenyl TPT: triphenyltin PCP: pentachlorophenol UDP: uridine diphosphate RIA: radioimmunoassay UGT: uridine diphosphate (glucuronosyl RXR: retinoid X receptor or glucosyl) transferase SHBG: sex steroid binding protein VDS Index: vas deference sequence SPE: solid phase extraction index StAR: steroidogenic acute regulatory WHO: world health organization protein XRE: xenobiotic response element STAT: signal transducers and activators of transcription INDEX INDEX Chapter 1. GENERAL INTRODUCTION 1 1.1 Global framework in endocrine disruption research and origin of the COMPRENDO project 2 1.2 Evidences of endocrine disruption 4 1.2.1 Evidences in vertebrates 4 1.2.2 Evidences in invertebrates 5 1.3 Endocrine disruptor compounds: definition and examples 9 1.4 Endocrine disruption escapes from classical toxicology 19 1.5 Potential pathways of endocrine disruption 21 1.5.1 Binding to steroid receptors 22 1.5.2 Interference with sex steroid binding proteins 23 1.5.3 Alteration of receptor levels. 23 1.5.4 Changes in hormone biosynthesis and metabolism 23 1.5.5 Interactions with the non-genomic action of steroid hormones 27 1.6 The endocrine system in invertebrates 27 1.6.1 Neuropeptides 30 1.6.2 Ecdysteroids and juvenoids 31 1.6.3 Sex steroid hormones 31 1.7 Sex steroids, steroidogenesis and steroid metabolism 34 1.7.1 Steroid structure and classes 34 1.7.2 Steroidogenesis in vertebrates 36 1.7.3 Steroidogenesis in invertebrates 39 1.7.4 Androstenedione and testosterone metabolic pathways 41 1.8 Investigating steroids and steroid metabolism. A methodological remark 48 1.8.1 The identification and quantitative determination of sex steroids 48 1.8.2 The investigation of sex steroid metabolism 52 1.8.3 Thin layer chromatography 53 1.8.4 High pressure liquid chromatography 54 1.9 The test species 54 1.9.1 Mollusca 54 1.9.2 Echinodermata 55 1.9.3 Arthropoda 56 1.10 Objectives of this thesis 56 Chapter 2. CHARACTERIZATION OF STEROID METABOLISM IN INVERTEBRATE SPECIES AND THE EFFECTS OF ENVIRONMENTAL POLLUTANTS IN VITRO. 59 2.1 Paper 1: A comparative study on androgen metabolism in three invertebrate 61 species 2.2 Paper 2: Esterification of vertebrate-like steroids in the oyster Crassostrea 75 virginica 2.3 Paper 3: Sulfatase activity in the oyster Crassostrea virginica: its potential interference with sulfotransferase determination 85 2.4 Paper 4: Steroid conjugating activities in invertebrates: are they target for endocrine disruption? 91 2.5 Complementary work on Chapter 2: phase II metabolism of steroids in molluscs 103 CHAPTER 3. IN VIVO EFFECTS OF XENOBIOTIC EXPOSURE ON STEROID LEVELS AND STEROID METABOLISM IN MOLLUSCS. 119 3.1 Paper 5: Effects of 17b-estradiol exposure in the mussel Mytilus galloprovincialis: the regulating role of steroid acyltransferases. 121 3.2 Paper 6: Steroid levels and steroid metabolism in the mussel Mytilus edulis: the modulating effect of dispersed crude oil and alkylphenols. 137 3.3 Paper 7: Sexual dimorphism in esterified steroid levels in the gastropod Marisa cornuarietis: the effect of xenoandrogenic compounds. 153 3.4 Paper 8: The effect of organotin compounds on gender specific androstenedione metabolism in Marisa cornuarietis 175 CHAPTER 4. SUMMARY OF RESULTS AND GENERAL DISCUSSION 195 4.1 Characterization of steroid metabolism in invertebrates 197 4.1.1 Phase I metabolism of androgens 197 4.1.2 Phase II metabolism of steroids 201 4.1.3 Implications for the sensibility to xenobiotics 203 4.2 Steroid levels in molluscs 204 4.3 In vitro effects of endocrine disruptors 208 4.4 In vivo effects of endocrine disruptors 209 4.4.1 In vivo effects of exposure to estradiol, crude oil and the mixture of crude oil and alkylphenolic compounds 211 4.4.2 In vivo effects of exposure to triphenyltin, tributyltin, fenarimol and methyltestosterone 212 CHAPTER 5. CONCLUSIONS 219 CHAPTER 6. REFERENCES 223 ANNEX. A MOLECULAR APPROACH TO STUDY ENDOCRINE DISRUPTION IN INVERTEBRATES 243 RESUM / ABSTRACT 263 CHAPTER 1.
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