The fish embryo as an alternative model for the assessment of endocrine active environmental chemicals: Elucidation of endocrine disruptive mechanisms and identification of relevant effect endpoints using transcriptomics Von der Fakult¨atf¨ur Mathematik, Informatik und Naturwissenschaften der RWTH Aachen University zur Erlangung des akademischen Grades einer Doktorin der Naturwissenschaften genehmigte Dissertation vorgelegt von Diplom-Oecotrophologin Viktoria Schiller aus Simferopol (Ukraine) Berichter: Universit¨atsprofessorDr. Rainer Fischer Universit¨atsprofessorDr. Henner Hollert Universit¨atsprofessorDr. J¨orgOehlmann Tag der m¨undlichen Pr¨ufung:02.12.2013 Diese Dissertation ist auf den Internetseiten der Hochschulbibliothek on- line verf¨ugbar. ii Contents Summary xv Zusammenfassung xviii 1 Introduction and objectives of the thesis1 1.1 Endocrine disruption in fish.................1 1.2 Endocrine disrupting chemicals in current regulation...3 1.3 Alternatives for endocrine disruption testing........5 1.4 Fundamental background of endocrine disruption.....8 1.4.1 Hypothalamic-pituitary-gonadal axis in fish....8 1.4.2 Mechanisms of endocrine disruption in brief.... 10 1.4.2.1 Receptor mediated signalling....... 12 1.4.2.2 Receptor-independent mechanisms.... 13 1.5 Key facts about zebrafish and medaka........... 13 1.5.1 Species- and embryo specific traits (or attributes). 15 1.5.1.1 Sexual differentiation of zebrafish and medaka 16 1.5.1.2 Role of aromatase in sex differentiation.. 17 1.5.1.3 Sex steroid receptors in fish embryos... 18 1.6 Objectives........................... 20 2 Material and methods 23 2.1 Fish maintenance and exposure............... 23 2.2 RNA extraction and reverse transcription.......... 25 2.3 Quantitative real-time PCR................. 26 2.4 Microarray analysis...................... 27 2.4.1 Microarray processing................ 27 iv CONTENTS 2.4.2 Functional analysis.................. 29 2.4.3 Visualization of gene set analysis.......... 30 3 Transcriptome pattern in zebrafish 33 3.1 Abstract............................ 33 3.2 Introduction.......................... 34 3.3 Material and methods.................... 38 3.3.1 Fish maintenance and exposure........... 38 3.3.2 Chemicals....................... 39 3.3.3 Fish Embryo Test (FET)............... 39 3.3.4 RNA extraction.................... 40 3.3.5 Microarray analysis.................. 41 3.3.6 Visualization of functional analysis......... 43 3.3.7 Intersection analysis of affected categories in non- reference and reference compounds......... 44 3.3.8 Quantitative real-time PCR............. 44 3.4 Results............................. 45 3.4.1 Morphological assessment and dose response rela- tionships........................ 45 3.4.2 Significance analysis of microarray data....... 46 3.4.3 Gene set analysis of microarray data........ 46 3.5 Discussion........................... 55 3.6 Conclusion........................... 65 3.A Appendix........................... 67 4 Zebrafish and Medaka Comparison 77 4.1 Abstract............................ 77 4.2 Introduction.......................... 78 4.3 Material and methods.................... 80 4.3.1 Fish maintenance and exposure........... 80 4.3.2 Chemicals....................... 81 4.3.3 Fish Embryo Toxicity Test (FET).......... 81 4.3.4 RNA extraction and reverse transcription...... 83 CONTENTS v 4.3.5 Quantitative real-time PCR............. 83 4.3.6 Microarray analysis.................. 85 4.4 Results............................. 85 4.4.1 Morphological effects................. 85 4.4.2 Gene expression analysis............... 86 4.5 Discussion........................... 93 5 Molecular mechanisms of genistein in fish embryos 103 5.1 Abstract............................ 103 5.2 Introduction.......................... 104 5.3 Methods............................ 108 5.3.1 Fish maintenance and exposure........... 108 5.3.2 Chemical analysis................... 109 5.3.3 Morphological assessments.............. 110 5.3.4 RNA extraction and reverse transcription...... 110 5.3.5 Quantitative real-time PCR............. 111 5.3.6 Microarray analysis.................. 112 5.4 Results............................. 113 5.4.1 Chemical analysis................... 113 5.4.2 Morphological defects................. 114 5.4.3 Zebrafish microarray................. 115 5.4.4 Medaka quantitative real-time PCR......... 119 5.5 Discussion........................... 120 5.6 Conclusion........................... 129 5.A Appendix........................... 131 5.A.1 Acridine orange staining............... 131 6 General Discussion and Conclusions 133 Bibliography 143 Annex 173 A Primer sequences....................... 174 A.1 Zebrafish primer................... 174 vi Contents A.2 Medaka primer.................... 175 B Morphological endpoints................... 176 C Entire transcriptome response in 48hpf zebrafish embryos. 177 C.1 17α-Ethinylestradiol................. 177 C.2 Flutamide....................... 179 C.3 Bisphenol A...................... 181 C.4 Genistein....................... 188 C.5 Linuron........................ 209 C.6 Methylparaben.................... 216 C.7 Propanil........................ 220 C.8 Prochloraz....................... 225 D Genistein gene enrichment analysis............. 232 Curriculum Vitae 273 List of Figures 1.1 Steroid feedback regulation.................. 11 1.2 Thesis workflow........................ 22 3.1 Estrogenic and anti-androgenic pathways in 48hpf zebrafish embryos............................ 48 3.2 Visualized estrogenic and anti-androgenic mode of action in 48hpf zebrafish embryos................... 50 3.3 Pathway intersections between reference and non-reference compounds.......................... 52 3.A-1Concentration{response to 48h exposure of tested compounds in zebrafish.......................... 68 3.A-2Concentration{response to 48h exposure of tested compounds in zebrafish.......................... 70 3.A-3Microarray significance validation.............. 76 4.4-1 Proportion of morphological effects in zebrafish and medaka 86 4.4-2 Gene response to EE2 and flutamide in 48hpf zebrafish em- bryos.............................. 88 4.4-3 Gene response to EE2 and flutamide in 96hpf zebrafish em- bryos.............................. 89 4.4-4 Gene response to EE2 and flutamide in 7dpf medaka embryos 90 4.4-5 Gene response to estrogenic compounds in 7dpf medaka em- bryos.............................. 92 4.4-6 Gene response to anti-androgenic compounds in 7dpf medaka embryos............................ 93 viii List of figures 5.4-1 Dose-response relationship for genistein in 48hpf zebrafish embryos............................ 114 5.4-2 Morphological effects in zebrafish embryos after genistein exposure............................ 115 5.4-3 Morphological effects in medaka embryos after genistein ex- posure............................. 115 5.4-4 Functional analysis of the genistein microarray data in ze- brafish............................. 116 5.4-5 qPCR validation zebrafish.................. 118 5.4-6 qPCR analysis medaka.................... 119 5.A-1Acridine orange staining................... 132 List of Tables 3.1 Effect concentrations and morphological effects in 48hpf ze- brafish embryos........................ 45 3.2 Comparison of embryonic and adult gene response to EE2 58 3.3 Target genes and primer................... 69 3.4 REVIGO EE2 attributes................... 71 3.5 REVIGO flutamide attributes................ 72 3.6 EE2 pathways included in intersection calculations.... 73 3.7 Flutamide pathways included in intersection calculations. 75 4.1 Effect concentrations in zebrafish and medaka....... 87 5.1 Additional genistein responsive endocrine genes...... 117 A-1 Zebrafish primer........................ 174 A-2 Medaka primer........................ 175 C-1 Entire set of pathways affected by EE2........... 177 C-2 Entire set of pathways affected by flutamide........ 179 C-3 Entire set of pathways affected by bisphenol A....... 181 C-4 Entire set of pathways affected by genistein......... 188 C-5 Entire set of pathways affected by linuron......... 209 C-6 Entire set of pathways affected by methylparaben..... 216 C-7 Entire set of pathways affected by propanil......... 220 C-8 Entire set of pathways affected by prochloraz........ 225 D-1 Differentially expressed genes in the genistein EC10 data set 232 D-2 Differentially expressed genes in the genistein EC20 data set 233 D-3 Differentially expressed genes in the solvent data set vs ISO control............................. 269 x List of tables D-4 Affected KEGG, GO and InterPro terms in the genistein EC20 data set......................... 269 List of Abbreviations 11β-hsd 11-beta-hydroxysteroid dehydrogenase 11β 11-beta-hydroxylase 3R's refinement, reduction, replacement of animal testing 3β-hsd 3-beta-hydroxysteroid dehydrogenase Amh anti-Mullerian hormone ANOVA analysis of variance Ar androgen receptor ARC arcuate nucleus AVPV anteroventral periventricular nucleus BPA bisphenol A cAMP cyclic adenosine monophosphate cDNA complementary DNA cRNA complementary RNA Ctgf Connective tissue growth factor Cy3 cyanine 3 Cyp1 Cytochrome P450, family 1, member A1 Cyp11b Cytochrome P450, subfamily 11b Cyp17 lyase Cytochrome P450, family 17, subfamily A Cyp19a1a Cytochrome P450, family 19, subfamily A, polypeptide 1a; aromatase a Cyp19a1b Cytochrome P450, family 19, subfamily A, polypeptide 1b; aromatase b Cyp3a65 Cytochrome P450, family 3, subfamily A, polypeptide 65 cyr61 cysteine-rich, angiogenic inducer, 6 DIN Deutsches Institut f¨urNormung xii List of abbreviations DMRT1 DM-related