Enhanced applications of the zebrafish (Danio rerio) embryo toxicity test as a model to mechanistically differentiate metal toxicity effects in fish. Von der Fakultät für Mathematik, Informatik und Naturwissenschaften der RWTH Aachen University zur Erlangung des akademischen Grades einer Doktorin der Naturwissenschaften genehmigte Dissertation vorgelegt von Laura Sonnack, M. Eng. aus Frankfurt am Main Berichter: Univ.-Prof. Dr. rer. nat. Henner Hollert Univ.-Prof. Dr. rer. nat. Andreas Schäffer Tag der mündlichen Prüfung: 19. Februar 2018 Diese Dissertation ist auf den Internetseiten der Universitätsbibliothek online verfügbar. ___________________________________________________________________ ___________________________________________________________________ Contents Contents Summary ..................................................................................................................................... i Zusammenfassung ..................................................................................................................... iv List of figures .......................................................................................................................... viii List of tables ............................................................................................................................... x Chapter 1: General Introduction ................................................................................................. 1 1.1. Metals in the environment ........................................................................................... 2 1.1.1. Cadmium ............................................................................................................... 6 1.1.2. Cobalt .................................................................................................................... 6 1.1.3. Copper ................................................................................................................... 7 1.2. The test organism zebrafish and the embryo toxicity test as a test system ................. 8 1.2.1. Assessment of neurotoxic effects using defects of motor neuron development . 10 1.2.2. Assessment of neuromast damage as asensitive indicator of peripheral neurotoxicity ..................................................................................................................... 12 1.2.3. Gene expression analyses.................................................................................... 13 1.3. Objectives .................................................................................................................. 15 Chapter 2: Material & Methods ............................................................................................... 17 2.1. Zebrafish husbandry .................................................................................................. 18 2.2. Metals, chemicals and reagents ................................................................................. 18 2.3. Zebrafish embryo toxicity tests ................................................................................. 19 2.3.1. Assessment of exposure concentration dependent morphological effects .......... 19 2.3.2. Assessment of motor neuron impairment using immunofluorescence staining . 20 2.3.3. Assessment of neuromast damage using a vital dye staining ............................. 22 2.3.4. Assessment of escape response ........................................................................... 23 2.3.5. Statistical analysis of the morphological and functional endpoints .................... 24 2.4. Gene transcription analysis ........................................................................................ 24 2.4.1. RNA Extraction................................................................................................... 24 2.4.2. Microarray experiments ...................................................................................... 25 2.4.3. Functional analysis .............................................................................................. 26 2.4.4. Real-time quantitative PCR ................................................................................ 27 Chapter 3 .................................................................................................................................. 29 Effects of metal exposure on motor neuron development, neuromasts and the escape response of zebrafish embryos ............................................................................................. 29 ___________________________________________________________________ Contents 3.1. Abstract ...................................................................................................................... 30 3.2. Introduction ............................................................................................................... 31 3.3. Material and Methods ................................................................................................ 33 3.3.1. Chemicals ............................................................................................................ 33 3.3.2. Analysis of metals ............................................................................................... 34 3.3.3. Exposure of zebrafish embryos ........................................................................... 35 3.4. Results ....................................................................................................................... 36 3.4.1. Morphological effects in zebrafish embryos ....................................................... 36 3.4.2. Effects on motor neuron development ................................................................ 39 3.4.3. Neuromast damage .............................................................................................. 41 3.4.4. Behavioral effects ............................................................................................... 42 3.5. Discussion .................................................................................................................. 43 Chapter 4 .................................................................................................................................. 51 Comparative analysis of the transcriptome responses of zebrafish embryos after exposure to low concentrations of cadmium, cobalt and copper ............................................................. 51 4.1. Abstract ...................................................................................................................... 52 4.2. Introduction ............................................................................................................... 53 4.3. Materials and Methods .............................................................................................. 54 4.3.1. Chemicals ............................................................................................................ 54 4.3.2. Analysis of metals ............................................................................................... 55 4.4. Results ....................................................................................................................... 56 4.4.1. Morphological effects ......................................................................................... 56 4.4.2. Microarray analysis ............................................................................................. 57 4.4.3. Real-time quantitative PCR ................................................................................ 62 4.5. Discussion .................................................................................................................. 64 Chapter 5 .................................................................................................................................. 71 Concentration dependent transcriptome responses of zebrafish embryos after exposure to cadmium, cobalt and copper. ................................................................................................ 71 5.1. Abstract .................................................................................................................. 72 5.2. Introduction ............................................................................................................ 73 5.3. Materials and Methods .............................................................................................. 74 5.3.1. Analysis of metals ............................................................................................... 74 5.4. Results .................................................................................................................... 75 5.4.1. Microarray-Analysis ........................................................................................... 75 5.4.2. Real-time quantitative PCR ................................................................................ 83 ___________________________________________________________________ Contents 5.5. Discussion .............................................................................................................. 84 Chapter 6: General discussion & conclusion ........................................................................... 93 6.1. Addressing limitations of the zebrafish embryo toxicity test ................................ 94 6.2. Special considerations regarding metals and toxicity ............................................ 98