List of Abbreviations ........................................... i
Chapter 1 General Introduction ................................. 1
Chapter 2 Methodology ......................................... 15
Chapter 3 Proteome Analysis During the Development of
Zebrafish ........................................... 29
Chapter 4 Proteomics Study of Toxic Stress in Early
Embryonal Stages of the Zebrafish ................... 51
Chapter 5 Proteomics Study of Toxic Stress in Zebrafish
Larvae .............................................. 67
Chapter 6 Cathepsins as a New Family of Biomarkers in
Zebrafish Embryos for Normal Embryonal Development
and Toxic Exposure ................................. 101
Chapter 7 Summary - Conclusion ............................... 119
Zusammenfassung ............................................... 125
References .................................................... 131
Appendix ........................................................ I
List of Abbreviations ........................................... i
1 General Introduction ......................................... 1
2 Methodology ................................................. 15
2.1 Physico-Chemical Properties of Characterised
Toxicants .............................................. 16
2.2 Fish Culture, Embryo Collection and Exposure
Conditions ............................................. 17
2.2.1 Fish Embryo Test (DarT) ......................... 17
2.2.2 Effect Determination in Larvae .................. 19
2.2.3 Exposure Conditions for the Analysis of Toxic
Responses at the Proteome Level ................. 20
2.3 Protein Analysis ....................................... 21
2.3.1 Protein Sample Preparation ...................... 21
2.3.2 One Dimensional SDS-PAGE ........................ 21
2.3.3 Two-Dimensional Gelelectrophoresis (2-DE) ....... 22
2.3.4 Image Analysis and Statistics ................... 23
2.3.5 Identification of Proteins ...................... 24
2.3.6 Immunoblot Analysis ............................. 24
2.4 Cathepsin Enzymatic Activity Analysis (Chapter 6) ......... 25
2.4.1 Danio rerio Embryo Collection for Enzymatic
Measurements at Different Developmental
Stages .......................................... 25
2.4.2 Exposure Conditions to Study in vivo Effects
of Leupeptin, DNOC, Ethanol and Rotenone on
Cathepsin Activities ............................ 25
2.4.3 Protein Isolation from Danio rerio Embryos ...... 26
2.4.4 Cathepsins Assays (CatC, CatH, CatL) ............ 26
3 Proteome Analysis During the Development of Zebrafish ....... 29
3.1 Introduction ........................................... 30
3.2 Results ................................................ 32
3.2.1 Characterisation of the Protein Composition of
Zebrafish Embryos at Different Developmental
Stages ......................................... 32
3.2.2 Identification of Proteins in 2 dpf old
Embryos ......................................... 35
3.2.3 Immunoblot Analysis with Anti-Vtg Antibody ...... 40
3.2.4 Identification of Proteins in Five Day old
Larvae .......................................... 42
3.3 Discussion ............................................. 44
3.3.1 Protein Pattern Changes During Embryonal
Development ..................................... 44
3.3.2 Ratio of Yolk-to Cellular Proteins Decreases
During Embryonal Development .................... 47
4 Proteomics Study of Toxic Stress in Early Embryonal Stages
of the Zebrafish ............................................ 51
4.1 Introduction ........................................... 52
4.2 Results ................................................ 54
4.2.1 Danio rerio Fish Embryo Test with Ethanol ....... 54
4.2.2 2-DE Analysis of Zebrafish Embryos Exposed to
two Different Ethanol Concentrations ............ 56
4.3 Discussion ............................................. 62
5 Proteomics Study of Toxic Stress Zebrafish Lavae ............ 67
5.1 Introduction ........................................... 68
5.2 Results ................................................ 69
5.2.1 Optimisation of 2-DE Method Concerning Age of
Larvae .......................................... 69
5.2.2 Analysis of Morphological and Physiological
Effects in Zebrafish Larvae Treated with DNOC,
Rotenone and Diclofenac ......................... 72
5.2.3 Toxicoproteomics Approaches with Zebrafish
Larvae Using three Toxicants: Rotenone, DNOC,
Diclofenac ...................................... 75
5.3 Discussion ............................................. 87
6 Cathepsins as a new Family of Biomarkers in Zebrafish
Embryos for Normal Embryonal Development and Toxic
Exposure ................................................... 101
6.1 Introduction .......................................... 102
6.2 Results ............................................... 103
6.2.1 Cathepsin Activities in Embryonal and Larval
Stages of the Zebrafish ........................ 103
6.2.2 Leupeptin as Positive Control for direct in
vivo Cathepsin Inhibition ...................... 106
6.2.3 Effect of three Chemicals on the in vivo
Activity of the Cathepsins L, С and H .......... 108
6.2.4 Concentration Dependence of Effects on
Cathepsin Activity ............................. 110
6.3 Discussion ............................................ 112
6.3.1 Cathepsin L, С and H Activity During
Embryogenesis .................................. 113
6.3.2 Toxicant Related Changes in Cathepsins
Activities ..................................... 115
7 Summary and Conclusions .................................... 119
Zusammenfassung ............................................... 125
References .................................................... 131
Appendix ........................................................ I
I Results of Toxicoproteomics Studies ..................... II
II Results of Effect Screening Assays with Embryonal and
Larval Stages of the Zebrafish ....................... XVIII
Publications/Conference Contributions
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