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Inspired by the promising applications of the zebrafish embryo model in toxicology research, with the objectives of developing analysis techniques and applying them[r]
(1)VIETNAM NATIONAL UNIVERSITY, HANOI
INSTITUTE OF MICROBIOLOGY AND BIOTECHNOLOGY and
UNIVERSITY OF LIÈGE -
Đinh Duy Thành
TOXICITY ASSESSMENT OF SMALL MOLECULES USING THE ZEBRAFISH AS A MODEL SYSTEM
Subject: Biotechnology Code: 60.42.02.01
MASTER’S THESIS
SUPERVISORS:
Prof Marc Muller Dr Nguyễn Lai Thành
(2)ACKNOWLEDGEMENT This thesis would not have been possible without all the support, guidance, inspiration, and patience of the following people and organisations during the course of my study It is a privilege to convey my gratefulness to them in my humble acknowledgements First and foremost, I own my deepest gratitude to Prof Marc Muller, who gave me the opportunity to pursue my own interests as a trainee in the GIGA-Research Your wisdom, guidance, support, and endurance enable me to develop and improve my expertise in both laboratory works and scientific writing Moreover, you did motivate me through my inner pressures as well as outer obstacles
I offer my thankfulness to my co-supervisor, Dr Nguyễn Lai Thành, for continuously encouraging me to explore my own ideas Your knowledge, gentleness, and trust have inspired me and other students to keep following the scientific path
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study with devoted professors and lectures within the course They not only gave me the knowledge but also a new vision to perceive the Science of Life
It is my great pleasure to thank Benoist, Yoann, and Audrey in the Toxicology team as well as the Mullerians and members of the BMGG: Thomas, Marie, David, and all others Your supports and helps during my stay in Liège crucially contributed to the completion of my research I would also like to express my thanks to my friends and colleagues: Lung, Tuấn, An, Loan, and others for their cares and encouragements in life and work
My research trip was co-sponsored by the Wallonia-Brussels International (WBI) and the Wallonia-Brussels delegation to Vietnam I would like to thank you for your commitment to supporting scientific innovations as well as strengthening the collaborations between the two laboratories and between our countries
(4)TABLE OF CONTENTS
TABLE OF CONTENTS i
LIST OF TABLES AND FIGURES v
ABBREVIATIONS vii
PREFACE 1
Chapter 1: BACKGROUND INFORMATION 2
1.1 Small molecules: safety concerns
1.1.1 Pharmaceuticals and personal care products (PPCPs) 3
1.1.2 Food additives 4
1.1.3 Household chemicals 5
1.2 The Zebrafish embryo toxicity test (ZET)
Chapter 2: METHODS 11
2.1 Substances 11
2.2 Zebrafish maintenance 12
2.3 Chemical exposure and embryo observation 12
2.4 Behavioural analysis 14
2.5 Gene expression analysis 14
2.5.1 Reverse transcription and quantitative polymerase chain reaction 14
2.5.2 Transgenic fluorescent lines 16
2.6 Statistical analysis 16
2.7 Quality control 17
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3.1 Morphological and lethal effects 18
3.2 Locomotor defects 29
3.3 Specific transgene expression in living embryos 33
3.4 Reverse transcriptive – qPCR 38
Chapter 4: CONCLUSIONS 41
(6)LIST OF TABLES AND FIGURES Tables
Table 2-1: List of studied chemicals 11
Table 2-2: Lethality endpoints 13
Table 2-3: Quantitative PCR primer set 15
Table 3-1: Concentration ranges selected for the main study 18
Table 3-2: Lethal concentrations, effective concentrations, teratogenic indices, and typical defects of studied substances 25
Figures Figure 1.1: Orthologous genes shared among the zebrafish, human, mouse and chicken genomes (reprinted from Howe et al [33]) 7
Figure 1.2: Literature analysis using the Scopus database in February 2014 8
Figure 1.3: Comparisons between the ZET test and the classical acute fish toxicity test (reprinted from Lammer et al [40]) 10
Figure 2.1: Normal morphological stages of zebrafish development at 28.5 C (photos excerpted from Kimmel et.al [39]) Scale bars = 250 M 13
Figure 3.1: Morphological phenotypes in hatched zebrafish larvae 19
Figure 3.2: Concentration-response curves and frequency of typical phenotypes caused by tested substances 22
Figure 3.3: LC50, EC50 Hill slope values of tested chemicals 27
Figure 3.4: Correlation between LC50s resulting from this study and those obtained using the procedure described in the OECD 236 guideline [59] 28
Figure 3.5: Larval motion measurements during the dark/light cycles 30
Figure 3.6: Comparative analysis of larval activity 31
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(8)ABBREVIATIONS
DCA 3,4-Dichloroaniline
DMSO Dimethyl sulfoxide
dpf Day post fertilisation
EtOH Ethanol
hpf Hour post fertilisation
MSG Monosodium glutamate
OECD Organisation for Economic Co-operation and
Development
PPCPs Pharmaceuticals and Personal Care Products
qPCR Quantitative polymerase chain reaction
QY Quinoline yellow
SB Sodium Benzoate
TTZ Tartrazine
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PREFACE
The human population are increasingly exposed to various chemicals whose beneficial or deleterious properties often remain unexplored The rising public concern about hazardous substances existing in foods and consumer products has forced legislators to tighten chemical management policy that requires extensive toxicity testing However, assessment of chemical toxicity is a challenging task, especially in terms of reliability and efficiency Ethical issues over the use of animal testing also add further complication to the task
The zebrafish (Danio rerio) embryo is an emerging model system for chemical testing that is attracting scientific and legal attention Its advantages including rapid development, high availability, and easy observation have made the model amenable to high-throughput assays Moreover, as a complex and independent organism retaining the “non-animal” status, the zebrafish embryo is the ideal vertebrate testing model
(10)Chapter 1: BACKGROUND INFORMATION 1.1 Small molecules: safety concerns
Chemicals have become an integral part of modern daily life They play an important role in almost all industries and economic sectors Consumer goods of our everyday-use are either containing chemicals, or involving them during production Global chemical production has increased from million tonnes in 1930 to 400 million tonnes in 2001 [25], with more than 143,000 substances in the European market* It is undeniable that these chemicals are progressively benefiting people’s life and economy
However, many chemicals are also posing potential deleterious effects on human and environment health, especially those with small molecular size (<900 Daltons) Amongst the most well-known examples is the thalidomide scandal which involved thousands of cases of stillborn and extreme congenital deformity [38], or the carcinogenic benzene [73] which may have claimed thousands of deaths around the world Another case is DDT, the insecticide whose extensive use and high accumulation have greatly threatened both wildlife species and human health [83] A common theme in these three instances is that large-scale application of these chemicals was conducted without having sufficient knowledge on their adverse impacts, and measures to restrict the uses were taken too late to prevent irreversible damages
Ironically, despite efforts to achieve the world governments’ agreement to use and produce chemicals “…in ways that not lead to significant adverse effects on human health and the environment…” by 2020 using scientific assessment procedures [85], the number of compounds and the complexity of the issue lead to the situation that unrecognised or unacknowledged toxic compounds in domestic
*
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