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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

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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.

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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 ... 2

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) ... 6

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

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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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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.

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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 1 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 do 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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NGHIÊN CỨU PHÂN LẬP, XÁC ĐỊNH CẤU TRÚC VÀ KHẢO SÁT HOẠT TÍNH SINH HỌC CỦA CÁC HỢP CHẤT PHENOLIC TỪ CÂY ALPINIA PINNANENSIS T. L. WU ET SENJEN (ZINGIBERACEAE)