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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 Hà Nội - 2014 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 Special thanks to Dr Nguyễn Huỳnh Minh Quyên, Prof Jacques Dommes, the Institute of Microbiology and Biotechnology (VNUIMBT), and the University of Liège (ULg) for organising this Master Program in Biotechnology It is also an honour for me to i 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 Last, but by no means least, my sincerest admiration and gratitude are dedicated to my dear family, particularly my beloved wife for unconditionally trusting and pushing me to overcome all kinds of difficulty I encounter in the past, present, and future ii TABLE OF CONTENTS TABLE OF CONTENTS i LIST OF TABLES AND FIGURES v ABBREVIATIONS vii PREFACE Chapter 1: BACKGROUND INFORMATION .2 1.1 Small molecules: safety concerns 1.1.1 Pharmaceuticals and personal care products (PPCPs) 1.1.2 Food additives 1.1.3 Household chemicals 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 Chapter 3: RESULTS AND DISCUSSION 18 iii 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 REFERENCES 43 iv 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]) Figure 1.2: Literature analysis using the Scopus database in February 2014 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 Figure 3.7: Motoneuron visualisation in dpf hb9:GFP embryos and larvae 33 v Figure 3.8: Vascularisation in dpf Tg[fli1:EGFP] embryos and larvae 36 Figure 3.9: Amplification plots of two reference candidates for this study 38 Figure 3.10: Relative expression of five tested genes using ef1α as internal control (mean SD) 39 Figure 3.11: Expression profiles of five substances on the selected genes 40 vi 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 ZET Zebrafish embryo toxicity test vii 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 Inspired by the promising applications of the zebrafish embryo model in toxicology research, with the objectives of developing analysis techniques and applying them in testing of different small molecular compounds, we decided to carry out the project “Toxicity assessment of small molecules using the zebrafish as a model system” 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 (50-fold) and EtOH (>10-fold) – suggesting long-lasting stress caused by all chemical treatments Interestingly, the -actin level generally considered as an invariant house-keeping gene, also increased up to more than 2fold in DCA-treated and more than 6-fold in EtOH-treated groups, raising a question on similar studies which used this gene as the reference Although less significant, the nerve cell markers foxd3 and mbpa were down regulated following EtOH treatment and up regulated in the MSG-treated group Another weak, but significant change was the down regulation of the vascularisation marker vegfr2 after EtOH-, DMSO-, SB-, and MSG-treatments, as these seem to correlate with minor haemostasis phenotypes observed in the corresponding treated groups Figure 3.11: Expression profiles of five substances on the selected genes In order to visualise patterns of chemical-induced regulation of our gene set, a radar graph representing the expression profile for the selected genes by different substances was built (Figure 3.11) This graph may also provide a valuable tool for chemical toxicity categorisation in further studies 40 Chapter 4: CONCLUSIONS Zebrafish embryos and larvae are increasingly utilised as a powerful tool to investigate the potential harmful effects of chemicals on an entire living vertebrate In this study, we employed a panel of developmental toxicity tests with emphasis on vasculogenic and neurobehavioural defects to assess seven substances representing different chemical classes of various physico-chemical properties In addition to using standard substances (i.e EtOH, DMSO, and DCA) to validate our approach, we also tested four common food additives (SB, MSG, TTZ, and QY) for their potential toxicity on the zebrafish embryo model Although more tests must be conducted on more standard substances, the correlation between LC50 values obtained using our established method and the validated OECD protocol (Figure 3.4) has initially verified our approach’s reliability Moreover, while the OECD guideline only considers lethal effects, our methodology allows detecting more specific effects as well as planning subsequent experiments Following morphological and lethal observation, behavioural tests and gene expression experiments have provided additional toxicological data as well as supportive information on observed defects Despite not being able to fully elucidate the mechanisms involved, the panel of tests has proven to be an effective screening tool for chemical toxicity assessment which may provide suggestions for subsequent studies In addition, the substance-specific pattern of effects based on our test panel may serve as a tool to categorise chemical toxicity using methods such as principal component analysis (PCA) [67] or hierarchical clustering A major finding of this study is the superior reliability of ef1α over -actin as a reference gene in toxicological applications and the need to re-validate consistency of other housekeeping genes in similar research EtOH and DMSO are among the most popular solvent carriers in toxicity testing of insoluble substances Results from our study showed that DMSO is 41 preferable in such tests due to its higher lethal and effective concentrations as well as its more consistent effects at different doses (Figure 3.2) Regarding their potential neurotoxicity and teratogenicity, care should be taken when interpreting toxicological results involving EtOH or DMSO as carrier, final concentrations for exposure should be kept below, respectively and 1.5 % for these solvents Our survival results showed that SB is a Cat aquatoxic compound (LC504d lies between 10-100 mg/L) with a neurotoxic effect, while there have been no other published data categorising SB as toxicant Regarding the extensive use of SB as preservative in food and cosmetic products, the safety of these products should be considered, especially for those being used during pregnancy and childhood Another major finding is the potential anti-angiogenic effect of TTZ, which needs to be verified by further investigation using convenient angiogenesis tests such as HUVEC culture or aortic ring assays [47] If the hypothesis is true, it may partially explain some symptoms occurring in people hypersensitive to TTZ and may as well lead to pharmaceutical applications for this common food colouring agent While being categorised as non-aquatoxic, QY was revealed as the most potent teratogenic compound (TI4d ~ 80), with an EC50 of 930 mg/L and a lowest effective concentration (LOEC) of 20 mg/L Additionally, it should be noted that “non-aquatoxic” does not legally nor scientifically guarantee safety to human health While no toxicity was previously described for QY when tested individually, QY- and MSG-containing mixtures were found to affect neural development [43] and child behaviour [51], probably through a synergistic effect Our subsequent study may involve testing whether similar mixtures could also affect zebrafish neural development and behaviour The obtained result may contribute to either raising concerns on, or confirming the safety of these controversial food additives 42 REFERENCES Aboel-Zahab H., el-Khyat Z., Sidhom G., Awadallah R., Abdel-al W., and Mahdy K (1997), “Physiological effects of some synthetic food colouring additives on rats”, 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zebrafish Tg(nkx2.2a:mEGFP) provides a highly sensitive monitoring tool for neurotoxins”, PLoS One, (2), pp e55474 92 Zurita J.L., Jos A., del Peso A., Salguero M., Lopez-Artiguez M., and Repetto G (2007), “Ecotoxicological effects of the antioxidant additive propyl gallate in five aquatic systems”, Water research, 41 (12), pp 25992611 50 in ... decreased as they adapted to darkness The habituation effect can also be seen as the decrease of active time toward the end of the dark phases, though the point of reaching maximal activity varied... CCGTCGTGGAGACGTCAA CGAGGAGAGGACACAAAGCT TCCACAACTGCTTCCTGATG CACACGACTCAATGCGTACC Subsequently, cDNA was amplified using the SensiMix SYBR Hi-ROX Kit (Bioline; Meridian Life Science) and the reaction... contributed to the dramatic boost in the distance that larvae moved in the dark Larvae responded to the onset of darkness with a strong startle, causing a maximal peak on the speed actogram, then their