Alternative developmental toxicity models for assessing the INVITATION Alternative developmental toxicity For attending the public models for assessing the defence of my thesis entitled: in vivo embryotoxicity of azoles Alternative developmental toxicity models for assessing the in vivo embryotoxicity of azoles On Monday 5th of March 2018 at 1:30 p.m. In the Aula of Wageningen University in vivo General Foulkesweg 1a Wageningen embryotoxicity of azoles Myrto Dimopoulou [email protected] Paranymphs: Marcia Bodero [email protected] Myrto Dimopoulou Ignacio Miro Estruch [email protected] Myrto Dimopoulou Alternative developmental toxicity models for assessing the in vivo embryotoxicity of azoles Myrto Dimopoulou Thesis committee Promotors Prof. Dr B. van Ravenzwaay Professor of Reproductive and Developmental Toxicology, Wageningen University & Research Senior Vice President of Experimental Toxicology and Ecology, BASF SE, Ludwigshafen, Germany Prof. Dr A.H. Piersma Professor of Reproductive and Developmental Toxicology Utrecht University & National Institute for Public Health and the Environment (RIVM), Centre for Health Protection, Bilthoven, the Netherlands Co-promotor Prof. Dr I.M.C.M. Rietjens Professor of Toxicology Wageningen University & Research Other members Prof. Dr A.J. Murk, Wageningen University & Research Prof. Dr J. Kleinjans, Maastricht University, the Netherlands Dr J. Louisse, KWR Water, Nieuwegein, the Netherlands Prof. Dr F.G.M. Russel, Radboud University, Nijmegen, the Netherlands This research was conducted under the auspices of the Graduate School VLAG (Advanced studies in Food Technology, Agrobiotechnology, Nutrition and Health Sciences). Alternative developmental toxicity models for assessing the in vivo embryotoxicity of azoles Myrto Dimopoulou Thesis submitted in fulfilment of the requirements for the degree of doctor at Wageningen University by the authority of Rector Magnificus, Prof. Dr A.P.J. Mol, in the presence of the Thesis Committee appointed by the Academic Board to be defended in public on Monday 05 March 2018 at 01.30 p.m. in the Aula. Myrto Dimopoulou Alternative developmental toxicity models for assessing the in vivo embryotoxicity of azoles, 200 pages. PhD thesis, Wageningen University, Wageningen, the Netherlands (2018) With references, with summary in English ISBN: 978-94-6343-731-8 DOI: https://doi.org/10.18174/430573 Contents Abbreviations 7 Chapter 1 General introduction 11 Chapter 2 Flusilazole induces spatio-temporal expression patterns of retinoic acid-, differentiation- and sterol biosynthesis- related genes in the rat Whole Embryo Culture 43 Chapter 3 Embryotoxic and pharmacologic potency ranking of six azoles in the rat Whole Embryo Culture by morphological and transcriptomic analysis 69 Chapter 4 A transcriptomic approach for evaluating the relative potency and mechanism of action of azoles in the rat Whole Embryo Culture 103 Chapter 5 A comparison of the embryonic stem cell test and whole embryo Culture assay combined with the BeWo placental passage model for predicting the embryotoxicity of azoles 131 Chapter 6 General discussion and future perspectives 163 Chapter 7 Summary 187 Appendix Acknowledgments 194 List of publications 196 Curriculum Vitae 197 Overview of completed training activities 198 Abbreviations Abbreviations ADME Absorption, distribution, metabolism and excretion AOP Adverse outcome pathway BMD Benchmark dose BMR Benchmark response CDF Chip Description File cDNA Complementary DNA CNS Central nervous system CTD Comparative Toxicology Database DAVID Database for Annotation, Visualization and Integrated Discovery DFZ Difenoconazole DMSO Dimethylsulfoxide DNA Deoxyribonucleic acid EB Embryonic body ECVAM European Centre for the Validation of Alternative Methods ESC Embryonic stem cells EST Embryonic stem cell test FC Fold Change FDR False discovery rate FEN Fenarimol FLU Flusilazole FR Fold Ratio GD Gestational day GO Gene Ontology GSEA Gene Set Enrichment Analysis ICxx Inhibitory concentration of malformations xx % IDxx Inhibitory concentration of differentiation xx % Abbreviations KEGG Kyoto Encyclopedia of Genes and Genomes KTZ Ketoconazole LEL Lowest effect level MCZ Miconazole MeHg Methylmercury mRNA Messenger ribonucleic acid NCBI National Center for Biotechnology Information NOAEL No observed adverse effect level OECD Organization for Economic Co-operation and Development PCA Principal component analysis PRO Propiconazole PTZ Prothioconazole QIVIVE Quantitative in vitro-in vivo extrapolation QSAR Quantitative structure-activity relationship RA Retinoic acid REACH Registration Authorization and Evaluation of Chemicals RMA Robust Multichip Average RNA Ribonucleic acid RT-PCR Real-time Polymerase Chain Reaction TDF Triadimefon TEB Tebuconazole TMS Total morphological score WEC Whole embryo culture ZET Zebrafish embryo test Chapter 1 General introduction General introduction 1. The reproductive cycle in mammals Reproduction in mammals is a complicated process including fertilization, prenatal and postnatal development. It includes a cascade of events such as sexual behaviour, pregnancy, birth, growth and subsequent sexual maturation. The reproductive process is organized and regulated through a variety of mechanisms at the biochemical, molecular, cellular, tissue and organ level, leading to embryonic differentiation and morphogenesis. These processes include the organization of different cells to build three-dimensional structures and, consequently, to form organs and the entire organism [1, 2]. The complexity and sensitivity of the reproductive cycle could be affected by many factors, including malnutrition and stressful environment, or exposure to xenobiotics, biotoxins and radiation [3]. 2. Reproductive and developmental toxicology Reproductive and developmental toxicology is the field of expertise that deals with effects on fertility and with birth defects that could occur during any stage of the reproductive cycle [4]. With the term “birth defects”, structural or functional abnormalities are described, which are identified at birth, can cause physical, intellectual and developmental disabilities and can lead to death of infants during their first year of life. Several birth defects are caused by xenobiotics, called teratogens, coming from the Greek word “teras” that means monster [5]. Teratology belongs to the field of developmental toxicology, and its research objective is to identify the cause of teratogenesis, or otherwise, the cause of morphological adverse outcomes of pregnancy [6]. Historically, I.G. de Saint-Hillaire firstly introduced the term of “teratology” during the 19th century [7]. However, rational biologically based theories of congenital developmental disorders caused by exogenous factors were firstly introduced by J. Warkany in the 1930s and 1940s [8]. Despite the birth defects observed during the first half of the 20th century due to exposure to teratogenic factors such as aminopterin, radiation, estrogens, androgens, cortisone, hypovitaminosis, folic acid, vitamin A and vitamin D supplements, not much attention was given to developmental toxicity studies [7]. The importance of developmental toxicity studies gained the scientific attention in the early 60s when the thalidomide tragedy occurred [9]. Thalidomide, or the commercially so-called Softenon, was prescribed as a sedative to pregnant women against morning sickness, causing severe congenital limb defects (phocomelia) in more than 10,000 children [9, 10]. In 1973, Wilson gave a more comprehensive definition of “teratology”, introducing the terms of mechanism of development, time of exposure, frequency and degree of dosing and different manifestations of deviant development (death, malformation, growth retardation and functional defect), with which he formulated the principles of teratology [6]. 12 Chapter 1 Due to the thalidomide incident in the 1960s, since 1980 worldwide guidelines for developmental toxicity testing were incorporated by the Organization for Economic Co- operation and Development (OECD) for improving the assessment of chemical safety and decreasing the risk of other tragedies such as the one caused by thalidomide. The OECD guidelines were designed to assess the developmental toxic effects of pharmaceuticals and pesticides during the entire reproductive cycle, including sexual maturation, fertilization, prenatal and postnatal development by implementing in vivo animal testing protocols [11]. The three key phases of reproduction, fertility, pre- and postnatal development, are covered by five OECD test guidelines, which include the prenatal developmental toxicity study (OECD 414), the one-generation (OECD 415) and two-generation studies (OECD 416), as well as the reproductive/developmental toxicity screening test (OECD 421) and the reproductive/developmental toxicity screening study (OECD 422) [12-16]. Later, the OECD 426 study was established for screening developmental neurotoxic effects of chemicals [17]. The application of these guidelines implied the increase of animal testing, experimental costs and time needed for conducting a complete hazard assessment. Additionally, in 2007 the European legislation for Registration, Evaluation, Authorization and Restriction of Chemicals (REACH) was implemented to improve the hazard identification of chemicals and to perform a risk assessment for all chemicals, which are produced or imported in the European Union [18, 19]. The resulting demand to assess the safety of almost 30,000 current and of even