Thyroid in a Jar: Towards an Integrated In Vitro Testing Strategy for Thyroid-Active Compounds Barae Jomaa Thesis committee Promotor Prof. Dr Ivonne M.C.M Rietjens Professor of Toxicology Wageningen University Co-promotors Dr Jac M.M.J.G. Aarts Senior researcher, Faculty of Archaeology, Human Origins Group Leiden University Dr Ad A.C.M. Peijnenburg DLO Group Head RIKILT Wageningen UR Other members Prof. Dr Jos C. Kleinjans, Maastricht University Prof. Dr Juliette Legler, VU University Amsterdam Dr Willem G.E.J. Schoonen Prof. Dr Geert F. Wegertjes, Wageningen University This research was conducted under the auspices of the Graduate School VLAG (Advanced studies in Food Technology, Agrobiotechnology, Nutrition and Health Sciences). Thyroid in a Jar: Towards an Integrated In Vitro Testing Strategy for Thyroid-Active Compounds Barae Jomaa Thesis submitted in fulfillment of the requirements for the degree of doctor at Wageningen University by the authority of the Academic Board, in the presence of the Thesis Committee appointed by the Academic Board to be defended in public on Friday 05 June 2015 at 4 p.m. in the Aula. Barae Jomaa Thyroid in a Jar: Towards an Integrated In Vitro Testing Strategy for Thyroid-Active Compounds. 188 pages. PhD thesis, Wageningen University, Wageningen, NL (2015) With references, with summaries in Dutch and English ISBN: 978-90-9029008-9 Contents Chapter 1 General introduction 7 Chapter 2 In vitro pituitary and thyroid cell proliferation assays and their relevance as alternatives to animal testing 41 Chapter 3 Simple and rapid in vitro assay for detecting human thyroid peroxidase disruption 69 Chapter 4 Identification of thyroid hormone receptor coregulator interactions and profiling of their modulation by T3, T4, amiodarone and compound 1-850 89 Chapter 5 Developmental toxicity of thyroid-active compounds in a zebrafish embryotoxicity test 121 Chapter 6 General discussion and future perspectives 147 Chapter 7 Summary 165 Chapter 8 Samenvatting 171 Appendix Acknowledgements, curriculum vitae, list of publications and overview of completed training activities 177 This page has been intentionally left empty Chapter 1 General introduction Barae Jomaa Based on Thyroid toxicogenomics: A multi-organ paradigm. In J. Kleinjans [ed.], Toxicogenomics-Based Cellular Models: Alternatives to Animal Testing for Safety Assessment, 159–180 (2014). Academic Press. GENERAL INTRODUCTION 9 1.1 The thyroid scare 1 HAPTER Disorders related to the thyroid have long been known to man owing to the visible C enlargement of the gland known as goiter. This condition can be due to either an overactive or an underactive thyroid gland, with the former increasing and the latter decreasing the rate at which the body breaks down nutrients and produces cellular components necessary to sustain life (Terris and Gourin, 2008). The successful treatment of goiter with animal thyroid glands was reported in China as early as 643 CE (Temple and Needham, 2007). That environmental contaminants, particularly lead, are associated with goiter had already been proposed by Paracelsus in the sixteenth century. In the nineteenth century, the essential element iodine that is a basic constituent of thyroid hormones (THs) was discovered (Anon, 2013; Kelly, 1961). Thyroid hormones were characterized in the twentieth century, starting with tetraiodothyronine (T4) in 1915, and later triiodothyronine (T3) in 1952 (Figure 1.1) (Edward Calvin Kendall, 1915; Gross and Pitt-Rivers, 1952). The twentieth century saw a chain of events that raised the level of public concern over compounds that affect the thyroid, and ultimately culminated in the inclusion of thyroid disruption endpoints by the Organisation for Economic Co-operation and Development (OECD) in guidelines for the testing of chemicals (Figure 1.2). The “cranberry scare” of 1959 brought a herbicide, aminotriazole, and its effect on the thyroid into US politics, and toxicology to the forefront of public life. At the very core of the debate was a rat study that showed a high incidence of thyroid cancer upon exposure to high amounts of the pesticide. The idea that chemicals affecting rodents at high doses will affect humans at moderate doses, also known as the “mouse-as-little-man” principle, was so entrenched in people’s minds at the time that it formed part of an amendment to the Federal Food, Drug, and Cosmetic Act known as the Delaney clause. To ease the newly created “chemophobia,” the then-vice-president Richard Nixon ate cranberry sauce during his presidential election campaign (Lieberman, Adam J., 2004). A decade later, Nixon became president and proposed the establishment of the US Environmental Protection Agency (USEPA), which was approved by congress. Today, the jury is still out on the human relevance of the adverse effect of aminotriazole that was detected in the rat study, even after a retrospective cohort study of exposed Swedish railroad workers found it to be a “suspicious” carcinogen (Axelson et al., 1980). On the one hand, epidemiological data are often mired with confounders such as the presence of a mixture of active compounds in pesticides, and on the other hand, rodents have been shown to be overly sensitive to thyroid hormone disruption (Alison et al., 1994). These uncertainties have led the USEPA to consider aminotriazole as a “probable” human carcinogen (Group B) instead of a definitive human carcinogen (Group A) (USEPA, 1996b). 10 THYROID IN A JAR: TOWARDS AN INTEGRATED IN VITRO TESTING STRATEGY FOR THYROID-ACTIVE COMPOUNDS C HAPTER HO HO O O 1 H2N H2N I I I HO O I HO O I I I Fig. 1: Chemical structures of T3 and T4, respectively. The establishment of the European Chemicals Agency (ECHA) in 2007 came on the heels of another public concern, that of endocrine disruption. Again, as one of the three main classes of endocrine disruptors, thyroid disruptors are at the very heart of modern toxicology—the other two classes being estrogen disruptors and androgen disruptors. The main fear today is that a change to thyroid homeostasis could be caused by man- made chemicals and affect the hormone system’s well-documented orchestration of sexual and mental development, and this at much smaller doses than predicted from the effects observed at high doses (Vandenberg et al., 2012). 1.2 The thyroid system The thyroid system is highly responsive to a wide range of external stressors. The hypothalamus responds to conditions requiring increased energy expenditure, such as pregnancy or prolonged periods of cold, by secreting thyrotropin-releasing hormone (TRH), which stimulates the anterior pituitary to produce thyroid-stimulating hormone (TSH), which in turn stimulates the thyroid to secrete thyroid hormones. The hypothalamus also responds to changes in diet by setting off a similar downstream chain of events. A diet that is high in carbohydrates promotes energy expenditure and leads to increased T3 levels, whereas fasting promotes energy conservation and therefore leads to a drop in T3 levels (Palmblad et al., 1977). In order to keep the rise in T3 production and resulting increase in energy consumption and thermogenesis under control, the body has a negative feedback loop, whereby high levels of T3 downregulate TRH and TSH production. The resultant drop in these tropic hormones leads to decreased stimulation of thyroid hormone production and secretion, and ultimately a drop in serum thyroid hormone levels. GENERAL INTRODUCTION 11 TBG 1 T4 OH H2 N O HO H C Synthesis 3 HAPTER R S C N H3 C C O thiocyanate Synthesis Cl CH CH Cl Cl 3 3 pH 5-9 O + R S HO Biomagnification DMIT NH O O Cl Cl HO S O S O– oxazolidine-2-thione Exposure N O Synergy pH 3-4 – DDT HO OH cyanide ( CN ) 60 days post R Disruption glucosinolate partum T4 Food toxicology: Thyreostatic Industrial toxicology: DDT competes with Prenatal exposure to thyromimetic compounds (Chesney, 1928) T4 for TBG binding sites (Marshall, 1968) DIMIT leads to permanent thyroid hormone disruption (Porterfield, 1985) 1900 1910 1920 1930 1940 1950 1960 1970 1980 1990 2000 2010 Feeding rabbits large amounts of EU ban on thyreostatic ECHA is cabbage leads to ‘cabbage goiter’ drugs (1981) Cranberry established (Chesney, 1928) scare Developmental exposure to PCBs (2007) (1959) ‘Brassica seed goitre’ less responsive causes thyroid hormone disruption to iodine therapy (Hercus, 1936) and leads to abnormal development Active component causing “brassica seed of the cochlea in rats (Goldey, 1995) goiter” identified as OZT (Astwood, 1949) EPA suggests inclusion of thyroid endpoints to Hershberger asssay (1998) Active component causing “cabbage goiter” identified as the thiociannate ion [SCN] (Gmelin, 1961) OECD suggests inclusion of thyroid endpoints to OECD 407 TG (1998) Fig. 2: Timeline of the events that have shaped our understanding of thyroid hormone disruption in the century preceding the establishment of the European Chemicals Agency in 2007. TBG stands for thyroxine binding globulin; thyroxine is another name for T4 [Source: Barae Jomaa/Wikimedia Commons]. The circulating levels of the hormones T3, T4, and TSH are constantly reacting to environmental factors that include temperature, oxygen levels, light, diet, and physical and emotional stress, in addition to chemicals and drugs (Capen and Martin, 1989; Chan et al., 1978; Hackney et al., 1995; Roth et al., 2002; Singh and Turner, 1969; Tingley et al., 1965). TH levels are also influenced by age and sex (Franklyn et al., 1985). Moreover, an individual’s range is half the width of the population reference