Zebrafish as a tool to study mechanisms of developmental toxicology of environmental chemicals. Jessica Legradi, P. Cenijn, R. Carvalho, J.
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1 Zebrafish as a tool to study mechanisms of developmental toxicology of environmental chemicals Jessica Legradi, P. Cenijn, R. Carvalho, J. Legler
2 Overview Introduce zebrafish as model organism Zebrafish embryo toxicity test (ZFET) Expanding ZFET Angiogenesis Neurodevelopment Energy metabolism EDA Zebrafish are a valuable system to study mechanisms of developmental toxicology of environmental chemicals 2
3 The Zebrafish (Danio Rerio) Freshwater fish Native to southeastern Himalaya region Inhabits streams, canals, ponds, and slow moving water bodies, including rice fields Length of the adults 3-4 cm Adult Zebrafish (4-5 month) 3
4 Zebrafish are much more then a tool One female can lay eggs per week Egg-diameter is around 1 mm Clear chorion allows to monitor the early development Quick development (hatch at day 3) Survive in 96 well plates till day 5 Till the end of the larva stage they are no animals (replace animal tests) Complex organism 1 hour Embryo (2 days) Larva (3 days) 4
5 But we have many tools to work with Gene Knock outs In situ hybridization Mutagenic zebrafish Transcriptomics/ Metabolomics Transgenic zebrafish 5
6 DOSE Zebrafish embryo toxicity test (ZFET) 6
7 ZFET (Zebrafish embryo toxicity test) Focus is not on Modes of Action or Adverse Outcome Pathways From Nagel et al 7
8 Expand the ZFET to better understand mechanisms of toxicity SMART ZFET Angiogenesis Neurodevelopment Energy metabolism 8
9 Phenotypic effects of anti-cancer drugs ZFET (4hpf-6dpf) Compound X 9
10 Heartbeat Cardiac edema 4dpf, y mm Compound X Control 10
11 Age in hpf Heart beat per minute Heartbeat Heart beat concentration mm 11
12 Blood vessel growth Cardiovascular toxicity (Casper::FLI) Control 48hpf CPA Compound 10mM X 48hpf 4dpf Control 12
13 Blood vessel growth Cardiovascular toxicity (Casper::FLI) Control 48hpf CPA 10mM 48hpf 13
14 Expand the ZFET to better understand mechanisms of toxicity SMART ZFET Angiogenesis Neurodevelopment Energy metabolism 14
15 Phenotypic effects of carbamates MOA 24 hpf 48hpf 72 hpf 144 hpf behavior aldicarb AChE inhibitor hyperactive pirimicarb AChE inhibitor methomyl AChE inhibitor carbaryl AChE inhibitor hyperactive tail malformation, curved dead shorter Bodylength normal edema 15
16 Viewpoint Zebrabox Behavior screen a startle reflex assay (light response), in 96 well plates Measure : Speed Distance Duration 16
17 Neurite outgrowth Rohon Beard neurons Mauthner neuron (ventral side) Acetylated alpha-tubulin Antibody 17
18 Expand the ZFET to better understand mechanisms of toxicity SMART ZFET Angiogenesis Neurodevelopment Energy metabolism 18
19 Phenotypic effects of 19 OH-PBDEs OXPHOSTOX Developmental delay Unspecific Cardiotoxic Pigmentation Tail development Compound X (48 hpf) Acute (1h exposure) LOEC µm Chronic (72 h exposure) LOEC µm 19
20 Aerobic energy metabolism -oxidative phosphorylation (OXPHOS) OXPHOSTOX Energy demand Oxygen consumption CO2 production up up up 20
21 New in vivo assays for OXPHOS disruption Mineral oil 50 µl Metabolic rate measurement in humans Larva 3 days old OXPHOSTOX 21
22 In vivo measurement of CO 2 production Measure the CO 2 production as acid (H 2 CO 3 ) in the medium via a phsensitive dye (phenol red) Metabolic rate assay in a 96 well plate Makky K. (2008), Journal of Biomolecular Screening OXPHOSTOX 22
23 O 2 (µm) In vivo measurement of oxygen consumption Negative Control 'OH-BDE49 (7uM) DMSO (5) time (minutes) OxoPlate oxygen sensitive dye at the well bottom developed for cell and bacteria assays measured with a standard multiplate reader OxoPlate (PreSens) OXPHOSTOX 23
24 Expand the ZFET to better understand mechanisms of toxicity SMART ZFET Angiogenesis Neurodevelopment Use SMART ZFET to expand the possibility of chemical analysis Energy metabolism Effect Directed Analysis (EDA) 24
25 Effect directed analysis Extraction (ASE DCM/Ac) Clean up (GPC) Fractionation (RP-HPLC) embryotoxicity Chemical screen (GC-MSD + LC Orbitrap MSD embryotoxicity Legler
26 HT-EDA complex contamination extraction Brack 2003 biotesting analysis fractionation biotesting toxicant 26
27 Conclusion We could link chemicals to mechanisms Pharmaceuticals-> Cardiotoxicity Pesticides -> Neurotoxicity Metabolites of Flameretardents -> Disruption of Energy metabolism We could also link mechanisms to chemicals EDA using mechanistic bioassays in vivo 27
28 Acknowledgement Project coordinator John Sumter, Brunel University, UK Benoit Roig, Advanced School of Public Health, France Pim Leonards, IVM, Netherlands Milou Dingemanns, Institute for Risk Assessment Sciences, Netherlands OXPHOSTOX Ake Bergman, University of Stockholm, Sweden
29 Acknowledgement Juliette Legler Peter Cenijn Ralph Carvalho IVM Marija Lamoree Jana Weiss 29
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