Introduction to alternatives to animal testing in toxicology

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  • The fish is named for the five uniform, pigmented, horizontal blue stripes on the side of the body, all of which extend to the end of the caudal fin. Its mouth directed upwards. Males are torpedo-shaped and have gold stripes between the blue stripes; females have a larger, whitish belly and have silver stripes instead of gold. Adult females will exhibit a small genital papilla in front of the anal fin origin. The zebrafish can grow to 6.4 centimetres (2.5 in), although it is uncommon for them to grow past 4 cm in captivity. Life-span in captivity is around 2–3 years, although in ideal conditions, may extend to 5 years.
    The zebrafish is omnivorous. It primarily eats zooplankton, insects, insect larvae, and phytoplankton. It can eat a variety of other foods, such as worms and small crustaceans if its preferred sources are not readily available.5 Most danios accept common food flakes and tubifex worms in the aquarium.
  • The fish is named for the five uniform, pigmented, horizontal blue stripes on the side of the body, all of which extend to the end of the caudal fin. Its mouth directed upwards. Males are torpedo-shaped and have gold stripes between the blue stripes; females have a larger, whitish belly and have silver stripes instead of gold. Adult females will exhibit a small genital papilla in front of the anal fin origin. The zebrafish can grow to 6.4 centimetres (2.5 in), although it is uncommon for them to grow past 4 cm in captivity. Life-span in captivity is around 2–3 years, although in ideal conditions, may extend to 5 years.
    The zebrafish is omnivorous. It primarily eats zooplankton, insects, insect larvae, and phytoplankton. It can eat a variety of other foods, such as worms and small crustaceans if its preferred sources are not readily available.5 Most danios accept common food flakes and tubifex worms in the aquarium.
  • The approximate generation time for the Danio is 3–4 months. A male must be present for ovulation and spawning to occur. Females are able to spawn at intervals of 2–3 days, laying hundreds of eggs in each clutch. Upon release, embryonic development begins; absent sperm, growth stops after the first few cell divisions. Fertilized eggs almost immediately become transparent, a characteristic that makes D. rerio a convenient research model species. Development progresses rapidly. Precursors to all major organs appear within 36 hours of fertilization, and hatching takes place 12-36 hours later, depending on the embryo's internal conditions and the external temperature, ideally . Swimming and feeding behavior begin about 36 hours later. The sex of juveniles cannot be distinguished except by dissection, and sex determinants are not clearly understood.
  • Chorion=
    The chorion is one of the membranes that exist during pregnancy between the developing fetus and mother. It is formed by extraembryonic mesoderm and the two layers of trophoblast and surrounds the embryo and other membranes
    Somites =
    a division of the body of an animal. In vertebrates this is mainly discernible in the embryostage. In the developing vertebrate embryo, somites are masses of mesoderm distributed along the two sides of the neural tube and that will eventually become dermis (dermatome), skeletal muscle (myotome), and vertebrae (sclerotome).
  • Notochord =
    The notochord is a flexible rod-shaped body found in embryos of all chordates. It is composed of cells derived from the mesoderm and defines the primitive axis of the embryo. In some chordates, it persists throughout life as the main axial support of the body, while in most vertebrates it becomes the vertebral body of the vertebral column. The notochord is found ventral to the neural tube.
  • Spinal Chord = Rückenmark
    The spinal cord is a long, thin, tubular bundle of nervous tissue and support cells that extends from the brain (the medulla oblongata specifically). The brain and spinal cord together make up the central nervous system (CNS).
  • Introduction to alternatives to animal testing in toxicology

    1. 1. Center for Alternatives to Animal Testing (CAAT) Johns Hopkins University, Baltimore, USA – University of Konstanz, Germany Francois Busquet, PhD Introduction to General Toxicology
    2. 2. WHAT IS MY BACKGROUND?
    3. 3. •Toxicologist - 10 years •Alternative test methods •Regulatory affairs •Ecotoxicology •PhD with Zebrafish egg model for teratogenicity screening test (humans) – Private sector •Post-Doc with Zebrafish egg model for ecotoxicology (environment) – Government
    4. 4. WHO I WORK FOR?
    5. 5. 1981 2010
    6. 6. WHAT IS TOXICOLOGY ABOUT?
    7. 7. TESTING PRODUCT SAFETY
    8. 8. PROTECTION FOR
    9. 9. TOXICOLOGY DOGMA
    10. 10. Paracelsus “Everything is poisonous, only the dose makes the poison” – XVIth Century
    11. 11. • How much? e.g. Dose • How long? e.g. Time Exposure • When? e.g. Critical Window • Which effects? e.g. Lethality TOXICOLOGY PRINCIPLES
    12. 12. WHAT ARE THE COMMON IN VIVO MODELS?
    13. 13. HUMAN ENV
    14. 14. WHAT ARE ALTERNATIVES TEST METHODS?
    15. 15. 3Rs REPLACEMENT REDUCTION REFINEMENT
    16. 16. WHY USING IN VITRO MODELS?
    17. 17. ANIMAL WELFARE DILEMMA
    18. 18. PREDICTIVITY
    19. 19. STATISTICS TG 203 TG 236 7 FISH X 6 GROUPS = 42 20 FISH EGGS X 7 GROUPS = 140
    20. 20. LOW COST & TIME =X 10 millions US dollars + 5 years time X = 10,000 US dollars + 2 weeks vs. 6
    21. 21. EU REGULATION + =+ =++
    22. 22. EU REGULATION + = = + ++ =
    23. 23. TRENDS IN US
    24. 24. An atmosphere of departure in toxicology 2007 New technologies from biotech and (bio-)informatics revolution Mapping of pathways of toxicity (PoT) NAS vision report Tox-21c
    25. 25. Tox-21c Reg. Outputs EDSP21 = in vitro HTS in silico Full replacement for Tier 1 EDSP20
    26. 26. STATUS OF IN VITRO MODELS ACCEPTANCE IN EU
    27. 27. EU Status – ACCEPTED
    28. 28. EU Status – ACCEPTED
    29. 29. EU Status – ON GOING
    30. 30. EU Status – ON GOING
    31. 31. EAWAG – INSTITUTE VIDEO DEMONSTRATION AT EAWAG - SWITZERLAND
    32. 32. ZebraFish Danio rerio Embryo as an Alternative Method for Eco and Human toxicology
    33. 33. WHY EVERYBODY WANT TO WORK WITH ZEBRAFISH EGGS?
    34. 34. •Easy maintenance and mating •High Fecundity rate = laying down transparent eggs all year long •Embryological devt is fast (96h) and well described (Kimmel et al. 1995)
    35. 35. •Low cost model •Genome sequenced •GFP •HTP transposable •A key model in Germany Nagel, 2002 •Regulatory RELEVANT •Academic model
    36. 36. ZEBRAFISH HANDLING & CARE IN THE LABORATORY
    37. 37. ZEBRAFISH ANATOMY • Male & Female • Age / Life Span / Size
    38. 38. ZEBRAFISH REPRODUCTION • Approximate generation time: 3-4 months • Spawning interval: 2-3 days • Up to 400 eggs per week • Eggs: non-adherent and fully transparent • Rapid embryonic development: 3 days = precursor organs (ideal 28.5° C, tolerated: 23 – 33° C)
    39. 39. •Water quality (Cl, NO3 2- ) •Number of fish •Size of the fish tank •Food 24/7 •Water renewal •Physico-chemical properties •Cleaning ZEBRAFISH HUSBANDRY
    40. 40. ZEBRAFISH AQUARIUM
    41. 41. • Male/Female ratio • Mass spawning vs. spawning unit • Spawning traps • Stimulus - plant • Light onset • No stones! • Live Food! ZEBRAFISH EGG PRODUCTION & COLLECTION
    42. 42. Acute toxicity in fish (Replacement) Testing of WW Developmental Toxicity (Teratogenicity) Safety Pharmacology Mammalian Organotoxicity General Model for vertebrate developmental biology Genomic ResearchDrug Discovery ZF AS A UNIVERSAL MODEL
    43. 43. Acute toxicity in fish (Replacement) Testing of WW Developmental Toxicity (Teratogenicity) Safety Pharmacology Mammalian Organotoxicity General Model for vertebrate developmental biology Genomic ResearchDrug Discovery ZF AS A UNIVERSAL MODEL
    44. 44. ZF Developmental Stages (26° C) 200 µm 8 cells stage 1 hpf Yolk sac
    45. 45. 200 µm 12 hpf Somites Eye Head Chorion ZF Developmental Stages (26° C)
    46. 46. 24 hpf Eye Sacculi/Otoliths Tail detached Cerebellum Notochord Brain Heart cavity Yolk ZF Developmental Stages (26° C)
    47. 47. 48 hpf Sacculi/Otoliths Pigmentation Spinal chord ZF Developmental Stages (26° C)
    48. 48. Sacculi/Otoliths Heart Fin Spinal chord 72 hpf Intestine ZF Developmental Stages (26° C)
    49. 49. Beginning coagulation 1 h 24 h Normal Lethal effects 48 h E = eye; S = somites; Ch = chorion; C = chorda; TD = tail detached; TND = tail not detached E ECh SC TD C Ch Lack of somites Tail bud not detached ETND S photosprovidedbyJ.Bachmann,UBA,Berlin
    50. 50. Acute toxicity in fish (Replacement) Testing of WW Developmental Toxicity (Teratogenicity) Safety Pharmacology Mammalian Organotoxicity General Model for vertebrate developmental biology Genomic ResearchDrug Discovery ZF AS A UNIVERSAL MODEL
    51. 51. •Testing of the toxicity of waste water: Replacement of fish test (Goldorfen-Test) by fish egg assay since 2005 (sewage tax law) DIN 38415, part 6 (safes 40.000 – 50.000 animals/year) 48 h/26°C Lethal endpoints: Coagulated egg ? No formation of somites ? No heartbeat ? No detachment of the tail ? Waste water TESTING OF WASTE WATER 0- CONTROL 1- UNDILUTED WW 2- 50% DILUTED WW 3- 33% DILUTED WW 4- PC
    52. 52. Acute toxicity in fish (Replacement) Testing of WW Developmental Toxicity (Teratogenicity) Safety Pharmacology Mammalian Organotoxicity General Model for vertebrate developmental biology Genomic ResearchDrug Discovery ZF A UNIVERSAL MODEL
    53. 53. • newly fertilised zebrafish embryos • 20 embryos/concentration/control • 5 test concentrations • 2 ml/well; 26 ± 1° C and light cycle • 96 h exposure with semi-static renewal procedure • 4 endpoints for acute lethality (24, 48, 72 and 96h) • LC50 calculation at 48 and 96h FET TG236
    54. 54. Acute toxicity in fish (Replacement) Testing of WW Developmental Toxicity (Teratogenicity) Safety Pharmacology Mammalian Organotoxicity General Model for vertebrate developmental biology Genomic ResearchDrug Discovery ZF A UNIVERSAL MODEL
    55. 55. MAMMALIAN ORGANOTOXICITY
    56. 56. MAMMALIAN ORGANOTOXICITY
    57. 57. Acute toxicity in fish (Replacement) Testing of WW Developmental Toxicity (Teratogenicity) Safety Pharmacology Mammalian Organotoxicity General Model for vertebrate developmental biology Genomic ResearchDrug Discovery ZF A UNIVERSAL MODEL
    58. 58. Tris-buffer pH 7.4 (0.5% DMSO) 8 hpf 1 dpf 2 dpf3 dpf 2.5 hpf DEVELOPMENTAL TOXICITY (TERATOGENICITY)
    59. 59. ZF SCORING TERATOGENICITY Category Physiological/ dysmorphogenic effect 8 hpf 24 hpf 48 hpf 72 hpf Lethal effect Coagulated egg + + + + No heart beat + Teratogenic effects Malformation of head + + + Malformation of eyes + + + Modification of the spinal chord + + + Modification of the chorda + + + Malformation of tail + + + Malformation of tail tip (end tail) + + + Scoliosis + + + Malformation of sacculi/otoliths + + + Malformation of heart + Deformity of yolk + + + Growth retardation + + +
    60. 60. 6 µM AAF, 1 dpf aYS aYS: additional yolk sac 2-Acetylaminofluorene (AAF)
    61. 61. 125 µM CBZ, 3 dpf T: Tail T control Carbamazepine (CBZ)
    62. 62. 5 mM TEG, 3 dpf 5 mM TEG, 3 dpf T: Tail; C: Chorda; H: Head C H T control Tefagur
    63. 63. S/O: Sacculi/otoliths; H: Head; E: Eye; C: Chorda; TT: Tail tip; T: Tail 250 µM WRF, 3 dpf 500 µM WRF, 3 dpf S/O E S/O C TT H T control Warfarin (WFR)
    64. 64. • http://zfin.org/ The Zebrafish Model Organism Database • http://zfatlas.psu.edu/ The Zebrafish Atlas • http://itgmv1.fzk.de/eufishbiomed/eufish_main.php EUFishBioMed (European Network on Fish Biomedical Models) ZEBRAFISH LINKS
    65. 65. • Research gate • http://www.bdpa-detroit.org/portal/index.php/comit • Massive Online Open Course (MOOC) RESEARCH LINKS
    66. 66. Acknowledgements Dr. Thomas Broschard Dr. Nicole Hübler Dr. Stefan Weigt Julia PohlAlex Küfner Andreas Gado Johann Gally
    67. 67. POSSIBLE FUTURE STEPS FOR UVA WELLASSA WITH ZEBRAFISH EGGS
    68. 68. Short term • Uva x Colombo = national ZF network? • Research topic on medium to allow fish egg transport by blocking ZF Egg Devt? • Student project to develop live food from the aquaculture Dpt for ZF? • ZF unit for student (training)? • Lectures in Zebrafish egg
    69. 69. Medium term • ZF – Research Topics –Heavy metals & water quality? –Field sampling - pesticides impact & kidney failure disorders? –Indigenous medicines toxicity? –Agriculture Dpt? Dvpt of substitutes for pesticides or best practices for farmers?

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