Introduction to alternatives to animal testing in toxicology
1. Center for Alternatives to Animal Testing (CAAT)
Johns Hopkins University, Baltimore, USA –
University of Konstanz, Germany
Francois Busquet, PhD
Introduction to General
Toxicology
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
25. 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
37. •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)
38. •Low cost model
•Genome sequenced
•GFP
•HTP transposable
•A key model in Germany
Nagel, 2002
•Regulatory RELEVANT
•Academic model
41. 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)
42. •Water quality (Cl, NO3
2-
)
•Number of fish
•Size of the fish tank
•Food 24/7
•Water renewal
•Physico-chemical
properties
•Cleaning
ZEBRAFISH HUSBANDRY
44. • Male/Female ratio
• Mass spawning vs. spawning unit
• Spawning traps
• Stimulus - plant
• Light onset
• No stones!
• Live Food!
ZEBRAFISH EGG PRODUCTION
& COLLECTION
45. 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
46. 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
52. 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
53. 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
54. •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
55. 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
56. • 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
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
60. 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
64. 125 µM CBZ, 3 dpf
T: Tail
T
control
Carbamazepine (CBZ)
65. 5 mM TEG, 3 dpf
5 mM TEG, 3 dpf
T: Tail; C: Chorda; H: Head
C
H
T
control
Tefagur
66. 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)
67. • 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
68. • Research gate
• http://www.bdpa-detroit.org/portal/index.php/comit
• Massive Online Open Course (MOOC)
RESEARCH LINKS
71. 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
72. 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?
Notas do Editor
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).