Post on 04-Jul-2015
Center for Alternatives to Animal Testing (CAAT)
Johns Hopkins University, Baltimore, USA – University of Konstanz, Germany
Francois Busquet, PhD
Introduction to General Toxicology
WHAT IS MY BACKGROUND?
•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
WHO I WORK FOR?
1981 2010
WHAT IS TOXICOLOGY ABOUT?
TESTING PRODUCT SAFETY
PROTECTION FOR
TOXICOLOGY DOGMA
Paracelsus “Everything is poisonous, only the dose makes the poison” – XVIth Century
• How much? e.g. Dose• How long? e.g. Time Exposure• When? e.g. Critical Window• Which effects? e.g. Lethality
TOXICOLOGY PRINCIPLES
WHAT ARE THE COMMON IN VIVO MODELS?
HUMAN ENV
WHAT ARE ALTERNATIVES TEST METHODS?
3Rs
REPLACEMENT
REDUCTION
REFINEMENT
WHY USING IN VITRO MODELS?
ANIMAL WELFARE DILEMMA
PREDICTIVITY
STATISTICS
TG 203 TG 236
7 FISH X 6 GROUPS = 42 20 FISH EGGS X 7 GROUPS = 140
LOW COST & TIME
=X
10 millions US dollars + 5 years time
X =
10,000 US dollars + 2 weeks vs. 6 months
EU REGULATION
+ =+
=++
EU REGULATION
+ =
=
+
++ =
TRENDS IN US
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
Tox-21c Reg. Outputs
EDSP21 =
in vitro HTSin silico
Full replacement
for Tier 1
EDSP20
STATUS OF IN VITRO MODELS ACCEPTANCE IN
EU
EU Status – ACCEPTED
EU Status – ACCEPTED
EU Status – ON GOING
EU Status – ON GOING
EAWAG – INSTITUTE
VIDEO DEMONSTRATION AT EAWAG
- SWITZERLAND
ZebraFish Danio rerio Embryo
as an Alternative Method for Eco and Human toxicology
WHY EVERYBODY WANT TO WORK WITH ZEBRAFISH
EGGS?
•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)
•Low cost model•Genome sequenced•GFP•HTP transposable•A key model in Germany Nagel, 2002•Regulatory RELEVANT•Academic model
ZEBRAFISH HANDLING & CARE IN THE
LABORATORY
ZEBRAFISH ANATOMY
• Male & Female• Age / Life Span / Size
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)
•Water quality (Cl, NO32-)
•Number of fish•Size of the fish tank•Food 24/7•Water renewal•Physico-chemical properties •Cleaning
ZEBRAFISH HUSBANDRY
ZEBRAFISH AQUARIUM
• Male/Female ratio• Mass spawning vs. spawning unit• Spawning traps• Stimulus - plant• Light onset• No stones!• Live Food!
ZEBRAFISH EGG PRODUCTION & COLLECTION
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
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
ZF Developmental Stages (26° C)
200 µm
8 cells stage
1 hpf
Yolk sac
200 µm
12 hpf
Somites
Eye
Head
Chorion
ZF Developmental Stages (26° C)
24 hpf
Eye
Sacculi/Otoliths
Taildetached
Cerebellum
Notochord
Brain
Heart cavity
Yolk
ZF Developmental Stages (26° C)
48 hpf Sacculi/Otoliths
Pigmentation
Spinal chord
ZF Developmental Stages (26° C)
Sacculi/Otoliths
Heart
Fin Spinal chord
72 hpf
Intestine
ZF Developmental Stages (26° C)
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
phot
os p
rovi
ded
by J
. Bac
hman
n, U
BA
, Ber
lin
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
•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- CONTROL1- UNDILUTED WW2- 50% DILUTED WW3- 33% DILUTED WW4- PC
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
• 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
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
MAMMALIAN ORGANOTOXICITY
MAMMALIAN ORGANOTOXICITY
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
Tris-buffer pH 7.4
(0.5% DMSO)
8 hpf
1 dpf
2 dpf3 dpf
2.5 hpf
DEVELOPMENTAL TOXICITY(TERATOGENICITY)
ZF SCORING TERATOGENICITY
CategoryPhysiological/ 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 + + +
6 µM AAF, 1 dpf
aYS
aYS: additional yolk sac
2-Acetylaminofluorene (AAF)
125 µM CBZ, 3 dpf
T: Tail
T
control
Carbamazepine (CBZ)
5 mM TEG, 3 dpf
5 mM TEG, 3 dpf
T: Tail; C: Chorda; H: Head
C
H
T
control
Tefagur
S/O: Sacculi/otoliths; H: Head; E: Eye; C: Chorda;
TT: Tail tip; T: Tail
250 µM WRF, 3 dpf500 µM WRF, 3 dpf
S/O
E
S/O
CTT
H
T
control
Warfarin (WFR)
• http://zfin.org/The Zebrafish Model Organism Database
• http://zfatlas.psu.edu/The Zebrafish Atlas
• http://itgmv1.fzk.de/eufishbiomed/eufish_main.phpEUFishBioMed (European Network on Fish Biomedical Models)
ZEBRAFISH LINKS
• Research gate
• http://www.bdpa-detroit.org/portal/index.php/comittees/high-school-computer-competition-hscc/29-education/57-moocs-top-10-sites-for-free-education-with-elite-universities.html• Massive Online Open Course (MOOC)
RESEARCH LINKS
Acknowledgements
Dr. Thomas Broschard
Dr. Nicole Hübler
Dr. Stefan Weigt
Julia PohlAlex Küfner
Andreas Gado
Johann Gally
POSSIBLE FUTURE STEPS FORUVA WELLASSA
WITH ZEBRAFISH EGGS
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 techniques for toxicity?
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?