Zebrafish as a Model System to Study Toxicology

1. Amol R. Padol
PhD Scholar
Division of Pharmacology and Toxicology, IVRI

2. What is Zebrafish..??
o Tropical fresh water
fish
o Origin- Ganges &
Brahmaputra river
basin
o popular aquarium fish
Kingdom Animalia
Phylum Chordata
Class Actinopterygii
Order Cypriniformes
Family Cyprinidae
Genus Danio
Species D. rerio

3. Developmental stages
Zygote
Cleavage
Blastula
Gastrula
Segmentation
Hatching
Larva
Juvenile
Adult

4. Zebrafish into Research
o 1970's- University of Oregon
o Dr George Streisinger
o Zebrafish Information Network (ZFIN)
o Over 5,000 researchers in 450 labs
throughout 31 countries
o NCBI- over 1400 publications

5. Genome sequencing
o The Wellcome Trust Sanger Institute - zebrafish
genome sequencing project
o 2009- Institute of Genomics & Integrative
Biology, New Delhi
o 17 April 2013- zebrafish reference genome
sequence was published

6. Zebrafish a model system
o Small size
o Short life cycle & generation time
o Good reproduction captivity
o External fertilization
o Optically transparent embryo
o Rapid embryonic development

7. o High similarity in cellular structure, signaling &
physiology with other high-order vertebrate
o Drug metabolising CYPs (1A, 3A) & phase II
enzymes (e.g. GST, sulfotransferases)
(Goldstone et al., 2010)
Zebrafish a model system
Orthologue genes between
Danio rerio & humans

8. Powerful model organism
o Genetics
o Developmental biology
o Toxicology
o Pharmacology
o DNA repair
o Cancer

9. Organizations that promotes
alternatives to animal testing
o European Centre for Validation of Alternative
Methods (ECVAM)
o Interagency Coordinating Committee on the
Validation of Alternative Methods (ICCVAM)
o Department of Pharmacology, Jawaharlal Nehru
Medical College, Aligarh Muslim University, Aligarh
o Japanese Centre for the Validation of Alternative
Methods (JACVAM)

10. Zebrafish use in ecotoxicology
o Eco-environmental monitoring
o Sensitivity to different contaminants
o Toxic heavy metals, endocrine disruptors &
organic pollutants
o Changes in morphology, gene expression,
behavior or physiology
(Dai et al., 2014)

11. Current OECD guidelines
o OECD TG 236: Fish Embryo Acute Toxicity (FET) Test
o OECD TG 203: Fish, Acute Toxicity Test
o OECD 204: Fish, Prolonged Toxicity Test: 14-day Study
o OECD 215: Fish, Juvenile Growth Test

12. o OECD TG 203: Fish,
acute toxicity test
Exposure period- 96 h
Mortalities are recorded at
24, 48, 72 & 96 h to
determine LC50
Zebrafish use in ecotoxicology

13. Zebrafish use in ecotoxicology
OECD TG 236: Fish embryo
toxicity
Test system- embryonic stages
Exposure period- 96 h
Indicators of lethality
 coagulation of fertilized eggs,
 lack of somite formation,
 lack of detachment of the tail-bud
from the yolk sac,
 lack of heart-beat.
LC 50 is determined

14. Biomarker Toxicant Reference
Cholinesterases Cadmium acetate, lead
acetate
Richetti et al., 2011
SOD, catalase Cadmium, zinc Ling et al., 2011
Adenosine
deaminase
mercury chloride Senger et al., 2010
Metallothionein Cu2+, Hg2+, Cd2+ Chan et al., 2007
Thyroid hormone
levels
Polybrominated
diphenyl ethers
Yu et al., 2010
Principal biomarkers used in
ecotoxicological evaluations with
zebrafish

15. Monitoring endocrine disruptors
o Polybrominated diphenyl ethers (PBDEs )-
disrupts transport, storage & metabolism of
retinoid in zebrafish (Chen et al., 2012)
o Dioxin inhibits follicle maturation by
disturbing estrogen biosynthesis & estrogen-
regulated signal transduction (Heiden et al., 2008)

16. Monitoring endocrine disruptors
o BPA exposure - brain-specific overexpression of
aromatase in early zebrafish embryos (Chung et al.,
2011)
o Vitellogenin 1 (vtg1) mRNA expression in male
zebrafish - BPA, endosulfan, heptachlor,
Methoxychlor
(Zhong et al., 2014)

17. Developmental Toxicology: In Vivo
Assays
Mammalian studies:
o Segment I: Fertility in males & females (rats)
o Segment II: Developmental toxicity/embryotoxicity (rats
& rabbits)
o Segment III: Peri-natal toxicity (rats)
Segment II protocol (rabbits)*:
6-18 days Day 30Day 0
Maternal Developmental
Body weight Implantation
Feed consumption Resorption rate
Clinical signs Fetal weight
Gross lesions External, visceral,
skeletal alterations

18. o Fertilized eggs
o Exposure period- 3 days
o Morphological endpoints:
Heart, spinal cord, somite, notochord,
brains, arches, jaws, tail, fins, face,
stomach, liver
o Growth measurements:
Body length, body shape, somite numbers,
yolk ball, swimming bladder, pigmentation
o Teratogenic index
Teratogenicity: Zebrafish
Brannen et al., 2010

19. Teratogenicity
o Evaluation of embryotoxicity of 12 compounds
• compared with teratogenicity of these
compounds in mammals
• non-teratogenic - 75 %
• teratogenic - 100 % (McGrath et al., 2008)
o Can detect both direct teratogens & proteratogens

20. o to develop a harmonized zebrafish developmental
toxicity assay
o 10 teratogens & 10 non-teratogens

21. cont…
o Inter-laboratory assessment achieved 60-70
% concordance
o Entire test set re-evaluated using the
optimized protocol- 85 % concordance was
achieved
o Zebrafish developmental toxicity assay can
be used for predictive classification of
teratogenic potential of drugs

22. o 10 % market withdrawals
o Current assessment
 mammalian models
 FOB
 neurohistopathology
 behavioral assessment
o laborious & time consuming
Neurobehavioral toxicity

23. Neurobehavioral toxicity:
Zebrafish
o Developing or adult zebrafish
o Impairment of swimming
o Locomotor activity
o Morphologic lesions
 neuronal apoptosis
 proliferation
 integrity of the myelin
sheath
(Muth-Köhne et al., 2012)

24. o Two chamber heart
o Voltage gated sodium channel, L & T-
type Ca channels & potassium
channels
o Clarity of embryos & larvae
o survive for several days without
circulation
o ECG pattern
Cardiotoxicity
A. Human

25. Cardiotoxicity
o Zebrafish larvae (3 dpf)
o Exposure period- 3 h
o ventricular & auricular heart rate, cardiac
output & stroke volume
o Cardiac morphology
Wu et al., 2013

26. Ototoxicity
o Common side effect of many drugs- no
standard screen
o Neuromasts- the zebrafish counterpart of hair
cells
o Neuromasts- sensitive to ototoxic agents like
gentamicin, cisplatin & quinine
(Buck et al., 2012)

27. o 5 day old embryo
o Exposure period- 24 h
o Endpoints:
• Microscopic evaluation
• Hair cell density
• reversiblity
Ototoxicity: Zebrafish
Chang et al., 2013

28. Organ-specific toxicity
Preparation Concentration Observed effect/toxicity
Doxorubicin 30 mg/l Teratogenicity, nephrotoxicity,
Hepatotoxicity, cardiotoxicity
Dexamethasone 324 mg/l Nephrotoxicity, hepatotoxicity,
GIT lesion
Methotrexate 454 mg/l Teratogenicity, nephrotoxicity,
Hepatotoxicity, cardiotoxicity,
GIT lesion
Fluorouracil 3.3 mg/l Nephrotoxicity, hepatotoxicity
Cyclosporin A 69 mg/l Teratogenicity, nephrotoxicity,
Hepatotoxicity, cardiotoxicity
Caffeine 108 mg/l Change of locomotor activity,
Muscular spasticity

29. Zebrafish Labs in India
o Institute of Genomics and Integrative Biology
(New Delhi)
o Center for Cellular and Molecular Biology
(Hyderabad)
o Indian Institute of Science (Bangalore)
o Tata Institute of Fundamental Research
(Mumbai)
o Institute of Life Sciences (Hyderabad)
o National Centre for Radio Astrophysics (Pune)

30. Limitations:
o The metabolizing enzymes are not fully
characterized
o Studies on zebrafish takes into consideration
only aqueous concentration of the TS
o Far more remote from humans than other animal
models such as rodents
o Anatomical differences with human

31. Conclusion
o Zebrafish as a model organism for toxicology has
low cost, ease of breeding & other advantages
o Certain known pathways leading to drug toxicity
in mammals are conserved in zebrafish
o Zebrafish model does not replace classical
mammalian models, but does represent an early
stage model for the toxicological assessment