zebrafish as a model system to study toxicology
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Zebrafish as a Model System to Study ToxicologyTRANSCRIPT
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Amol R. PadolPhD Scholar
Division of Pharmacology and Toxicology, IVRI
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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
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Developmental stages
Zygote
Cleavage
Blastula
Gastrula
Segmentation
Hatching
Larva
Juvenile
Adult
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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
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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
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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
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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
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Powerful model organism
o Genetics
o Developmental biology
o Toxicology
o Pharmacology
o DNA repair
o Cancer
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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)
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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)
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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
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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
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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
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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
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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)
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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)
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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 0Maternal Developmental
Body weight Implantation
Feed consumption Resorption rate
Clinical signs Fetal weight
Gross lesions External, visceral,
skeletal alterations
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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
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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
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o to develop a harmonized zebrafish developmental
toxicity assay
o 10 teratogens & 10 non-teratogens
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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
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o 10 % market withdrawals
o Current assessment
mammalian models
FOB
neurohistopathology
behavioral assessment
o laborious & time consuming
Neurobehavioral toxicity
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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)
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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
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Cardiotoxicity
o Zebrafish larvae (3 dpf)
o Exposure period- 3 h
o ventricular & auricular heart rate, cardiac
output & stroke volume
o Cardiac morphologyWu et al., 2013
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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)
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o 5 day old embryo
o Exposure period- 24 h
o Endpoints:
• Microscopic evaluation
• Hair cell density
• reversiblity
Ototoxicity: Zebrafish
Chang et al., 2013
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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
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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)
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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
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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
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