Rapid In Vivo Assessment of Bioactivity in Zebrafish: High Content Data for Predictive Toxicology
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Transcript of Rapid In Vivo Assessment of Bioactivity in Zebrafish: High Content Data for Predictive Toxicology
Rapid In Vivo Assessment of Bioactivity in Zebrafish: High Content Data for Predictive
Toxicology
Robert Tanguay
Environmental and Molecular Toxicology Sinnhuber Aquatic Research Laboratory Environmental Health Sciences Center
Oregon State University
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Funding NIEHS T32 ES7060 P30 ES00210, RC4ES019764 P42 ES016465, R01 ES016896
Acknowledgements Tanguay Lab Lisa Truong, PhD Mike Simonich, PhD Jane LaDu Britton Goodale Andrea Knecht David Mandrell Annika Swanson
PNNL Susan Tilton, PhD Katrina Waters, PhD
SARL Staff Cari Buchner Carrie Barton Greg Gonnermann Eric Johnson, MS
Kolluri Lab - OSU Siva Kolluri, PhD William Bisson, PhD Dan Koch Edmond O’Donnell
NC State David Reif, PhD
Outline
Working Assumptions
Challenges for predictive toxicology
Need for rapid robust phenotype discovery
Need to crank it up! Process engineering
Putting it Into Action – Examples EPA ToxCast I and II
Environmental mixtures
Comparative PAH toxicity “binning”
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Key Assumptions
(Some) environmental exposure negatively impact human and environmental health
These chemicals interact with “genomes” to cause harm
We can identify the hazardous agents
It is possible to identify the “targets” of these chemicals
Using structural and mechanistic information we can predict future toxicity
It will be possible to proactively design inherently safer products
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Linking EARLY Molecular Responses to Phenotype
Exposure Tissue Dose
Biologically Effective Dose
Early Responses
Late Responses
Pathology/ Disease
Goal is to identify causality – In Vivo
Evaluate global molecular resposnes following exposure
Focus on the early responses…when the endpoints are not visible
Use whole genome arrays, RNA-seq (including small RNAs), proteomics
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Conceptual Framework
Chemical Information
- Chemical Structure - Mixture
Composition
Genomic Responses - mRNA Expression - miRNA Expression - Protein Expression - Metabolomics
Phenotypic Responses - Morphology - Behavior
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Why Zebrafish?
Share many developmental, anatomical, and physiological characteristics with mammals
Molecular signaling is conserved across species
Technical advantages of cell culture – power of in vivo
Amendable to rapid whole animal mechanistic evaluations
Genetically tractable-mutants, KO, transgenics, TALEN, ZFN, etc.
Focus on responses, then identify the “AOP”
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Systems Biological Approach - Early Embryonic Development -
Generally more responsive to insult…
… most dynamic life stage … most conserved fundamental process/mechanisms … full signaling repertoire is expressed & active … highest potential to detect adverse interactions
If a chemical or nanomaterial is developmentally toxic, it
must influence the activity of a molecular pathway or process… i.e. hit or influence a “Toxicity Pathway”
Use the phenotypic response as anchor for pathway and target identifications
Explore targets in other system
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Example: Acute Exposures - Early Responses in Zebrafish -
Multiple levels of interrogation
Challenge the complex system as soon as possible
Embryonic development serves as a “biological sensor and amplifier”
Look for “any” difference related to exposure
The more we measure, the higher the sensitivity
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Expose
5 days
Developmental Stages of Assessments
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6 hr 24 hr 120 hr 10 min
Typical Experimental Design
Rapid Assessments (Phenotype Discovery)
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Test Materials
Nano, mixtures, Libraries, Mixtures
Screening for responses 1-5 days
1 Embryo/well
A large adult colony is required to support testing laboratory SPF Facility
Remove Chorions Multiple Replicates Multiple Concentrations QA/QC -Negative -Controls
High Content Endpoints (Assessed between 24 and 120 hpf)
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MORPHOLOGICAL - Common, but highly specific Malformations i.e. pericardial edema, body axis angle, fin malformations, eye diameter Circulation Heart beat (rate) Developmental progression Embryo viability
OMICS
BEHAVIORAL Spontaneous movement (18-24 hpf) Touch response (27 hpf) Motility, learning and memory (adults)
What Do We Look For?
• MORPHOLOGICAL Malformations i.e. pericardial edema, body axis angle, fin malformations, eye diameter Circulation Heart beat (rate) Developmental progression Embryo viability
• OMICS
• BEHAVIORAL Spontaneous movement (18-24 hpf) Touch response (27 hpf) Motility, learning and memory
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Some Examples of What We Look For
14 Snout/Jaw Pericardial
Edema
Yolk Sack Edema
Caudal Fin
Axis/Trunk
Notochord Control
Automation: To Increase Throughput
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Automation developed and implemented; throughput is no longer a barrier
Embryo Production – unlimited
Embryo Handling
Chorion Removal
Microinjections
Automated Imaging
Behavioral Assays – Multiple Platforms
Bulk Spawning
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Tanks contain ~1,200 brood stock fish
Fish are spawned in place, via an internal apparatus, that is plumbed to an external embryo collection unit
Embryos can be collected at intervals throughout the morning with minimal interruptions to the fish
40,000/tank/day
• Chorion removal is necessary for exposure consistency
• Increase bioavailability
• Allows for:
o Up to 8000 embryos per 16 min/cycle o Greater consistency than by hand o Removal of debris from plates
• Better image analysis
Mandrell, D., Truong, L., et al . 2012. Automated zebrafish chorion removal and single embryo placement: Optimizing throughput of zebrafish developmental toxicity screens. Journal of Laboratory Automation 17 (1) 66-74.
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Automated Chorion Removal
Robotic Embryo Handling - Plate Loading -
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Greater consistency
Efficiently Load 96/384 well plates with embryos
Automated Embryo Placement System (AEPS)
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PhotoMotor Response Assay Tool (PRAT)
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Single embryo output
Behavioral Testing
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Assesses motor behavior responses simultaneously in 400 animals
Expandable…
Larval Behavioral Responses
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Larval Behavior Testing Distance Moved During Alternating Periods of Light and Dark
23 Time (min)0 10 20 30 40 50 60 70
Dist
ance
Mov
ed (m
m)
0
20
40
60
80
100
Rest 1 2 3
0 20 10 30 40 50 60 minutes
BPA Exposure Leads to Hyperactivity
24
Time (min)0 5 10 15 20 25 30 35 40
Burs
t Acti
vity (
>5 p
ixels/
sec)
0
1
2
3
4
5
Control 0.1 uM BPA
Ex.
Putting it Into Action
25
ToxCast I, II, (1,072 compounds)
Concentrations (64 µM, 6.4 µM, 640 nM, 64 nM, and 6.4 nM)
N=32 animal/group
22 endpoints
2 Behavioral Assays
Data Analysis and integration
Bin compounds by structure and responses
Fertilization 6 h 24 h (1 day)
Chemical Exposure
120 h (5 day)
[uM]
Light Pulse Exposure
Behavioral Assessment Developmental Assessment And Motor Responses
= 1060 unique chemicals x 6 concentrations x 32 biological (well)
replicates
Integrated Screening Approach for Developmental and Neurotoxicity
HTS: High Throughput Screening
1060 chemicals x 18 endpoints Analysis considerations • Correlation structure • Global patterns and “hit”
distributions • Chemical property covariates • Relationship between mortality
endpoint (MORT) and other specific endpoints
• Comparison to related datasets
Zebrafish 5dpf Development: Analysis
[Truong et al. Tox Sci (2014)]
28
Summary of ToxCast I, II
Clustered Summary of ToxCast I, II
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Control
Hit Compound
Exposure-induced Notochord Distortion
Notochord Hits (I)
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Notochord Hits (II)
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At ~18 hpf, embryos begin to spontaneously move. The photomotor response assay measures this movement in response to flashes of light. Normal fish (in the absence of chemical) will respond in the excitatory period (after 1st light pulse) but not after the 2nd light pulse. 1,060 chemicals were screened in concentration-response format {0.0064 … 64 uM} to identify chemicals that alter this normal response.
Background Refractory Excitatory
1st Light Pulse 2nd Light Pulse
Time (seconds)
24 hpf behavioral assay screen for neuromodulator chemicals
…
…
Summarize the concentration-response profiles for 1,060 unique chemicals into a countable set of prototype patterns
Characterizing behavioral response patterns in a neuromodulator chemical screen
Hits Identified in PRAT (24 hpf)
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Larval Behavioral Responses (5 days old)
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Time (min)0 10 20 30 40 50 60 70
Dist
ance
Mov
ed (m
m)
0
20
40
60
80
100
37
120 motor activity DARK RESPONSE
44’4"-Ethane-111-triyltriphenol
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120 motor activity DARK RESPONSE
44’4"-Ethane-111-triyltriphenol
Biological Response Indicator Devices for Gauging Environmental Stressors
(BRIDGES)
39
Kim Anderson – OSU SRP
Example #2
PAHs in Portland Harbor passive sampler extracts
Water Passive Sampling • Bioavailable fraction
• Before and after remediation
Willamette River Basin
Sampling SitePortland HarborSuperfund
• Anderson, et al; ES&T, 2008 • Allan, et al; Bridging environmental mixtures and toxic effects.
ET&C 2012 • Allan, et al; Estimating risk at a Superfund site using passive
sampling devices as biological surrogates in human health risk models. Chemosphere 2011.
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Superfund Deployment Sites
Spatial and Temporal PAHs in a Model Harbor
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• Water quality data for the carcinogenic EPA PP PAHs.
• = wet season • = dry season • The red dashed
lines represent the EPA Water Quality Guidelines for human health for consumption of water and organism (3.8 ng/L).
Site-specific Biological Responses Abnormal developmental morphological endpoints observed in embryonic zebrafish exposed to
contaminant mixtures from extracts of LFTs deployed at Superfund Sites
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Control
30 h
pf12
6 hp
f
1% LFT ExtractNot
T
PEYSE
Not= notochord waviness; PE= pericardial edema; YSE= yolk sac edema; T= bent tail
PSD Successfully Bridged to Full Organism Bio-Assay
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• Positive control trimethyltin
• Negative control 1% DMSO
• PSD dose response 0.8 to 100x extract 1% max in fishwater
• River Mile = 8.0 • Sept 2009 • N=32 each dose
SRP A09000012
Percent of Total (%)0 20 40 60 80 100 120
1% DMSO
0.8x
4x
20x
100x
5uM TMT
Mortality Adversely Affected Unaffected
Site-Specific Biological Responses
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• 6 of 18 biological responses were significantly different in exposed embryos compared to controls
• MLR, likelihood ratio, p<0.05; n=941
M30
1 2 3 4 5 60
20
40
60
80
M126
1 2 3 4 5 60
20
40
60
80126 hpf mortality
Stubby
1 2 3 4 5 60
20
40
60
80 stubby body
Tail
1 2 3 4 5 60
20
40
60
80bent tail
YSE
1 2 3 4 5 60
20
40
60
80 yolk sac edema
Notochord 126 hpf
1 2 3 4 5 60
20
40
60
80wavy notochord
% In
cide
nce
Control Embryos
RM 1
RM 3.5
RM 7E
RM 7W
RM 17
Downriver Superfund Upriver
30 hpf mortality
X
X
X
X
Hillwalker et al, 2010
Testing numerous “real world samples” and Effects Driven Analysis much more to come…
Polycyclic Aromatic Hydrocarbons
46
•PAHs are ubiquitous in the environment Fossil fuels, combustion etc.
•PAH exposures occur primarily via inhalation and ingestion •Known carcinogens in humans Soot, coal tars
•PAHs measured in placental tissue
•Recent concern about developmental effects
Polycyclic aromatic hydrocarbons and human health
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Mechanisms of Toxicity for Most PAHs are Unknown
48 Challenge: how can we efficiently assess the developmental toxicity of
these compounds and define mechanisms of action?
Air particulate matter can contain over 100 PAHs
Environmentally Dynamic
Parent, substituted compounds
Toxicity data is scarce for substituted PAHs
PAHs induce AHR-dependent and AHR-independent developmental toxicity, dependent on structure -Incardona, J. P., T. K. Collier, et al. (2004)
Toxicol Appl Pharmacol
AHR HSP 90
HSP 90 AIP
AHR Binding
AHR ARNT
Transcription
CYP Induction
No metabolism
Metabolites
Disruption of endogenous binding/pathways
AHR Independent Toxicity
The AHR and PAH pathways of toxicity
AHR HSP
90 AIP
AHR Binding
AHR ARNT
Transcription
CYP1A Induction
Disruption of endogenous binding/pathways
No CYP1A induction
CYP1A is a marker of AHR activation
Zebrafish have three AHRs, AHR2 is functionally conserved with human
HSP 90
Modeling a “Target” Zebrafish AHRs
51 Bisson, W.H. et al. 2009, J Med Chem. O’Donnell, E.F. et al. 2010, PLOS One
Zebrafish have three AHRs •AHR2 primary mediator of toxicity •AHR1A deficient in TCDD binding and transactivation
activity •AHR1B functional but no known toxicological
mechanism
AHR Homology Model •AHR ligand binding domain models built using NMR
structure of HIF2α (PAS domain) •Mouse, rat, human, zebrafish •Performed molecular docking of putative AHR ligands
TCDD Molecular Docking with the Zebrafish AHRs
52
AHR2 AHR1B AHR1A
Unable to dock
-3.97 -4.86
Predicted binding energy (kcal/mole)
Bisson, W.H. et al. 2009, J Med Chem.
The ahr2hu3335 Zebrafish Line
BHLH PAS A PAS B Q- Rich
T → A mutation in residue 534 resulting in a premature stop
•Truncated protein is predicted to be non-functional
•Basal mRNA expression suggests mutant ahr2hu3335 transcript is degraded
Edwin Cuppen, PhD The Hubrecht Institute Goodale et al. PloS one 2012 53
Ahr2hu3335 Mutants Are Resistant to TCDD-Induced Developmental Toxicity
A ahr2+ ahr2hu3335
54
ahr2 Mutants Are Resistant to TCDD-induced CYP Expression Changes
ahr2+ ahr2hu3335 1 nM TCDD 1 nM TCDD 55
Leflunomide Molecular Docking
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AHR2 AHR1B AHR1A
-2.13 -1.97 -2.19
Predicted binding energy (kcal/mole)
O’Donnell, E.F. et al. 2010, PLOS One
Leflunomide-induced CYP1A expression is partially AHR2 dependent
ahr2+/hu3335
ahr2hu3335
10 uM Lef
10 uM Lef
1a 1b 2
1a 1b 2
57
AHR1A Dependent CYP1A Expression
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ahr2+/hu3335
ahr2hu3335
ahr2hu3335 ahr2hu3335
ahr2hu3335 ahr2hu3335
Control morpholino
10 uM Lef 10 uM Lef
10 uM Lef 1% DMSO
AHR1B + AHR1A morpholino
Control morpholino AHR1B morpholino
1a 1b 2 1a 1b 2
1a 1b 2 1a 1b 2
Model PAHs with Different Response Profiles
Control (1% DMSO)
BAA
DBT
PYR
PAH Phenotype (5 dpf) CYP1A (5 dpf) AHR2 dependent toxicity1?
Yes
No
Partial
25 uM
25 uM
25 uM
Control
No
1. Incardona et al. 2004 Toxicology and Applied Pharmacology
Early Transcriptional Responses
Expose to 25 uM BAA, DBT, PYR or Control (4 replicates)
Collect RNA
Microarray analysis of RNA expression
(Agilent zebrafish V2 microarray)
Functional annotation clustering (DAVID) Transcription factor prediction (Metacore)
6 hpf 24 hpf 120 hpf 10 min 48 hpf
Significantly different than control, One-way ANOVA, 5% FDR adjusted p < 0.05
Significantly Misexpressed Transcripts (24 and 48 hpf)
Transcriptional profiles are PAH- and time-dependent
BAA 24hr
BAA 48hr
DBT 48hr
PYR 48hr
DBT 24hr
PYR 24hr
p < 0.05, ANOVA with 5% FDR
Robust BAA response
Goodale, B.C. et al. in press, Toxicology and Applied Pharmacology
Embryonic Uptake Is Structure-Dependent
PAH body burden (umol/g) at microarray concentration (25 uM)
DBT PYR BAA 24 hpf 3.4 1.0 0.1 48 hpf 5.3 2.9 0.2
Goodale, B.C. et al. in press, Toxicology and Applied Pharmacology
PYR Response Is Less Robust But Highly correlated with DBT
Direct statistical comparison between DBT and PYR (1.5 FC, p < 0.05)
Common transcriptional response analyzed for
biological functions and regulatory networks
BAA Enriched Biological Functions
Biological Process (GO Term level 4)
Gene Count
P value
24 h
pf
hormone metabolic process 3 5.1E-03
tissue development 4 2.8E-02
48 h
pf
cellular homeostasis 10 4.5E-04 chemotaxis 5 2.2E-03
hormone metabolic process 4 1.3E-02 tetrapyrrole metabolic process 3 1.2E-02
vasculature development 6 1.0E-02 hydrogen peroxide metabolic process 3 5.6E-03
cation transport 7 3.8E-02 organ development 15 4.1E-02
DBT/PYR enriched biological functions Biological Process (GO Term level 4) Gene
Count P value
24 h
pf
fatty acid biosynthetic process 8 6.10E-04 ion transport 22 7.86E-03 skeletal muscle contraction 4 1.10E-03 steroid biosynthetic process 8 9.43E-04 oxoacid metabolic process 19 1.27E-02 intermediate filament organization 3 6.71E-03 negative regulation of cell proliferation 13 1.67E-02 muscle cell development 5 1.89E-02
sterol biosynthetic process 5 5.49E-03 cellular amide metabolic process 5 2.64E-02
48 h
pf
oxoacid metabolic process 34 2.66E-05 embryonic development ending in birth or egg hatching
24 1.01E-04
regionalization 17 2.75E-04 neurogenesis 31 3.27E-03 embryonic organ development 14 2.40E-03 positive regulation of macromolecule metabolic process
38 2.19E-03
negative regulation of cell communication 14 1.01E-02 cellular component morphogenesis 21 9.16E-03 central nervous system development 22 1.27E-02 hormone metabolic process 8 1.51E-02
PAHs Disrupt Distinct Regulatory Networks
DBT/PYR
BAA
Goodale, B.C. et al. in press, Toxicology and Applied Pharmacology
Load embryos into 96-well plate
6 hpf 24 hpf 120 hpf
Evaluate for malformations
Evaluate for malformations Fix in 4% PFA for immunohistochemisty
38 Oxy PAHs screened for developmental Toxicity and CYP1A expression
68
Differential Response Profiles Induced by OPAHs
Xanthone exposure activates AHR1A
Control MO AHR1A MO 20 uM xanthone 20 uM xanthone
Benz(a)anthracene-7,12-dione exposure activates AHR2
ahr2hu3335 ahr2+ 4 uM BADO 4 uM BADO
Benzanthrone does not induce CYP1A
ahr2hu3335
ahr2+
20 uM
Diagnostic Binning of OPAHs
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To Summarize High throughput in vivo data is now feasible
Phenotypic anchoring – highly relevant for “predictions”
Platform for structure based predictions
Translating zebrafish data:
Benchmark for in vitro data
- Bridging data for extrapolations
Prioritizing further testing
Deal with mixtures
Now in a position to understand the imitations of model
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