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

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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)]

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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)

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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

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•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

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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

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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

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AHR1A Dependent CYP1A Expression

58

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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