Dr. Robert Tanguay's presentation on April 30, 2014 with the 21st Century Toxicology Seminar Series of the California Dept. of Pesticide Regulation. https://www.facebook.com/media/set/?set=a.766268766739722.1073741858.440748475958421&type=3&uploaded=5 For more information about the research of Robert Tanguay, visit the Superfund Research Program: http://superfund.oregonstate.edu and the Environmental Health Science Center: http://ehsc.oregonstate.edu

1. 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

2. 2
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

3. 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”
3

4. 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
4

5. 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
5

6. Conceptual Framework
Chemical Information
- Chemical Structure
- Mixture
Composition
Genomic Responses
- mRNA Expression
- miRNA Expression
- Protein Expression
- Metabolomics
Phenotypic Responses
- Morphology
- Behavior
6

7. 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”
7

8. 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
8

9. 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
9
Expose
5 days

10. Developmental Stages of
Assessments
10
6 hr 24 hr 120 hr10 min
Typical Experimental Design

11. Rapid Assessments
(Phenotype Discovery)
11
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

12. High Content Endpoints
(Assessed between 24 and 120 hpf)
12
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)

13. 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
13

14. Some Examples of What We Look For
14
Snout/Jaw Pericardial
Edema
Yolk Sack Edema
Caudal Fin
Axis/Trunk
Notochord
Control

15. Automation: To Increase Throughput
15
 Automation developed and implemented;
throughput is no longer a barrier
 Embryo Production – unlimited
 Embryo Handling
 Chorion Removal
 Microinjections
 Automated Imaging
 Behavioral Assays – Multiple Platforms

16. Bulk Spawning
16
 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

17. • 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.
17
Automated Chorion Removal

18. Robotic Embryo Handling
- Plate Loading -
18
Greater consistency
Efficiently Load 96/384 well
plates with embryos

19. Automated Embryo Placement
System (AEPS)
19

20. PhotoMotor Response Assay Tool
(PRAT)
20
Single
embryo
output

21. Behavioral Testing
21
 Assesses motor behavior responses simultaneously in 400
animals
 Expandable…

22. Larval Behavioral Responses
22

23. Larval Behavior Testing
Distance Moved During Alternating Periods of Light and Dark
23Time (min)
0 10 20 30 40 50 60 70
DistanceMoved(mm)
0
20
40
60
80
100
Rest 1 2 3
0 2010 30 40 50 60 minutes

24. BPA Exposure Leads to Hyperactivity
24
Time (min)
0 5 10 15 20 25 30 35 40
BurstActivity(>5pixels/sec)
0
1
2
3
4
5
Control
0.1 uM BPA
Ex.

25. 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

26. 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

27. 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. 28
Summary of ToxCast I, II

29. Clustered Summary of ToxCast I, II
29

30. Control
Hit
Compound
Exposure-induced Notochord Distortion

31. Notochord Hits (I)
31

32. Notochord Hits (II)
32

33. 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 RefractoryExcitatory
1st Light Pulse 2nd Light Pulse
Time (seconds)
24 hpf behavioral assay screen for
neuromodulator chemicals

34. …
…
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

35. Hits Identified in PRAT (24 hpf)
35

36. Larval Behavioral Responses
(5 days old)
36
Time (min)
0 10 20 30 40 50 60 70
DistanceMoved(mm)
0
20
40
60
80
100

37. 37
120 motor activity
DARK RESPONSE
44’4"-Ethane-111-triyltriphenol

38. 38
120 motor activity
DARK RESPONSE
44’4"-Ethane-111-triyltriphenol

39. Biological Response Indicator Devices for
Gauging Environmental Stressors
(BRIDGES)
39
Kim Anderson – OSU SRP
Example #2

40. PAHs in Portland Harbor passive sampler extracts
Water Passive Sampling
• Bioavailable fraction
• Before and after remediation
Willamette
River Basin
Sampling Site
Portland Harbor
Superfund
• 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.

41. 41
Superfund Deployment Sites

42. Spatial and Temporal PAHs in a
Model Harbor
42
• 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).

43. Site-specific Biological Responses
Abnormal developmental morphological endpoints observed in embryonic zebrafish exposed to
contaminant mixtures from extracts of LFTs deployed at Superfund Sites
43
Control
30hpf126hpf
1% LFT Extract
Not
T
PE
YSE
Not= notochord waviness; PE= pericardial edema;
YSE= yolk sac edema; T= bent tail

44. PSD Successfully Bridged to Full Organism
Bio-Assay
44
• 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

45. Site-Specific Biological Responses
45
• 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 6
0
20
40
60
80
M126
1 2 3 4 5 6
0
20
40
60
80
126 hpf mortality
Stubby
1 2 3 4 5 6
0
20
40
60
80
stubby body
Tail
1 2 3 4 5 6
0
20
40
60
80
bent tail
YSE
1 2 3 4 5 6
0
20
40
60
80
yolk sac edema
Notochord 126 hpf
1 2 3 4 5 6
0
20
40
60
80
wavy notochord
%Incidence
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…

46. Polycyclic Aromatic Hydrocarbons
46

47. •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
47

48. 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

49. AHRHSP
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

50. 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

51. Modeling a “Target” Zebrafish AHRs
51Bisson, 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

52. 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.

53. 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

54. Ahr2hu3335 Mutants Are Resistant to TCDD-
Induced Developmental Toxicity
A ahr2+ ahr2hu3335
54

55. ahr2 Mutants Are Resistant to TCDD-induced
CYP Expression Changes
ahr2+ ahr2hu33351 nM TCDD 1 nM TCDD55

56. Leflunomide Molecular Docking
56
AHR2 AHR1B AHR1A
-2.13 -1.97 -2.19
Predicted binding energy (kcal/mole)
O’Donnell, E.F. et al. 2010, PLOS One

57. Leflunomide-induced CYP1A expression is
partially AHR2 dependent
ahr2+/hu3335
ahr2hu3335
10 uM Lef
10 uM Lef
1a 1b 2
1a 1b 2
57

58. 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

59. 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
Contro
l
No
1. Incardona et al. 2004 Toxicology and Applied Pharmacology

60. 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 hpf10 min 48 hpf

61. Significantly different than control, One-way ANOVA, 5% FDR adjusted p < 0.05
Significantly Misexpressed Transcripts
(24 and 48 hpf)

62. 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

63. 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

64. 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

65. BAA Enriched Biological Functions
Biological Process (GO Term level 4) Gene
Count
P value
24hpf
hormone metabolic process 3 5.1E-03
tissue development 4 2.8E-02
48hpf
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

66. DBT/PYR enriched biological functions
Biological Process (GO Term level 4) Gene
Count
P value
24hpf
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
48hpf
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

67. PAHs Disrupt Distinct Regulatory Networks
DBT/PYR
BAA
Goodale, B.C. et al. in press, Toxicology and Applied Pharmacology

68. 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

69. Differential Response Profiles Induced by OPAHs

70. Xanthone exposure activates AHR1A
Control MO AHR1A MO20 uM xanthone 20 uM xanthone

71. Benz(a)anthracene-7,12-dione exposure
activates AHR2
ahr2hu3335ahr2+
4 uM BADO 4 uM BADO

72. Benzanthrone does not induce CYP1A
ahr2hu3335
ahr2+
20 uM

73. Diagnostic Binning of OPAHs
73

74. 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 model74