0
Rapid In Vivo Assessment of
Bioactivity in Zebrafish: High
Content Data for Predictive
Toxicology
Robert Tanguay
Environme...
2
Funding
NIEHS T32 ES7060 P30
ES00210, RC4ES019764 P42
ES016465, R01 ES016896
Acknowledgements
Tanguay Lab
Lisa Truong, P...
Outline
 Working Assumptions
 Challenges for predictive toxicology
 Need for rapid robust phenotype discovery
 Need to...
Key Assumptions
 (Some) environmental exposure negatively impact
human and environmental health
 These chemicals interac...
Linking EARLY Molecular
Responses to Phenotype
Exposure Tissue
Dose
Biologically
Effective Dose
Early
Responses
Late
Respo...
Conceptual Framework
Chemical Information
- Chemical Structure
- Mixture
Composition
Genomic Responses
- mRNA Expression
-...
Why Zebrafish?
 Share many developmental, anatomical, and
physiological characteristics with mammals
 Molecular signalin...
Systems Biological Approach
- Early Embryonic Development -
 Generally more responsive to insult…
… most dynamic life sta...
Example: Acute Exposures
- Early Responses in Zebrafish -
 Multiple levels of interrogation
 Challenge the complex syste...
Developmental Stages of
Assessments
10
6 hr 24 hr 120 hr10 min
Typical Experimental Design
Rapid Assessments
(Phenotype Discovery)
11
Test
Materials
Nano,
mixtures,
Libraries,
Mixtures
Screening for responses 1-5 ...
High Content Endpoints
(Assessed between 24 and 120 hpf)
12
MORPHOLOGICAL - Common, but highly specific
Malformations
i.e....
What Do We Look For?
• MORPHOLOGICAL
Malformations
i.e. pericardial edema, body axis angle, fin
malformations, eye diamete...
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
15
 Automation developed and implemented;
throughput is no longer a barrier
 Embryo P...
Bulk Spawning
16
 Tanks contain ~1,200 brood stock fish
 Fish are spawned in place, via an internal apparatus,
that is p...
• Chorion removal is necessary for exposure
consistency
• Increase bioavailability
• Allows for:
o Up to 8000 embryos per ...
Robotic Embryo Handling
- Plate Loading -
18
Greater consistency
Efficiently Load 96/384 well
plates with embryos
Automated Embryo Placement
System (AEPS)
19
PhotoMotor Response Assay Tool
(PRAT)
20
Single
embryo
output
Behavioral Testing
21
 Assesses motor behavior responses simultaneously in 400
animals
 Expandable…
Larval Behavioral Responses
22
Larval Behavior Testing
Distance Moved During Alternating Periods of Light and Dark
23Time (min)
0 10 20 30 40 50 60 70
Di...
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...
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)
...
Fertilization 6 h 24 h (1 day)
Chemical Exposure
120 h (5 day)
[uM]
Light Pulse Exposure
Behavioral Assessment Development...
HTS: High Throughput Screening
1060 chemicals x 18 endpoints
Analysis considerations
• Correlation structure
• Global patt...
28
Summary of ToxCast I, II
Clustered Summary of ToxCast I, II
29
Control
Hit
Compound
Exposure-induced Notochord Distortion
Notochord Hits (I)
31
Notochord Hits (II)
32
At ~18 hpf, embryos begin to
spontaneously move.
The photomotor response assay
measures this movement in
response to flash...
…
…
Summarize the concentration-response profiles for 1,060 unique
chemicals into a countable set of prototype patterns
Ch...
Hits Identified in PRAT (24 hpf)
35
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
120 motor activity
DARK RESPONSE
44’4"-Ethane-111-triyltriphenol
38
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 remedia...
41
Superfund Deployment Sites
Spatial and Temporal PAHs in a
Model Harbor
42
• Water quality data
for the
carcinogenic EPA
PP PAHs.
•  = wet season
• ...
Site-specific Biological Responses
Abnormal developmental morphological endpoints observed in embryonic zebrafish exposed ...
PSD Successfully Bridged to Full Organism
Bio-Assay
44
• Positive control
trimethyltin
• Negative control
1% DMSO
• PSD do...
Site-Specific Biological Responses
45
• 6 of 18 biological
responses were
significantly different in
exposed embryos
compa...
Polycyclic Aromatic Hydrocarbons
46
•PAHs are ubiquitous in the environment
Fossil fuels, combustion etc.
•PAH exposures occur primarily via
inhalation and i...
Mechanisms of Toxicity for Most PAHs are
Unknown
48
Challenge: how can we efficiently assess the developmental toxicity of...
AHRHSP
90
HSP
90
AIP
AHR Binding
AHR
ARNT
Transcription
CYP
Induction
No metabolism
Metabolites
Disruption of
endogenous
b...
AHR
HSP
90
AIP
AHR Binding
AHR
ARNT
Transcription
CYP1A
Induction
Disruption of
endogenous
binding/pathways
No CYP1A
induc...
Modeling a “Target” Zebrafish AHRs
51Bisson, W.H. et al. 2009, J Med Chem. O’Donnell, E.F. et al. 2010, PLOS One
Zebrafish...
TCDD Molecular Docking with the
Zebrafish AHRs
52
AHR2 AHR1B AHR1A
Unable to
dock
-3.97 -4.86
Predicted binding energy
(kc...
The ahr2hu3335 Zebrafish Line
BHLH PAS A PAS B Q- Rich
T → A mutation in residue 534 resulting in a premature stop
•Trunca...
Ahr2hu3335 Mutants Are Resistant to TCDD-
Induced Developmental Toxicity
A ahr2+ ahr2hu3335
54
ahr2 Mutants Are Resistant to TCDD-induced
CYP Expression Changes
ahr2+ ahr2hu33351 nM TCDD 1 nM TCDD55
Leflunomide Molecular Docking
56
AHR2 AHR1B AHR1A
-2.13 -1.97 -2.19
Predicted binding energy (kcal/mole)
O’Donnell, E.F. e...
Leflunomide-induced CYP1A expression is
partially AHR2 dependent
ahr2+/hu3335
ahr2hu3335
10 uM Lef
10 uM Lef
1a 1b 2
1a 1b...
AHR1A Dependent CYP1A Expression
58
ahr2+/hu3335
ahr2hu3335
ahr2hu3335 ahr2hu3335
ahr2hu3335 ahr2hu3335
Control morpholino...
Model PAHs with Different Response Profiles
Control
(1% DMSO)
BAA
DBT
PYR
PAH Phenotype (5 dpf) CYP1A (5 dpf)AHR2 dependen...
Early Transcriptional Responses
Expose to 25
uM BAA, DBT,
PYR or Control
(4 replicates)
Collect RNA
Microarray analysis of...
Significantly different than control, One-way ANOVA, 5% FDR adjusted p < 0.05
Significantly Misexpressed Transcripts
(24 a...
Transcriptional profiles are PAH- and time-dependent
BAA
24hr
BAA
48hr
DBT
48hr
PYR
48hr
DBT
24hr
PYR
24hr
p < 0.05, ANOVA...
Embryonic Uptake Is Structure-Dependent
PAH body burden (umol/g) at microarray concentration (25 uM)
DBT PYR BAA
24 hpf 3....
PYR Response Is Less Robust
But Highly correlated with DBT
Direct statistical
comparison between DBT
and PYR (1.5 FC, p < ...
BAA Enriched Biological Functions
Biological Process (GO Term level 4) Gene
Count
P value
24hpf
hormone metabolic process ...
DBT/PYR enriched biological functions
Biological Process (GO Term level 4) Gene
Count
P value
24hpf
fatty acid biosyntheti...
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% P...
Differential Response Profiles Induced by OPAHs
Xanthone exposure activates AHR1A
Control MO AHR1A MO20 uM xanthone 20 uM xanthone
Benz(a)anthracene-7,12-dione exposure
activates AHR2
ahr2hu3335ahr2+
4 uM BADO 4 uM BADO
Benzanthrone does not induce CYP1A
ahr2hu3335
ahr2+
20 uM
Diagnostic Binning of OPAHs
73
To Summarize
 High throughput in vivo data is now feasible
 Phenotypic anchoring – highly relevant for “predictions”
 P...
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Rapid In Vivo Assessment of Bioactivity in Zebrafish: High Content Data for Predictive Toxicology

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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

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Transcript of "Rapid In Vivo Assessment of Bioactivity in Zebrafish: High Content Data for Predictive Toxicology"

  1. 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. 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. 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. 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. 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. 6. Conceptual Framework Chemical Information - Chemical Structure - Mixture Composition Genomic Responses - mRNA Expression - miRNA Expression - Protein Expression - Metabolomics Phenotypic Responses - Morphology - Behavior 6
  7. 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. 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. 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. 10. Developmental Stages of Assessments 10 6 hr 24 hr 120 hr10 min Typical Experimental Design
  11. 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. 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. 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. 14. Some Examples of What We Look For 14 Snout/Jaw Pericardial Edema Yolk Sack Edema Caudal Fin Axis/Trunk Notochord Control
  15. 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. 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. 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. 18. Robotic Embryo Handling - Plate Loading - 18 Greater consistency Efficiently Load 96/384 well plates with embryos
  19. 19. Automated Embryo Placement System (AEPS) 19
  20. 20. PhotoMotor Response Assay Tool (PRAT) 20 Single embryo output
  21. 21. Behavioral Testing 21  Assesses motor behavior responses simultaneously in 400 animals  Expandable…
  22. 22. Larval Behavioral Responses 22
  23. 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. 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. 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. 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. 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. 28 Summary of ToxCast I, II
  29. 29. Clustered Summary of ToxCast I, II 29
  30. 30. Control Hit Compound Exposure-induced Notochord Distortion
  31. 31. Notochord Hits (I) 31
  32. 32. Notochord Hits (II) 32
  33. 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. 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. 35. Hits Identified in PRAT (24 hpf) 35
  36. 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. 37 120 motor activity DARK RESPONSE 44’4"-Ethane-111-triyltriphenol
  38. 38. 38 120 motor activity DARK RESPONSE 44’4"-Ethane-111-triyltriphenol
  39. 39. Biological Response Indicator Devices for Gauging Environmental Stressors (BRIDGES) 39 Kim Anderson – OSU SRP Example #2
  40. 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. 41 Superfund Deployment Sites
  42. 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. 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. 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. 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. 46. Polycyclic Aromatic Hydrocarbons 46
  47. 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. 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. 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. 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. 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. 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. 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. 54. Ahr2hu3335 Mutants Are Resistant to TCDD- Induced Developmental Toxicity A ahr2+ ahr2hu3335 54
  55. 55. ahr2 Mutants Are Resistant to TCDD-induced CYP Expression Changes ahr2+ ahr2hu33351 nM TCDD 1 nM TCDD55
  56. 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. 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. 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. 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. 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. 61. Significantly different than control, One-way ANOVA, 5% FDR adjusted p < 0.05 Significantly Misexpressed Transcripts (24 and 48 hpf)
  62. 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. 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. 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. 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. 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. 67. PAHs Disrupt Distinct Regulatory Networks DBT/PYR BAA Goodale, B.C. et al. in press, Toxicology and Applied Pharmacology
  68. 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. 69. Differential Response Profiles Induced by OPAHs
  70. 70. Xanthone exposure activates AHR1A Control MO AHR1A MO20 uM xanthone 20 uM xanthone
  71. 71. Benz(a)anthracene-7,12-dione exposure activates AHR2 ahr2hu3335ahr2+ 4 uM BADO 4 uM BADO
  72. 72. Benzanthrone does not induce CYP1A ahr2hu3335 ahr2+ 20 uM
  73. 73. Diagnostic Binning of OPAHs 73
  74. 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
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