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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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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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. …

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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  • 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

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