A high throughput in vivo model to understand PAH toxicity L ISA T - - PowerPoint PPT Presentation

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A high throughput in vivo model to understand PAH toxicity L ISA T - - PowerPoint PPT Presentation

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A high throughput in vivo model to understand PAH toxicity L ISA T RUONG Department of Environmental Molecular Toxicology Sinnhuber Aquatic Research Laboratory Environmental Health Sciences Center May 14, 2018 Polycyclic aromatic hydrocarbons and

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A high throughput in vivo model to understand PAH toxicity

LISA TRUONG

Department of Environmental Molecular Toxicology Sinnhuber Aquatic Research Laboratory Environmental Health Sciences Center May 14, 2018

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Polycyclic aromatic hydrocarbons and human health effects

  • PAHs are ubiquitous in the environment, fossil fuels, combustion,

food etc.

  • PAH exposures occur primarily

via inhalation and ingestion

  • Known carcinogens in humans
  • PAHs measured in placental tissue
  • Concern about developmental

effects

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Toxicity Mechanisms for Most PAHs are Unknown

  • Environmental samples can contain

100’s PAHs

  • Parent, substituted PAHs
  • Toxicity data is limited but growing

for substituted PAHs

  • PAHs induce AHR-dependent and

AHR-independent developmental toxicity, dependent on structure

  • We

lack the structural basis for developmental and neurotoxicity

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Why Zebrafish?

  • Molecular signaling is conserved with humans
  • High degree of homology with humans
  • 71% human proteins have orthologue in zebrafish
  • Well suited to discover

gene functions

  • Metabolically competent by 72 hpf
  • Amendable to rapid whole animal mechanistic evaluations

Chemical Information Genomic Response Phenotypic Response

Chemical Structure mRNA | miRNA | protein Expression/ Morphology/Functional/ Mixture Compositions Metabolomics Behavior/Epigentics

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The High Throughput Screening Platform

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  • Specific Pathogen Free Facility

10 min

Truong et al. (2014) Toxicol Sci 137: 212-233. 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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The High Throughput Screening Platform

6 6 hr 1 day 5 days 10 min

Embryo Collection

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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The High Throughput Screening Platform

10 min

Embryo Collection

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The High Throughput Screening Platform

6 hr 10 min

Embryo Collection

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.

Chemical Exposure

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The High Throughput Screening Platform

6 hr 1 day 10 min

Embryo Collection Photomotor Response Chemical Exposure

30 40 Time (s)

Truong et al. (2014) Toxicol Sci 137: 212-233.

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The High Throughput Screening Platform

6 hr 1 day 5 days 10 min

Embryo Collection

Time (s) 30 40

Photomotor Response

Truong et al. (2014) Toxicol Sci 137: 212-233.

Developmental Assessments

Time (min) 3 - 9 10 - 17

Locomotor Behavior Chemical Exposure

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Zebrafish Acquisition and Analysis (ZAAP)

  • Custom-build laboratory information

system (LIMS)

  • Stores chemical inventory, and allows

real-time data acquisition

  • Tracks individual well information from

96-well plates

  • Built in data analysis
  • Ensures rigor in data generated
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Comparative PAH Screening

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Developed a Library of 123 PAHs for Comparative Analysis

  • Dr. Kim Anderson
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High Throughput Screening of PAH Library

6 hpf 120 hpf 24 hpf

  • 5 concentrations
  • 50-1 µM
  • 5-0.1 µM
  • N=32
20 40 60 80 100 Time Movement Blank D 21 32 36 42 29 35 39 48 loess [uM] 0.000128 0.00064 0.0032 0.016 0.08 0.4 2 10 50 1e+05
  • 4 morphological

endpoints

  • Behavioral assay
  • Morphology
  • Behavior
  • CYP1A Localization
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CYP1a Expression Pattern as a Biomarker of AHR Activation

CYTOPLASM NUCLEUS

Geier et al. 2017

Figure 2. Representative images illustrating CYP1A expression patterns in 120 hpf larvae. a None, b vasculature, c liver, d yolk, e skin and neuromasts, f skin

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Comparative Profile of 123 PAHs

Any.DevTox.Effect Any.Except.Mort Any.Effect 24 hpf Mortality 24 hpf Developmental Progression 24 hpf Spontaneous Movement 24 hpf Notochord 120 hpf Mortality Yolk Sac Edema Axis Eye Snout Jaw Otolith Pericardial Edema Brain Somites Pectoral Fin Caudal Fin Pigmentation Circulation Trunk Swim Bladder Notochord Touch−Response EPR Basline EPR Excitation EPR Recovery LPR Dark LPR Light CYP: Vasculature CYP: Liver CYP: Skin CYP: Neuromasts CYP: Yolk Transcriptomics 3−hydroxyphenanthrene Quinoline 9−Nitroanthracene 2,6−Dimethylnaphthalene 1,6−dihydroxynaphthalene 3−hydroxybenzo[e]pyrene Pyrene−4,5−dione Benzo[g,h,i]perylene 5,6−Benzoquinoline Benz[a]anthracene−7,12−dione 9−aminophenanthrene Chromone Benzanthrone Anthracene 1,8−Dimethylnaphthalene 3−Nitrodibenzofuran 2,3−Dimethylanthracene 4−hydroxychrysene 1,5−dimethylnaphthalene Triphenylene Benzo[c]phenanthrene[1,4]dione Dibenzo[j,l]fluoranthene Thianaphthene 4H−cyclopenta[lmn]phenanth ridine−5,9−dione 9−anthracene carboxylic acid 2−Nitronaphthalene 6−Nitrobenzo[a]pyrene 1,4−anthraquinone 2−hydroxynaphthalene 2−Methylanthracene 1,2−naphthoquinone Xanthene 9,10−phenanthrenequinone Acenaphthene 2−Methylbenzofuran 2−Nitropyrene 2,7−dihydroxynaphthalene 1−Nitronaphthalene Perinaphthenone 6H−benzo[cd]pyren−6−one Naphtho[2,3−j]fluoranthene Benzo[e]pyrene acenaphthenequinone anthraquinone Naphtho[1,2−b]fluo ranthene Naphtho[2,3−b]fluo ranthene 9−hydroxyphenanthrene 1,8−Dinitropyrene 2−methylnaphthalene Fluorene 9−fluorenone 3−hydroxyfluoranthene 1,6−Dimethylnaphthalene 1−Methylnaphthalene Naphthalene 4−hydroxyphenanthrene 4h−cyclopenta[def]phenanthren−4−one Acenaphthylene 1,2−Dimethylnaphthalene Phenanthrene Dibenzothiophene Dibenzo[a,l]pyrene 2−Nitroanthracene Indole 2−Nitrofluoranthene 2−ethylanthraquinone 1,3−Dinitropyrene 8−methylquinoline Acridine 1−Nitropyrene 2,8−Dinitrodibenzothiophene 1−hydroxyphenanthrene Pyrene Xanthone 2−Nitrofluorene 2−Nitrodibenzothiophene Anthrathrene 5−Nitroacenaphthalene 1,3−dihydroxynaphthalene 3−Nitrophenanthrene 9−Nitrophenanthrene 1−hydroxypyrene 3−Nitrobenzanthrone 9−anthracene carbonitrile Dibenz[a,c]anthracene 7−Nitrobenz[a]anthracene Dibenzo[a,k]fluoranthene 6−Nitrochrysene Benzo[a]pyrene 1,5−dihydroxynaphthalene 3−hydroxybenz[a]anthracene 1,4−phenanthrenedione 10−hydroxybenzo[a]pyrene 2,3−dihydroxynaphthalene 1,4−Dimethylnaphthalene 5,12−naphthacenequinone Coronene Retene 6−Methylchrysene 3−hydroxyfluorene Benzo[a]anthracene Benzo[b]fluorene Dibenzofuran Chrysene Benzo[b]fluoranthene Indeno[1,2,3− c,d]pyrene 1−hydroxyindeno[1,2,3−c,d]pyrene 3,7−dinitrobenzo[k]fluoranthene 7−nitrobenzo[k]fluoranthene 1,6−Dinitropyrene 9−methylanthracene Carbazole 11−H−benzo[b]fluoren−11−one Fluoranthene 3,6−Dimethylphenanthrene 1−hydroxynaphthalene 3−nitrofluoranthene 1−Aminopyrene Benzo[k]fluoranthene Dibenzo[b,k]fluoranthene Dibenz[a,h]anthracene Benzo[j]fluoranthene Dibenzo[a,h]pyrene Dibenzo[a,i]pyrene Naphtho[2,3−e]pyrene Naphtho[2,3−k]fluoranthene 1 20 30 50 Heterocyclic Oxygenated Parent Nitrated Methylated Hydroxylated Aminated 40 10 LEL (uM)

Morphology EPR LPR

CYP Expression

RNA seq Any Effect

There are no association of morphological or behavioral endpoints and CYP expression

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Differential responses in parent and derivatives

More mechanistic insight is need to explain why

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Mechanistic insight of 16 PAHs (transcriptomics)

  • 16 PAHs were selected from the

screen by:

  • developmental bioactivity

(morphological and behavioral)

  • their ability to activate AHR
  • the spatial expression of CYP1A
  • Anchored to 120 hpf phenotype

Collect RNA RNAseq analysis EC80 of PAH 1% DMSO control Exposure

24 hp f 6 hp f 48 hpf 120 hpf

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Overview of Differentially Expressed Genes (DEGs)

1,5−dimethylnaphthalene Anthracene Acenaphthene 2−methylnaphthalene 4h−cyclopenta[def]phenanthren−4−one Phenanthrene Retene 9−methylanthracene Carbazole Fluoranthene 3−nitrofluoranthene Benzo[j]fluoranthene Benzo[k]fluoranthene Benzo[b]fluoranthene Dibenzo[a,h]pyrene Dibenzo[a,i]pyrene

Morphology EPR LPR

CYP Expression

Any Effect Any.DevTox.Effect Any.Except.Mort Any.Effect 24 hpf Mortality 24 hpf Developmental Progression 24 hpf Spontaneous M ovement 24 hpf Notochord 120 hpf Mortality Yolk Sac Edema Axis Eye Snout Jaw Otolith Pericardial Edema Brain Somites Pectoral Fin Caudal Fin Pigmentation Circulation Trunk Swim Bladder Notochord Touch−Response EPR Basline EPR Excitation EPR Recovery LPR Dark LPR Light CYP: Vasculature CYP: Liver CYP: Skin CYP: Neuromasts CYP: Yolk

DEG 44 236 130 77 64 21 55 43 89 10 51 1 4 1

* * * * * *

* denotes PAH with body burden data

Morphological and behavioral responses is not directly associated with # of DEGs

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No Association between DEGs, body burden and Log Kow

50 µM 50 µM 12.2 µM 50 µM

Compound Log Kow Log10(Conc Uptake)

2Methylnaphthalene 3.86 1.79 Acenapthene 3.85 2.24 Phenanthrene 4.46 3.17 Fluoranthene 5.16 3.71 Retene 6.35 2.86 Benzo(b)fluoranthene 6.6 1.81

Acenapthene (4) 2Methylnaphthalene (1) Phenanthrene (10) 50 µM Fluoranthene (21) 50 µM Retene (89) Benzo[b]fluoranthene (89)

Embryos were exposed to 3 concentrations (5.39, 11.6, and 25 µM) from 6 to 48 hpf. Using the measured values, a concentration uptake ratio was computed from the ratio of the concentration inside the embryo and to the nominal media concentration. The number of DEGs are annotated near the chemical name, along with the test concentration (in blue). Data points in red represent PAHs with <5.5 log Kow, and green being >5.5.

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Comparing transcriptomic profiles

0.1 0.2 0.3 0.4

Benzo[j]fluoranthene Dibenzo[a,h]pyrene Retene Dibenzo[a,i]pyrene Carbazole Benzo[k]fluoranthene 4h-Cyclopenta[def] phenanthren-4-one Benzo[b]fluoranthene Acenapthene Fluoranthene 9-methylanthracene Phenanthrene B e n z

  • [

j ] f l u

  • r

a n t h e n e Dibenzo[a,h]pyrene Retene Dibenzo[a,i]pyrene C a r b a z

  • l

e Benzo[k]fluoranthene 4h-Cyclopenta[def]phenanthren-4-one Benzo[b]fluoranthene Acenapthene Fluoranthene 9

  • m

e t h y l a n t h r a c e n e Phenanthrene

  • Using Jaccard similarity analysis for

DEGs >1.5 fold change, and p<0.05, a correlation matrix was generated. The lighter the color, the higher correlation.

  • The black indicates overlap not

significant by Fisher’s exact test (pvalue > 0.05)

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Transcriptomic clustering predicted adverse

  • utcome later in development

Treated Control

Benzo[j]fluoranthene Benzo[k]fluoranthene

Chloride Ion homeostasis Cellular response to xenobiotic stimulus Monooxygenase activity Testosterone 6betahydroxylase activity Common Enriched Pathways

Bin 1

Dibenzo[a,h]pyrene Benzo[b]fluoranthene

Common Enriched Pathways Ion transport Neuromast primordium migration Lateral line/ sensory system development

Bin 2

Transcriptomics is a powerful tool to provide mechanistic insight

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Developed Tools to Measure Complex Central Nervous System Changes in Responses to Developmental PAH Exposures

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Adult Behavioral Measures

  • Fitness
  • Swimming activity
  • Anxiety
  • Fear
  • Social Interactions
  • Learning
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Fitness

Training to this side

Oxygen Consumption (mg O2/kg/hr) Swim Speed

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Swimming Activity Over Time

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Anxiety

2 4 6 8 10 12 14 1 11 31 51 71 91 111

10 secs 10 secs 10 secs 10 mins Total Distance Moved (mm) Time (s)

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Fear

Predator video % Time near video Time (s) Acclimation Video

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Learning

Chem

Training to this side

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Determine which PAHs and Mixtures Produce Transgenerational Adverse Outcomes

PAH or vehicle Waterborne exposure

F0

x

F1

germline exposed

x

F2

epigenetic generation

6 hpf 120 hpf >90 dpf +90 dpf +90 dpf

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31 Figure 7. LPR graphs from F0, F2 and F4 generations. At 120 hpf B[a]P-exposed generations (blue line) exhibit significant hyperactivity in the dark, when compared to vehicle controls (black line). Initial exposed generation (F0) and two epigenetic generations shown. F1 and F3 generations exhibited phenotype as well (data not shown).

Knecht AL, Truong L, Marvel SW, Reif DM, Garcia A, Lu C, Simonich MT, Teeguarden JG, Tanguay RL. Transgenerational inheritance of neurobehavioral and physiological deficits from developmental exposure to benzo[a]pyrene in zebrafish. Toxicology and applied pharmacology. 2017;329:148-57.

Example Transgenerational Impacts – B[a]P

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To Summarize

  • Collect high content bioactivity data in a vertebrate model
  • Phenotypic anchoring – for pathway discovery
  • Platform for structure-based predictions
  • Rapid data for decision making
  • Translating zebrafish data:
  • Prioritizing further testing
  • Highly amenable for mixture assessments - Major effort
  • f the OSU Superfund Research Program
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Tanguay Lab

  • Dr. Robert Tanguay
  • Dr. Andrea Knecht
  • Dr. Mitra Geier
  • Dr. Gloria Garcia
  • Dr. Michael Simonich

Mike Garland Laura Holden Kim Hayward Prarthana Shankar Jane LaDu Hao Truong Eric Johnson Greg Gonnerman Carrie Barton

Funding (NIEHS)

  • P42 ES016465
  • RC4 ES019764
  • P30 ES000210

Collaborators

NC State University

  • Dr. David Reif
  • Dr. Skylar Marvel

OSU

  • Dr. Susan Tilton
  • Dr. Kim Anderson
  • Dr. Staci Simonich

PNNL

  • Dr. Katrina Waters
  • Dr. Ryan Mcclure
  • Dr. Paritosh Pande

Acknowledgements

Engineering Team

Corwin Perrin Dylan Thrush David Mandrell Mushfiq Sarker Caleb Jephson Chris Lang Drew Gabler

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Questions?

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Back up slides

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16 EPA Priority PAHs Do Not Reflect Full Range of Effects

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New Biomarkers for AHR2 Activation

Control MO AHR2 MO

Benzo[k] fluoranthene Dibenzo[a,h] pyrene

GeneSymbol Product BbF BjF BkF DBahP DBaiP Retene CYP1C1 cytochrome P450 2.38 2.51 3.10 1.40 1.36 4.15 CYP1C2 cytochrome P450 1.26 1.45 1.95 0.44 0.53 3.17 WFIKKN1 WAP, follistatin/kazal, immunoglobulin, kunitz and netrin domain containing 1 1.21 1.96 2.09 1.18 1.19 2.63 CYP1B1 cytochrome P450 1.18 2.13 2.67 1.08 1.32 2.13 CYP1A cytochrome P450 1.16 2.08 2.18 1.22 1.37 2.06 CABZ01103755.1 N/A 0.59 1.10 1.61 0.62 0.84 2.05 SULT6B1 sulfotransferase family, cytosolic, 6b, member 1 1.40 2.16 2.07 1.24 1.27 1.96 GSTP1 glutathione Stransferase pi 1 1.10 1.82 0.99 0.59 0.54 1.96 DHRS13L1 dehydrogenase/reductase (SDR family) member 13 like 1.16 1.37 0.99 0.82 0.83 1.71 AHRRB arylhydrocarbon receptor repressor b 0.64 1.28 1.40 0.70 1.10 1.70

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The AHR and PAH pathways of toxicity

Signaling functions:

  • 1. Developmental & homeostatic
  • 2. Adaptive (cyp1a)
  • 3. Toxic (adverse effects)

Phenotypic impacts:

  • 1. Development
  • 2. Cardiac
  • 3. Cognitive
  • 4. Reproductive

AHR

HSP 90 HSP 90 AIP

AHR Binding

AHR ARNT Transcription

CYP Induction No metabolism Metabolites

Disruption of endogenous binding/pathways

AHR Independent Toxicity

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3 Aryl Hydrocarbon Receptor (AHR) in Zebrafish

AHR Role CYP1A Expression

AHR2 Primary mediator of toxicity Vasculature AHR1A Deficient in TCDD binding and transactivation activity Liver AHR1B Functional, but no known toxicological role TBD

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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

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AHR2 importance confirmed in CRISPR/Cas9 line

Garcia GR, Bugel SM, Truong L, Spagnoli S, Tanguay RL. AHR2 required for normal behavioral responses and proper development of the skeletal and reproductive systems in zebrafish. PloS

  • ne. 2018;13(3)