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

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 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 2

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 3

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 Phenotypic Response Chemical Information Genomic Response Chemical Structure mRNA | miRNA | protein Expression/ Morphology/Functional/ 4 Mixture Compositions Metabolomics Behavior/Epigentics

The High Throughput Screening Platform 10 min • Specific Pathogen Free Facility 5 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.

The High Throughput Screening Platform 10 min 6 hr 1 day 5 days Embryo Collection 6 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.

The High Throughput Screening Platform 10 min Embryo Collection 7

The High Throughput Screening Platform 10 min 6 hr Embryo Collection Chemical Exposure 8 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.

The High Throughput Screening Platform 10 min 6 hr 1 day Photomotor Response Embryo Collection 30 40 Time (s) Chemical Exposure 9 Truong et al. (2014) Toxicol Sci 137: 212-233.

The High Throughput Screening Platform 10 min 6 hr 1 day 5 days Locomotor Behavior 3 - 9 10 - 17 Photomotor Response Time (min) Embryo Collection 30 40 Developmental Time (s) Assessments Chemical Exposure 10 Truong et al. (2014) Toxicol Sci 137: 212-233.

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 11

Comparative PAH Screening 12

Developed a Library of 123 PAHs for Comparative Analysis 13 Dr. Kim Anderson

High Throughput Screening of PAH Library Blank D loess [uM] 0 100 0.000128 0.00064 0.0032 0.016 0.08 0.4 2 10 80 50 1e+05 Movement 60 40 20 0 21 29 32 35 36 39 42 48 Time 6 hpf 24 hpf 120 hpf • 5 concentrations • 4 morphological • Morphology • 50-1 µM endpoints • Behavior • 5-0.1 µM • Behavioral assay • CYP1A Localization • N=32 14

CYP1a Expression Pattern as a Biomarker of AHR Activation CYTOPLASM NUCLEUS Figure 2. Representative images illustrating CYP1A expression patterns in 120 hpf larvae. a Geier et al. 2017 None, b vasculature, c liver, d yolk, e skin and neuromasts, f skin 15

Comparative Profile of 123 PAHs LEL (uM) 1 10 20 30 40 50 Heterocyclic Oxygenated Parent Nitrated CYP Morphology EPR LPR RNA Methylated Any Hydroxylated Effect Expression seq Aminated Naphtho[2,3 − k]fluoranthene Naphtho[2,3 − e]pyrene Dibenzo[a,i]pyrene Dibenzo[a,h]pyrene Benzo[j]fluoranthene Dibenz[a,h]anthracene Dibenzo[b,k]fluoranthene Benzo[k]fluoranthene 1 − Aminopyrene 3 − nitrofluoranthene 1 − hydroxynaphthalene 3,6 − Dimethylphenanthrene Fluoranthene 11 − H − benzo[b]fluoren − 11 − one Carbazole 9 − methylanthracene 1,6 − Dinitropyrene 7 − nitrobenzo[k]fluoranthene 3,7 − dinitrobenzo[k]fluoranthene 1 − hydroxyindeno[1,2,3 − c,d]pyrene Indeno[1,2,3 − c,d]pyrene Benzo[b]fluoranthene Chrysene Dibenzofuran Benzo[b]fluorene Benzo[a]anthracene 3 − hydroxyfluorene 6 − Methylchrysene Retene Coronene 5,12 − naphthacenequinone 1,4 − Dimethylnaphthalene 2,3 − dihydroxynaphthalene 10 − hydroxybenzo[a]pyrene 1,4 − phenanthrenedione 3 − hydroxybenz[a]anthracene 1,5 − dihydroxynaphthalene Benzo[a]pyrene 6 − Nitrochrysene Dibenzo[a,k]fluoranthene 7 − Nitrobenz[a]anthracene Dibenz[a,c]anthracene 9 − anthracene carbonitrile 3 − Nitrobenzanthrone 1 − hydroxypyrene 9 − Nitrophenanthrene 3 − Nitrophenanthrene 1,3 − dihydroxynaphthalene 5 − Nitroacenaphthalene Anthrathrene 2 − Nitrodibenzothiophene 2 − Nitrofluorene Xanthone Pyrene 1 − hydroxyphenanthrene 2,8 − Dinitrodibenzothiophene 1 − Nitropyrene Acridine 8 − methylquinoline 1,3 − Dinitropyrene 2 − ethylanthraquinone 2 − Nitrofluoranthene Indole 2 − Nitroanthracene Dibenzo[a,l]pyrene Dibenzothiophene Phenanthrene 1,2 − Dimethylnaphthalene Acenaphthylene 4h − cyclopenta[def]phenanthren − 4 − one 4 − hydroxyphenanthrene Naphthalene 1 − Methylnaphthalene 1,6 − Dimethylnaphthalene 3 − hydroxyfluoranthene 9 − fluorenone Fluorene 2 − methylnaphthalene 1,8 − Dinitropyrene 9 − hydroxyphenanthrene Naphtho[2,3 − b]fluo ranthene Naphtho[1,2 − b]fluo ranthene anthraquinone acenaphthenequinone Benzo[e]pyrene Naphtho[2,3 − j]fluoranthene 6H − benzo[cd]pyren − 6 − one Perinaphthenone 1 − Nitronaphthalene 2,7 − dihydroxynaphthalene 2 − Nitropyrene 2 − Methylbenzofuran Acenaphthene 9,10 − phenanthrenequinone Xanthene 1,2 − naphthoquinone 2 − Methylanthracene 2 − hydroxynaphthalene 1,4 − anthraquinone 6 − Nitrobenzo[a]pyrene 2 − Nitronaphthalene 9 − anthracene carboxylic acid 4H − cyclopenta[lmn]phenanth ridine − 5,9 − dione Thianaphthene Dibenzo[j,l]fluoranthene Benzo[c]phenanthrene[1,4]dione Triphenylene 1,5 − dimethylnaphthalene 4 − hydroxychrysene 2,3 − Dimethylanthracene 3 − Nitrodibenzofuran 1,8 − Dimethylnaphthalene Anthracene Benzanthrone Chromone 9 − aminophenanthrene Benz[a]anthracene − 7,12 − dione 5,6 − Benzoquinoline Benzo[g,h,i]perylene Pyrene − 4,5 − dione 3 − hydroxybenzo[e]pyrene 1,6 − dihydroxynaphthalene 2,6 − Dimethylnaphthalene 9 − Nitroanthracene Quinoline 3 − hydroxyphenanthrene Any.DevTox.Effect Any.Except.Mort Any.Effect 24 hpf Mortality 24 hpf Spontaneous Movement 120 hpf Mortality Axis Eye Snout Jaw Otolith Pericardial Edema Brain Somites Pectoral Fin Caudal Fin Pigmentation Circulation Trunk Notochord EPR Basline EPR Excitation EPR Recovery LPR Dark LPR Light CYP: Vasculature CYP: Liver CYP: Skin CYP: Neuromasts CYP: Yolk Transcriptomics 24 hpf Developmental Progression 24 hpf Notochord Yolk Sac Edema Swim Bladder Touch − Response There are no association of morphological or behavioral endpoints and CYP expression 16

Differential responses in parent and derivatives More mechanistic insight is need to explain why 17

Mechanistic insight of 16 PAHs (transcriptomics) • 16 PAHs were selected from the screen by: 48 hpf 24 120 hpf 6 hp hp • developmental bioactivity f f (morphological and behavioral) • their ability to activate AHR • the spatial expression of CYP1A Collect RNA • Anchored to 120 hpf phenotype Exposure EC80 of PAH RNAseq 1% DMSO analysis control 18

Overview of Differentially Expressed Genes CYP Morphology EPR LPR (DEGs) Any DEG Effect Expression Dibenzo[a,i]pyrene 44 Dibenzo[a,h]pyrene 236 * Benzo[b]fluoranthene 130 77 Benzo[k]fluoranthene 64 Benzo[j]fluoranthene 3 − nitrofluoranthene 0 * Fluoranthene 21 Carbazole 55 9 − methylanthracene 43 * Retene 89 * Phenanthrene 10 51 4h − cyclopenta[def]phenanthren − 4 − one * 2 − methylnaphthalene 1 * Acenaphthene 4 1 Anthracene 0 1,5 − dimethylnaphthalene 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 Morphological and behavioral responses is not directly associated with # of DEGs 19 * denotes PAH with body burden data