Final Report: System Toxicological Approaches to Define Flame Retardant Adverse Outcome Pathways

EPA Grant Number: R835796
Title: System Toxicological Approaches to Define Flame Retardant Adverse Outcome Pathways
Investigators: Tanguay, Robert , Du, Jane La , Reif, David , Simonich, Mike , Sullivan, Chris
Institution: Oregon State University , North Carolina State University at Raleigh
EPA Project Officer: Lasat, Mitch
Project Period: June 1, 2015 through May 31, 2018 (Extended to May 31, 2019)
Project Amount: $798,661
RFA: Systems-Based Research for Evaluating Ecological Impacts of Manufactured Chemicals (2014) RFA Text |  Recipients Lists
Research Category: Ecological Indicators/Assessment/Restoration , Ecosystems , Safer Chemicals

Objective:

Research Objectives:

  • Expose embryonic zebrafish to a comprehensive list of flame retardant chemicals (FRCs) and observe their morphology and behavior for signs of toxicity.
  • Grow exposed zebrafish to adulthood and measure their physiology and behavior for signs of persistent toxicity.
  • Bin FRC outcomes using multiple levels of biological organization, i.e., chemical structure, the similarity of gene expression profiles, early and adult life stage adverse outcomes, and thereby define adverse outcome pathways (AOP) for mechanistic FRC hazard prediction.
  • Share our data with EPA and the broader research community.

Summary/Accomplishments (Outputs/Outcomes):

In the past three years,researchers have built a library of 61 FRCs from various sources. Usinga high throughput zebrafish toxicity model,ivestigators assessed the 61 FRCs by exposing embryos from 6-120 hours post-fertilization (hpf) to 11 concentrations (0.1 – 80 µM). At 24 and 120 hpf, 22 morphological endpoints were evaluated. Additionally, exposed embryos underwent 2 behavioral assays.Investigators selected a subset for genome-wide transcriptomics. The criteria used to select this subset was 1) the FRC stock was analyzed for purity and integrity soinvestigators were confident of its identity, 2) the FRC had a robust in vitro bioactivity profile, 3)researchers had conducted behavioral analysis from developmentally exposed adult zebrafish and 4) each of the various classes of FRCs had at least one representative. Embryos exposed from 6 – 48 hpf were exposed to a concentration that caused 80% effects (EC80) to 10 FRCs using RNA-sequencing technology (Table 1).

Table 1. Candidate list for genome-wide transcriptomic and validated EC80

Chemical Name

Abbreviation

EC80 (uM)

2,2,4,4-tetra-bromodiphenyl ether

BDE-47

85

3,3',5,5'-tetrabromobisphenol A (TBBPA)

TBBPA

4

Bis(2-ethylhexyl) tetrabromophthalate

TBPH

72

Tetrabromobisphenol A-2-3-dibromopropyl ether (TBBPA-DBPE)

TBBPA-DBPE

4

Triisobutyl phosphate

TiBP

158

Triisopropylated phenyl phosphate

IPP

19.8

Triphenyl phosphate

TPP

8

Tris(2,3-dibromopropyl) phosphate

TDBPP

3

Tris(2-chloroethyl) phosphate

TCEP

85

Tris(2-chloroisopropyl)phosphate

TCPP

85

The EC80 was anchored to morphological changes for 3 FRCs (Triisobutyl phosphate [TiBP], Triisopropylated phenyl phosphate [IPP], and 3,3',5,5'-tetrabromobisphenol A [TBBPA]) that are classified as aryl phosphate ester and brominated phenols. There were 2 FRCs, Triphenyl phosphate [TPP], and Tris(2,3-dibromopropyl) phosphate [TDBPP] caused mortality in the embryos. Exposure to TDBPP caused the most statistically significant genes (p<0.05, log2 fold change > 1.5) in the embryonic zebrafish, 1534. When comparing the transcriptional profile of the top 30 significant genes of TDBPP to the 9 other FRCs, 3 other FRCs clustered nicely together – TiBP, IPP and TBBPA (Figure 1).

Figure 1. Top 30 statistically significant genes with a log2 Fold change (1.5) from TDBPP.

Interestingly, these 3 FRCs induced > 500 statistically significant genes (1060, 764, and 484). To investigate the role of these misregulated genes, a pathway analysis software, Metacore was applied to the gene lists. Enrichment analysis was conducted, and the most enriched network processes were related to inflammation complement system/ Jak-STAT pathway and neurophysiological process related to visual perception.

miRNA-sequencing

In addition to conducting mRNA-sequencing,researchers conducted micro RNA-sequencing for the same 10 FRCs. These samples were collected from the same 4 biological replicates for each FRC. Initial evaluation of these 40 samples shows the biological replicates for each treatment were tightly associated (Figure 2).

Figure 2. A PCA plot of top 100 genes based on gene variance (miRNA-seq)

Two FRCs (TBPH, and TCPP) grouped with the controls and were also the ones that did not cause any malformations or only at high concentrations. The number of differentially expressed mRNA genes was also low for these samples (<50). These results provide confidence that these FRCs were the least bioactive of the suite. The TDBPP samples were more variable and did not group with any other FRCs. This FRC induced the largest number of differentially expressed genes. The most misregulated micro-RNA was dre-miR-34a. This microRNA has a number of predicted target genes involved in nervous system and brain development, neuron differentiation, and neurogenesis (Figure 3).

Figure 3. miR-34a differentially expressed in TDBPP and its predicted mRNA target genes

Evaluation of the bioactivity of TDBPP revealed morphological malformations at 120 hpf, also behavioral deficits were observed at 24hpf in response to the light pulses. Additionally, at 120 hpf, the exposed larvae did not exhibit the typical behavior response when alternating between light and dark cycles. A typical response is less activity in the light and more in the dark. These behavioral effects occur at higher TDBPP concentrations (less bioactivity), demonstrating the power of utilizing miRNA and mRNA transcriptional assessments to identify neurologically active FRCs. Researchersare on the path to identifying genes of interest and adverse outcome pathways related to the bioactive FRCs: TDBPP, TiBP, IPP and TBBPA.

 


Journal Articles on this Report : 7 Displayed | Download in RIS Format

Other project views: All 31 publications 8 publications in selected types All 8 journal articles
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Journal Article Chen J, Tanguay RL, Simonich M, Nie S, Zhao Y, Li L, Bai C, Dong Q, Huang C, Lin K. TBBPA chronic exposure produces sex-specific neurobehavioral and social interaction changes in adult zebrafish. Neurotoxicology and Teratology 2016;56:9-15. R835796 (2015)
R835796 (2016)
R835796 (Final)
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  • Journal Article Chen J, Tanguay RL, Xiao Y, Haggard DE, Ge X, Jia Y, Zheng Y, Dong Q, Huang C, Lin K. TBBPA exposure during a sensitive developmental window produces neurobehavioral changes in larval zebrafish. Environmental Pollution 2016;216:53-63. R835796 (2015)
    R835796 (2016)
    R835796 (Final)
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  • Journal Article Noyes PD, Haggard DE, Gonnerman GD, Tanguay RL. Advanced morphological-behavioral test platform reveals neurodevelopmental defects in embryonic zebrafish exposed to comprehensive suite of halogenated and organophosphate flame retardants.Toxicological Sciences 2015;145(1):177-195. R835796 (2015)
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    R835796 (Final)
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  • Journal Article Truong L, Bugel SM, Chlebowski A, Usenko CY, Simonich MT, Simonich SLM, Tanguay RL. Optimizing multi-dimensional high throughput screening using zebrafish. Reproductive Toxicology 2016;65:139-147. R835796 (2017)
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  • Journal Article Zhang G, Truong L, Tanguay RL, Reif DM. A new statistical approach to characterize chemical-elicited behavioral effects in high-throughput studies using zebrafish. PLoS One 2017;12(1):e0169408 (16 pp.). R835796 (2017)
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  • Journal Article Zhang G, Marvel S, Truong L, Tanguay RL, Reif DM. Aggregate entropy scoring for quantifying activity across endpoints with irregular correlation structure. Reproductive Toxicology 2016;62:92-99. R835796 (2017)
    R835796 (Final)
    R835168 (Final)
    R835802 (2015)
    R835802 (2016)
    R835802 (2017)
    R835802 (2018)
    R835802C003 (2015)
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  • Journal Article Zhang G, Roell KR, Truong L, Tanguay RL, Reif DM. A data-driven weighting scheme for multivariate phenotypic endpoints recapitulates zebrafish developmental cascades. Toxicology and Applied Pharmacology 2017;314:109-117. R835796 (Final)
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  • Progress and Final Reports:

    Original Abstract
  • 2015 Progress Report
  • 2016 Progress Report
  • 2017 Progress Report