2014 Progress Report: Toxicity Screening with Zebrafish AssayEPA Grant Number: R835168
Title: Toxicity Screening with Zebrafish Assay
Investigators: Tanguay, Robert L.
Institution: Oregon State University
EPA Project Officer: Klieforth, Barbara I
Project Period: May 1, 2012 through April 30, 2016
Project Period Covered by this Report: May 1, 2014 through April 30,2015
Project Amount: $1,199,999
RFA: Developing High-Throughput Assays for Predictive Modeling of Reproductive and Developmental Toxicity Modulated Through the Endocrine System or Pertinent Pathways in Humans and Species Relevant to Ecological Risk Assessment (2011) RFA Text | Recipients Lists
Research Category: Computational Toxicology , Endocrine Disruptors , Health , Ecosystems , Safer Chemicals
The objectives of our project have not changed and are:
- Expose zebrafish embryos to at least 1,000 chemicals.
- Study the morphology and behavior of the embryos to look for evidence of developmental toxicity.
- When an adverse effect occurs, conduct whole-transcriptome RNA sequencing studies to observe gene expression patterns, identify the initial biochemical target, and begin to elucidate the toxicity pathway.
- Conduct statistical modeling to compare the results of the zebrafish assay with the results of in vitro assays and other in vivo assays where they exist.
- Share our data with EPA and the broader research community.
As identified on the last progress report, the following were the focus of this year’s efforts:
- Complete the array analysis for the 25 selected EDC compounds
- Begin the comparative bioinformatic analysis.
- Complete the Triclosan gene expression analysis
- Broadly disseminate results.
1) Complete the array analysis for the 25 selected EDC compound.
Due to variability in RNA sequencing quality, we decided to use high density micro arrays for the gene expression studies. A summary of the experimental plan that was developed is summarized below in Figure 1. We selected the ArrayXS Zebrafish microarrays as robust high-density platform for assessing the impact of EDCs in developing zebrafish. Since we were not primarily interested in identifying novel transcripts, these 60K high density arrays are ideally suited for these studies. The replicates were nicely clustered using Principle Component analysis.
2) Begin the comparative bioinformatic analysis of the EDC 48 hpf gene expression analysis.
All of the initial gene expression analysis and identified clear separation of the patterns of gene expression (Figure 2) has been done. The three thyroid compounds and the unknown compound, 4-7-hydroxy, had the most similar transcriptome profiles of all chemicals tested. In this year we have focused efforts on the thyroid active compounds. A total of 343 unique transcripts were DE for all four compounds (Figure 3). Regression analysis showed good correlation between the treatments and a small grouping of highly expressed transcripts. The 12 significant differentially expressed transcripts in common with all four compounds are listed in Table 1. It is noteworthy that these transcripts may represent new diagnostic biomarkers of thyroid active compounds in this model. The validation studies are underway. Finally, pathway analysis is underway and will be focus of the next year.
3) Complete the Triclosan gene expression analysis
Detailed gene expression analysis following Triclosan exposures is complete. Of all of the tested chemicals in this study, Triclosan led to the largest number of genes expression changes. Detailed analysis will be reported in the final progress report. We are currently comparing the identified differentially expressed genes with the assays “hit” by Triclosan in the Tox Cast. Additionally, we are working with Ruben Abagyan (UC San Diego) to mine the targets predicted to be bound by Triclosan. A number, of compelling hits have been identified and are being pursued. This manuscript will be submitted to TAAP in early 2016.
4) Broadly disseminate results.
Ongoing effort, several manuscripts and public presentations are planned.
The primary results have been disseminated in several venues by the PI.
- Exploiting zebrafish as a sensor for chemical bioactivity, Boston University, Boston MA, October 30, 2015.
- Using Multidimensional Zebrafish Data to Advance Environmental Health Science. The University of Illinois, October 6, 2015.
- Using Zebrafish as a High-Throughput Non-Mammalian In Vivo Model to Identify DNT Compounds the American Association for Laboratory Animal Science (AALAS), October 3, 2015 Phoenix AZ.
- Coupling the power of high throughput zebrafish screening and synthetic chemistry to design safer chemicals. American Chemical Society, August 16, 2015 Boston MA.
- Using Multidimensional Zebrafish Data to Advance Environmental Health, Environmental Defense Fund Meeting Elucidating Environmental Dimensions of Neurological Disorders and Diseases: Understanding New Tools from Federal Chemical Testing Programs. UC Davis, Davis CA June 20, 2015.
- A Strategy for the Design of Safer Products. Design of Safer Chemicals and Products: The Nexus of Toxicology and Chemistry McMenamins' Edgefield, Troutdale, OR April 23-24, 2015
- High Throughput Assessment of Bioactivity Using Zebrafish: High Content Data for the 21st Century. Oklahoma State University, Stillwater Oklahoma February 19, 2015
- Submit the Thyroid focused manuscript
- Complete the Triclosan gene expression and comparative activity manuscript
- Complete comparative bioinformatic analysis for all compounds
- Submit the Estrogenic and Androgenic focused manuscripts
- Broadly disseminate results.