Prof. Dr. Dries Knapen
University of Antwerp
Universiteitsplein 1, UC.173
Tel: +32 3 265 27 24
Dr. Lucia Vergauwen, Postdoctoral researcher, University of Antwerp (UA), Wilrijk, Belgium
Dr. Daniel Villeneuve, Research Toxicologist, Environmental Protection Agency (EPA), USA
Prof. Dr. Gerald Ankley, Senior Research Toxicologist, Adjunct Professor, Environmental Protection Agency (EPA), USA
Dr. Hilda Witters, Project manager, Flemish Institute for Technological Research (VITO), Mol, Belgium
Prof. Dr. Ronny Blust, Full professor, University of Antwerp (UA), Antwerpen, Belgium
CO20.2 AT A GLANCE
This project demonstrates the use of in chemico assays targeting specific key events of an established Adverse Outcome Pathway (AOP) to predict higher biological endpoints. One of the case studies in this project focused on the role of thyroid hormones in the embryonic development of fish.
An AOP network linking thyroid hormone disruption to impaired swim bladder inflation in fish was developed. This network was used to select alternative in chemico assays measuring thyroperoxidase and deiodinase inhibition, key enzymes in the thyroid hormone metabolism. A set of 51 compounds was screened, and data were used to predict acute and chronic effects on swim bladder inflation.
Predictions were validated using acute (FET) and chronic (FELS) in vivo experiments in zebrafish and fathead minnow, and in chemico to in vivo extrapolation thresholds were established. A tiered testing strategy for the identification of thyroid hormone disrupting compounds was then proposed.
The goals of ECO20.2 were (1) to apply the alternative assays which were developed during ECO20 on a set of selected compounds, (2) to predict acute and chronic toxicity based on the assay results, and (3) to perform acute (FET) and chronic (FELS) toxicity experiments to validate the predictions. The ECO20.2 output is intended to serve as a proof-of-concept of using AOPs for assay development for screening and prioritization of chemicals.
For the AOP network on thyroid hormone disruption, we used the TPO, DIO1 and DIO2 in chemico enzyme inhibition assays, optimized during ECO20, to measure the molecular initiating events (MIEs) for an array of 51 potential thyroid hormone disrupting compounds. Acute effects on posterior swim bladder inflation were predicted based on the information captured by the AOPs. These predictions were evaluated for 20 compounds by performing acute zebrafish embryo tests. By linking the in chemico DIO1 and DIO2 datasets to the in vivo results and setting thresholds, we demonstrated that the DIO in chemico inhibition assays can be used as a tool to predict the biological effects on posterior chamber inflation, with only few outliers. By performing specific case studies considering (1) species sensitivity differences in the in chemico DIO enzyme inhibition assay, and (2) the effects of compounds on posterior chamber surface area as a more sensitive endpoint in addition to the binary scoring of posterior chamber inflation, we were able to further reduce the number of false negative predictions.
Based on life stage specificity in the AOP network, both the TPO and DIO inhibition assays were used as a basis to predict chronic effects on anterior swim bladder inflation. These predictions were evaluated by performing chronic (FELS) experiments for 4 compounds, of which 2 compounds were tested in fathead minnow as well as in zebrafish. We showed that the combined information of the three enzyme inhibition assays could be used to make reliable predictions of the occurrence of chronic thyroid hormone disruption effects based on the information in the AOP network.
We propose a tiered testing strategy for the identification of thyroid hormone disrupting compounds. The decision tree starts by performing Tier 1 in chemico assays, followed by Tier 2 FET experiments if results are inconclusive. If compounds appear to be thyroid-active, testing ends at this stage. Only in specific cases will Tier 3 FELS experiments be needed to rule out thyroid hormone disruption occurring only in a chronic time frame. Further expanding our thyroid AOP network, especially to include additional thyroid related MIEs and taxa, will be helpful to more clearly define and delineate the applicability domain of the enzyme inhibition assays and amend the decision scheme.
Our results show how information organized using the AOP framework can be employed to develop or select alternative assays, and successfully predict downstream key events along the AOP. Our proposed tiered AOP-based testing strategy is aimed at limiting the use of animal testing and could serve as a proof of concept to expand this approach to other modes of action in the future.
Our thyroid hormone disruption AOP network is part of the AOP development programme workplan of the OECD (project 1.35). OECD review is planned for late 2018. Pending OECD endorsement, the assays developed in this project may be considered for addition to existing OECD testing guidelines. As part of their endocrine disruptor screening program, the US EPA included our work while assembling a conceptual thyroid AOP network spanning different taxonomic groups (fish, amphibians, mammals) to assist high throughput assay development. The currently ongoing JRC EURL ECVAM validation effort of in vitro assays for thyroid disruption screening, to which VITO is contributing as one of the EU-NETVAL laboratories, is making use of this project’s data to ensure synergies and overlap.
Download the project flyer here.
Stinckens, E., Vergauwen, L., Cavallin, JE., Blackwell, BR., Witters, H., Blust, R., Ankley, GT., Volz, DC., Villeneuve, DL., Knapen D. (2017). Development of an alternative testing strategy for the fish early life-stage test using the AOP network “thyroperoxidase and deiodinase inhibition leading to impaired swim bladder inflation”. SETAC Europe 27th Annual Meeting, Brussels, Belgium. May 7-11, 2017.
Stinckens, E., Vergauwen, L., Cavallin, JE., Schroeder, AL., Blackwell, BR., Witters, H., Blust, R., Ankley, GT., Villeneuve, DL., Knapen D. (2017). Cross-species assay validation using the AOP “deiodinase inhibition leading to impaired posterior chamber inflation”. SETAC Europe 27th Annual Meeting, Brussels, Belgium. May 7-11, 2017.
Hagenaars A., Stinckens E., Vergauwen L., Bervoets L., Knapen D. (2014). PFOS affects posterior swim bladder chamber inflation and swimming performance of zebrafish larvae. Aquatic Toxicology. 157, 225-235.
Bagci E., Heijlen M., Vergauwen L., Hagenaars A., Houbrechts A.M., Esguerra C.V., Blust R., Darras V.M., Knapen D. (2015). Deiodinase knockdown during early zebrafish development affects growth, development, energy metabolism, motility and phototransduction. PLoS One. 10, e0123285.
Vergauwen L., Schmidt S.N., Stinckens E., Maho W., Blust R., Mayer P., Covaci A., Knapen D. (2015) A high throughput passive dosing format for the Fish Embryo Acute Toxicity test. Chemosphere 139, 9-17.
Knapen D., Vergauwen L., Villeneuve D.L., Ankley G.T. (2015). The potential of AOP networks for reproductive and developmental toxicity assay development. Reproductive Toxicology 56, 52-55.
Nelson KR, Schroeder AL, Ankley GT, Blackwell BR, Blanksma C, Degitz SJ, Flynn KM, Jensen KM, Johnson RD, Kahl MD, Knapen D, Kosian PA, Milsk RY, Randolph EC, Saari T, Stinckens E, Vergauwen L, Villeneuve DL. 2016. Impaired anterior swim bladder inflation following exposure to the thyroid peroxidase inhibitor 2-mercaptobenzothiazole – Part I: fathead minnow. Aquatic Toxicology 173:192-203
Stinckens E, Vergauwen L, Schroeder AL, Maho W, Blackwell BR, Witters H, Blust R, Ankley GT, Covaci A, Villeneuve DL, Knapen D. 2016. Impaired anterior swim bladder inflation following exposure to the thyroid peroxidase inhibitor 2-mercaptobenzothiazole – Part II: zebrafish. Aquatic Toxicology 173:204-217
Villeneuve D.L., Crump D., Garcia-Reyero N., Hecker M., Hutchinson T.H., LaLone C.A., Landesmann B., Lettieri T., Munn S., Nepelska M., Ottinger M.A., Vergauwen L., Whelan M. 2014. Adverse Outcome Pathway (AOP) Development I: Strategies and Principles. Toxicological Sciences 142, 312-320.
Villeneuve D.L., Crump D., Garcia-Reyero N., Hecker M., Hutchinson T.H., LaLone C.A., Landesmann B., Lettieri T., Munn S., Nepelska M., Ottinger M.A., Vergauwen L., Whelan M. 2014. Adverse Outcome Pathway Development II: Best Practices. Toxicological Sciences 142, 321-330.
Perkins E, Antczak P, Burgoon L, Falciani F, Garcia-Reyero N, Gutsell S, Hodges G, Kienzler A, Knapen D, McBride M, Willett C. 2015. Adverse Outcome Pathways for Regulatory Applications: examination of four case studies with different degrees of completeness and scientific confidence. Toxicological Sciences 148(1):14-25.
Cavallin, J.E., Ankley, G.T., Blackwell, B.R., Blanksma, C.A., Fay, K.A., Jensen, K.M., Kahl, M.D., Knapen, D., Kosian, P.A., Poole, S., Randolph, E.C., Schroeder, A.L., Vergauwen, L., Villeneuve, D.L. 2017. Impaired swim bladder inflation in early-life stage fathead minnows exposed to a deiodinase inhibitor, iopanoic acid. Environ. Toxicol. Chem. 36, 2942–2952.
Evelyn Stinckens, Lucia Vergauwen, Gerald T. Ankley, Ronny Blust, Veerle M. Darras, Daniel L. Villeneuve, Hilda Witters, David C. Volz, Dries Knapen. 2018. An AOP-based alternative testing strategy to predict the impact of thyroid hormone disruption on swim bladder inflation in zebrafish. Aquatic Toxicology 200, 1-12.
Lucia Vergauwen & Jenna E. Cavallin, Gerald T. Ankley, Chloé Bars, Isabelle J. Gabriëls, Ellen D.G. Michiels, Krysta R. Fitzpatrick, Jelena Periz-Stanacev, Eric C. Randolph, Serina L. Robinson, Travis W. Saari, Anthony L. Schroeder, Evelyn Stinckens, Joe Swintek, Steven J. Van Cruchten, Evy Verbueken, Daniel L. Villeneuve, Dries Knapen. 2018. Gene transcription ontogeny of hypothalamic-pituitary-thyroid axis development in early-life stage fathead minnow and zebrafish. General and Comparative Endocrinology. https://doi.org/10.1016/j.ygcen.2018.05.001
Mathieu Vinken & Dries Knapen, Lucia Vergauwen, Jan Hengstler, Michelle Angrish, Maurice Whelan. 2017. Adverse outcome pathways: a concise introduction for toxicologists. Arch. Toxicol. 91, 3697-3707.
Dries Knapen, Michelle Angrish, Marie Fortin, Ioanna Katsiadaki, Marc Leonard, Luigi Margiotta-Casaluci, Sharon Munn, Jason O’Brien, Cody Smith, Xiaowei Zhang, Daniel L. Villeneuve. 2018. Adverse Outcome Pathway Networks I: Development and Applications. Environ. Toxicol. Chem. https://doi.org/ 10.1002/etc.4125
Daniel L. Villeneuve, Michelle Angrish, Marie Forti, Ioanna Katsiadaki, Marc Leonard, Luigi Margiotta-Casaluci, Sharon Munn, Jason O’Brien, Nathan Pollesch, Cody Smith, Xiaowei Zhang, Dries Knapen. 2018. Adverse Outcome Pathway Networks II: Network Analytics. Environ. Toxicol. Chem. https://doi.org/10.1002/etc.4124