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Cross-species assay validation using the AOP “deiodinase inhibition leading to impaired posterior chamber inflation”
Citation:
Stinckens, E., L. Vergauwen, J. Cavallin, A. Schroeder, B. Blackwell, H. Witters, R. Blust, G. Ankley, Dan Villeneuve, AND D. Knapen. Cross-species assay validation using the AOP “deiodinase inhibition leading to impaired posterior chamber inflation”. EU SETAC, Brussels, BELGIUM, May 07 - 11, 2017.
Impact/Purpose:
Deiodinase inhibition is one important mechanism through which chemicals can disrupt thyroid hormone signaling. In support of the Endocrine Disruptor Screening Program, has developed high throughput assays to screen for deiodinase inhibition. A key question in the use of a mammalian-based screening assay, is whether it is reasonably predictive of deiodinase inhibition in other species. The current work establishes that with just a few exceptions, a deiodinase inhibition assay based on rat or pig liver homogenates is effective for detecting the same chemicals that impair deiodinase inhibition in fish liver homogenates (in this case zebrafish). This lends confidence that, for this endpoint, a prioritization approach based on high throughput screening should be adequately protective for fish and incorporation of a fish-specific deiodinase assay into the prioritization strategy is probably unnecessary. Results of this research directly support the aims of OCSPP to modernize the EDSP.
Description:
High throughput screening assays able to detect chemical interactions with specific biological targets are increasingly being used to identify chemicals that could be hazardous to humans or wildlife. Most of these assays examine interaction with mammalian proteins. The present work demonstrates that mammalian-based assays designed to screen for interactions of chemicals with deiodinase, an enzyme important to thyroid hormone signaling provides results that are generally consistent with those obtained when a fish-specific deiodinase assay was employed. This gives confidence, that in most cases, a mammalian-based screening assay should detect chemicals that could act as thyroid disrupting chemicals (through this particular mode of action) in fish as well as mammals. Thus, this work helps support implementation of more efficient and cost effective approaches to chemical safety assessment.Abstract: The Adverse Outcome Pathway (AOP) concept is increasingly being recognized as a promising conceptual framework for describing toxicity pathways, which contains information that is sufficient to predict an adverse outcome of regulatory importance. Previously, we assessed the feasibility of developing an alternative, mechanistically informative testing strategy to replace the chronic Fish Early-Life Stage test (FELS, OECD TG 210), using an AOP-based approach. We developed an AOP encompassing deiodinase (DIO) inhibition resulting in decreased T3 concentrations leading to impaired swim bladder inflation in fish.In vitroassays were used to measure DIO enzyme activity of 51 relevant compounds. Using these results,in vivoeffects on swim bladder inflation were predicted. These predictions were biologically validated for a set of 14 compounds, with the exception of only 2 false positives and 1 false negative, using zebrafish as model organism. Our results were in line with our AOP and illustrate how AOP-derived information can be used for assay development and refinement. In a next step, we assessed the cross-species applicability of our AOP-based assays. In order to predict an AO based on a molecular initiating event (MIE) or key event, one needs to take into account the fact that the affinity of certain compounds to interact with receptors, enzymes etc. can differ among species. As DIO1 inhibition is the MIE of our AOP on which anin vitroassay was based, we investigated whether the use of porcine, rat or fish liver as starting material would result in similarin vitroDIO1 inhibition patterns for a set of 22 compounds, and thus whether predictions in one species can be made based on assays using tissues of other species as the starting material. Results show that the DIO1 inhibitory potential compared to a reference compound is nearly identical between the three selected species. However, a set of bisphenol A derivatives showed lower inhibition potential in fish and rat compared to pig. A reassessment of the validation experiments based on zebrafish inhibition data shows that TCBPA is no longer a false positive prediction, while BPA appears to become a false negative prediction. These results show that for most compounds, tissue originating from different species can be used in our DIO1 assay to predict apical outcomes in fish. Furthermore, it demonstrates that AOPs can support cross-species extrapolation after investigating their taxonomic applicability.