An AOP-based alternative testing strategy to predict the impact of thyroid hormone disruption on swim bladder inflation in zebrafish

Abstract

The adverse outcome pathway (AOP) framework can be used to help support the development of alternative testing strategies aimed at predicting adverse outcomes caused by triggering specific toxicity pathways. In this paper, we present a case-study demonstrating the selection of alternative in chemico assays targeting the molecular initiating events of established AOPs, and evaluate use of the resulting data to predict higher level biological endpoints. Based on two AOPs linking inhibition of the deiodinase (DIO) enzymes to impaired posterior swim bladder inflation in fish, we used in chemico enzyme inhibition assays to measure the molecular initiating events for an array of 51 chemicals. Zebrafish embryos were then exposed to 14 compounds with different measured inhibition potentials. Effects on posterior swim bladder inflation, predicted based on the information captured by the AOPs, were evaluated. By linking the two datasets and setting thresholds, we were able to demonstrate that the in chemico dataset can be used to predict biological effects on posterior chamber inflation, with only two outliers out of the 14 tested compounds. Our results show how information organized using the AOP framework can be employed to develop or select alternative assays, and successfully forecast downstream key events along the AOP. In general, such in chemico assays could serve as a first-tier high-throughput system to screen and prioritize chemicals for subsequent acute and chronic fish testing, potentially reducing the need for long-term and costly toxicity tests requiring large numbers of animals.

Functions of the Thyroid-Stimulating Hormone on Key Developmental Features Revealed in a Series of Zebrafish Dyshormonogenesis Models

“…However, TH signaling may be necessary for the proper developmental remodeling process in zebrafish. Originally, the role of THs in zebrafish development was studied using goitrogen treatments [44][45][46], which biologically block TH synthesis in thyrocytes and induce the hypothyroid state. The conclusions of the LTJT studies with goitrogen treatments can be confusing as the chemicals used in these studies also generate risks due to toxicity associated with non-TH-related side effects [47].…”

Assessing the Ecological Risks of Per‐ and Polyfluoroalkyl Substances: Current State‐of‐the Science and a Proposed Path Forward

“…Specifically, when observed bioactivities/targets can be linked to known pathways/processes, for example using AOPs, insights as to susceptible species and sensitive endpoints can be gained. As a simple illustration of this, Stinckens et al (2018) reported that PFOA caused significant inhibition of the activity of deiodinases involved in thyroid hormone synthesis using in vitro assays with liver tissue from pigs.…”

Toward an AOP Network-Based Tiered Testing Strategy for the Assessment of Thyroid Hormone Disruption

“…The AOP network used in this case example links TH disruption to impaired swim bladder inflation in fish and is mainly based on experimental evidence from studies on zebrafish and fathead minnow including chemical exposures, knockdowns, knockouts and rescue experiments [15][16][17][18][19][20][21][22][23][24][25][26][27][28][29][30][31][32] . The swim bladder is an internal gas-filled organ found in many bony fish species and typically consists of two gas-filled chambers.…”

“…Specifically, a positive result in the in vitro Tpo and/or the Dio inhibition assay could trigger fish in vivo testing to confirm the occurrence of downstream events along the AOP network including altered TH levels, impaired swim bladder inflation, and altered swimming performance (see sections 3.2 and 3.3). An initial validation case study showed that adverse swim bladder effects could be predicted along the AOP network based on in vitro Dio inhibition data 22 . The further development of such predictive approaches, including expanding the quantitative understanding of the relationships depicted in the AOPs where possible 42 , would significantly reduce the need for in vivo testing in the future.…”