An AOP-based alternative testing strategy to predict the impact of thyroid hormone disruption on swim bladder inflation in zebrafish
Introduction
Industry and regulatory bodies have expressed the need for developing alternative testing strategies for risk assessment and hazard identification, focusing on non-animal alternatives and the use of mechanistic information (Ankley et al., 2010). Fish are ideal sentinels for evaluating aquatic toxicity to vertebrates, but testing for chronic fish toxicity is resource and animal intensive. Although a number of in vitro assays based on fish cells or cell lines have been developed (Bols et al., 2005; Segner, 2004, Segner, 1998; Stadnicka-Michalak et al., 2014; Tan et al., 2008), fish embryos have also become a popular alternative model system in aquatic ecotoxicology (Braunbeck and Lammer, 2006; Scholz et al., 2008). The publication of OECD Testing Guideline (TG) 236, the “Fish Embryo Acute Toxicity (FET) Test” (OECD, 2013a), describing a 96 h fish embryo test, has greatly facilitated the use of fish embryos in toxicity studies. The testing guideline is currently limited to observations of lethal endpoints and hatching, but research has shown that more subtle toxic effects can also be reliably investigated using fish embryos (Braunbeck et al., 2014; Hagenaars et al., 2014; Hill et al., 2005; Michiels et al., 2017; Pype et al., 2015; Scholz et al., 2008; Selderslaghs et al., 2013; Stinckens et al., 2016; Verstraelen et al., 2016; Voelker et al., 2007). However, the development of alternative assays capable of capturing and representing the mechanisms underlying toxicity pathways at sub-organismal levels of biological organization requires a targeted approach. Adverse outcome pathways (AOPs) can assist in the identification of measurable processes at specific levels of biological organization, termed key events (KEs), that are essential in a given toxicity pathway (Ankley et al., 2010). In this way, the AOP framework can directly assist in assay development by guiding the selection of specific KEs which are likely to have a high predictive value for an AO of interest.
The aim of the present study was to demonstrate how in chemico assays targeting specific KEs of an established AOP were selected and used to predict higher biological endpoints. The selected AOP focuses on the role of thyroid hormones in embryonic development in fish. Thyroid hormones (THs) have been shown to play an important role in a wide range of biological processes in vertebrates and disruption of the thyroid axis can lead to ecologically relevant adverse outcomes. For example, THs are involved in development, especially in amphibian metamorphosis (Callery and Elinson, 2000), embryonic-to-larval transition (Liu and Chan, 2002) and larval-to-juvenile transition (Brown, 1997) in fish. The two primary THs are the prohormone thyroxin (T4) and the biologically more active 3,5,3′-triiodothyronine (T3) (Hulbert, 2000). The synthesis of these THs is a process that involves several steps, with thyroperoxidase (TPO) playing an essential role in the production of T4, and to a lesser extent of T3. The bioavailability of T3 in developing cells is regulated by several processes, including deiodination by enzymes called iodothyronine deiodinases (DIOs) (Darras and Van Herck, 2012; Gereben et al., 2008; Orozco and Valverde-R, 2005). To date, three types of iodothyronine deiodinases (DIO1-3) have been described in vertebrates. Type 2 deiodinase (encoded by the DIO2 gene) is capable of activating T4 into T3, as well as of converting reverse T3 (rT3) into 3,3′ T2. Deiodinase 3 can convert T4 and T3 to the inactive TH forms rT3 and 3,3′ T2 respectively. Type 1 deiodinase (encoded by the DIO1 gene) is capable of both outer and inner ring deiodination, and can therefore catalyze all four TH deiodination reactions (Darras and Van Herck, 2012; Gereben et al., 2008; Orozco and Valverde-R, 2005).
Numerous chemicals are known to disturb thyroid-related processes, for example by inhibiting the TPO and/or DIO enzymes, by upregulating metabolization pathways, or by inhibiting sodium/iodide symporter (NIS) mediated iodide uptake (Butt et al., 2011; Hallinger et al., 2017; Hornung et al., 2010; Kim et al., 2015; Paul et al., 2014; Visser et al., 1979). Previous work suggests that chemicals interfering with the conversion of (maternal) T4 to T3 (a reaction catalyzed by either Dio1 or Dio2) could inhibit inflation of the posterior swim bladder in fish, which may result in reduced swimming capacity, an adverse outcome that can affect feeding behavior and predator avoidance, ultimately resulting in lower survival probability and population trajectory decline (Czesny et al., 2005; Woolley and Qin, 2010). The swim bladder of the zebrafish is a gas-filled structure that consists of a posterior and an anterior chamber. While the posterior chamber inflates during early development (96–120 h post fertilization, hpf), the anterior chamber only inflates around 20–21 days post fertilization (dpf). Both chambers are important for regulating buoyancy and body density, and the anterior chamber additionally has a role in hearing in fish species of the superorder Ostariophysi (which possess a Weberian apparatus) (Dumbarton et al., 2010; Lindsey et al., 2010; Roberston et al., 2007).
Building on our previous work focused on AOP development related to TH disruption in fish (Cavallin et al., 2017; Nelson et al., 2016; Stinckens et al., 2016), we aligned the present study with AOPs that are publicly available in the AOP-Wiki (aopwiki.org), an online database organizing the available knowledge and published research into individual AOP descriptions using a user friendly Wiki interface. More specifically, we focused on two AOPs linking the molecular initiating events (MIEs) Dio1 and Dio2 inhibition to impaired inflation of the posterior swim bladder chamber in fish during early life-stages (Fig. 1; Bagci et al., 2015; Cavallin et al., 2017; Knapen et al., 2018; Nelson et al., 2016; Stinckens et al., 2016; Villeneuve et al., 2018). These AOPs were used to optimize in chemico assays to measure the potential of compounds to inhibit DIO1 and DIO2 enzyme activity, using porcine tissue. We then selected 14 compounds with different DIO inhibitory potencies to assess potential effects on posterior chamber inflation, using the zebrafish embryo as a model system. By linking both datasets, we evaluated the use of in chemico data to explain and ultimately predict the in vivo biological effects on posterior chamber inflation.
Section snippets
Ethics statement
The EU Directive 2010/63/EU and the Commission Implementing Decision 2012/707/EU state that fish are non-protected animals until they are free feeding, i.e. 120 h post fertilization (hpf) for zebrafish (Strähle et al., 2012). All experiments of this study executed at the University of Antwerp (UA) exceeding 120 hpf were approved by the Ethical Committee for Animals of the University of Antwerp (project IDs 2014-29 and 2016-46). According to the Animal Research Advisory Committee Guidelines for
Results and discussion
As a case study of using the AOP framework for the development of alternative assays, we selected an AOP that describes the effects of Dio inhibition on posterior swim bladder inflation in fish. We first used in chemico assays for measuring the MIE (i.e., DIO inhibition). We then assessed the effects of exposure to a selection of DIO inhibiting compounds on posterior chamber inflation using zebrafish embryo assays. We linked both datasets to evaluate the potential for using AOP-based in chemico
Conclusion
We have performed a set of experiments to evaluate in chemico assays for predicting acute swim bladder inflation effects based on the AOP framework. Our results suggest that Dio2 may play a more important role in swim bladder inflation compared to Dio1. By linking the in chemico and in vivo datasets and setting threshold values, we were able to demonstrate that the DIO2 in chemico dataset can be used as a predictive tool for the biological effects on posterior chamber inflation, with only few
Acknowledgements
The authors would like to thank Lobke Claes for her assistance with carrying out the deiodinase inhibition assays. This work was funded by the Cefic Long-range Research Initiative (http://www.cefic-lri.org/) project LRI-ECO20.2-UA (Development of an alternative testing strategy for the fish early life-stage test for predicting chronic toxicity: assay validation) with support of ECETOC. This work was further supported by the Society of Environmental Toxicology and Chemistry (SETAC)/Procter &
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