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

Highlights

• AOPs linking deiodinase inhibition to impaired swim bladder inflation in fish were previously developed.

• Enzyme inhibition assays were selected based on these AOPs, targeting two molecular initiating events.

• An array of 51 compounds was screened using these assays.

• The in chemico dataset was used to accurately predict in vivo effects on swim bladder inflation in zebrafish.

• This study illustrates how AOPs can guide alternative assay development.

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.

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.