Two adverse outcomes pathways (AOPs), entitled “Ecdysone receptor (EcR) agonism leading to incomplete ecdysis associated mortality” (https://aopwiki.org/aops/4) and “Juvenile hormone (JHR) receptor agonism leading to male offspring induction associated population decline” (https://aopwiki.org/aops/201) have been defined and submitted to the AOPWiki. The EcR AOP describes the molecular initiating event (MiE) of EcR activation by EcR agonists leading to a series of key events, such as suppression of the Ftz-f1 gene and loss of competence for secretion of the ecdysis triggering hormone (ETH), causing incomplete ecdysis behavior and ultimately mortality in arthropods. The JHR AOP is an additional AOP developed recently by EDRISK to describe the MIE of JHR activation leading to abnormal sex determination associated population decline in arthropods.
A third AOP that focuses on “Ionotropic gamma-aminobutyric acid receptor (GABAR) activation mediated neurotransmission inhibition leading to mortality”, that addresses neurotoxic effects of GABA receptor agonists has been developed (https://aopwiki.org/aops/160). This AOP has been associated with incomplete ecdysis and thus a potential interaction pathway in a AOP network related to molting-related increase in lethality in crustaceans. Review papers describing the development of the AOPs have been published (Song et al., 2016, 2017a).
In WP1, the sequence similarity comparison tool SeqAPASS has now been publicly released and available for cross-species extrapolations. This tool can be accessed at: https://seqapass.epa.gov/seqapass. Additionally, the SeqAPASS work has been published recently (Lalone et al. 2016). A number of computational tools for identifying conserved sequence orthologs/homologs and ligand-binding/docking models have been developed to characterize the taxonomic applicability domain of the EcR (Evenseth et al. 2014). A specific structural alert (KNIME) workflow to identify potential EcR agonists and antagonists have been developed (Mellor et al. 2014).
In WP2, a number of experimental studies have been conducted to characterize the key events in the three conceptual AOPs. For EcR AOP, a functional bioassay was developed to measure the chitobiase activity as an indicator of molting activity in D. magna (Song et al., 2017b). Short-term exposure studies on the well-known EcR agonists 20-hydroxyecdysone (20E, endogenous hormone) and tebufenozide (TEB, commercial pesticide) showed that both chemicals reduced the molting frequency and chitobiase activity, caused incomplete ecdysis and associated mortality in juvenile D. magna after 96h exposure, thus verifying that EcR agonists cause incomplete ecdysis (Song et al., 2017a, 2017b). In addition to known EcR agonists, emamectin benzoate (EMB), a commercially available anti-sea lice chemical was studied (Song et al. 2016). This chemical was chosen as it is known to cause molting defect and mortality in other crustacean species such as the lobsters. Results from this study showed that EMB reduced the molting frequency and caused mortality in D. magna after 48h exposure. Global gene expression analysis showed that EMB potentially stimulated the 20E titer and subsequently activated the EcR pathway leading to molting defect without being a direct agonist for the EcR (Song et al. 2016). For the JHR AOP, adult female D. magna were exposed to methylfarnesoate (MF, endogenous juvenile hormone) and fenoxycarb (FCB, commercial pesticide) for 7d using a short-term screening (STS) assay that was developed by collaborator Iguchi (Abe et al., 2015). The results from the STS assay clearly showed that the two JH mimics caused male offspring formation and reduction of reproduction in a concentration-dependent manner, confirming one of the key event proposed in the conceptual AOP. Moreover, studies on D. magna embryos revealed that the Neverland (Nvd) genes were necessary for the synthesis of the endogenous hormone 20E (Sumiya et al., 2016). A few studies on the JH signaling pathways also showed that environmental cues and EDCs may affect the sex determination and reproduction of D. magna (Toyota et al., 2015a; 2015b).
In WP3, ultraviolet (UV) B radiation was used as an environmental stressor to challenge the EcR AOP. The results were compared to an earlier exposure study with 20E, the endogenous molting hormone. The robustness testing showed that UV-B, dissimilarly to 20E, neither affected the molting frequency nor led to incomplete ecdysis, albeit this stressor still caused concentration-dependent mortality in D. magna, indicating that the lethal adverse outcome may be caused through a different mode of action (MoA) than those studied earlier. Follow-up studies will be performed to obtain in-depth knowledge on the effects and mechanisms of UV-B in D. magna.
In WP4, predictive approaches are under development using gene expression profiles. Gene expression signatures representing the responses of D. magna to both molting and juvenile hormones were developed (Basili, 2017). A panel of chemicals (including EDCs) were then compared to the expression profiles of the reference hormones. Compounds having a similar functional profile to either molting or juvenile hormones were taken as evidence for common MoA between the different compounds. Although this correlational approach was able to identify potential endocrine disrupting compounds in D. magna, it remains to verify that the toxicity pathways triggered in common are specifically associated with ED mechanisms and assess if this is associated with adverse effects relevant for ED. An effect-based database (www.niva.no/radb) has been developed to support WoE considerations along the AOP continuum, and will be used to support linking changes to MiE and KE to higher level effects (e.g. the AO). EDRISK has supported workshop attendance and reporting of topics related to developing and using the AOP concept, and how the AOP and supporting methods can be used to advance research and regulatory processes (Tollefsen et al., 2014; Villeneue et al., 2014a,b; Garccia-Reyero, 2015; Groh and Tollefsen, 2015; Brockmeier et al., 2017; Buesen et al., 2017; Gant et al., 2017; Bridges et al., 2017, Kauffmann et al., 2017; Fay et al., 2017).
The project has led to the successful publication of more than 20 scientific papers (see Publications), over 30 presentations at national and international scientific meetings (see Conference presentations) and almost 10 popular science dissemination activities (see Popular science disseminations).