Overall, the work on developing adverse outcomes pathways (AOPs) for endocrine disruption in Daphnia magna is well underway. Two AOPs, entitled “Ecdysone receptor (EcR) agonism leading to incomplete ecdysis associated mortality” (https://aopwiki.org/wiki/index.php/Aop:4) and “Juvenile hormone (JHR) receptor agonism leading to male offspring induction associated population decline” (https://aopwiki.org/wiki/index.php/Aop:201) have been defined and submitted to the AOP Wiki. 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 (AOP no. 201) 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 focus on Ionotropic gamma-aminobutyric acid receptor (GABAR) activation mediated neurotransmission inhibition leading to mortality, that address neurotoxic effects of GABA receptor agonists has been developed as 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. Qualitative and quantitative weight of evidence (WoE) assessment of the AOPs are currently ongoing. One review paper describing the development of the EcR AOP is close to finalization (Song et al., 2016a), a second review paper on the JHR AOP is under development (Song et al., 2016b) and a paper describing the GABAR-mediated effects has been submitted (Song et al., 2016c).
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, and a manuscript is currently being compiled for publication (Evenseth et al. 2016). A specific structural alert (KNIME) workflow to identify potential EcR agonists and antagonists have been developed (Mellor et al. 2016). The work is currently being summarized into a manuscript for submission in 2016.
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. 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., In prep.). In addition to known EcR agonists, emamectin benzoate (EMB), a commercially available anti-sea lice chemical was studied (Song et al. 2016, In press). This chemical was chosen as it’s 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. 2016c). 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 (developed by the collaborator Iguchi, T. et al., NIBB, Japan). 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 (Song et al., In prep.). These studies are in the process of being compiled for publication. 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). A number of papers has been generated from these studies and published in peer-review journals.
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 to support development of an AOP for UV-B.
In WP4, predictive approaches are under development using gene expression profiles. Gene expression signatures representing the response of D. magna to both molting and juvenile hormones were developed. 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 were 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 (Danilo et al. 2016). An effect-based database has been constructed to support WoE considerations, and will be considered used to support linking changes to MiE and KE to higher level effects (e.g. the AO). Two scientific papers will be submitted in 2016 to present how different methods/approaches can be used to develop AOPs (Fay et al., 2016, Brockmeier et al. 2016).
A number of scientific papers have been submitted for publication in peer-review journals (see publications).