Exploring endocrine effects of microcystins on mussels by the adverse outcome pathway approach

Cyanobacterial blooms, driven by human activity in aquatic ecosystems, are increasingly reported in estuarine environments and often accompanied by the release of cyanotoxins. Among these, microcystins (MCs) are the most extensively studied due to their widespread occurrence and high toxicity. MCs are known as potent and specific inhibitors of eukaryotic protein serine or threonine phosphatases 1 and 2A (PP1 and PP2A). Beyond their hepatotoxic effects, several studies have reported reproductive toxicity of MCs in aquatic and terrestrial organisms, including fish, frogs, and birds. In female zebrafish, for example, MCs exposure has been shown to interfere with endocrine function and oogenesis, ultimately impairing reproductive performance. Mussels are globally recognized as sentinel species in coastal monitoring programs because of their filter-feeding behavior and capacity to bioaccumulate environmental pollutants over time. However, this also makes them potential vectors for trophic transfer of toxins to predators and humans. The adverse outcome pathway (AOP) framework offers a structured way to link molecular-level interactions of toxicants with apical endpoints relevant to risk assessment and regulatory decision-making. AOPs consist of a sequence of key events (KEs), connected through key event relationships (KERs), spanning from the molecular initiating event (MIE) to the adverse outcome (AO). In mussels, the nongenomic estrogen signaling pathway and the steroidogenesis pathway have been proposed as potential mechanisms of action for endocrine-disrupting compounds (EDCs), and may serve as MIEs relevant to MC exposure. Published AOs following EDC exposure include reduced fertilization success and decreased production of viable eggs. However, despite these insights, information on intermediate key events in mussels exposed to EDCs, including MCs, remains limited. Therefore, the aim of this study is to investigate the endocrine toxicity of MCs in mussels, using published MIEs and AOs of EDCs as a foundation. By identifying and characterizing missing KEs, we seek to complete the AOPs for endocrine disruption in mussels and contribute to the development of integrated approaches to testing and assessment (IATA) for environmental risk evaluation.