Mechanistic insights into the effects of Fluoxetine in Mytilus galloprovincialis using in vivo and in vitro approaches

• Effects and mechanisms of action of the model SSRI Fluoxetine-FLX in mussels • FLX affects immune and lysosomal parameters of hemocytes in vitro • In vivo exposure to environmental concentrations of FLX and its metabolite NFL • In the digestive gland, tissue accumulation and changes in gene expression • FLX acts through multiple molecular pathways, lysosomal system as a main target Antidepressants such as selective serotonin reuptake inhibitors (SSRIs) are pseudo-persistent contaminants in aquatic ecosystems. While their effects and molecular targets are relatively well understood in vertebrates, less information is available in aquatic invertebrates. In this work, the effects and mechanisms of action of Fluoxetine (FLX), a widely prescribed SSRI, were investigated in the model marine bivalve Mytilus galloprovincialis. In vitro exposure of isolated hemocytes to FLX (0.03-0.3-3 µg/mL) induced significant alterations in immune and lysosomal parameters. In mussels exposed to FLX or its active metabolite norfluoxetine NFL (0.5-5-10-50 ng/L, 7 days), a concentration dependent accumulation of either compound was observed in the digestive gland. NFL was also detected in FLX-exposed mussels, indicating biotransformation. FLX and NFL affected transcription of monoamine receptors, and of lysosomal, autophagy, and ceramide related genes, with a distinct pattern for each compound, with FLX mainly inducing downregulation of gene expression. The results demonstrate that in mussels both FLX and NFL act through multiple molecular pathways, pointing at the lysosomal system as a main target for both compounds. These data provide novel mechanistic insights into antidepressant toxicity in a non-target marine invertebrate and contribute to draw Adverse Outcome Pathways for SSRIs in bivalves, that represent foundation species in coastal environments.