Beyond opioid and SNRI-like actions: Proteomic insights into the effects of tramadol and O-desmethyltramadol in zebrafish larvae

The presence of pharmaceuticals in natural habitats is an increasing concern. In particular, the antidepressant tramadol (TRA) and its metabolite o-desmethyltramadol (OTRA) have become ubiquitous compounds in aquatic ecosystems. However, investigation of their impact on fish, particularly on their proteome, still needs attention. Therefore, this work assesses the alterations in the proteome of zebrafish larvae caused by TRA or OTRA exposure. Zebrafish larvae (0-3 h post-fertilization, hpf) were exposed to 0.1 or 100 μg/L of TRA or OTRA until reaching 168 hpf. The larvae proteome was then investigated through shotgun proteomics, employing the filter-aided sample preparation method (FASP) followed by high-throughput LC-MS/MS analysis. Coupling mass spectrometry quantification with a saturated orthogonal multiple linear regression analysis provided identification of differentially expressed proteins (DEPs). Finally, the functional analysis of these DEPs was performed with STRING-DB online tool to identify possible molecular pathways affected by the treatments. A total of 162 DEPs were identified, with the highest number observed in larvae exposed to the lower TRA concentration. These highlights possible non-monotonic responses and supports previously described effects for other endpoints, which may affect the serotonergic, noradrenergic and opioid systems. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis also indicated putative alterations in metabolic pathways e.g., Glycolysis/Gluconeogenesis, Tricarboxylic Acid Cycle (TCA), Oxidative phosphorylation, Pyruvate metabolism, and Carbon metabolism. Our results suggest that TRA and OTRA exposure may impact vital biological processes in fish larvae, emphasizing the need for adequate integrated monitoring of tramadol, its metabolites, and effects in aquatic ecosystems.