Neurotoxicity-Based Toxicometabolomics of N‑Ethyl Pentedrone Using Zebrafish as an In Vivo Model.

New psychoactive substances (NPS), particularly synthetic cathinones, have gained increasing attention due to their widespread recreational use and associated public health risks. Within this class, N-ethyl pentedrone (NEP) has been linked to cases of severe intoxication; however, its metabolism and neurobiological effects have remained poorly characterized. This study aimed to investigate the metabolism and neurotoxicological effects of NEP using the Zebrafish Water Tank protocol, an established alternative model for evaluating the toxicological properties of psychoactive substances. Liquid chromatography-high-resolution mass spectrometry (LC-HRMS) was employed to characterize NEP metabolites in exposure water and zebrafish brain tissue, complemented by a toxicometabolomic approach to elucidate adverse events triggered in the central nervous system. The analysis revealed metabolic processes of NEP primarily through N-dealkylation, β-ketone reduction, hydroxylation, and O-glucuronidation. Metabolites were identified in exposure water (n = 3) and in brain tissue (n = 7). Untargeted toxicometabolomics revealed six statistically significant differentially expressed metabolites between the exposed and control animals. Four annotated metabolites were found upregulated in NEP-exposed zebrafish: propionylcarnitine (p = 0.001, fold change (FC) = 2.2), l-kynurenine (p = 0.024, FC = 2.9), adenylyl(3'-5')-cytidine (p = 0.027, FC = 2.1), and cytidine (p = 0.028, FC = 2.5), whereas two were downregulated: putatively PI-(19:1-(9Z)/0:0) (p = 0.032, FC = 0.2) and an identified compound (p = 0.034, FC = 0.3). Altogether, these findings suggest neurochemical alterations induced by NEP exposure involving disruptions in neurotransmitter biosynthesis and function, energy metabolism, and oxidative stress responses. Furthermore, changes in lipid metabolism and mitochondrial function highlight the potential mechanisms underlying the observed neurotoxicity. Overall, our findings provide new insights into the metabolism and neurobiological effects of NEP, underscoring its potential neurotoxicity and associated mechanisms. Additionally, this study reinforces the utility of zebrafish as a model for investigating the pharmacokinetics and toxicodynamics of NPS.