Zebrafish larvae as an alternative animal model to study toxicity of New Synthetic Opioids

In forensic toxicology, a problem of global concern is represented by the continuous rapid emergence of New Psychoactive Substances (NPS) due to several factors, including multiplicity of chemical structures, poorly known activity, short half-life in the illicit drug market, and lack of pure standards. Among these problems, the lack of information about metabolism and adverse effects is also of the highest relevance. Nowadays, opioids and, in particular, synthetic opioids are of high concern since they represent one of the fastest-growing groups of NPS. For this reason, a new rapid screening tool is needed to keep up with the timing of releasing these new compounds in the drug market. In the present thesis work, zebrafish larvae have been tested as an alternative animal model with the effort to study the NPS, exploiting the advantages that this model offers compared to other in vivo studies (i.e., high reproducibility, low cost, and less ethical issues). To this aim, the first step of this study was dedicated to developing a new protocol for evaluating the toxicity of new synthetic opioids. Thus, zebrafish larvae have been exposed to a relatively known opioid (i.e., fentanyl) to test the validity of the zebrafish larvae (ZL) model, which had been rarely used in forensic toxicology so far. The investigation involved behavioral and metabolic assay and morphological defect evaluation. In parallel, the mice model was used for comparison. To test behavioral effects, larvae were exposed to 1 and 10 μM concentrations on the fourth-day post-fertilization. The locomotor activity was evaluated as distance moved in the unit of time and registered with a tracking system. Using ZL, it was possible to highlight the presence of several morphological abnormalities when exposed to high fentanyl doses (50, 100 μM). For the metabolic assay, a 1260 Infinity LC coupled to a 6540 Accurate-Mass QTOF spectrometer was used for identification of metabolites after exposing ZL for 24 hours to fentanyl (1 and 10 μM). In a second step, ocfentanil and 2-furanylfentanyl were selected as target compounds. These drugs belong to the class of synthetic opioids, but only a few data were available in the literature regarding their toxicity and remarkably, no data about their metabolic fate. The same behavioral and metabolic assay protocols were applied, and data were compared with those obtained in mice. Overall, the obtained results confirm the validity of ZL for identifying metabolites, and only minor differences in metabolism were observed when compared to mice. The last part of the thesis project was focused on the metabolic assay, and on the analysis of a panel of n=9 New Synthetic Opioids (NSO), namely, AP-237 and 2- methyl-AP-237, isotonitazene, flunitazene, etodesnitazene, metonitazene, metodesnitazene, N-pyrrolidino etonitazene, and butonitazene. On the fourth day post-fertilization, larvae were exposed for 24 hours to 1 μM concentration and analyzed with a Vanquish UPLC coupled with an Orbitrap Fusion™ Lumos™ Tribrid™ Mass Spectrometer. Moreover, an in vivo cell death assay was performed to test and quantify the toxicity level of these compounds: the acridine orange was applied after 24h exposure to 0.1 μM of each drug. The number of fluorescent dots (corresponding to a cluster of apoptotic cells) was quantified: all the tested compounds exhibited a toxicity level significantly higher than the control. The results of this thesis project, clearly support the suitability of the zebrafish larvae model as an effective platform to rapidly assess synthetic opioid toxicity.