Biotransformation studies and behavioral assays of new psychoactive substances through in vivo models using Zebrafish (Danio rerio)

• Grant number: 22/00037-0
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)
• Start date: April 01, 2023
• Status: Discontinued
• Field of knowledge: Health Sciences - Pharmacy - Toxicological Analysis
• Principal Investigator: José Luiz da Costa
• Grantee: Leonardo Costalonga Rodrigues
• Host Institution: Faculdade de Ciências Farmacêuticas (FCF). Universidade Estadual de Campinas (UNICAMP). Campinas , SP, Brazil
• Associated scholarship(s): 25/00898-4 - In vitro toxicokinetics of cyclopropyl amphetamines (CPAs), BE.EP.DD 23/07372-2 - Steroids screening for reducing opioid self-administration and their possible depressogenic effects in adult zebrafish, BE.EP.DD

Abstract

Currently, the New Psychoactive Substances (NPS) are gaining prominence among the substances used for recreational use. The emergence of NPS brings a challenge to toxicological analysis, since the demand for the identification of such substances is increasing. In toxicological studies, assays using zebrafish (Danio rerio) have been gaining prominence, due to several advantages over other in vivo models. Acute toxicity, biotransformation and behavior studies observing specific responses, according to the pattern of position in the aquarium, fish movement and aggressiveness, can be conducted. Thus, the aim is to study the acute toxicity, biotransformation and effects on the behavior of synthetic cannabinoids, synthetic cathinones, ketamine derivatives and new benzodiazepines, using in vivo experiments with zebrafish. In this way, the project will follow the recommendations of the OECD 236 and the manual for raising zebrafish in vivarium for obtaining, maintaining and using the animals. To determine the toxicity of NPS in stages of embryonic development, an embryo toxicity test will be performed, consisting of early exposure of the embryo to the substance. During this period, up to four apical changes are recorded as indicators of toxicity. The larval stage toxicity test will also be carried out, with the objective of determining the maximum tolerated concentration to be used in metabolism and behavioral studies. For studies of metabolism in the larval stage, ten larvae 4 dpf will be placed per well, with assays in triplicates. The larvae will be euthanized in ice water and, both these and the medium in which they are, will be subjected to a method of extraction and analysis by liquid chromatography tandem mass spectrometry (LC-MS/MS) and liquid chromatography-high resolution mass spectrometry (LC-HRMS). For the study of metabolism in the adult stage, the zebrafish water tank metabolism (ZWT) procedure will be followed, with the addition of the solution containing the substance of interest to the tank. The samples will be submitted to the extraction method and analysis by LC-MS/MS and LC-HRMS. Finally, behavioral tests will be conducted with the larval stage (thigmotaxis, swimming test and light-dark test) and with the adult stage (light-dark test, mirror test, open field, new aquarium test and social preference). Control groups (without exposure to the substance) will be used for comparison purposes, the exposure groups will be exposed to each substance of interest for 20 minutes and submitted to the procedures. All larval stage behavioral tests will be performed in triplicate and adult tests will use 12 animals per exposure substance each. Through the acute toxicity test in embryos, it is expected to determine the level of toxicity of the NPS tested in the stages of embryonic development of zebrafish, as well as the determination of the LC50 for them. In turn, with the acute toxicity test in the larval stage, it is expected to define the working test dose, being it, the highest concentration tested in which there are no signs of toxicity such as sedation and acute locomotor impairment. With the biotransformation assays in larval and adult stages, it is expected to identify, by the LC-MS/MS and LC-HRMS techniques, the possible biotransformation products formed from the exposure of zebrafish to the NPS under study. For this, we seek to develop, optimize, and validate an analytical methodology for the substances in question and their biotransformation products. Finally, with behavioral studies carried out with zebrafish in larval and adult stages, we seek to identify the acute behavioral pattern in the face of exposure to such NPS, according to preferred location in the aquatic environment, position pattern in the aquarium, movement of fish and aggression. (AU)