Human Stem Cell-Derived Neurospheroids and a Multi-Assay Strategy Provide Brain Cytotoxicity New Insights from ADB-FUBIATA Exposure

Synthetic cannabinoids (SCs) are chemical and inhomogeneous broad group of new psychoactive substances associated with severe toxicity and death. This study aims to characterize neurotoxicological profile of ADB-FUBIATA, third-generation indole-3-acetamide-type SCRA, applying 3D human stem-cell-derived neuronal (3D-hNLCs) spheroids testing at different concentrations (25-2000 nM) and exposure times. i)Single exposure to ADB-FUBIATA induced decrease of: cell viability (2000 nM after 24-h and ≥ 800 nM after 48-h), spheroid compactness (≥ 1600 nM after 24-h and ≥ 200 nM after 48-h), collagen I fibre content (25–35%; 400–800 nM; after 24-48-h), E-cadherin expression (40% from 400 nM after 48-h), enolase (800 nM after 24-h and 400 nM after 48-h) and MAP-2 expression (400–800 nM after 48-h) and increase of interstitial space between cells (400–800 nM). NF-κB p50 and CB1 receptor were not modified. ii)Repeated administration at fixed doses caused at the end of treatment (day 10) several concentration-dependent effects: cell mortality (≥ 200 nM), spheroid compactness loss (≥ 50 nM) and disaggregation, cells detachment from the main body (400–800 nM) and spheroid diameter/size reduction (400–800 nM). 3D-hNLC spheroids reproduce and simulate microenvironment conditions more closely the native human brain and provide relevant information about mechanism-based and biological responses induced by ADB-FUBIATA after both short and long-term repeated exposure, contributing to the development of a more accurate toxicological evaluation method for different SCs. Findings show a translational relevance since data may be transferable to human context expanding the mechanistic understanding of the cellular responses to understand the clinical effects associated with persistence SC use.