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

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.