The impact of AMB-FUBINACA on beta-galactosidase: contrasting responses in different neuronal in vitro models

Synthetic cannabinoids (SC) have emerged as a prominent category of new psychoactive drugs, popular among users due to their diversity and low price. While sharing similarities with D9-tetrahydrocannabinol (THC), the primary psychoactive principle of cannabis, SC display heightened potency in their interaction with the cannabinoid receptors. Recent findings suggesting accelerated ageing in cannabis consumers, coupled with THC’s documented increased ß-galactosidase activity in human fibroblasts, a first-line marker of cellular senescence, prompt the inquiry into whether SC share this ability.

Two in vitro neuronal models were used for this investigation, the human neuroblastoma cell line SH-SY5Y, and primary hippocampal cultures (PHC) isolated from the hippocampus of Wistar rat embryos on embryonic day 18-19. For SH-SY5Y cells, the SC AMB-FUBINACA (AMB-FUB) was tested at 1 nM and 1 µM for 10 cell-doubling passages (between passages 24 and 34), with the exposure to the drug renovated at each passage; ß-galactosidase activity and cell cycle analysis were assessed at passages 24, 28, and 34. PHC were exposed to 1 µM, 1 nM, and 1 pM AMB-FUB starting at either day-in-vitro (DIV) 3 or 7, and continued until DIV 21, when ß-galactosidase activity was assayed. ß-galactosidase activity was determined with a commercially available kit (Enzo Life Sciences), and cell cycle analysis was conducted by flow cytometry in cells labeled with propidium iodide (only in SH-SY5Y).

Exposure of PHC to AMB-FUB led to a reduction in ß-galactosidase of up to ~30% activity, compared to solvent control (0.02% DMSO), in both exposure protocols. In contrast, in SH-SY5Y cells, AMB-FUB exposure did not significantly alter either of the measured endpoints.
The reasons for these differential effects require further investigation. Of particular interest would be to determine the dependence on cannabinoid receptor-activation. Additional senescence markers need to be assayed and a comparison with THC is also deemed necessary for a more detailed understanding on the potential neuronal aging effects of AMB-FUB and other SC.

Acknowledgements:
This work was supported by FCT—Fundação para a Ciência e a Tecnologia (projects 
UIDB/04378/2020 and UIDP/04378/2020 of the Applied Molecular Biosciences—UCIBIO), the project LA/P/0140/2020 of the Associate Laboratory Institute for Health and Bioeconomy—i4HB, the PhD grant 2020.04493.BD (RRB) and research contract (under Scientific Employment Stimulus) 2021.01789.CEECIND/CP1662/CT0014 (JPS)