The synthetic cannabinoid ADB-FUBINACA modulates epigenetic mechanisms during in vitro neuronal differentiation at biologically relevant concentrations

ADB-FUBINACA is a synthetic cannabinoid (SC) commonly detected in seizures within the EU. It displays a 140-fold higher affinity to cannabinoid receptor 1 (CB1R) than the phytocannabinoid tetrahydrocannabinol (THC), and has already been associated with acute intoxications and deaths. Previously, we showed that this SC increased in vitro neurodifferentiation of NG108-15 neuroblastoma x glioma cells. Here, we assessed the effects of ADB-FUBINACA on epigenetic modifications, namely global DNA methylation and histone H3 acetylation, in differentiating neuronal cells, as these mechanisms play a crucial role during neurogenesis.

Differentiation of NG108-15 cells was induced in serum-starved (1% FBS) cell culture medium supplemented with 30μM forskolin and 10μM retinoic acid. Global DNA methylation and histone H3 acetylation levels were determined using colorimetric and fluorometric kits, respectively, in 3 distinct SC exposure settings: (A) one-shot SC addition at day 0, sample collection at day 3; (B) one-shot addition at day 3, sample collection at day 6; (C) SC addition at days 0 and 3, sample collection at day 6. ADB-FUBINACA (TicTac Communications Ltd,UK) was tested at non-toxic, in vivo-relevant concentrations (1pM – 1µM).

ADB-FUBINACA increased global DNA methylation following treatment (A) (1nM, one-shot addition), but decreased this parameter at day 6 in treatments (B) (1µM, one-shot addition) and (C) (1µM, multiple additions). Global histone H3 acetylation levels were increased at all concentrations tested in treatment (A) and in treatment (B) following exposure to 1nM ADB-FUBINACA, and decreased in treatment (C) in response to 1pM ADB-FUBINACA.

Our data suggest that global changes in DNA methylation and histone H3 acetylation patterns may contribute to the ADB-FUBINACA-mediated enhancement of neuronal differentiation, thus highlighting epigenetic changes as likely drivers of SC-triggered neurogenesis. However, further research is required to clarify the mechanisms underlying such effects, as well as the specific targets epigenetically modified upon SC exposure.

This work was funded by FEDER and by national funds from Fundação para a Ciência e a Tecnologia (FCT) in the scope of the project NeuroSCANN (POCI-01-0145-FEDER-029584) and the grants UIDB/04378/2020 (Research Unit on Applied Molecular Biosciences-UCIBIO) and LA/P/0140/2020 (Associate Laboratory Institute for Health and Bioeconomy – i4HB). RFM acknowledges FCT for his Ph.D. grant 2020.07135.BD.