Synthetic cannabinoids ADB-FUBINACA and THJ-2201 dysregulate osteogenesis-related mechanisms in vitro

Background: Synthetic cannabinoids (SCs) are popular new psychoactive substances (NPS) designed to emulate, with higher potency, the pharmacological action of phytocannabinoids, like tetrahydrocannabinol (THC). These substances target the endocannabinoid system, which in turn is involved in regulating several biological processes, including bone remodeling. However, their impact on bone tissue dynamics remains mostly unexplored. Here, we hypothesized that SCs interfere with osteogenesis, having assessed the effects of two SCs commonly identified in seizures and intoxication reports, ADB-FUBINACA (ADB) and THJ-2201 (THJ), on in vitro osteogenesis-related mechanisms, at in vivo-relevant concentrations.Methods: MC3T3-E1 mouse pre-osteoblasts were exposed to in vivo-relevant concentrations (1nM and 1μM) of ADB and THJ during the cells’ differentiation into osteoblasts (induced with 50µM ascorbic acid and 20mM β-glycerophosphate). Alkaline phosphatase (ALP) activity (an early marker of osteoblast differentiation) was colorimetrically measured after 7, 10, and 14 days using a colorimetric assay. Vehicle (0.1% DMSO) and positive (300 μM naproxen) controls were also tested. To ascertain the CB1 and CB2 involvement in SC-induced ALP activity, 500nM SR141716A or SR144258 (selective CB1 and CB2 antagonists, respectively), were added 20 min before SCs’ addition. The effect of THJ-2201 on MC3T3-E1 cell proliferation was measured by sulforhodamine B (SRB) staining during 72h.Results: We observed a significant increase in ALP activity on day 10 when cells were exposed to both 1nM and 1μM ADB, showing approximately 2.5- and 2-fold enhancements, respectively, compared to the vehicle control. However, by day 14, only the cells exposed to 1nM ADB exhibited a continued rise in ALP activity, about 1.5-fold higher than the control. Notably, both CB1 and CB2 appeared to play a role in mediating these changes, as pre-incubation of cells with SR141716 and SR144258 effectively restored ALP activity to basal levels. Interestingly, the impact of THJ on ALP activity differed, as it did not induce any noticeable changes in ALP activity. However, it's worth noting that MC3T3-E1 cells exposed to both THJ concentrations (1nM and 1μM) exhibited reduced proliferation, compared to the vehicle control.Conclusions: Further research is ongoing to elucidate the molecular mechanisms underlying the impact of ADB and THJ on bone remodeling. Nevertheless, these initial findings are indicative that structurally different SCs may interfere with osteogenesis through distinct mechanisms, underpinning the need to better understand the toxicological outcomes of these substances on bone tissue dynamics. Funding: This work was funded by Fundação para a Ciência e a Tecnologia (FCT) in the scope of grants UIDB/04378/2020 (UCIBIO) and LA/P/0140/2020 (i4HB). JPS acknowledges FCT for research contract (under Scientific Employment Stimulus) 2021.01789.CEECIND/CP1662/CT0014.