New Insights into the neurological effects of chloro-cathinones: Impact in the dopaminergic and cholinergic systems

The consumption of illicit drugs is commonly linked to the dopaminergic system, governed by the neurotransmitter dopamine. This system plays a role in various brain functions such as reward, cognition, and motivation. Nevertheless, drug dependence is intricately connected to the interplay between the cholinergic and dopaminergic systems. The cholinergic system, which is associated to nicotine dependence, hints at its potential involvement in the effects associated with illicit drug consumption. This work aims to disclose new insights into the neurological effects of chloro-cathinones by evaluating their cytotoxic potential on a differentiated human neuroblastoma cell line (SH‑SY5Y) as a model of dopaminergic neurons, and their impact in the cholinergic system by evaluating their capacity to inhibit the enzyme acetylcholinesterase (AChE). 
Twenty cathinones were synthesised, including some of those already reported to the EMCDDA, along with some new isomers that may soon emerge on the illicit drug markets. The viability of differentiated SH‑SY5Y cells, assessed by MTT colorimetry assay after 24 h exposure at different concentrations (0.05-5 mM), showed a concentration-dependent cytotoxicity of all tested cathinones with IC50 values ranging from 0.6 to 2.5 mM, being 4-CBC the most cytotoxic. It should be highlighted that some of these new cathinones tested were more toxic than mephedrone and other controlled drugs such as 4-CMC. 
To understand the mechanism underlying neurotoxicity, whether it correlates with oxidative stress or with mitochondrial events, the production of reactive oxygen species (ROS) and the mitochondrial membrane potential (MMP) were evaluated for the most cytotoxic cathinones at 3 different doses (IC25, IC50 and IC75) after 6 h of treatment. The results suggested, that for the majority of the tested cathinones, the cytotoxic effects seem to be link to an increase in the ROS production. However, for cathinones with 4 carbon atoms in the amino group, such as 3-CBC, 4‑CBC and 3-Cl-DEC, the toxicity is also related with an increase of MMP. 
All tested cahinones demonstrated the ability to inhibit AChE activity, with EC50 values ranging from 0.1 to 2 mM. It is noteworthy that these results are the first evidence of the effect of cathinones in the cholinergic system. 
These findings demonstrate the importance of taking a proactive approach to highlight the health risks associated with NSP consumption.
Fundação para a Ciência e Tecnologia (FCT) is acknowledged for funding the projects: UIDB/04046/2020 (DOI:10.54499/UIDB/04046/2020) and UIDP/04046/2020 (DOI: 10.54499/UIDP/04046/2020) to BioISI-Biosystems & Integrative Sciences Institute; UIDB/00100/2020 (DOI: 10.54499/UIDB/00100/2020) and UIDP/00100/2020 (DOI:10.54499/UIDP/00100/2020) to Centro de Química Estrutural, LA/P/0056/2020 (DOI 10.54499/LA/P/0056/2020) to Institute of Molecular Sciences; UIDP/04292/2020 and UIDB/04292/2020 to MARE-Marine and Environmental Sciences Centre; ARNET (LA/P/0069/2020) and NEURONS4 (2022.09196.PTDC, DOI: 10.54499/2022.09196.PTDC).