From ionic homeostasis modulation to oxidative stress: toxicity pathways of 4th-generation synthetic cathinones

New Psychoactive Substances (NPS), particularly synthetic cathinones, represent a growing toxicological concern due to their rapid emergence and poorly understood mechanisms of action. This study explores the neurotoxic and cytotoxic effects of two structurally related cathinones—3-methylmethcathinone (3-MMC) and 3-chloromethcathinone (3-CMC)—in differentiated SH-SY5Y human neuroblastoma cells, used as an in-vitro neuronal model. Through combined biochemical, electrophysiological, and imaging approaches, cell viability (MTT, LDH), intracellular calcium dynamics (Fura-2AM), ion current modulation (patch- and current-clamp), and stress responses (Western blot) were evaluated, alongside mitochondrial reactive oxygen species (ROS) production. Results indicate distinct toxicity mechanisms. 3-MMC primarily induces functional neuronal alterations without immediate cytotoxicity. At sub-toxic doses, it causes sustained depolarization, enhanced sodium currents, reduced potassium currents, and selective inhibition of the reverse Na⁺/Ca²⁺ exchanger mode, leading to elevated intracellular Ca²⁺ and neuronal hyperexcitability. These alterations trigger endoplasmic reticulum (ER) stress and activation of apoptotic signaling via BiP and caspase-9. In contrast, 3-CMC shows a strongly cytotoxic profile, significantly reducing cell viability and increasing LDH release even at low concentrations. Its toxic action is linked to marked mitochondrial ROS accumulation, indicating that oxidative stress and mitochondrial dysfunction are central to its mechanism. In summary, while 3-MMC acts mainly as a neurofunctional disruptor, 3-CMC exerts direct oxidative cytotoxicity. These findings highlight how minor structural differences among synthetic cathinones profoundly influence their toxicological outcomes and emphasize the need for integrated functional and viability-based assessments in evaluating emerging NPS.