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Background: Piperazine derivatives such as BZP and TFMPP have been used as “party pill” substitutes for MDMA and are associated with neurological and cardiovascular toxicity. While their psychoactive effects are largely attributed to monoaminergic mechanisms, the cellular pathways underlying their toxicity are less well defined. Previous in vitro studies indicate mitochondrial dysfunction and oxidative stress, but potential effects on the neuronal cytoskeleton, specifically microtubules, have not been systematically investigated.

Methods: The effects of MeOPP, BZP, pFPP and TFMPP were evaluated in retinoic acid–differentiated P19 mouse embryonal carcinoma–derived neurons using complementary assays of cell viability (calcein-AM), metabolic activity (MTT), membrane integrity (LDH), mitochondrial membrane potential (TMRE) and βIII-tubulin immunofluorescence. Key findings were evaluated in differentiated human SH-SY5Y neuroblastoma cells and in Caco-2 human colorectal adenocarcinoma cells. Tubulin polymerization was assessed in a complementary cell-free assay.

Results: All compounds induced concentration-dependent toxicity, with marked differences in potency and efficacy. TFMPP was the most active compound across endpoints, producing early and sustained loss of mitochondrial membrane potential followed by reduced viability, cytoskeletal changes and increased membrane damage. BZP and pFPP showed moderate toxicity at higher concentrations, whereas MeOPP had limited effects. Time-course analysis demonstrated that mitochondrial depolarization preceded membrane damage. Reduced βIII-tubulin immunofluorescence in neuronal cells, together with inhibition of tubulin polymerization in a cell-free system, is consistent with effects on microtubule-related processes. Similar toxicity patterns were observed in SH-SY5Y cells, and cytotoxic effects of BZP and TFMPP were also detected in Caco-2 cells.

Conclusions: Piperazine derivatives are associated with cellular toxicity characterized by early mitochondrial dysfunction and subsequent effects on the neuronal cytoskeleton. TFMPP showed the most consistent activity across models. The findings indicate that microtubule-related processes may contribute to toxicity and support further mechanistic studies beyond monoaminergic pathways.