In vitro hepatotoxicity of 'Legal X': the combination of 1-benzylpiperazine (BZP) and 1-(m-trifluoromethylphenyl)piperazine (TFMPP) triggers oxidative stress, mitochondrial impairment and apoptosis

N-Benzylpiperazine (BZP) and 1-(3-trifluoromethylphenyl)piperazine (TFMPP) are two synthetic phenylpiperazine analogues that have been frequently commercialized in combination as an alternative to ecstasy ('Legal X'). Despite reports of several clinical complications following the use of these drugs in association, few studies have been conducted so far to elucidate their combined toxicity. The present study was aimed at clarifying the cytotoxic effects of mixtures of BZP and TFMPP in vitro. Human-derived HepaRG cells and primary rat hepatocytes were exposed to the drugs, individually or combined at different mixture ratios, and cytotoxicity was assessed by the MTT assay. Mixture additivity expectations were calculated by the independent action and the concentration addition (CA) models and compared with the experimental outcomes. To delineate the mechanisms underlying the elicited effects, a range of stress endpoints was evaluated, including oxidative stress, energetic imbalance, and metabolic interactions. It was observed that primary rat hepatocytes are more sensitive than HepaRG cells to the toxicity of BZP (EC50 2.20 and 6.60 mM, respectively) and TFMPP (EC50 0.14 and 0.45 mM, respectively). For all BZP-TFMPP combinations tested, CA was the most appropriate model to predict the mixture effects. TFMPP proved to act additively with BZP to produce significant hepatotoxicity (p < 0.01). Remarkably, substantial mixture effects were observed even when each drug was present at concentrations that were harmless individually. In primary hepatocytes, a small deviation from additivity (antagonism) was observed toward the upper range of the concentration-response curve. GC/MS data suggest that a metabolic interaction may be at a play, as the mixture favors the metabolism of both substances, to a significant extent in the case of BZP (p < 0.05). Also, our results demonstrate the influence of oxidative stress and energetic imbalance on these effects (increase in RNS and ROS production, decrease in intracellular GSH/GSSG, ATP depletion and mitochondrial Delta psi m disruption). The present work clearly demonstrates that potentially harmful interactions among BZP and TFMPP are expected when these drugs are taken concomitantly.