title: 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.
abstract: 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 (EC502.20 and 6.60 mM, respectively) and TFMPP (EC500.14 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 Δψm disruption). The present work clearly demonstrates that potentially harmful interactions among BZP and TFMPP are expected when these drugs are taken concomitantly.
authors.affiliation: UCIBIO, REQUIMTE, Laboratory of Toxicology, Departament of Biological Sciences, Faculty of Pharmacy, University of Porto, Rua de Jorge Viterbo Ferreira, 228, 4050-313, Porto, Portugal. diana.dds@gmail.com.
authors.forename: Diana
authors.identifier: http://orcid.org/0000-0002-7331-9157
authors.initials: D
authors.lastname: Dias da Silva
authors.affiliation: UCIBIO, REQUIMTE, Laboratory of Toxicology, Departament of Biological Sciences, Faculty of Pharmacy, University of Porto, Rua de Jorge Viterbo Ferreira, 228, 4050-313, Porto, Portugal.
authors.forename: Maria João
authors.initials: MJ
authors.lastname: Silva
authors.affiliation: UCIBIO, REQUIMTE, Laboratory of Toxicology, Departament of Biological Sciences, Faculty of Pharmacy, University of Porto, Rua de Jorge Viterbo Ferreira, 228, 4050-313, Porto, Portugal.
authors.forename: Patrícia
authors.initials: P
authors.lastname: Moreira
authors.affiliation: UCIBIO, REQUIMTE, Laboratory of Toxicology, Departament of Biological Sciences, Faculty of Pharmacy, University of Porto, Rua de Jorge Viterbo Ferreira, 228, 4050-313, Porto, Portugal.
authors.forename: Maria João
authors.initials: MJ
authors.lastname: Martins
authors.affiliation: UCIBIO, REQUIMTE, Laboratory of Toxicology, Departament of Biological Sciences, Faculty of Pharmacy, University of Porto, Rua de Jorge Viterbo Ferreira, 228, 4050-313, Porto, Portugal.
authors.forename: Maria João
authors.initials: MJ
authors.lastname: Valente
authors.affiliation: UCIBIO, REQUIMTE, Laboratory of Toxicology, Departament of Biological Sciences, Faculty of Pharmacy, University of Porto, Rua de Jorge Viterbo Ferreira, 228, 4050-313, Porto, Portugal.
authors.forename: Félix
authors.initials: F
authors.lastname: Carvalho
authors.affiliation: UCIBIO, REQUIMTE, Laboratory of Toxicology, Departament of Biological Sciences, Faculty of Pharmacy, University of Porto, Rua de Jorge Viterbo Ferreira, 228, 4050-313, Porto, Portugal.
authors.forename: Maria de Lourdes
authors.initials: ML
authors.lastname: Bastos
authors.affiliation: UCIBIO, REQUIMTE, Laboratory of Toxicology, Departament of Biological Sciences, Faculty of Pharmacy, University of Porto, Rua de Jorge Viterbo Ferreira, 228, 4050-313, Porto, Portugal. helenacarmo@ff.up.pt.
authors.forename: Helena
authors.initials: H
authors.lastname: Carmo
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D004347:Drug Interactions; D004357:Drug Synergism; D004734:Energy Metabolism / Q000187:drug effects; D022781:Hepatocytes / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D006801:Humans; D008297:Male; D008928:Mitochondria / Q000187:drug effects*; D018384:Oxidative Stress / Q000187:drug effects*; D010879:Piperazines / Q000008:administration & dosage / Q000633:toxicity*; D017208:Rats, Wistar; D013045:Species Specificity
chemical_list: C461488:N-benzylpiperazine; D010879:Piperazines; C018406:1-(3-trifluoromethylphenyl)piperazine
keywords: 1-(3-Trifluoromethylphenyl)piperazine (TFMPP); HepaRG cells; Liver toxicity; Mixture effects; N-Benzylpiperazine (BZP); Primary rat hepatocytes