Piperazine designer drugs induce toxicity in cardiomyoblast h9c2 cells through mitochondrial impairment

Abstract

Abuse of synthetic drugs is widespread among young people worldwide. In this context, piperazine derived drugs recently appeared in the recreational drug market. Clinical studies and case-reports describe sympathomimetic effects including hypertension, tachycardia, and increased heart rate. Our aim was to investigate the cytotoxicity of N-benzylpiperazine (BZP), 1-(3-trifluoromethylphenyl) piperazine (TFMPP), 1-(4-methoxyphenyl) piperazine (MeOPP), and 1-(3,4-methylenedioxybenzyl) piperazine (MDBP) in the H9c2 rat cardiac cell line. Complete cytotoxicity curves were obtained at a 0-20 mM concentration range after 24 h incubations with each drug. The EC50 values (μM) were 343.9, 59.6, 570.1, and 702.5 for BZP, TFMPP, MeOPP, and MDBP, respectively. There was no change in oxidative stress markers. However, a decrease in total GSH content was noted for MDBP, probably due to metabolic conjugation reactions. All drugs caused significant decreases in intracellular ATP, accompanied by increased intracellular calcium levels and a decrease in mitochondrial membrane potential that seems to involve the mitochondrial permeability transition pore. The cell death mode revealed early apoptotic cells and high number of cells undergoing secondary necrosis. Among the tested drugs, TFMPP seems to be the most potent cytotoxic compound. Overall, piperazine designer drugs are potentially cardiotoxic and support concerns on risks associated with the intake of these drugs.

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

“…Similar differences across different hepatic models were already observed and discussed for amphetamines (Dias da Silva et al 2013c). Comparisons with results of other in vitro studies showed that immortalized hepatocytes display a lower threshold for susceptibility to BZP than CAKI-2 renal cells (EC 50 1.57 mg/mL, i.e., 8.91 mM) (Vaughan and Gautam 2011) but higher than H9c2 cardiomyocytes (EC 50 2.33 mM) (Arbo et al 2014) and SH-SY5Y neurons (EC 50 4.92 mM) (Arbo et al 2016). A similar trend was also found for TFMPP in those cell systems, but this drug displayed a significantly higher toxicity toward all cellular models (HepG2 EC 50 310 μM; HepaRG EC 50 450 μM; rat hepatocytes EC 50 140 μM; H9c2 cardiomyocytes EC 50 186 μM; SH-SY5Y neurons EC 50 386 μM) (Arbo et al 2014(Arbo et al , 2016Dias da Silva et al 2015).…”

“…In the future, it would be interesting to test the possible influence of the combination of these drugs in the homeostasis of glutathione (e.g., inhibition of GSH biosynthesis by interaction with γ-glutamylcysteine ligase or GSH synthase). A previous work has already shown that the piperazine drugs have no impact on GSH reductase activity (Arbo et al 2014).…”

“…Our data are consistent with depolarization of mitochondria, ATP depletion and downstream caspase-3 activation (triggering the apoptosis common pathway). Previous studies with piperazines have also demonstrated exaggerated increases in cytosolic Ca 2+ , which ultimately sentences cell to death (Arbo et al 2014(Arbo et al , 2016. Whether the stimuli that condemns cells to death is originated from the inside (mitochondrial pathway) or the outside is yet to be unveiled, but studies conducted on this matter excluded the involvement of caspase-8 in BZP toxicity (Persona et al 2016).…”

“…Recent in vitro studies conducted in cardiomyocytes (Arbo et al 2014), hepatocytes (Dias da and neuronal cells (Arbo et al 2016;Persona et al 2016) have shed some light on the molecular mechanisms implicit in piperazine-elicited toxicity, providing evidence that some derivatives of the group disturb the oxidative and energetic intracellular homeostasis and disrupt the mitochondrial inner membrane potential, which ultimately culminates in the activation of programmed cell death.…”

Hepatotoxicity of piperazine designer drugs: Comparison of different in vitro models

“…A lethal intoxication case with death after massive brain oedema (Balmelli et al, 2001) has also been reported. Recent preliminary evidence suggest that BZP is highly toxic to heart and kidney cell lines, which may explain the symptoms of renal and cardiovascular toxicity in users Monteiro et al, 2013, Arbo et al, 2014. Hepatic liver failure has also been reported after BZP intake (Cole, 2011;Monteiro et al, 2013).…”