In vitro neurotoxicity evaluation of piperazine designer drugs in differentiated human neuroblastoma SH‐SY5Y cells

Abstract

Abuse of synthetic drugs is widespread worldwide. Studies indicate that piperazine designer drugs act as substrates at dopaminergic and serotonergic receptors and/or transporters in the brain. This work aimed to investigate the cytotoxicity of N-benzylpiperazine, 1-(3-trifluoromethylphenyl)piperazine, 1-(4-methoxyphenyl)piperazine and 1-(3,4-methylenedioxybenzyl)piperazine in the differentiated human neuroblastoma SH-SY5Y cell line. Cytotoxicity was evaluated after 24 h incubations through the MTT reduction and neutral red uptake assays. Oxidative stress (reactive oxygen and nitrogen species production and glutathione content) and energetic (ATP content) parameters, as well as intracellular Ca(2+), mitochondrial membrane potential, DNA damage (comet assay) and cell death mode were also evaluated. Complete cytotoxicity curves were obtained after 24 h incubations with each drug. A significant decrease in intracellular total glutathione content was noted for all the tested drugs. All drugs caused a significant increase of intracellular free Ca(2+) levels, accompanied by mitochondrial hyperpolarization. However, ATP levels remained unchanged. The investigation of cell death mode revealed a predominance of early apoptotic cells. No genotoxicity was found in the comet assay. Among the tested drugs, 1-(3-trifluoromethylphenyl)piperazine was the most cytotoxic. Overall, piperazine designer drugs are potentially neurotoxic, supporting concerns on risks associated with the abuse 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

“…Degradation of DNA following oxidative stress provoked by BZP and TFMPP was already reported (Arbo et al 2016;Persona et al 2016). On the other hand, reactive species are also able to target mitochondria, disturbing the sequestration of Ca 2+ within the mitochondrial matrix and impairing Δψm that is crucial for the functional integrity of mitochondrial electron transport chain and, therefore, for production of ATP by oxidative phosphorylation (Dias da Silva et al 2013a, c).…”

“…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). Accordingly, the results from the current study indicate that TFMPP is approximately 7× more detrimental than BZP to primary cells and 15× to HepaRG cells.…”

“…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).…”

“…In either case, excessive apoptotic activation might lead to serious complications (e.g., autoimmune deregulation, neurodegenerative diseases, cancer, etc.). It is also possible that piperazine designer drugs preferentially die by apoptosis (Arbo et al 2016), but necrotic pathways activation under more stressful cell conditions should not be excluded (Dias da Silva et al 2014a;Dias da Silva et al 2013a).…”

“…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.…”