The mixture of “ecstasy” and its metabolites is toxic to human SH-SY5Y differentiated cells at in vivo relevant concentrations

Abstract

The neurotoxicity of "ecstasy" (3,4-methylenedioxymethamphetamine, MDMA) is thought to involve hepatic metabolism, though its real contribution is not completely understood. Most in vitro neurotoxicity studies concern isolated exposures of MDMA or its metabolites, at high concentrations, not considering their mixture, as expected in vivo. Therefore, our postulate is that combined deleterious effects of MDMA and its metabolites, at low micromolar concentrations that may be attained into the brain, may elicit neurotoxicity. Using human SH-SY5Y differentiated cells as dopaminergic neuronal model, we studied the neurotoxicity of MDMA and its MDMA metabolites α-methyldopamine and N-methyl-α-methyldopamine and their correspondent glutathione and N-acetylcysteine monoconjugates, under isolated exposure and as a mixture, at normothermic or hyperthermic conditions. The results showed that the mixture of MDMA and its metabolites was toxic to SH-SY5Y differentiated cells, an effect potentiated by hyperthermia and prevented by N-acetylcysteine. As a mixture, MDMA and its metabolites presented a different toxicity profile, compared to each compound alone, even at equimolar concentrations. Caspase 3 activation, increased reactive oxygen species production, and intracellular Ca(2+) raises were implicated in the toxic effect. The mixture increased intracellular glutathione levels by increasing its de novo synthesis. In conclusion, this study demonstrated, for the first time, that the mixture of MDMA and its metabolites, at low micromolar concentrations, which represents a more realistic approach of the in vivo scenario, elicited toxicity to human SH-SY5Y differentiated cells, thus constituting a new insight into the context of MDMA-related neurotoxicity.

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

“…The MTT reduction assay was performed as previously described (Barbosa et al, 2014b). This assay measures cellular dehydrogenases activity, an indicator of metabolically active mitochondria, and therefore of cell viability.…”

“…The intracellular reactive oxygen and nitrogen species production was monitored by means of the dichloro-dihydrofluorescein diacetate assay as previously described (Barbosa et al, 2014b). Cells were seeded as described for cytotoxicity and differentiated for 6 days.…”

“…After centrifugation (210 g, 5 min, 4°C), the supernatant was removed and the cell pellet was lysed with 5% HClO 4 and centrifuged (16 000 g, 10min, 4°C). The total GSH levels were evaluated by the DTNB/oxidized GSH (GSSG) reductase assay, as previously described (Barbosa et al, 2014b). The stoichiometric formation of 5-thio-2-nitrobenzoic acid was followed every 10 s for 3 min at 415 nm and at 30°C, and then compared with a standard curve.…”

Piperazine designer drugs elicit toxicity in the alternative in vivo model Caenorhabditis elegans

“…Oxidative stress, which occurs when ROS generation leads to an imbalance between ROS and antioxidants, is involved in the mechanism of toxicity of a large number of compounds (Sies, ). It is a well described mechanism that plays an essential role in the toxic effects of various amphetamine derivatives that induce the formation of highly reactive species (Barbosa et al, ; Silva, Silva, & Carmo, ). Interestingly, piperazine designer drugs did not induce ROS formation in the in vitro models using rat cardiomyoblasts H9c2 (Arbo et al, ) and human neuroblastoma SH‐SY5Y cells (Arbo et al, 2016b), but increased free radicals in primary rat hepatocytes (Dias‐da‐Silva et al, ; Dias‐da‐Silva et al, ).…”

Monoaminergic toxicity induced by cathinone phthalimide: An in vitro study

“…However, it is not likely that the preparation used in the present study generated any metabolites. Even though metabolites of CP have not been investigated to date, it is reasonable investigate their presence and impact on toxicity as MDMA metabolites are reported to induce toxicity at lower concentrations than MDMA alone [8, 16, 39]. Because of the structural similarity between CP and MDMA, potential effects of CP metabolites cannot be ruled out and also warrant further investigation.…”