Pro-oxidant effects of Ecstasy and its metabolites in mouse brain synaptosomes

REQUIMTE (Rede de Química e Tecnologia), Laboratório de Toxicologia, Departamento de Ciências Biológicas, Faculdade de Farmácia, Universidade do Porto, Porto, Portugal.
British Journal of Pharmacology (Impact Factor: 4.84). 04/2011; 165(4b):1017-33. DOI: 10.1111/j.1476-5381.2011.01453.x
Source: PubMed


3,4-Methylenedioxymethamphetamine (MDMA or 'Ecstasy') is a worldwide major drug of abuse known to elicit neurotoxic effects. The mechanisms underlying the neurotoxic effects of MDMA are not clear at present, but the metabolism of dopamine and 5-HT by monoamine oxidase (MAO), as well as the hepatic biotransformation of MDMA into pro-oxidant reactive metabolites is thought to contribute to its adverse effects.
Using mouse brain synaptosomes, we evaluated the pro-oxidant effects of MDMA and its metabolites, α-methyldopamine (α-MeDA), N-methyl-α-methyldopamine (N-Me-α-MeDA) and 5-(glutathion-S-yl)-α-methyldopamine [5-(GSH)-α-MeDA], as well as those of 5-HT, dopamine, l-DOPA and 3,4-dihydroxyphenylacetic acid (DOPAC).
5-HT, dopamine, l-DOPA, DOPAC and MDMA metabolites α-MeDA, N-Me-α-MeDA and 5-(GSH)-α-MeDA, concentration- and time-dependently increased H(2) O(2 ) production, which was significantly reduced by the antioxidants N-acetyl-l-cysteine (NAC), ascorbic acid and melatonin. From experiments with MAO inhibitors, it was observed that H(2) O(2) generation induced by 5-HT was totally dependent on MAO-related metabolism, while for dopamine, it was a minor pathway. The MDMA metabolites, dopamine, l-DOPA and DOPAC concentration-dependently increased quinoproteins formation and, like 5-HT, altered the synaptosomal glutathione status. Finally, none of the compounds modified the number of polarized mitochondria in the synaptosomal preparations, and the compounds' pro-oxidant effects were unaffected by prior mitochondrial depolarization, excluding a significant role for mitochondrial-dependent mechanisms of toxicity in this experimental model.
MDMA metabolites along with high levels of monoamine neurotransmitters can be major effectors of neurotoxicity induced by Ecstasy.

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Available from: Daniel José Barbosa,
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    • "Among the putative aspects that may contribute to the described toxicity is MDMA metabolism. In humans, MDMA is principally cleared by hepatic metabolism, which results in production of redox-active metabolites (Antolino-Lobo et al., 2011; Barbosa et al., 2012; Carvalho et al., 2010, 2012; de la Torre & Farre, 2004). These metabolites formed in vivo may act jointly with the parent compound to produce an overall effect that can be different than that elicited by MDMA alone. "
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    ABSTRACT: Hepatic injury after 3,4-methylenedioxymethamphetamine (MDMA; ecstasy) intoxications is highly unpredictable and does not seem to correlate with either dosage or frequency of use. The mechanisms involved include the drug metabolic bioactivation and the hyperthermic state of the liver triggered by its thermogenic action and exacerbated by the environmental circumstances of abuse at hot and crowded venues. We became interested in understanding the interaction between ecstasy and its metabolites generated in vivo as users are always exposed to mixtures of parent drug and metabolites. With this purpose, Hep G2 cells were incubated with MDMA and its main human metabolites methylenedioxyamphetamine (MDA), α-methyldopamine (α-MeDA) and N-methyl-α-methyldopamine (N-Me-α-MeDA), individually and in mixture (drugs combined in proportion to their individual EC01 ), at normal (37 °C) and hyperthermic (40.5 °C) conditions. After 48 h, viability was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Extensive concentration-response analysis was performed with single drugs and the parameters of the individual non-linear logit fits were used to predict joint effects using the well-founded models of concentration addition (CA) and independent action (IA). Experimental testing revealed that mixture effects on cell viability conformed to CA, for both temperature settings. Additionally, substantial combination effects were attained even when each substance was present at concentrations that individually produced unnoticeable effects. Hyperthermic incubations dramatically increased the toxicity of the tested drug and metabolites, both individually and combined. These outcomes suggest that MDMA metabolism has hazard implications to liver cells even when metabolites are found in low concentrations, as they contribute additively to the overall toxic effect of MDMA. Copyright © 2013 John Wiley & Sons, Ltd.
    Journal of Applied Toxicology 06/2014; 34(6). DOI:10.1002/jat.2885 · 2.98 Impact Factor
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    • "The loss of synaptic terminals in MDMA-administrated rats is by far the most reported neurotoxic event associated with this drug of abuse (Capela et al., 2009; Rudnick and Wall, 1992). Therefore, as neuronal mitochondrial trafficking and targeting to neuronal compartments and synapses are critical for many neuronal functions, including energy production and correct synaptic transmission (Barbosa et al., 2014c; López- Doménech et al., 2012; Sheng and Cai, 2012), we postulate that disrupted mitochondrial trafficking may contribute to the MDMA's neurotoxic and/or psychostimulant effects. Additionally , because alterations in mitochondrial morphology resulting from a failure of mitochondrial quality control mechanisms, like fusion/fission events, may ultimately result in a collapse of mitochondrial network and neuronal injury (Rugarli and Langer, 2012), these data describe a potential new mechanism underlying the effects of MDMA and its metabolites at the neuronal level. "
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    ABSTRACT: 3,4-Methylenedioxymethamphetamine (MDMA; "ecstasy"), is a potentially neurotoxic recreational drug of abuse. Though the mechanisms involved are still not completely understood, formation of reactive metabolites and mitochondrial dysfunction contribute to MDMA-related neurotoxicity. Mitochondrial neuronal trafficking, and their targeting to synapses, is essential for proper neuronal function and survival, rendering neurons particularly vulnerable to mitochondrial dysfunction. Indeed, MDMA-associated disruption of Ca(2+) homeostasis and ATP depletions have been described in neurons, thus suggesting possible MDMA interference on mitochondrial dynamics. In this study, we performed real-time functional experiments of mitochondrial trafficking to explore the role of in situ mitochondrial dysfunction in MDMA's neurotoxic actions. We show that the mixture of MDMA and its 6 major in vivo metabolites, each compound at 10 μM, impaired mitochondrial trafficking and increased the fragmentation of axonal mitochondria in cultured hippocampal neurons. Furthermore, the over-expression of mitofusin 2 (Mfn2) or dynamin-related protein 1 (Drp1) K38A constructs almost completely rescued the trafficking deficits caused by this mixture. Finally, in hippocampal neurons over-expressing a Mfn2 mutant, Mfn2 R94Q, with impaired fusion and transport properties, it was confirmed that a dysregulation of mitochondrial fission/fusion events greatly contributed to the reported trafficking phenotype. In conclusion, our study demonstrated, for the first time, that the mixture of MDMA and its metabolites, at concentrations relevant to the in vivo scenario, impaired mitochondrial trafficking and increased mitochondrial fragmentation in hippocampal neurons, thus providing a new insight in the context of "ecstasy"-induced neuronal injury.
    Toxicological Sciences 03/2014; 139(2). DOI:10.1093/toxsci/kfu042 · 3.85 Impact Factor
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    • "Exposure to MDMA's metabolites 5-(GSH)-N-Me-α- MeDA (Figure 2) or 5-(NAC)-N-Me-α-MeDA (Figure 3 and Supplementary Movie S2), at the concentration of 400 μM for 90 min, dramatically reduced the percentage of motile mitochondria and velocity, in both anterograde and retrograde directions. These data, in accordance with previous published results [4] [5] [6] [14] [15], support a major role for metabolism in MDMA-induced neuronal effects. "
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    ABSTRACT: Drugs of abuse cause a variety of complex neuronal events at the cellular level, including changes in membrane excitability and neurotransmission, activation of complex signaling pathways, altered synaptic physiology and structural changes, and drug-evoked synaptic plasticity and neurotoxicity, which mediate both acuteand long-lasting effects and addiction. Neuronal mitochondria are highly dynamic organelles that, by undergoing fusion and fission events, are efficiently translocated along the neuronal processes, frequently changing direction, pausing or switching to persistent docking. The neuronal integrity and functionality are dependent upon the proper maintenance of a healthy mitochondrial population and their efficient distribution. There is a general consensus that mitochondrial-dependent pathways can provide a major understanding concerning pathological processes underlying neurotoxicity of drugs of abuse. As such, it is plausible to consider that alterations on mitochondrial trafficking may be key players on the neuronal effects mediated by these drugs. This work aims to provide a comprehensive and up-to-date review of the data linking mitochondrial trafficking impairment to amphetamine-like drugs, and, thus, contribute to a better understanding of their neuronal effects. Additionally, new research data describing alterations in neuronal mitochondrial trafficking for 3,4-methylenedioxymethamphetamine (MDMA; “ecstasy”) conjugated metabolites 5-(glutathion-S-yl)-N-methyl-α-methyldopamine [5-(GSH)-N-Me-α-MeDA] and 5-(N-acetylcystein-S-yl)-N-methyl-α- methyldopamine [5-(NAC)-N-Me-α-MeDA] are also provided.
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