Comparative neurochemical profile of 3,4-methylenedioxymethamphetamine and its metabolite alpha-methyldopamine on key targets of MDMA neurotoxicity

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Abstract

The neurotoxicity of MDMA or “Ecstasy” in rats is selectively serotonergic, while in mice it is both dopaminergic and serotonergic. MDMA metabolism may play a key role in this neurotoxicity. The function of serotonin and dopamine transporter and the effect of MDMA and its metabolites on them are essential to understand MDMA neurotoxicity. The aim of the present study was to investigate and compare the effects of MDMA and its metabolite alpha-methyldopamine (MeDA) on several molecular targets, mainly the dopamine and serotonin transporter functionality, to provide evidence for the role of this metabolite in the neurotoxicity of MDMA in rodents.

MeDA had no affinity for the serotonin transporter but competed with serotonin for its uptake. It had no persistent effects on the functionalism of the serotonin transporter, in contrast to the effect of MDMA. Moreover, MeDA inhibited the uptake of dopamine into the serotonergic terminal and also MAOB activity.

MeDA inhibited dopamine uptake with a lower IC50 value than MDMA. After drug washout, the inhibition by MeDA persisted while that of MDMA was significantly reduced. The effect of MDMA on the dopamine transporter is related with dopamine release from vesicular stores, as this inhibition disappeared in reserpine-treated animals. However, the effect of MeDA seems to be a persistent conformational change of this transporter.

Moreover, in contrast with MDMA, MeDA did not show affinity for nicotinic receptors, so no effects of MeDA derived from these interactions can be expected. The metabolite reduced cell viability at lower concentrations than MDMA. Apoptosis plays a key role in MDMA induced cellular toxicity but necrosis is the major process involved in MeDA cytotoxicity.

We conclude that MeDA could protect against the serotonergic lesion induced by MDMA but potentiate the dopaminergic lesion as a result of the persistent blockade of the dopamine transporter induced this metabolite.

Research highlights

Methyldopamine constitutes the major first line of metabolites from MDMA and MDA. ▶ Methyldopamine induces a persistent change of the dopamine transporter conformation. ▶ Methyldopamine competes with serotonin for its uptake. ▶ Methyldopamine can reduce the neurotoxicity induced by MDMA at serotonergic neurons. ▶ Methyldopamine seems to potentiate the dopaminergic lesion induced by MDMA.

Introduction

3,4-Methylenedioxymethamphetamine, also known as MDMA or “Ecstasy” is an amphetamine derivative that is widely abused in Western countries, particularly at dance parties (“raves”) and on college campuses. High environmental and core body temperature and muscular exertion, such as that which accompanies marathon dancing, seem to lower the threshold for serious MDMA-associated adverse effects (Schwartz and Miller, 1997).

In the central nervous system, MDMA has acute effects on serotonin (5-HT) pathways. It induces a rapid increase in extracellular 5-HT in the striatum, frontal-cortex and hippocampus via the release of 5-HT and the inhibition of its uptake (Green et al., 2003). The acute increase in serotonergic neurotransmission markedly amplifies the concentration of extracellular dopamine (DA) (Gudelsky and Nash, 1996) through the activation of postsynaptic 5-HT2A\2C receptors (Sprague et al., 1998). Additionally, Iravani et al. (2000) established that MDMA is a better inhibitor of 5-HT and DA uptake than stimulator of the release of these neurotransmitters.

Neurotoxicity is the main long-term effect induced by MDMA in the central nervous system. The patterns of neurotoxicity of MDMA in mice and rats differ in that mice typically exhibit neurotoxicity to both DA- and 5-HT-containing neurons, whereas rats commonly display selective neurotoxicity to 5-HT-containing neurons (Pubill et al., 2003, Sprague and Nichols, 2005).

Two theories account for this neurotoxicity. Firstly, the neurotoxicity induced by MDMA may at least partially be a consequence of its metabolism (de la Torre et al., 2000, Mueller et al., 2009). This hypothesis is based on the fact that a direct intracerebral injection of MDMA failed to reproduce the neurotoxicity profile that appears after its peripheral administration (Esteban et al., 2001). It has been proposed that some quinone thioether adducts that result from the peripheral metabolism of MDMA might be the ultimate mediators of its neurotoxicity (Capela et al., 2007). However, the specific neurotoxic profile of the different metabolites is controversial (McCann and Ricaurte, 1991, Johnson et al., 1992) and remains to be well characterized.

In cultured rat cortical neurons, Capela et al. (2006) found that some metabolites are more neurotoxic than the parent compound MDMA. The glutathionyl adduct, 5-(glutathion-S-yl)-α-methyldopamine was found to be the most toxic metabolite. This suggests that MDMA metabolites contribute to MDMA-induced neurotoxicity especially under hyperthermic conditions.

The other main theory involves reactive oxygen species, although the two theories cannot be considered mutually exclusive. Oxidative stress appears to be one of the main factors involved in the serotonergic and dopaminergic terminal injury induced by MDMA (Jayanthi et al., 1999, Chipana et al., 2008a). Despite the general agreement that oxidative stress is one of the main causes of MDMA-induced 5-HT toxicity, the source of reactive oxygen species is still a matter of discussion.

Several authors have suggested that the depletion of 5-HT in the rat brain is dependent, in part, on DA metabolism/oxidation inside serotonergic terminals. DA can enter the serotonergic terminal by means of the 5-HT transporter (SERT). This effect may be particularly relevant when DA is present in abnormally high amounts and 5-HT terminals are depleted (Jones et al., 2004, Saldana and Barker, 2004). Sprague and Nichols (1995) showed that MAOB metabolizes DA inside the serotonergic terminal. This produces hydrogen peroxide which could lead to lipid peroxidation and general oxidative stress.

DA is also involved in the theory that explains the source of reactive oxygen species in dopaminergic terminals. Some evidence supports the contention that when MDMA concentration is high, it enters the dopaminergic terminal by diffusion and not via the DA uptake site (Camarero et al., 2002, Chipana et al., 2008b). Inside these terminals, MDMA may redistribute DA from the reducing environment within vesicles to extravesicular intracellular oxidizing environments. Using a semipurified synaptosomal preparation from the striatum of mice or rats we have demonstrated that methamphetamine and MDMA induce the formation of radical oxygen species inside dopaminergic terminals that is dependent on DA contents, DA transporter (DAT) functionality and calcium influx (Pubill et al., 2005, Chipana et al., 2006, Chipana et al., 2008a). Moreover, inside the terminal, the oxidant effect of these compounds can inhibit DAT functionality (Park et al., 2002). This inhibition traps DA inside the terminal and potentiates reactive oxygen species formation, which can finally damage dopaminergic nerve terminals. Therefore, it can be deduced that stronger inhibition of DAT by MDMA could lead to greater formation of reactive oxygen species, which potentiates the drug's toxicity. Additionally, blocking DA reuptake to dopaminergic terminals increases the extracellular levels of this neurotransmitter and the possibility that it will be oxidized and taken up by the depleted 5-HT terminals.

Therefore, the role of SERT and DAT functionality and the effect of MDMA and its metabolites on them are essential to understand MDMA-induced neurotoxicity.

In previous studies we demonstrated that MDMA interacts with nicotinic acetylcholine receptors (heteromeric receptors containing beta2 subunits and homomeric alpha7). From affinity studies, it is possible to conclude that this interaction is involved in some of the actions of amphetamine derivatives, which are found in vivo, such as neurotoxicity (García-Ratés et al., 2007). Moreover, isolated synaptosomes from rat and mice can be used as an in vitro model of MDMA-induced neurotoxicity (Pubill et al., 2005). Using this preparation, we have demonstrated that calcium entry through the alpha-7 nicotinic acetylcholine receptor is involved in the oxidative effect of MDMA (Chipana et al., 2008a). Furthermore, antagonists of this receptor type significantly prevent the in vivo neurotoxicity that is induced by MDMA (Chipana et al., 2006, Camarasa et al., 2008).

Different quantitative MDMA metabolic pathways have been identified in rats (Jones et al., 2005, Goñi-Allo et al., 2008) and in humans (de la Torre et al., 2004, Abraham et al., 2009). In rats, the major pathway implies the N-demethylation of MDMA to 3,4-methylenedioxyamphetamine (also a recreational drug known as MDA) and the subsequent O-demethylenation, which leads to 3,4-dihydroxyamphetamine (alpha-methyldopamine, MeDA). Catecholic metabolites can be oxidized to their corresponding orthoquinones and can undergo further conjugation with sulphate, glucuronide and other thiol-containing endogenous substances.

Accordingly, MeDA constitutes the major first line of metabolites that originate from MDMA after its transformation to MDA, a metabolite of MDMA that has been yet described as neurotoxic (Colado et al., 1995).

The aim of the present study was to investigate and compare the effects of MDMA and MeDA on several molecular targets, mainly the dopamine and serotonin transporter functionality, to provide evidence for the role of this metabolite in the neurotoxicity of MDMA in rodents.

Section snippets

Animals

Male Sprague–Dawley rats (125–150 g) (Janvier, France) were used. The animals were housed in a regulated environment (21 ± 1 °C; 12 h light/dark cycle, lights on at 08:00 h) with free access to food and water. Experimental protocols for the use of animals in this study were approved by the Animal Ethics Committee of the University of Barcelona under the supervision of the Autonomous Government of Catalonia, following the guidelines of the European Communities Council (86/609/EEC). Efforts were made

Competitive inhibition of 5-HT uptake

In synaptosomes isolated from rat hippocampus, MDMA and MeDA, ranging from 10−8 to 5 × 10−6 M, inhibited SERT function, in a concentration-dependent manner, as indicated by a significant decrease in [3H]5-HT uptake (Fig. 1A). IC50 calculated values were 4.13 ± 2.49 μM for MDMA and 1.58 ± 0.34 μM for MeDA, respectively. The difference between these two values was not statistically significant.

Persistent inhibition of 5-HT uptake

In another set of experiments, to evaluate the long-term effects of MDMA and MeDA on 5-HT uptake, rat

Discussion

Neurotoxicity from MDMA abuse is a matter of continuous research. Several factors may contribute to MDMA-induced neurotoxicity, including MDMA metabolism. To our knowledge, this is the first study to report a comparative neurochemical profile of MDMA and its metabolite MeDA in terms of 5-HT and DA transporter functionality, which play a key role in the neurotoxicity process. We demonstrate that the serotonergic and dopaminergic pathways are differentially affected by these two compounds.

Results

Conclusions

Finally, we can conclude that MeDA, a main metabolite of MDMA, competes with 5-HT for its uptake to the serotonergic terminal but has no persistent effects on the functionalism of the SERT, in contrast to the effect of MDMA. Moreover MeDA inhibits the uptake of DA into the serotonergic terminal and also MAOB activity, which could result in a reduction of the neurotoxicity induced by MDMA at the serotonergic neurons.

By contrast, MeDA induces persistent inhibition of DAT and competitively

Acknowledgments

We are grateful to Dr J. Serratosa (CSIC-IDIBAPS, Barcelona, Spain) for gift of the PC12 cell line, to Drs. M. Amat, J. Bosch and N. Llor, for spectral analysis of MDMA to demonstrate its chemical purity, and to the Language Advisory Service of the University of Barcelona for revising the manuscript. This work was supported by grants from the Plan Nacional sobre Drogas (Ministerio de Sanidad y Política Social) (2008/003), from the Ministerio de Ciencia e Innovación (SAF2010-15948) and from the

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