"Ecstasy"-induced toxicity in SH-SY5Y differentiated cells: role of hyperthermia and metabolites.

3,4-Methylenedioxymethamphetamine (MDMA; "ecstasy") is a recreational hallucinogenic drug of abuse known to elicit neurotoxic properties. Hepatic formation of neurotoxic metabolites is thought to play a major role in MDMA-related neurotoxicity, though the mechanisms involved are still unclear. Here, we studied the neurotoxicity mechanisms and stability of MDMA and 6 of its major human metabolites, namely α-methyldopamine (α-MeDA) and N-methyl-α-methyldopamine (N-Me-α-MeDA) and their correspondent glutathione (GSH) and N-acetyl-cysteine (NAC) conjugates, under normothermic (37 °C) or hyperthermic conditions (40 °C), using cultured SH-SY5Y differentiated cells. We showed that MDMA metabolites exhibited toxicity to SH-SY5Y differentiated cells, being the GSH and NAC conjugates more toxic than their catecholic precursors and MDMA. Furthermore, whereas the toxicity of the catechol metabolites was potentiated by hyperthermia, NAC-conjugated metabolites revealed higher toxicity under normothermia and GSH-conjugated metabolites-induced toxicity was temperature-independent. Moreover, a time-dependent decrease in extracellular concentration of MDMA metabolites was observed, which was potentiated by hyperthermia. The antioxidant NAC significantly protected against the neurotoxic effects of MDMA metabolites. MDMA metabolites increased intracellular glutathione levels, though depletion in thiol content was observed in MDMA-exposed cells. Finally, the neurotoxic effects induced by the MDMA metabolite N-Me-α-MeDA involved caspase 3 activation. In conclusion, this study evaluated the stability of MDMA metabolites in vitro, and demonstrated that the catechol MDMA metabolites and their GSH and NAC conjugates, rather than MDMA itself, exhibited neurotoxic actions in SH-SY5Y differentiated cells, which were differently affected by hyperthermia, thus highlighting a major role for reactive metabolites and hyperthermia in MDMA's neurotoxicity.

Mixtures of 3,4-methylenedioxymethamphetamine (ecstasy) and its major human metabolites act additively to induce significant toxicity to liver cells when combined at low, non-cytotoxic concentrations.

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.

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

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.

Effects of MDMA (ecstasy) and two of its metabolites on rat embryos in vitro.

MDMA consumers are young people of childbearing age. Consequently, developmental exposure to this drug is a potential public health concern. Several studies have addressed MDMA neurotoxicity in adults; however, knowledge of the effects of MDMA on developing embryos is limited. After administration, MDMA is metabolized species specifically via two main pathways. One leads to the formation of MDA and the other to the formation of HHMA. Here we evaluated the embryotoxic effects of MDMA, and also those of MDA, a main metabolite of MDMA in rats, and HHMA, a main metabolite in humans. For this purpose, we used the whole embryo culture (WEC). Our results show a concentration-dependent embryotoxic effect of MDMA, MDA and HHMA at a concentration range of 25-50µg/ml. The embryotoxic potential of the parent compound and the two metabolites was comparable in vitro.

Induction of glutathione synthesis and conjugation by 3,4-methylenedioxymethamphetamine (MDMA) and 3,4-dihydroxymethamphetamine (HHMA) in human and rat liver cells, including the protective role of some antioxidants.

MDMA (3,4-methylenedioxymethamphetamine) metabolism is a major cause of MDMA-mediated hepatotoxicity. In this study the effects of MDMA and its metabolites on the glutathione system were evaluated. Glutathione (GSH/GSSG) levels and gene expression of glutamate cysteine ligase catalytic subunit (GCLC), glutathione-S-transferase (GST) and pregnane X receptor (PXR) were compared in the immortalized human liver epithelial cell line THLE-Neo lacking phase I metabolism and primary rat hepatocytes expressing both phase I and II metabolism. Furthermore, we evaluated the potential protective effects of two antioxidants, N-acetyl-cysteine (NAC) and sulforaphane (SFN) in these cell systems. In THLE-Neo cells, the MDMA metabolite 3,4-dihydroxymetamphetamine (HHMA) significantly decreased cell viability and depleted GSH levels, resulting in an increased expression of GCLC and GST up to 3.4- and 2.2-fold, respectively. In primary rat hepatocytes, cell viability or GSH levels were not significantly affected upon MDMA exposure. GCLC expression levels where not significantly altered either, although GST expression was increased 2.3-fold. NAC counteracted MDMA-induced cytotoxicity and restored GSH levels. Phase II enzyme expression was also reverted. Conversely, SFN increased MDMA-induced cytotoxicity and GSH depletion, while GCLC and GST expression were significantly induced. In addition, PXR expression decreased after HHMA and MDMA exposure, while co-exposure to SFN induced it up to 3.6- and 3.9-fold compared to vehicle-control in the THLE-Neo cells and rat hepatocytes, respectively. Taken together, these data indicate that HHMA is a major factor in the MDMA-mediated hepatotoxicity through interaction with the glutathione system. The results of our study show that for MDMA intoxication the treatment with an antioxidant such as NAC may counteract the potentially hepatotoxicity. However, SFN supplementation should be considered with care because of the indications of possible drug-drug interactions.

Inhibition of 3,4-methylenedioxymethamphetamine metabolism leads to marked decrease in 3,4-dihydroxymethamphetamine formation but no change in serotonin neurotoxicity: implications for mechanisms of neurotoxicity.

3,4-Methylenedioxymethamphetamine (MDMA)'s O-demethylenated metabolite, 3,4-dihydroxymethamphetamine (HHMA), has been hypothesized to serve as a precursor for the formation of toxic catechol-thioether metabolites (e.g., 5-N-acetylcystein-S-yl-HHMA) that mediate MDMA neurotoxicity. To further test this hypothesis, HHMA formation was blocked with dextromethorphan (DXM), which competitively inhibits cytochrome P450 enzyme-mediated O-demethylenation of MDMA to HHMA. In particular, rats were randomly assigned to one of four treatment groups (n = 9-12 per group): (1) Saline/MDMA; (2) DXM/MDMA; (3) DXM/Saline; (4) Saline/Saline. During drug exposure, time-concentration profiles of MDMA and its metabolites were determined, along with body temperature. One week later, brain serotonin (5-HT) neuronal markers were measured in the same animals. DXM did not significantly alter core temperature in MDMA-treated animals. A large (greater than 70%) decrease in HHMA formation had no effect on the magnitude of MDMA neurotoxicity. These results cast doubt on the role of HHMA-derived catechol-thioether metabolites in the mechanism of MDMA neurotoxicity.

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

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 MAO(B) activity. MeDA inhibited dopamine uptake with a lower IC(50) 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.

Synthesis and in vitro cytotoxicity profile of the R-enantiomer of 3,4-dihydroxymethamphetamine (R-(-)-HHMA): comparison with related catecholamines.

(+/-)-3,4-Methylenedioxymethamphetamine (MDMA, also known as "ecstasy") is a chiral drug that is essentially metabolized in humans through O-demethylenation into 3,4-dihydroxymethamphetamine (HHMA). There has recently been a resurgence of interest in the possibility that MDMA metabolites, especially 5-(N-acetylcystein-S-yl)-N-methyl-alpha-methyldopamine (designated as 5-NAC-HHMA), might play a role in MDMA neurotoxicity. However, the chirality of MDMA was not considered in previously reported in vivo studies because HHMA, the precursor of the 5-NAC-HHMA metabolite, was used as the racemate. Since the stereochemistry of this chiral drug needs to be considered, the first total synthesis of R-(-)-HHMA is reported. Using L-DOPA as the chiral source, the preparation of R-(-)-HHMA is achieved through seven steps, in 30% overall yield and 99.5% enantiomeric excess. The cytotoxicity of R-(-)-HHMA and related catecholamines has been further determined by flow cytometric analysis of propidium iodide uptake in human dopaminergic neuroblastoma SH-SY5Y cells and by an Escherichia coli plate assay, specific for the detection of oxidative toxicity. The good correlation between the toxicities observed in both systems suggests that SH-SY5Y cells are sensitive to oxidative toxicity and that cell death (necrosis) would be mediated by reactive oxygen species mainly generated from redox active quinonoid centers. In contrast, apoptosis was detected for 3,4-dimethoxymethamphetamine (MMMA), the synthetic precursor of HHMA possessing a protected catechol group. MMMA was not toxic in the bacterial assay, indicating that its toxicity is not related to increased oxidative stress. Finally, we can conclude that there is a need to distinguish the toxicity ascribed to MDMA itself, also bearing a protected catechol moiety, from that depending on MDMA biotransformation leading to catechol metabolites such as HHMA and the thioether conjugates.

Metabolites of MDMA induce oxidative stress and contractile dysfunction in adult rat left ventricular myocytes.

Repeated administration of 3,4-methylenedioxymethamphetamine (MDMA) (ecstasy) produces eccentric left ventricular (LV) dilation and diastolic dysfunction. While the mechanism(s) underlying this toxicity are unknown, oxidative stress plays an important role. MDMA is metabolized into redox cycling metabolites that produce superoxide. In this study, we demonstrated that metabolites of MDMA induce oxidative stress and contractile dysfunction in adult rat left ventricular myocytes. Metabolites of MDMA used in this study included alpha-methyl dopamine, N-methyl alpha-methyl dopamine and 2,5-bis(glutathion-S-yl)-alpha-MeDA. Dihydroethidium was used to detect drug-induced increases in reactive oxygen species (ROS) production in ventricular myocytes. Contractile function and changes in intracellular calcium transients were measured in paced (1 Hz), Fura-2 AM loaded, myocytes using the IonOptix system. Production of ROS in ventricular myocytes treated with MDMA was not different from control. In contrast, all three metabolites of MDMA exhibited time- and concentration-dependent increases in ROS that were prevented by N-acetyl-cysteine (NAC). The metabolites of MDMA, but not MDMA alone, significantly decreased contractility and impaired relaxation in myocytes stimulated at 1 Hz. These effects were prevented by NAC. Together, these data suggest that MDMA-induced oxidative stress in the left ventricle can be due, at least in part, to the metabolism of MDMA to redox active metabolites.

[Metabolites of ecstasy and cytotoxicity effects]. / Métabolites de l'ecstasy et phénomènes de cytotoxicité

Intracerebroventricular injection of methylenedioxymethamphetamine (MDMA, ecstasy) in rats fails to reproduce long-term toxic effects observed after peripheral administration. Therefore, systemic metabolites would play an essential role in the development of cytotoxicity. In humans, the major metabolite is the 3,4-dihydroxymethamphetamine derivative (HHMA), which is easily oxidizable to the orthoquinone species. This can either participate to redox cycling generating semiquinone radicals and reactive oxygen species (ROS), or react with endogenous thiol derivatives yielding catechol-thioether conjugates whose the toxicity is not well established. A one pot electrochemical procedure has been developed allowing the synthesis of several catechol-thioether metabolites. Two in vitro assays have been used for evaluating their specific cytotoxicity. The first one is a bacterial assay, which shows that HHMA and some catechol-thioether conjugates can induce toxic phenomena leading to the formation of ROS, through redox cycling processes involving o-quinonoid species. The second one is an assay of cellular viability, performed on rat hippocampal pyramidal neurons. It confirms that some of these metabolites exhibit a noticeable cytotoxicity by markedly eliciting both necrosis and apoptosis markers.

The toxicity of N-methyl-alpha-methyldopamine to freshly isolated rat hepatocytes is prevented by ascorbic acid and N-acetylcysteine.

In the past decade, clinical evidence has increasingly shown that the liver is a target organ for 3,4-methylenedioxymethamphetamine (MDMA, "ecstasy") toxicity. The aims of the present in vitro study were: (1) to evaluate and compare the hepatotoxic effects of MDMA and one of its main metabolites, N-methyl-alpha-methyldopamine (N-Me-alpha-MeDA) and (2) to investigate the ability of antioxidants, namely ascorbic acid and N-acetyl-L-cysteine (NAC), to prevent N-Me-alpha-MeDA-induced toxic injury, using freshly isolated rat hepatocytes. Cell suspensions were incubated with MDMA or N-Me-alpha-MeDA in the final concentrations of 0.1, 0.2, 0.4, 0.8, and 1.6 mM for 3 h. To evaluate the potential protective effects of antioxidants, cells were preincubated with ascorbic acid in the final concentrations of 0.1 and 0.5 mM, or NAC in the final concentrations of 0.1 and 1 mM for 15 min before treatment with 1.6 mM N-Me-alpha-MeDA for 3 h (throughout this incubation period the cells were exposed to both compounds). The toxic effects were evaluated by measuring the cell viability, glutathione (GSH) and glutathione disulfide (GSSG), ATP, and the cellular activities of GSH peroxidase (GPX), GSSG reductase (GR), and GSH S-transferase (GST). MDMA induced a concentration- and time-dependent GSH depletion, but had a negligible effect on cell viability, ATP levels, or on the activities of GR, GPX, and GST. In contrast, N-Me-alpha-MeDA was shown to induce not only a concentration- and time-dependent depletion of GSH, but also a depletion of ATP levels accompanied by a loss in cell viability, and decreases in the antioxidant enzyme activities. For both compounds, GSH depletion was not accompanied by increases in GSSG levels, which seems to indicate GSH depletion by adduct formation. Importantly, the presence of ascorbic acid (0.5 mM) or NAC (1 mM) prevented cell death and GSH depletion induced by N-Me-alpha-MeDA. The results provide evidence that MDMA and its metabolite N-Me-alpha-MeDA induce toxicity to freshly isolated rat hepatocytes. Oxidative stress may play a major role in N-Me-alpha-MeDA-induced hepatic toxicity since antioxidant defense systems are impaired and administration of antioxidants prevented N-Me-alpha-MeDA toxicity.

Metabolism is required for the expression of ecstasy-induced cardiotoxicity in vitro.

Cardiovascular complications associated with 3,4-methylenedioxymethamphetamine (MDMA, ecstasy) abuse have increasingly been reported. The indirect effect of MDMA mediated by a sustained high level of circulating biogenic amines may contribute to the cardiotoxic effects, but other factors, like the direct toxic effects of MDMA and its metabolites in cardiac cells, remain to be investigated. Thus, the objective of the present in vitro study was to evaluate the potential cardiotoxic effects of MDMA and its major metabolites 3,4-methylenedioxyamphetamine (MDA), N-methyl-alpha-methyldopamine (N-Me-alpha-MeDA), and alpha-methyldopamine (alpha-MeDA) using freshly isolated adult rat cardiomyocytes. The cell suspensions were incubated with these compounds in the final concentrations of 0.1, 0.2, 0.4, 0.8, and 1.6 mM for 4 h. alpha-MeDA, N-Me-alpha-MeDA, and their respective aminochromes (oxidation products) were quantified in cell suspensions by HPLC-DAD. The toxic effects were evaluated at hourly intervals for 4 h by measuring the percentage of cells with normal morphology, glutathione (GSH), and glutathione disulfide (GSSG); intracellular Ca(2+), ATP, and ADP; and the cellular activities of glutathione peroxidase, glutathione reductase, and glutathione-S-transferase. No toxic effects were found after exposure of rat cardiomyocytes to MDMA or MDA at any of the tested concentrations for 4 h. In contrast, their catechol metabolites N-Me-alpha-MeDA and alpha-MeDA induced significant toxicity in rat cardiomyocytes. The toxic effects were characterized by a loss of normal cell morphology, which was preceded by a loss of GSH homeostasis due to conjugation of GSH with N-Me-alpha-MeDA and alpha-MeDA, sustained increase of intracellular Ca(2+) levels, ATP depletion, and decreases in the antioxidant enzyme activities. The oxidation of N-Me-alpha-MeDA and alpha-MeDA into the toxic compounds N-methyl-alpha-methyldopaminochrome and alpha-methyldopaminochrome, respectively, was also verified in cell suspensions incubated with these MDMA metabolites. The results obtained in this study provide evidence that the metabolism of MDMA into N-Me-alpha-MeDA and alpha-MeDA is required for the expression of MDMA-induced cardiotoxicity in vitro, being N-Me-alpha-MeDA the most toxic of the studied metabolites.

Hepatotoxicity of 3,4-methylenedioxyamphetamine and alpha-methyldopamine in isolated rat hepatocytes: formation of glutathione conjugates.

The amphetamine designer drugs 3,4-methylenedioxymethamphetamine (MDMA or "ecstasy") and its N-demethylated analogue 3,4-methylenedioxyamphetamine (MDA or "love") have been extensively used as recreational drugs of abuse. MDA itself is a main MDMA metabolite. MDMA abuse in humans has been associated with numerous reports of hepatocellular damage. Although MDMA undergoes extensive hepatic metabolism, the role of metabolites in MDMA-induced hepatotoxicity remains unclear. Thus, the aim of the present study was to evaluate the effects of MDA and alpha-methyldopamine (alpha-MeDA), a major metabolite of MDA, in freshly isolated rat hepatocyte suspensions. The cells were incubated with MDA or alpha-MeDA at final concentrations of 0.1, 0.2, 0.4, 0.8, or 1.6 mM for 3 h. The toxic effects induced following incubation of hepatocyte suspensions with these metabolites were evaluated by measuring cell viability, the extent of lipid peroxidation, levels of glutathione (GSH) and glutathione disulfide (GSSG), the formation of GSH conjugates, and the activities of GSSG reductase (GR), GSH peroxidase (GPX), and GSH S-transferase (GST). MDA induced a concentration- and time-dependent GSH depletion, but had a negligible effect on lipid peroxidation, cell viability, or on the activities of GR, GPX, and GST. In contrast, alpha-MeDA (1.6 mM, 3 h) induced a marked depletion of GSH accompanied by a loss on cell viability, and decreases in GR, GPX and GST activities, although no significant effect on lipid peroxidation was found. For both metabolites, GSH depletion was not accompanied by increases in GSSG levels; rather, 2-(glutathion- S-yl)-alpha-MeDA and 5-(glutathion- S-yl)-alpha-MeDA were identified by HPLC-DAD/EC within cells incubated with MDA or alpha-MeDA. The results provide evidence that one of the early consequences of MDMA metabolism is a disruption of thiol homeostasis, which may result in loss of protein function and the initiation of a cascade of events leading to cellular damage.

Glutathione and N-acetylcysteine conjugates of alpha-methyldopamine produce serotonergic neurotoxicity: possible role in methylenedioxyamphetamine-mediated neurotoxicity.

Direct injection of either 3,4-(+/-)-methylenedioxymethamphetamine (MDMA) or 3,4-(+/-)-methylenedioxyamphetamine (MDA) into the brain fails to reproduce the serotonergic neurotoxicity seen following peripheral administration. The serotonergic neurotoxicity of MDA and MDMA therefore appears to be dependent upon the generation of a neurotoxic metabolite, or metabolites, the identity of which remains unclear. alpha-Methyldopamine (alpha-MeDA) is a major metabolite of both MDA and MDMA. We have shown that intracerebroventricular (icv) injection of 2,5-bis(glutathion-S-yl)-alpha-methyldopamine [2, 5-bis(glutathion-S-yl)-alpha-MeDA] causes decreases in serotonin concentrations in the striatum, cortex, and hippocampus, and neurobehavioral effects similar to those seen following MDA and MDMA administration. In contrast, although 5-(glutathion-S-yl)-alpha-methyldopamine [5-(glutathion-S-yl)-alpha-MeDA] and 5-(N-acetylcystein-S-yl)-alpha-methyldopamine [5-(N-acetylcystein-S-yl)-alpha-MeDA] produce neurobehavioral changes similar to those seen with MDA and MDMA, and acute changes in brain 5-HT and dopamine concentrations, neither conjugate caused long-term decreases in 5-HT concentrations. We now report that direct intrastriatal or intracortical administration of 5-(glutathion-S-yl)-alpha-MeDA (4 x 200 or 4 x 400 nmol), 5-(N-acetylcystein-S-yl)-alpha-MeDA (4 x 7 or 4 x 20 nmol), and 2, 5-bis(glutathion-S-yl)-alpha-MeDA (4 x 150 or 4 x 300 nmol) causes significant decreases in striatal and cortical 5-HT concentrations (7 days following the last injection). Interestingly, intrastriatal injection of 5-(glutathion-S-yl)-alpha-MeDA or 2, 5-bis(glutathion-S-yl)-alpha-MeDA, but not 5-(N-acetylcystein-S-yl)-alpha-methyldopamine, also caused decreases in 5-HT concentrations in the ipsilateral cortex. The same pattern of changes was seen when the conjugates were injected into the cortex. The effects of the thioether conjugates of alpha-MeDA were confined to 5-HT nerve terminal fields, since no significant changes in monoamine neurotransmitter levels were detected in brain regions enriched with 5-HT cell bodies (midbrain/diencephalon/telencephalon and pons/medulla). In addition, the effects of the conjugates were selective with respect to the serotonergic system, as no significant changes were seen in dopamine or norepinephrine concentrations. The results indicate that thioether conjugates of alpha-MeDA are selective serotonergic neurotoxicants. Nonetheless, a role for these conjugates in the toxicity observed following systemic administration of MDA and MDMA remains to be demonstrated, and requires further experimentation.

The neurotoxic effects of methylenedioxymethamphetamine (MDMA) and its metabolites on rat brain spheroids in culture.

Rat whole-brain spheroids were used to assess the intrinsic neurotoxicity of methylenedioxy-methamphetamine (MDMA, Ecstasy) and two of its metabolites, dihydroxymethamphetamine (DHMA) and 6-hydroxy-MDMA (6-OH MDMA). Exposure of brain spheroids to MDMA or the metabolite 6-OH MDMA (up to 500 micromol/L) for 5 days in culture did not alter intracellular levels of glutathione (GSH), glial fibrillary acidic protein (GFAP) or serotonin (5-HT). In contrast, exposure to the metabolite DHMA, which can deplete intracellular thiols, significantly increased GSH levels (up to 170% of control) following exposure to 50 and 100 micromol/L DHMA. There was also a significant reduction in the levels of glial fibrillary acidic protein (GFAP) and GSH by DHMA at the highest concentration tested (500 micromol/L) but there was no effect on 5HT. This may constitute a sublethal neurotoxic compensatory response to DHMA in an attempt to replenish depleted intraneural GSH levels following metabolite exposure. Rat whole-brain spheroids may thus be a useful in vitro model to delineate mechanisms and effects of this class of neurotoxin.

2,5-Bis-(glutathion-S-yl)-alpha-methyldopamine, a putative metabolite of (+/-)-3,4-methylenedioxyamphetamine, decreases brain serotonin concentrations.

3,4-(+/-)-Methylenedioxyamphetamine (MDA) and 3,4-(+/-)-methylenedioxymethamphetamine (MDMA) are serotonergic neurotoxicants. However, when injected directly into brain, MDA and MDMA are not neurotoxic, suggesting that systemic metabolism plays an important role in the development of neurotoxicity. The nature of the metabolite(s) responsible for MDA- and MDMA-mediated neurotoxicity is unclear. alpha-Methyldopamine is a major metabolite of MDA and is readily oxidized to the o-quinone, followed by conjugation with glutathione (GSH). Because the conjugation of quinones with GSH frequently results in preservation or enhancement of biological (re)activity, we have been investigating the role of quinone-thioethers in the acute and long-term neurochemical changes observed after administration of MDA. Although intracerebroventricular (i.c.v.) administration of 5-(glutathion-S-yl)-alpha-methyldopamine (4 x 720 nmol) and 5-(N-acetylcystein-S-yl)-alpha-methyldopamine (1 x 7 nmol) to Sprague-Dawley rats produced overt behavioral changes similar to those seen following administration of MDA (93 mumol/kg, s.c.) they did not produce long-term decreases in brain serotonin (5-hydroxytryptamine, 5-HT) concentrations. In contrast, 2,5-bis-(glutathion-S-yl)-alpha-methyldopamine (4 x 475 nmol) decreased 5-HT levels by 24%, 65% and 30% in the striatum, hippocampus and cortex, respectively, 7 days after injection. The relative sensitivity of the striatum, hippocampus and cortex to 2,5-bis-(glutathion-S-yl)-alpha-methyldopamine was the same as that observed for MDA; the absolute effects were greater with MDA. The effects of 2,5-bis-(glutathion-S-yl)-alpha-methyldopamine were also selective for serotonergic nerve terminal fields, in that 5-HT levels were unaffected in regions of the cell bodies. Because 2,5-bis-(glutathion-S-yl)-alpha-methyldopamine caused long-term depletion in 5-HT without adversely affecting the dopaminergic system, it also mimics the selectivity of MDA/MDMA. The data imply a possible role for quinone-thioethers in the neurobehavioral and neurotoxicological effects of MDA/MDMA.

Effects of intracerebroventricular administration of 5-(glutathion-S-yl)-alpha-methyldopamine on brain dopamine, serotonin, and norepinephrine concentrations in male Sprague-Dawley rats.

alpha-Methyldopamine (alpha-MeDA) is a metabolite of the serotonergic neurotoxicants 3,4-(+/-)-(methylenedioxy)amphetamine (MDA) and 3,4-(+/-)-(methylenedioxy)methamphetamine (MDMA). alpha-MeDA readily oxidizes, and in the presence of glutathione (GSH) it forms 5-(glutathion-S-yl)-alpha-methyldopamine [5-(glutathion-S-yl)-alpha-MeDA]. Since GSH conjugates of many polyphenols are biologically (re)active, we investigated the role of 5-(glutathion-S-yl)-alpha-MeDA in the acute and long-term neurochemical changes observed after administration of MDA. Intracerebroventricular (icv) administration of 5-(glutathion-S-yl)-alpha-MeDA (720 nmol) to male Sprague-Dawley rats produced behavioral changes similar to those reported after subcutaneous administration of MDA. Thus, animals became hyperactive and aggressive and displayed forepaw treading and Straub tails, behaviors usually seen after administration of serotonin (5-HT) releasers, and consistent with a role for 5-(glutathion-S-yl)-alpha-MeDA in some of the behavioral alterations seen after administration of MDA and MDMA. In addition to the behavioral changes, 5-(glutathion-S-yl)-alpha-MeDA also caused short-term alterations in the dopaminergic, serotonergic, and noradrenergic systems. An increase in dopamine synthesis appears to be a prerequisite for the long-term depletion of brain 5-HT following MDMA administration. However, although 5-(glutathion-S-yl)-alpha-MeDA reproduced some of the effects of MDA on the dopaminergic system and was capable of causing acute increases in 5-HT turnover, a single icv injection of 5-(glutathion-S-yl)-alpha-MeDA did not result in long-term serotonergic toxicity. Thus, although acute stimulation of dopamine turnover may be necessary for long-term serotonergic toxicity, such changes are not sufficient to produce these effects. The effects of a multiple dosing schedule of 5-(glutathion-S-yl)-alpha-MeDA will therefore require investigation before we can define a role for this metabolite in MDA and MDMA mediated neurotoxicity. MDA also produces a pressor response that is related to its ability to release neuronal norepinephrine stores, and 5-(glutathion-S-yl)-alpha-MeDA caused comparable depletions of brain norepinephrine concentrations, indicating that both compounds produce similar effects on the noradrenergic system.

Metabolism of 5-(glutathion-S-yl)-alpha-methyldopamine following intracerebroventricular administration to male Sprague-Dawley rats.

5-(Glutathion-S-yl)-alpha-methyldopamine [5-(GSyl)-alpha-MeDA] is a putative metabolite of the serotonergic neurotoxicants 3,4-(+/-)-(methylenedioxy)amphetamine and 3,4-(+/-)-(methylenedioxy)methamphetamine. Glutathione (GSH) conjugates of several polyphenols are biologically (re)active. Therefore, as part of our studies on the role of 5-(GSyl)-alpha-MeDA in MDA-mediated neurotoxicity, we determined the regional brain metabolism of 5-(GSyl)-alpha-MeDA (720 nmol) following intracerebroventricular administration to male Sprague-Dawley rats. 5-(GSyl)-alpha-MeDA was rapidly cleared from all brain regions examined, and regional differences in the distribution of gamma-glutamyl transpeptidase (gamma-GT) correlated with the formation of 5-(cystein-S-yl)-alpha-methyldopamine (5-[CYS]-alpha-MeDA). We also observed the formation of 5-(N-acetyl-L-cystein-S-yl)-alpha-MeDA (5-[NAC]-alpha-MeDA) in all brain regions, indicating that the brain has the ability to synthesize mercapturic acids. Peak concentrations of 5-(NAC)-alpha-MeDA were found in the order: hypothalamus > midbrain/diencephalon/telencephalon > pons/medulla > hippocampus > cortex > striatum. In contrast to 5-(GSyl)-alpha-MeDA and 5-(CYS)-alpha-MeDA, 5-(NAC)-alpha-MeDA was eliminated relatively slowly from the brain. Differences were also found in cystein conjugate N-acetyltransferase activity in microsomes prepared from the various brain regions, but little difference was observed in brain cytosolic N-acetyl-L-cysteine conjugate N-deacetylase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Assessment of the role of alpha-methylepinine in the neurotoxicity of MDMA.

To assess the potential involvement of metabolism of 3,4-methylenedioxymethamphetamine (MDMA) to the catechol alpha-methylepinine in producing serotonergic neurotoxicity, we attempted to correlate aspects of this reaction with the neurotoxicity profile of MDMA. In contrast to the stereoselectivity of S-(+)-MDMA in causing persistent declines in rat brain 5-hydroxyindole levels, no stereochemical component to the metabolic reaction was apparent. Rat liver microsomes generated a significantly greater amount of alpha-methylepinine than did mouse microsomes, but similar amounts of metabolite were produced by brain microsomes from the two species. Formation of alpha-methylepinine by hepatic, but not brain, microsomes was inhibited by SKF 525A and induced by phenobarbital, possibly indicating a tissue specificity in cytochrome P-450-dependent metabolism of MDMA. To directly assess whether alpha-methylepine is a likely mediator of MDMA neurotoxicity, the compound was administered intracerebroventricularly. No persistent declines in biogenic amines or their metabolites were observed one week following treatment. These data suggest that alpha-methylepinine alone is not responsible for the neurotoxic effects of MDMA.