Sulfation of the 3,4-methylenedioxymethamphetamine (MDMA) metabolites 3,4-dihydroxymethamphetamine (DHMA) and 4-hydroxy-3-methoxymethamphetamine (HMMA) and their capability to inhibit human sulfotransferases

3,4-Methylenedioxymethamphetamine (MDMA, Ecstasy) is excreted in human urine mainly as conjugates of its metabolites 3,4-dihydroxymethamphetamine (DHMA) and 4-hydroxy-3-methoxymethamphetamine (HMMA). The glucuronidation kinetics of HMMA showed high capacities, but also high K_m values, unlikely to be reached after recreational user's doses. Therefore, the aim of the present work was to investigate the sulfation of DHMA and HMMA by human sulfotransferases (SULTs) in pooled human liver cytosol (pHLC). The kinetic data showed deviation from typical Michaelis-Menten kinetics. The overall efficiency for HMMA sulfation was calculated to be 2-10 times higher than for glucuronidation. As the sulfation of both MDMA metabolites showed substrate inhibition effects, their inhibitory potential towards typical sulfation reactions in pHLC was tested. The following substrates for typical sulfation reactions were used: nitrophenol, dopamine, estradiol, and dehydroepi androsten dione. Inhibition was observed towards dopamine sulfation by DHMA and HMMA, but not by MDMA. The 1/V vs. 1/S plots indicated a mixed-type or competitive inhibition model for DHMA and HMMA, respectively. In conclusion, the presented data indicated that sulfation of HMMA should be the major conjugation reaction observed in humans. Furthermore, both, DHMA and HMMA, were identified as inhibitors of dopamine sulfation.     

Identification of a possible role for atrial natriuretic peptide in MDMA-induced hyperthermia

MDMA (3,4-methylenedioxymethamphetamine) induces thermogenesis in a mitochondrial uncoupling protein 3-dependent manner. There is evidence that this hyperthermia is mediated in part by the lipolytic release of free fatty acids, that subsequently activate uncoupling protein 3 in skeletal muscle mitochondria. We hypothesize that atrial natriuretic peptide (ANP), a strong lipolytic mediator, may contribute to the induction and maintenance of MDMA-induced thermogenesis. The specific aims of this study were to (1) determine if ANP is released following MDMA administration, and (2) use the ANP receptor antagonist, Anantin, to ascertain the role of ANP in MDMA-induced hyperthermia. ANP levels were measured in plasma at baseline, 10, 20, 30 and 60 min following MDMA (40 mg/kg, sc) administration in 16 male Sprague-Dawley rats. A robust increase in ANP was seen within 10 min of MDMA administration. ANP levels returned to baseline at 20 min and then gradually rose over the 60 min monitoring period. The administration of Anantin (40 mg, ip), 15 min before and after MDMA, significantly attenuated the MDMA-induced hyperthermia. We conclude that ANP signaling contributes to the hyperthermia induced by MDMA.     

Persistent Nigrostriatal Dopaminergic Abnormalities in Ex-Users of MDMA (‘Ecstasy'): An 18F-Dopa PET Study

Ecstasy (±3,4-methylenedioxymethamphetamine, MDMA) is a popular recreational drug with known serotonergic neurotoxicity. Its long-term effects on dopaminergic function are less certain. Studying the long-term effects of ecstasy is often confounded by concomitant polydrug use and the short duration of abstinence. We used ¹⁸F-dopa positron emission tomography (PET) to investigate the long-term effects of ecstasy on nigrostriatal dopaminergic function in a group of male ex-recreational users of ecstasy who had been abstinent for a mean of 3.22 years. We studied 14 ex-ecstasy users (EEs), 14 polydrug-using controls (PCs) (matched to the ex-users for other recreational drug use), and 12 drug-naive controls (DCs). Each participant underwent one ¹⁸F-dopa PET, cognitive assessments, and hair and urinary analyses to corroborate drug-use history. The putamen ¹⁸F-dopa uptake of EEs was 9% higher than that of DCs (p=0.021). The putamen uptake rate of PCs fell between the other two groups, suggesting that the hyperdopaminergic state in EEs may be due to the combined effects of ecstasy and polydrug use. There was no relationship between the amount of ecstasy used and striatal ¹⁸F-dopa uptake. Increased putaminal ¹⁸F-dopa uptake in EEs after an abstinence of >3 years (mean) suggests that the effects are long lasting. Our findings suggest potential long-term effects of ecstasy use, in conjunction with other recreational drugs, on nigrostriatal dopaminergic functions. Further longitudinal studies are required to elucidate the significance of these findings as they may have important public health implications.     

Repeated intermittent MDMA binges reduce DAT density in mice and SERT density in rats in reward regions of the adolescent brain

The popular recreational drug, 3,4-methylenedioxymethamphetamine (MDMA) is often taken as intermittent binges by adolescents at dance clubs. The neurobiological mechanisms that underlie MDMA-induced psychiatric conditions are still poorly understood. In the present study, mimicking adolescent patterns of administration, repeated intermittent MDMA binges (3x5 mg/(kg day) given 3h apart, every 7th day for 4 weeks) were given to adolescent mice and rats. Behavioral responses in the open-field and autoradiographic ligand-binding to dopamine (DAT) and serotonin (SERT) transporters in reward regions of the brain were measured. In the open-field, total horizontal activity (HA) was significantly increased in both mice and rats following the first and third weekly administered MDMA binge. However, rats, but not mice, exhibited an enhanced activity in the centre of the open-field arena, indicating on reduced anxiety or enhanced impulsivity, which is known to be associated with altered serotonin activity. Specific binding of DAT, but not SERT, was significantly reduced in the mouse AcbSh and CPU using in vitro autoradiography. On the contrary, SERT, but not DAT density was significantly reduced in the AcbSh of rats. Taken together, our data provide evidence for differential regulation of DAT and SERT densities in reward-related brain regions of rats and mice after long-term intermittent administration of MDMA.     

Single dose of MDMA causes extensive decrement of serotoninergic fibre density without blockage of the fast axonal transport in Dark Agouti rat brain and spinal cord

Prolonged neurotoxicity of the recreational drug, MDMA (3,4-methylenedioxymethamphetamine) on serotoninergic axon terminals has been suggested. The effect of a single (15 mg/kg) dose of intraperitoneally administered MDMA on serotoninergic fibre density, defined by tryptophan hydroxylase (TpH) and serotonin transporter (5-HTT) immunoreactivity, has been evaluated in the spinal cord and brain areas in Dark Agouti rats, 7 and 180 days after MDMA applications. Immunostaining for amyloid precursor protein (APP) has been performed to examine possible defects of the fast axonal transport, and 5-HTT mRNA expressions were quantified in neurones of medullary raphe nuclei. Seven days after MDMA treatment, a substantial decrease in the density of TpH-immunoreactive fibres was detectable in the frontal cortex, the caudate-putamen, the CA1 region of the hippocampus, and marked decreases were found in the spinal cord. These changes in TpH density showed a high correlation with 5-HTT densities. In contrast, APP-immunoreactive axonal bulbs were not detected in any of the brain regions studied. Seven days after MDMA administrations, significantly elevated 5-HTT mRNA expressions were found in the raphe pallidus and obscurus. Our results suggest that a single dose of MDMA elicits widespread depletion of TpH and 5-HTT immunoreactivity in serotoninergic axons without morphological sign of the blockage of the fast anterograde axonal transport. Our results do not support the notion of MDMA-induced axotomy of serotoninergic neurones. The up-regulation of 5-HTT mRNA expressions 1 week after MDMA injections might indicate the potential recovery of the serotonin system.     

Developmental effects of +/-3,4-methylenedioxymethamphetamine on spatial versus path integration learning: effects of dose distribution

We previously demonstrated that postnatal day 11-20 +/-3,4-methylenedioxymethamphetamine (MDMA) exposure reduces locomotor activity and impairs path integration and spatial learning independent of the effects on activity. The effects were seen when the drug was administered twice per day, but the optimal dosing regimen is unknown. We tested whether the same total daily dose of MDMA administered in different patterns would equally affect later behavior. A split-litter design (15 litters) was used with one male/female pair per litter receiving one of four treatment regimens. All offspring received four injections per day on P11-20 as follows: 40 x 1 (40 mg/kg MDMA x 1 + saline x 3), 20 x 2 (20 mg/kg MDMA x 2 + saline x 2), 10 x 4 (10 mg/kg MDMA x 4), or Saline (saline x 4). Doses were spaced 2 h apart. Group 40 x 1 received MDMA as the first daily dose followed by three saline doses; group 20 x 2 received MDMA as the first and last dose and saline for the middle two doses; group 10 x 4 received MDMA for all four doses; and the saline group received saline for all four doses. Regardless of dose schedule, all groups treated with MDMA exhibited reduced locomotor activity. No MDMA effects were found on swimming ability in a straight channel. Modest MDMA effects were found on Barnes maze performance. The major findings were that the 40 x 1 and 20 x 2 MDMA groups showed impaired Cincinnati multiple T-water-maze learning and the 10 x 4 and 20 x 2 MDMA groups showed impaired Morris water maze learning. The results suggest that MDMA dose distribution has a long-term differential effect on different types of learning. Dose distribution warrants greater attention in the design of developmental drug studies along with the standard considerations of dose and age.     

Heating induces aggregation and decreases detection of serotonin transporter protein on western blots

In recent years, there has been growing interest in the use of Western blot analysis to monitor changes in the abundance of the serotonin transporter (SERT) protein. In the Western blot procedure, heat denaturation is a common, early step. We now report that heating samples to 90 degrees C decreases the abundance of the SERT protein band and causes dispersion of a majority of the SERT signal to a high molecular weight smear. These observations are in keeping with the fact that heating can influence the electrophoretic behavior of some proteins. By omitting the heat denaturation step in the Western blot procedure, better detection of the SERT protein is achieved.     

Effects of MDMA on blood glucose levels and brain glucose metabolism

Purpose This study was designed to assess changes in glucose metabolism in rats administered single or repeated doses of MDMA. Methods Two different experiments were performed: (1) A single-dose study with four groups receiving 20 mg/kg, 40 mg/kg, saline or heat, and (2) a repeated-dose study with two groups receiving three doses, at intervals of 2 h, of 5 mg/kg or saline. Rats were imaged using a dedicated small-animal PET scanner 1 h after single-dose administration or 7 days after repeated doses. Glucose metabolism was measured in 12 cerebral regions of interest. Rectal temperature and blood glucose were monitored. Results Peak body temperature was reached 1 h after MDMA administration. Blood glucose levels decreased significantly after MDMA administration. In the single-dose experiment, brain glucose metabolism showed hyperactivation in cerebellum and hypo-activation in the hippocampus, amygdala and auditory cortex. In the repeated-dose experiment, brain glucose metabolism did not show any significant change at day 7. Conclusion These results are the first to indicate that MDMA has the potential to produce significant hypoglycaemia. In addition, they show that MDMA alters glucose metabolism in components of the motor, limbic and somatosensory systems acutely but not on a long-term basis.     

Cardiac effects of MDMA on the metabolic profile determined with 1H-magnetic resonance spectroscopy in the rat

Despite the potential for deleterious (even fatal) effects on cardiac physiology, 3,4-methylenedioxymethamphetamine (MDMA; ecstasy) abuse abounds driven mainly by its euphoric effects. Acute exposure to MDMA has profound cardiovascular effects on blood pressure and heart rate in humans and animals. To determine the effects of MDMA on cardiac metabolites in rats, MDMA (0, 5, or 10 mg/kg) was injected every 2 h for a total of four injections; animals were sacrificed 2 h after the last injection (8 h drug exposure), and their hearts removed and tissue samples from left ventricular wall dissected. High resolution magic angle spinning proton magnetic resonance spectroscopy ((1)H-MRS) at 11.7 T, a specialized version of MRS aptly suited for analysis of semi-solid materials such as intact tissue samples, was used to measure the cardiac metabolomic profile, including alanine, lactate, succinate, creatine, and carnitine, in heart tissue from rats treated with MDMA. MDMA effects on MR-visible choline, glutamate, glutamine, and taurine were also determined. Body temperature was measured following each MDMA administration and serotonin and norepinephrine (NE) levels were measured by high pressure liquid chromatography (HPLC) in heart tissue from treated animals. MDMA significantly and dose-dependently increased body temperature, a hallmark of amphetamines. Serotonin, but not NE, levels were significantly and dose-dependently decreased by MDMA in the heart wall. MDMA significantly altered the MR-visible profile with an increase in carnitine and no change in other key compounds involved in cardiomyocyte energy metabolomics. Finally, choline levels were significantly decreased by MDMA in heart. The results are consistent with the notion that MDMA has significant effects on cardiovascular serotonergic tone and disrupts the metabolic homeostasis of energy regulation in cardiac tissue, potentially increasing utilization of fatty acid metabolism. The contributions of serotonergic signaling on MDMA-induced changes in cardiac metabolism remain to be determined.     

Oxidative stress in rat kidneys due to 3,4-methylenedioxymetamphetamine (ecstasy) toxicity

AIM: The mechanism of MDMA (3,4-methylenedioxymethamphetamine)-induced toxicity is believed to be, in part, due to enhanced oxidative stress. As MDMA is eliminated via the kidney, the aim of this study was to investigate whether MDMA created conditions of oxidative stress within rat kidney. METHODS: Adult male Wistar rats were divided into three groups, control treatment (water), acute MDMA administration (single oral dose: 5, 10, 20 or 40 mg/kg body weight) and subacute MDMA administration (5, 10, or 20 mg/kg body weight per day during 14 days). Animals were sacrificed 8 h after the single oral MDMA administration in the acute MDMA administration group and after the last MDMA administration in the subacute MDMA administration group. Rectal temperature measurements, oxidative stress status parameters and histological examinations were performed. RESULTS: In all MDMA-administered rats, rectal temperature markedly increased peaking approximately 1 h after MDMA ingestion. Superoxide dismutase activity and thiobarbituric acid reactive substances increased after MDMA administration. Histological examinations of the kidney revealed dose-dependent disruption of tissue structure in subacute MDMA-administered rats. The latter was not observed in acute MDMA-administered rats.