Effects of 3,4-methylenedioxymethamphetamine (MDMA) and its main metabolites on cardiovascular function in conscious rats.

BACKGROUND AND PURPOSE: The cardiovascular effects produced by 3,4-methylenedioxymethamphetamine (MDMA; 'Ecstasy') contribute to its acute toxicity, but the potential role of its metabolites in these cardiovascular effects is not known. Here we examined the effects of MDMA metabolites on cardiovascular function in rats. EXPERIMENTAL APPROACH: Radiotelemetry was employed to evaluate the effects of s.c. administration of racemic MDMA and its phase I metabolites on BP, heart rate (HR) and locomotor activity in conscious male rats. KEY RESULTS: MDMA (1-20 mg·kg(-1)) produced dose-related increases in BP, HR and activity. The peak effects on HR occurred at a lower dose than peak effects on BP or activity. The N-demethylated metabolite, 3,4-methylenedioxyamphetamine (MDA), produced effects that mimicked those of MDMA. The metabolite 3,4-dihydroxymethamphetamine (HHMA; 1-10 mg·kg(-1)) increased HR more potently and to a greater extent than MDMA, whereas 3,4-dihydroxyamphetamine (HHA) increased HR, but to a lesser extent than HHMA. Neither dihydroxy metabolite altered motor activity. The metabolites 4-hydroxy-3-methoxymethamphetamine (HMMA) and 4-hydroxy-3-methoxyamphetamine (HMA) did not affect any of the parameters measured. The tachycardia produced by MDMA and HHMA was blocked by the ß-adrenoceptor antagonist propranolol. CONCLUSIONS AND IMPLICATIONS: Our results demonstrate that HHMA may contribute significantly to the cardiovascular effects of MDMA in vivo. As such, determining the molecular mechanism of action of HHMA and the other hydroxyl metabolites of MDMA warrants further study.

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.

"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.

Determination of MDMA and its three metabolites in the rat perfused liver.

3,4-Methylenedioxymethamphetamine (MDMA) is one of the most commonly abused illicit drugs in the world. We developed a rapid and simple high-performance liquid chromatography with a fluorescence (FL) detector method to determine MDMA and its metabolites, such as 3,4-methylenedioxyamphetamine (MDA), 4-hydroxy-3-methoxyamphetamine (HMA) and its main unstable metabolite 3,4-dihydroxymethamphetamine (HHMA) besides the internal standards, in a perfusion medium. The separation of analytes was performed at 25°C on a Chromolith® C18 (100 × 4.6 mm) column from Merck (Darmstadt, Germany) without any derivatization. The FL detector wavelength was fixed at 285 nm for excitation and at 320 nm for emission. Acetonitrile:phosphate buffer (0.02 M) at pH = 3 (5:95 v/v) was used as the mobile phase. The elution order was HHMA, HMA, MDA and MDMA with a retention time of 1.7, 2.6, 6.1 and 7.4 min, respectively. The method was validated according to the FDA bioanalytical method validation guideline. The limits of quantifications (LOQs) obtained for MDMA, MDA, HMA and HHMA were 1, 1, 1.5 and 5 ng/mL, respectively. The repeatability of relative standard deviation was  <11% (except for LOQs). This method was applied successfully to determine MDMA and its metabolites in rat liver perfusion samples. To our knowledge, this is the first method introduced for the determination of HHMA as a free form with an FL detector.

A novel G protein-coupled receptor of Schistosoma mansoni (SmGPR-3) is activated by dopamine and is widely expressed in the nervous system.

Schistosomes have a well developed nervous system that coordinates virtually every activity of the parasite and therefore is considered to be a promising target for chemotherapeutic intervention. Neurotransmitter receptors, in particular those involved in neuromuscular control, are proven drug targets in other helminths but very few of these receptors have been identified in schistosomes and little is known about their roles in the biology of the worm. Here we describe a novel Schistosoma mansoni G protein-coupled receptor (named SmGPR-3) that was cloned, expressed heterologously and shown to be activated by dopamine, a well established neurotransmitter of the schistosome nervous system. SmGPR-3 belongs to a new clade of "orphan" amine-like receptors that exist in schistosomes but not the mammalian host. Further analysis of the recombinant protein showed that SmGPR-3 can also be activated by other catecholamines, including the dopamine metabolite, epinine, and it has an unusual antagonist profile when compared to mammalian receptors. Confocal immunofluorescence experiments using a specific peptide antibody showed that SmGPR-3 is abundantly expressed in the nervous system of schistosomes, particularly in the main nerve cords and the peripheral innervation of the body wall muscles. In addition, we show that dopamine, epinine and other dopaminergic agents have strong effects on the motility of larval schistosomes in culture. Together, the results suggest that SmGPR-3 is an important neuronal receptor and is probably involved in the control of motor activity in schistosomes. We have conducted a first analysis of the structure of SmGPR-3 by means of homology modeling and virtual ligand-docking simulations. This investigation has identified potentially important differences between SmGPR-3 and host dopamine receptors that could be exploited to develop new, parasite-selective anti-schistosomal drugs.

Kinetic cooperativity of tyrosinase. A general mechanism.

Tyrosinase shows kinetic cooperativity in its action on o-diphenols, but not when it acts on monophenols, confirming that the slow step is the hydroxylation of monophenols to o-diphenols. This model can be generalised to a wide range of substrates; for example, type S(A) substrates, which give rise to a stable product as the o-quinone evolves by means of a first or pseudo first order reaction (α-methyl dopa, dopa methyl ester, dopamine, 3,4-dihydroxyphenylpropionic acid, 3,4-dihydroxyphenylacetic acid, α-methyl-tyrosine, tyrosine methyl ester, tyramine, 4-hydroxyphenylpropionic acid and 4-hydroxyphenylacetic acid), type S(B) substrates, which include those whose o-quinone evolves with no clear stoichiometry (catechol, 4-methylcatechol, phenol and p-cresol) and, lastly, type S(C) substrates, which give rise to stable o-quinones (4-tert-butylcatechol/4-tert-butylphenol).

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.

Further studies on the role of metabolites in (+/-)-3,4-methylenedioxymethamphetamine-induced serotonergic neurotoxicity.

The mechanism by which the recreational drug (+/-)-3,4-methylenedioxymethamphetamine (MDMA) destroys brain serotonin (5-HT) axon terminals is not understood. Recent studies have implicated MDMA metabolites, but their precise role remains unclear. To further evaluate the relative importance of metabolites versus the parent compound in neurotoxicity, we explored the relationship between pharmacokinetic parameters of MDMA, 3,4-methylenedioxyamphetamine (MDA), 3,4-dihydroxymethamphetamine (HHMA), and 4-hydroxy-3-methoxymethamphetamine (HMMA) and indexes of serotonergic neurotoxicity in the same animals. We also further evaluated the neurotoxic potential of 5-(N-acetylcystein-S-yl)-HHMA (5-NAC-HHMA), an MDMA metabolite recently implicated in 5-HT neurotoxicity. Lasting serotonergic deficits correlated strongly with pharmacokinetic parameters of MDMA (C(max) and area under the concentration-time curve), more weakly with those of MDA, and not at all with those of HHMA or HMMA (total amounts of the free analytes obtained after conjugate cleavage). HHMA and HMMA could not be detected in the brains of animals with high brain MDMA concentrations and high plasma HHMA and HMMA concentrations, suggesting that HHMA and HMMA do not readily penetrate the blood-brain barrier (either in their free form or as sulfate or glucuronic conjugates) and that little or no MDMA is metabolized to HHMA or HMMA in the brain. Repeated intraparenchymal administration of 5-NAC-HHMA did not produce significant lasting serotonergic deficits in the rat brain. Taken together, these results indicate that MDMA and, possibly, MDA are more important determinants of brain 5-HT neurotoxicity in the rat than HHMA and HMMA and bring into question the role of metabolites (including 5-NAC-HHMA) in MDMA neurotoxicity.

Concerted action of the cytosolic sulfotransferase, SULT1A3, and catechol-O-methyltransferase in the metabolism of dopamine in SK-N-MC human neuroblastoma cells.

Conjugation reactions catalyzed by the cytosolic sulfotransferase, SULT1A3, or catechol-O-methyltransferase (COMT) are known to be involved in the regulation and homeostasis of dopamine and other monoamine neurotransmitters. Whether different conjugation reactions may act in a concerted manner, however, remains unclear. The current study aimed to investigate the concerted action of SULT1A3 and COMT in dopamine metabolism. Analysis of the medium of SK-N-MC cells, metabolically labeled with [(35)S]sulfate in the presence of dopamine, revealed the generation and release of predominantly [(35)S]sulfated 3-methyldopamine and, to a lesser extent [(35)S]sulfated dopamine. Addition to the labeling medium of tropolone, a COMT inhibitor, enhanced the production of [(35)S]sulfated dopamine, with a concomitant decrease of [(35)S]sulfated 3-methyldopamine. Enzymatic assays using the eleven known human cytosolic SULTs revealed SULT1A3 as the major enzyme responsible for the sulfation of both dopamine and 3-methyldopamine. Kinetic analysis showed that the catalytic efficiency of SULT1A3 with 3-methyldopamine was 1.6 times than that with dopamine. Using subcellular fractions prepared from SK-N-MC cells, the majority of COMT dopamine-methylating activity was found to be present in the cytosol. Collectively, these results imply a concerted action of sulfation and methylation in the irreversible inactivation and disposal of excess dopamine in SK-N-MC cells.

Cross-functioning between the extraneuronal monoamine transporter and multidrug resistance protein 1 in the uptake of adrenaline and export of 5-(glutathion-S-yl)adrenaline in rat cardiomyocytes.

Isolated heart cells are highly susceptible to the toxicity of catecholamine oxidation products, namely, to catecholamine-glutathione adducts. Although cellular uptake and/or efflux of these products may constitute a crucial step, the knowledge about the involvement of transporters is still very scarce. This work aimed to contribute to the characterization of membrane transport mechanisms, namely, extraneuronal monoamine transporter (EMT), the multidrug resistant protein 1 (MRP1), and P-glycoprotein (P-gp) in freshly isolated cardiomyocytes from adult rats. These transporters may be accountable for uptake and/or efflux of adrenaline and an adrenaline oxidation product, 5-(glutathion-S-yl)adrenaline, in cardiomyocyte suspensions. Our results showed that 5-(glutathion-S-yl)adrenaline efflux was mediated by MRP1. Additionally, we demonstrated that the adduct formation occurs within the cardiomyocytes, since EMT inhibition reduced the intracellular adduct levels. The classical uptake2 transport in rat myocardial cells was inhibited by the typical EMT inhibitor, corticosterone, and surprisingly was also inhibited by low concentrations of another drug, a well-known P-gp inhibitor, GF120918. The P-gp activity was absent in the cells since P-gp-mediated efflux of quinidine was not blocked by GF120918. In conclusion, this work showed that freshly isolated cardiomyocytes from adult rats constitute a good model for the study of catecholamines and catecholamines metabolites membrane transport. The cardiomyocytes maintain EMT and MRP1 fully active, and these transporters contribute to the formation and efflux of 5-(glutathion-S-yl)adrenaline. In the present experimental conditions, P-gp activity is absent in the isolated cardiomyocytes.

Liquid chromatographic-electrospray ionization mass spectrometric assay for simultaneous determination of 3,4-methylenedioxymethamphetamine and its metabolites 3,4-methylenedioxyamphetamine, 3,4-dihydroxymethamphetamine, and 4-hydroxy-3-methoxymethamphetamine in rat brain.

3,4-Methylenedioxymethamphetamine (MDMA) is a psychoactive drug with abuse liability and neurotoxic potential. Mechanisms by which MDMA produces behavioral and neurotoxic effects have yet to be elucidated. By measuring concentrations of MDMA and its metabolites in relevant brain sites, it may be possible to gain insight into mechanisms underlying MDMA actions. For this purpose, an LC-MS assay with electrospray ionization was developed after homogenization of rat brain and enzymatic conjugate cleavage. The method was successfully validated with respect to selectivity, linearity, accuracy, precision, recovery, and matrix effect and its use should help to delineate the neurotoxic mechanism of action of MDMA.

Stereochemical analysis of 3,4-methylenedioxymethamphetamine and its main metabolites in human samples including the catechol-type metabolite (3,4-dihydroxymethamphetamine).

3,4-Methylenedioxymethamphetamine (MDMA; "ecstasy") is a designer drug commonly misused in large segments of young populations. MDMA is usually formulated in tablets of its racemate (1:1 mixture of its enantiomers) in doses ranging from 50 to 200 mg. MDMA has an enantioselective metabolism, the (S)-enantiomer being metabolized faster than the (R)-enantiomer. Different pharmacologic properties have been attributed to each enantiomer. The carbon responsible for MDMA chirality is preserved along its metabolic disposition. An analytical method has been developed to determine MDMA enantiomers and those from its major metabolites, 3,4-methylenedioxyamphetamine (MDA), 3,4-dihydroxymeth-amphetamine (HHMA), and 4-hydroxy-3-methoxymethamphet-amine (HMMA). It has been applied to the analysis of plasma and urine samples from healthy recreational users of MDMA who participated voluntarily in a clinical trial and received 100 mg (R,S)-MDMA. HCl orally. (R)/(S) ratios both in plasma (0-48 h) and urine (0-72 h) for MDMA and MDA were >1 and <1, respectively. Ratios corresponding to HHMA and HMMA, close to unity, deviate from theoretical expectations and are most likely explained by the ability of MDMA to autoinhibit its own metabolism. The short elimination half-life of (S)-MDMA (4.8 h) is consistent with the subjective effects and psychomotor performance reported in subjects exposed to MDMA, whereas the much longer half-life of the (R)-enantiomer (14.8 h) correlates with mood and cognitive effects experienced on the next days after MDMA use.

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.

Synthesis, in vitro formation, and behavioural effects of glutathione regioisomers of alpha-methyldopamine with relevance to MDA and MDMA (ecstasy).

Administration of 3,4-methylenedioxymethamphetamine (MDMA) or 3,4-methylenedioxyamphetamine (MDA) to rats produces serotonergic nerve terminal degeneration. However, they are not neurotoxic when injected directly into the brain, suggesting the requirement for peripheral metabolism of MDMA to a neurotoxic metabolite. Alpha-methyldopamine (alpha-MeDA) is a major metabolite of MDA. There are indications that a glutathione metabolite of alpha-MeDA and/or 3,4-dihydroxymethamphetamine may be responsible for the neurotoxicity and some of the behavioural effects produced by MDMA and/or MDA. The present study details the synthesis, purification and separation of the 5-(glutathion-S-yl)-alpha-MeDA and 6-(glutathion-S-yl)-alpha-MeDA regioisomers of alpha-MeDA. Incubation of MDA with human liver microsomes demonstrated that production of both glutathione adducts are related to cytochrome P450 2D6 isoform activity. Following intracerebroventricular administration (180 nmol) of either GSH adduct into Dark Agouti or Sprague-Dawley rats only 5-(glutathion-S-yl)-alpha-MeDA produced behavioural effects characterised by hyperactivity, teeth chattering, tremor/trembling, head weaving, splayed posture, clonus and wet dog shakes. Pre-treatment with a dopamine receptor antagonist (haloperidol, 0.25 mg/kg; i.p.) attenuated hyperactivity, teeth chattering, low posture and clonus and potentiated splayed postural effects. These results indicate that MDA can be converted into two glutathione regioisomers by human liver microsomes, but only the 5-(glutathion-S-yl)-alpha-MeDA adduct is behaviourally active in the rat.

Synthesis and capillary electrophoretic analysis of enantiomerically enriched reference standards of MDMA and its main metabolites.

Enantiomerically-enriched (S)-3,4-methylenedioxymethamphetamine (MDMA) and its main metabolites (S)-4-hydroxy-3-methoxymethamphetamine (HMMA) and (S)-3,4-dihydroxymethamphetamine (HHMA) were prepared for unequivocal identification of the differential enantioselective metabolism of these compounds as well as for its application in the analysis of biological samples. Capillary electrophoresis with cyclodextrin derivatives and a chemical correlation of (S)-MDMA, (S)-HMMA and (S)-HHMA has been performed to assign the absolute stereochemistry of major isomers in analytical standards enriched with such enantiomers.

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.

The oxidation of dopamine and epinine by the two forms of monoamine oxidase from rat liver.

Information on the "in vitro" oxidation of epinine by monoamine oxidase (MAO) compared to dopamine is very poor. The aim of this work was to study the oxidative deamination of epinine and dopamine by rat liver MAO-A and MAO-B. The contributions of MAO-A and B to the metabolism of dopamine (55% and 45%, respectively) and epinine (70% and 30%, respectively) were similar. The results of this study show that epinine is a substrate for both forms of MAO in rat liver, although the contribution of MAO A to the deamination of this secondary amine appears to be slightly more important than that of MAO B.

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)