Esteban B, O'Shea E, Camarero J, Sanchez V, Green AR, Colado MI.
3,4-Methylenedioxymethamphetamine induces monoamine release, but not toxicity, when administered centrally at a concentration occurring following a peripherally injected neurotoxic dose.
Psychopharmacology (Berl). 2001 Mar;154(3):251-60.
"RATIONALE: There is good evidence that 3,4-methylenedioxymethamphetamine (MDMA)-induced neurotoxicity results from free radical formation. However, it is unclear whether it is the presence of MDMA or a metabolite in the brain that initiates this process. OBJECTIVE: We wished to measure the concentration of MDMA in the brain following peripheral administration of neurotoxic doses and examine the effect on acute monoamine release and the subsequent neurotoxic loss in 5-hydroxytryptamine (5-HT) content when a high concentration of MDMA was infused into cerebral tissue. METHODS: Selectively placed microdialysis probes were used to determine both the concentration of MDMA in the brain following peripheral injection and the degree of 5-HT release. Monoamines in dialysate and tissue were measured with standard HPLC techniques. RESULTS: MDMA, administered intraperitoneally, at doses of 10 and 15 mg/kg, which produce neurodegeneration, resulted in an estimated cerebral extracellular concentration of MDMA of 11 and 20 microM, respectively. When MDMA (100-400 microM) was perfused through a selectively placed microdialysis probe it dose-dependently increased 5-HT release in the hippocampus and dopamine release in the striatum. Seven days after perfusion of MDMA the concentration of 5-HT and its metabolite, 5-hydroxyindoleacetic acid was unchanged in the ipsilateral side of the brain of normothermic rats and also in the brains of animals made hyperthermic to mimic the acute effect of MDMA given peripherally. In contrast, perfusion with 5,7-dihydroxytryptamine (400 microM) markedly decreased the cerebral 5-HT content. A second probe, also placed in the hippocampus at a distance of 1 mm from the main probe, revealed that during the perfusion of MDMA (400 microM) the estimated extracellular concentration of MDMA in the hippocampus was between 10.4 and 19.5 microM, i.e. in the range of concentrations observed after systemic injection of neurotoxic doses of MDMA. CONCLUSIONS: These data demonstrate that MDMA when injected directly into the brain produces 5-HT release but no neurotoxicity, suggesting that it must be metabolised peripherally in order to produce compounds that induce free radical formation and neurotoxicity in the brain." [Abstract]

Ramamoorthy Y, Yu AM, Suh N, Haining RL, Tyndale RF, Sellers EM.
Reduced (+/-)-3,4-methylenedioxymethamphetamine ("Ecstasy") metabolism with cytochrome P450 2D6 inhibitors and pharmacogenetic variants in vitro.
Biochem Pharmacol. 2002 Jun 15;63(12):2111-9.
""Ecstasy" [(+/-)-3,4-methylenedioxymethamphetamine or MDMA] is a CNS stimulant, whose use is increasing despite evidence of long-term neurotoxicity. In vitro, the majority of MDMA is demethylenated to (+/-)-3,4-dihydroxymethamphetamine (DHMA) by the polymorphic cytochrome P450 2D6 (CYP2D6). We investigated the demethylenation of MDMA and dextromethorphan (DEX), as a comparison drug, in reconstituted microsomes expressing the variant CYP2D6 alleles (*)2, (*)10, and (*)17, all of which have been linked to decreased enzyme activity. With MDMA, intrinsic clearances (V(max)/K(m)) in CYP2D6.2, CYP2D6.17, and CYP2D6.10 were reduced 15-, 13-, and 135-fold, respectively, compared with wild-type CYP2D6.1. With DEX, intrinsic clearances were reduced by 37-, 51-, and 164-fold, respectively. It was evident that CYP2D6.17 displayed substrate-specific changes in drug affinity (K(m)). Compounds potentially used with MDMA [fluoxetine, paroxetine, (-)-cocaine] demonstrated significant inhibition of MDMA metabolism in both human liver and CYP2D6.1-expressing microsomes. These data demonstrate that individuals possessing the CYP2D6(*)2, (*)17, and, particularly, (*)10 alleles may show significantly reduced MDMA metabolism. These individuals, and those taking CYP2D6 inhibitors, may demonstrate altered acute and/or long-term MDMA-related toxicity." [Abstract]

Chu T, Kumagai Y, DiStefano EW, Cho AK.
Disposition of methylenedioxymethamphetamine and three metabolites in the brains of different rat strains and their possible roles in acute serotonin depletion.
Biochem Pharmacol. 1996 Mar 22;51(6):789-96.
"3,4-Methylenedioxymethamphetamine (MDMA) affects both dopamine and serotonin (5-HT) systems. One of its acute actions is to cause a reversible fall in steady-state brain 5-HT concentrations. To investigate the chemical basis of this acute effect, the brain levels of the parent compound and three major metabolites, 3,4- 3,4-methylenedioxyamphetamine (MDA), 3,4-dihydroxymethamphetamine (DHMA) and 6-hydroxy-3,4-methylenedioxymethamphetamine (6-OHMDMA), were monitored, together with 5-HT levels, over a period of 6 hr in male Sprague-Dawley (SD) rats. The temporal relationships between drug concentrations of both stereoisomers and depletions were evaluated first. There was no correlation between the concentrations of the compounds measured and the extent of 5-HT depletion. Brain levels of MDMA and MDA were higher than plasma levels and exhibited a stereoselectivity in that (-)-MDMA and (+)-MDA levels were higher than those of enantiomers. The relationship between the dose of ((+)-MDMA and reduction in 5-HT levels was next investigated in SD male, SD female, and Dark Agouti (DA) female rats. These animals exhibit different capabilities of MDMA metabolism. There is a lower level of MDA, the N-demethylated metabolite of MDMA, in female SD rats than in males. Female DA rats are deficient in CYP2D isozymes, one of the enzymes responsible for demethylenation of MDMA to DHMA at pharmacological concentrations of substrate. there was a significant accuulation of MDMA in the brain and plasma of DA rats, but their 5-HT depletion was somewhat attenuated. The results indicated that MDMA ++ was apparently not the single, causative agent for the acute 5-HT depletion, which may also involve a metabolite formed by CYP2D." [Abstract]

Elayan I, Gibb JW, Hanson GR, Lim HK, Foltz RL, Johnson M.
Short-term effects of 2,4,5-trihydroxyamphetamine, 2,4,5-trihydroxymethamphetamine and 3,4-dihydroxymethamphetamine on central tryptophan hydroxylase activity.
J Pharmacol Exp Ther. 1993 May;265(2):813-8.
"In previous studies, we have reported the long-term effects of several metabolites of 3,4-methylenedioxymethamphetamine (MDMA) on tryptophan hydroxylase (TPH) activity. In this study, the short-term effects of three metabolites of MDMA. 2,4,5-trihydroxyamphetamine (THA), 2,4,5-trihydroxymethamphetamine (THM) and 3,4-dihydroxymethamphetamine, and the in vitro effect of THA on TPH activity are reported. After short-term treatment, hippocampal TPH activity was decreased to 8 and 54% of control in response to THA and THM, respectively, but was unaltered after 3,4-dihydroxymethamphetamine. Incubating TPH from THM-treated rats with dithiothreitol under nitrogen failed to reverse the decrease in enzyme activity induced by THM treatment. THA also decreased tyrosine hydroxylase activity to 75% of control, whereas the enzyme activity remained unaltered by THM. The structural analog of THA, 6-hydroxydopamine, failed to reproduce the effect of THA on TPH activity; however, 5,6-dihydroxytryptamine decreased hippocampal TPH activity to 18% of control. In the in vitro study, the hippocampus and the striatum were incubated in varying concentrations of THA. After a 1-h incubation at 37 degrees C, hippocampal TPH activity was decreased to 83, 71, 68, 47 and 3% of control after exposure to 0.001, 0.01, 0.1, 0.5 or 5.0 mM THA, respectively; striatal TPH activity was reduced to 98, 95, 70, 54 and 17% of control, respectively. Incubating the enzyme under reducing conditions failed to restore the enzyme activity to control levels." [Abstract]

Elayan I, Gibb JW, Hanson GR, Foltz RL, Lim HK, Johnson M.
Long-term alteration in the central monoaminergic systems of the rat by 2,4,5-trihydroxyamphetamine but not by 2-hydroxy-4,5-methylenedioxymethamphetamine or 2-hydroxy-4,5-methylenedioxyamphetamine.
Eur J Pharmacol. 1992 Oct 20;221(2-3):281-8.
"The long-term effects of three metabolites of 3,4-methylenedioxymethamphetamine (MDMA) on the central monoaminergic systems of the rat were examined. Seven days after the intracerebroventricular administration of 0.25 and 0.5 mumol 2,4,5-trihydroxyamphetamine, hippocampal tryptophan hydroxylase (TPH) activity was reduced to 5 and 1% of control, respectively, while norepinephrine (NE) concentration was depressed to 10 and 18% of control. These two respective dosages also decreased striatal tyrosine hydroxylase (TH) activity to 67 and 10% of control, respectively, while nigral TH activity was reduced to 59 and 20% of control. Striatal TPH activity was reduced to 74 and 81% of control, respectively, while the activity in the dorsal and median raphe remained unaltered. The intracerebroventricular administration of 1 mumol 2-hydroxy-4,5-methylenedioxymethamphetamine (6-OH-MDMA) failed to alter TPH activity, TH activity or NE concentration after 14 days. In contrast, 1 mumol of 2-hydroxy-4,5-methylenedioxyamphetamine (6-OH-MDA) induced a 30% increase in striatal TPH activity and a 50% increase in nigral TH activity. The study of the formation of 2,4,5-trihydroxyamphetamine after MDMA treatment may provide insight as to how MDMA destroys serotonergic nerve terminals." [Abstract]

Johnson M, Elayan I, Hanson GR, Foltz RL, Gibb JW, Lim HK.
Effects of 3,4-dihydroxymethamphetamine and 2,4,5-trihydroxymethamphetamine, two metabolites of 3,4-methylenedioxymethamphetamine, on central serotonergic and dopaminergic systems.
J Pharmacol Exp Ther. 1992 May;261(2):447-53.
"The effects of 3,4-dihydroxymethamphetamine (DHM) and 2,4,5-trihydroxymethamphetamine (THM) on central serotonergic and dopaminergic systems were investigated to determine if these metabolites share the neurochemical properties of 3,4-methylenedioxymethamphetamine. THM (50-200 micrograms) or DHM (135 micrograms) was administered i.c.v. to rats; 5 days later, cortical, striatal and hippocampal tryptophan hydroxylase (TPH) activity were decreased by THM in a dose-dependent manner, whereas DHM was without effect in these brain structures. The concentration of serotonin in the brain structures contralateral to the side of THM injection was also decreased, but to a lesser degree. THM (100 and 200 micrograms) increased TPH activity to 155% of control in the dorsal raphe, whereas a dose of 50 micrograms increased TPH activity to 132% of control in the median raphe nucleus. THM also markedly reduced striatal tyrosine hydroxylase activity, but did not alter enzyme activity in the substantia nigra; DHM increased striatal tyrosine hydroxylase activity to 115% of control. These results suggest that THM, but not DHM, is toxic to both dopaminergic and serotonergic nerve terminals. Although THM could not be established as the neurotoxic metabolite explaining 3,4-methylenedioxymethamphetamine (MDMA) toxicity, its properties may prove useful in elucidating amphetamine toxicity." [Abstract]

Chen JC, Fine RE, Squicciarini J, Volicer L.
Neurotoxicity of free-radical-mediated serotonin neurotoxin in cultured embryonic chick brain neurons.
Eur J Pharmacol. 1996 May 6;303(1-2):109-14.
"Exposure of serotonin (5-HT) to oxygen-derived free-radical-generating system, xanthine oxidase-hypoxanthine or to a Fenton reaction results in the formation of the neurotoxin, tryptamine-4,5-dione. In cultured embryonic chick brain neurons, incubation of tryptamine-4,5-dione or its ethyl carbonate derivative resulted in a dose-dependent neurotoxicity (1-100 microM). The addition of sulfhydryl compound, glutathione at 2 or 10 microM significantly enhanced the toxicity induced by 10 microM tryptamine-4,5-dione. On the contrary, glutathione at 10 microM decreased the neurotoxic effect caused by 10 microM 5,6- and 5,7-dihydroxytryptamine in the cultured neurons. The toxicity resulted from 5,6- and 5,7-dihydroxytryptamine could be fully prevented by a 5-HT uptake inhibitor, fluoxetine. However, the toxicity caused by tryptamine-4,5-dione and glutathione conjugate could not be blocked by fluoxetine (10 or 100 microM) or by a glutathione transferase inhibitor, boric acid/serine. The results indicate a different molecular mechanism among 5-HT derived neurotoxins and suggest that tryptamine-4,5-dione and/or its glutathione conjugate would cause neuronal damage, if they are formed in vivo." [Abstract]

Chen JC, Schnepper PW, To A, Volicer L.
Neurochemical changes in the rat brain after intraventricular administration of tryptamine-4,5-dione.
Neuropharmacology. 1992 Mar;31(3):215-9.
"Tryptamine-4,5-dione (4,5-DKT) a neurotoxic derivative of serotonin (5-HT), was injected into the lateral ventricle of the rat in order to evaluate its biochemical effects. The levels of 8 substances in the hippocampus, striatum and prefrontal cortex were examined 3, 7 and 14 days after treatment with 4,5-DKT. 5-Hydroxytryptamine and 5-hydroxyindoleacetic acid (5-HIAA) levels were decreased in all three regions by days 7 and 14, respectively. Tryptamine-4,5-dione had no significant effect on dopaminergic or adrenergic systems or on the levels of L-tryptophan and L-tyrosine, in any of the three areas of brain examined. Reduced activity of tryptophan hydroxylase in the cortex was observed 14 days after administration of 4,5-DKT. However, administration of 4,5-DKT did not alter the binding of [3H]paroxetine, a specific antagonist of the uptake of 5-HT, to nerve terminals. These results indicate that 4,5-DKT produced depletion of 5-HT without eliminating serotoninergic nerve terminals." [Abstract]

Jiang XR, Dryhurst G.
Inhibition of the alpha-ketoglutarate dehydrogenase and pyruvate dehydrogenase complexes by a putative aberrant metabolite of serotonin, tryptamine-4,5-dione.
Chem Res Toxicol. 2002 Oct;15(10):1242-7.
"A transient energy impairment with resultant release and subsequent reuptake of 5-hydroxytryptamine (5-HT) and NMDA receptor activation with consequent cytoplasmic superoxide (O(2)(-)(*)), nitric oxide (NO(*)), and peroxynitrite (ONOO(-)) generation have all been implicated in a neurotoxic cascade which ultimately leads to the degeneration of serotonergic neurons evoked by methamphetamine (MA) and 3,4-methylenedioxymethamphetamine (MDMA). Such observations raise the possibility that the O(2)(-)(*)/NO(*)/ONOO(-)-mediated oxidation of 5-HT, as it returns via the plasma membrane transporter to the cytoplasm of serotonergic neurons when the MA/MDMA-induced energy impairment begins to subside, may generate an endogenous neurotoxin. In vitro the O(2)(-)(*)/NO(*)/ONOO(-)-mediated oxidation of 5-HT forms tryptamine-4,5-dione (T-4,5-D). When incubated with intact rat brain mitochondria, T-4,5-D strongly inhibits state 3 respiration with pyruvate or alpha-ketoglutarate as substrates at concentrations which do not affect succinate-supported (complex II) respiration. Experiments with freeze-thawed rat brain mitochondria reveal that T-4,5-D inhibits the pyruvate dehydrogenase and alpha-ketoglutarate dehydrogenase complexes. These and other properties of T-4,5-D raise the possibility that it may be an endogenously formed intraneuronal metabolite of 5-HT that contributes to the serotonergic neurotoxicity of MA and MDMA." [Abstract]

Helmlin HJ, Bracher K, Bourquin D, Vonlanthen D, Brenneisen R.
Analysis of 3,4-methylenedioxymethamphetamine (MDMA) and its metabolites in plasma and urine by HPLC-DAD and GC-MS.
J Anal Toxicol. 1996 Oct;20(6):432-40.
"In Europe, the compound 3,4-methylenedioxymethamphetamine (MDMA, Ecstasy, Adam), in addition to cannabis, is the most abused illicit drug at all-night "techno" parties. Methods for the determination of MDMA and its metabolites, 4-hydroxy-3-methoxymethamphetamine (HMMA), 3,4-dihydroxy-methamphetamine (HHMA), 3,4-methylenedioxyamphetamine (MDA), 4-hydroxy-3-methoxyamphetamine (HMA), and 3,4-dihydroxyamphetamine (HHA), in biological fluids were established. Plasma and urine samples were collected from two patients in a controlled clinical study over periods of 9 and 22 h, respectively. MDMA and MDA were determined in plasma and urine by reversed-phase high-performance liquid chromatography with diode array detection (HPLC-DAD) after solid-phase extraction on cation-exchange columns. Acidic or enzymatic hydrolysis was necessary to detect HMMA, HMA, HHMA, and HHA, which are mainly excreted as glucuronides. Gas chromatography-mass spectrometry (GC-MS) was used for confirmation. Sample extraction and on-disc derivatization with heptafluorobutyric anhydride (HFBA) were performed on Toxi-Lab SPEC solid-phase extraction concentrators. After administration of a single oral dose of 1.5 mg/kg body weight MDMA, peak plasma levels of 331 ng/ml MDMA and 15 ng/mL MDA were measured after 2 h and 6.3 h, respectively. Peak concentrations of 28.1 micrograms/mL MDMA in urine appeared after 21.5 h. Up to 2.3 micrograms/mL MDA, 35.1 micrograms/mL HMMA, and 2.1 micrograms/mL HMA were measured within 16-21.5 h. Conjugated HMMA and HHMA are the main urinary metabolites of MDMA." [Abstract]

Escobedo I, O'shea E, Orio L, Sanchez V, Segura M, de la Torre R, Farre M, Green AR, Colado MI
A comparative study on the acute and long-term effects of MDMA and 3,4-dihydroxymethamphetamine (HHMA) on brain monoamine levels after i.p. or striatal administration in mice.
Br J Pharmacol. 2004 12 13;
This study investigated whether the immediate and long-term effects of 3,4-methylenedioxymethamphetamine (MDMA) on monoamines in mouse brain are due to the parent compound and the possible contribution of a major reactive metabolite, 3,4-dihydroxymethamphetamine (HHMA), to these changes. The acute effect of each compound on rectal temperature was also determined. MDMA given i.p. (30 mg kg(-1), three times at 3-h intervals), but not into the striatum (1, 10 and 100 microg, three times at 3-h intervals), produced a reduction in striatal dopamine content and modest 5-HT reduction 1 h after the last dose. MDMA does not therefore appear to be responsible for the acute monoamine release that follows its peripheral injection. HHMA does not contribute to the acute MDMA-induced dopamine depletion as the acute central effects of MDMA and HHMA differed following i.p. injection. Both compounds induced hyperthermia, confirming that the acute dopamine depletion is not responsible for the temperature changes. Peripheral administration of MDMA produced dopamine depletion 7 days later. Intrastriatal MDMA administration only produced a long-term loss of dopamine at much higher concentrations than those reached after the i.p. dose and therefore bears little relevance to the neurotoxicity. This indicates that the long-term effect is not attributable to the parent compound. HHMA also appeared not to be responsible as i.p. administration failed to alter the striatal dopamine concentration 7 days later. HHMA was detected in plasma, but not in brain, following MDMA (i.p.), but it can cross the blood-brain barrier as it was detected in the brain following its peripheral injection. The fact that the acute changes induced by i.p. or intrastriatal HHMA administration differed indicates that HHMA is metabolised to other compounds which are responsible for changes observed after i.p. administration. [Abstract]

Walker TM, Davenport-Jones JE, Fox RM, Atterwill CK.
The neurotoxic effects of methylenedioxymethamphetamine (MDMA) and its metabolites on rat brain spheroids in culture.
Cell Biol Toxicol. 1999 Jun;15(3):137-42.
"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." [Abstract]

Yeh SY.
Effects of salicylate on 3,4-methylenedioxymethamphetamine (MDMA)-induced neurotoxicity in rats.
Pharmacol Biochem Behav. 1997 Nov;58(3):701-8.
"The drug 3,4-methylenedioxymethamphetamine (MDMA) is a serotonergic neurotoxicant that causes hyperthermia and depletion of serotonin (5-HT) and 5-hydroxy-indole-3-acetic acid (5-HIAA) in the central nervous system. Formation of neurotoxic metabolites of MDMA, e.g., 2,4,5-trihydroxy-methamphetamine and 2,4,5-trihydroxyamphetamine, involves hydroxyl and/or superoxide free radicals. The present study was designed to determine whether the hydroxyl free-radical-trapping agent salicylate could provide protection against MDMA neurotoxicity in rats. In the acute studies, sodium salicylate (12.5-400 mg/kg, calculated as free acid) was injected interperitoneally (i.p.) 1 h before subcutaneous (s.c.) injections of MDMA (20 mg/kg as base). In the chronic studies, sodium salicylate (3.1-100 mg/kg) was injected i.p. 1 h before repeated s.c. injections of MDMA (10 mg/kg as base, twice daily, at 0830 and 1730 h for 4 consecutive days). Repeated MDMA administration depleted contents of 5-HT and 5-HIAA in the frontal cortex, hippocampus and striatum. Coadministration of salicylate plus MDMA did not significantly alter MDMA-induced depletion of 5-HT and 5-HIAA in these tissues. Thus, salicylate, a hydroxyl free-radical-trapping agent, does not protect against MDMA-induced hyperthermia and depletion of 5-HT and 5-HIAA. These observations suggest that MDMA-induced neurotoxicity may occur mainly through the production of superoxide or other radicals rather than hydroxyl free radicals. Salicylate actually potentiated MDMA-induced hyperthermia and lethality, findings that might be of clinical relevance." [Abstract]

O'Shea E, Easton N, Fry JR, Green AR, Marsden CA.
Protection against 3,4-methylenedioxymethamphetamine-induced neurodegeneration produced by glutathione depletion in rats is mediated by attenuation of hyperthermia.
J Neurochem. 2002 May;81(4):686-95.
"3,4-Methylenedioxymethamphetamine (MDMA) administration produces neurotoxic degeneration of serotonin terminals in rat brain. These effects occur only after systemic administration and not after central injection, suggesting that peripheral metabolism, possibly hepatic, is required for toxicity. Glutathione is one of the principal cellular defence mechanisms, but conjugation with glutathione can, on some occasions, increase the reactivity of certain molecules. Previous studies have shown that central administration of glutathione adducts of a MDMA metabolite produces a neurotoxicity profile similar to that of systemic MDMA. In the present study, depletion of peripheral (hepatic) glutathione by 43% with dl-buthionine-(S,R)-sulfoximine (an inhibitor of glutathione synthesis) did not attenuate MDMA-induced neurotoxicity as indicated by the 34% loss of [(3) H]paroxetine binding to the serotonin uptake sites in Dark Agouti rats treated with the inhibitor. However, a more profound depletion (92%) of glutathione by diethylmaleate (direct conjugation) administration significantly reduced the serotonergic neurotoxicity produced by MDMA. This depletion protocol also attenuated the hyperthermic response to MDMA. A combination protocol utilising both buthionine-(S,R)-sulfoximine and diethylmaleate that did not alter the hyperthermic response of the rats given MDMA also failed to attenuate the neurotoxicity. These findings indicate that glutathione depletion does not offer specific protection against MDMA-induced serotonin neurotoxicity in Dark Agouti rats." [Abstract]

Jones DC, Duvauchelle C, Ikegami A, Olsen CM, Lau SS, de la Torre R, Monks TJ
Serotonergic Neurotoxic Metabolites of Ecstasy Identified in Rat Brain.
J Pharmacol Exp Ther. 2005 Jan 5;
The selective serotonergic neurotoxicity of 3,4-methylenedioxyamphetamine (MDA) and 3,4-methylenedioxymethamphetamine (MDMA, ecstasy) is dependent on their systemic metabolism. We have recently shown that inhibition of brain endothelial cell g-glutamyl transpeptidase (gamma-GT) potentiates the neurotoxicity of both MDMA and MDA, indicating that metabolites that are substrates for this enzyme contribute to the neurotoxicity. Consistent with this view, glutathione (GSH) and N-acetylcysteine conjugates of alpha-methyl dopamine (alpha-MeDA) are selective neurotoxicants. However, neurotoxic metabolites of MDMA or MDA have yet to be identified in brain. Using in vivo microdialysis coupled to LC-MS/MS and HPLC-CEAS we now show that GSH and N-acetylcysteine conjugates of N-methyl-alpha-MeDA are present in the striatum of rats administered MDMA by subcutaneous injection. Moreover, inhibition of gamma-GT with acivicin increases the concentration of GSH and N-acetylcysteine conjugates of N-methyl-alpha-MeDA in brain dialysate, and there is a direct correlation between the concentrations of metabolites in dialysate and the extent of neurotoxicity, measured by decreases in serotonin (5-HT) and 5-hydroxyindole acetic (5-HIAA) levels. Importantly, the effects of acivicin are independent of MDMA-induced hyperthermia, since acivicin-mediated potentiation of MDMA neurotoxicity occurs in the context of acivicin-mediated decreases in body temperature. Finally, we have synthesized 5-(N-acetylcystein-S-yl)- N-methyl-alpha-MeDA and established that it is a relatively potent serotonergic neurotoxicant. Taken together the data support the contention that MDMA-mediated serotonergic neurotoxicity is mediated by the systemic formation of GSH and N-acetylcysteine conjugates of N-methyl-alpha-MeDA (and alpha-MeDA). The mechanisms by which such metabolites access the brain and produce selective serotonergic neurotoxicity remain to be determined. [Abstract]

Jones DC, Lau SS, Monks TJ
Thioether metabolites of 3,4-methylenedioxyamphetamine and 3,4-methylenedioxymethamphetamine inhibit human serotonin transporter (hSERT) function and simultaneously stimulate dopamine uptake into hSERT-expressing SK-N-MC cells.
J Pharmacol Exp Ther. 2004 Oct;311(1):298-306.
3,4-Methylenedioxyamphetamine (MDA) and 3,4-methyl-enedioxymethamphetamine (MDMA, ecstasy) are widely abused amphetamine derivatives that target the serotonin system. The serotonergic neurotoxicity of MDA and MDMA seems dependent on their systemic metabolism. 5-(Glutathion-S-yl)-alpha-methyldopamine [5-(GSyl)-alpha-MeDA] and 2,5-bis(glutathion-S-yl)-alpha-methyldopamine [2,5-bis(GSyl)-alpha-MeDA], metabolites of MDA and MDMA, are also selective serotonergic neurotoxicants and produce behavioral and neurochemical changes similar to those seen with MDA and MDMA. We now show that 5-(GSyl)-alpha-MeDA and 2,5-bis(GSyl)-alpha-MeDA are more potent than MDA and MDMA (K(i) = 69, 50, 107, and 102 microM, respectively) at inhibiting 5-hy-droxytryptamine (serotonin) transport into SK-N-MC cells transiently transfected with the human serotonin transporter (hSERT). Moreover, 5-(GSyl)-alpha-MeDA and 2,5-bis(GSyl)-alpha-MeDA simultaneously stimulated dopamine (DA) transport into the hSERT-expressing cells, an effect attenuated by fluoxetine, indicating that stimulated DA transport was hSERT-dependent. Finally, 5-(GSyl)-alpha-MeDA and 2,5-bis(GSyl)-alpha-MeDA, and to a lesser extent MDA and MDMA, induced a concentration and time-dependent increase in reactive oxygen species (ROS) in both hSERT and human dopamine transporter-transfected cells. Fluoxetine attenuated the increase in ROS generation in hSERT-expressing cells. The results are consistent with the view that the serotonergic neurotoxicity of MDA and MDMA may be mediated by the metabolism-dependent stimulation of DA transport into hSERT-expressing cells and ROS generation by redox active catechol-thioether metabolites and DA. [Abstract]

Bai F, Lau SS, Monks TJ.
Glutathione and N-acetylcysteine conjugates of alpha-methyldopamine produce serotonergic neurotoxicity: possible role in methylenedioxyamphetamine-mediated neurotoxicity.
Chem Res Toxicol. 1999 Dec;12(12):1150-7.
"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." [Abstract]

Miller RT, Lau SS, Monks TJ.
2,5-Bis-(glutathion-S-yl)-alpha-methyldopamine, a putative metabolite of (+/-)-3,4-methylenedioxyamphetamine, decreases brain serotonin concentrations.
Eur J Pharmacol. 1997 Apr 4;323(2-3):173-80.
"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." [Abstract]

Bai F, Jones DC, Lau SS, Monks TJ.
Serotonergic neurotoxicity of 3,4-(+/-)-methylenedioxyamphetamine and 3,4-(+/-)-methylendioxymethamphetamine (ecstasy) is potentiated by inhibition of gamma-glutamyl transpeptidase.
Chem Res Toxicol. 2001 Jul;14(7):863-70.
"Reactive metabolites play an important role in 3,4-(+/-)-methylenedioxyamphetamine (MDA) and 3,4-(+/-)-methylenedioxymethamphetamine (MDMA; ecstasy)-mediated serotonergic neurotoxicity, although the specific identity of such metabolites remains unclear. 5-(Glutathion-S-yl)-alpha-methyldopamine (5-GSyl-alpha-MeDA) is a serotonergic neurotoxicant found in the bile of MDA-treated rats. The brain uptake of 5-GSyl-alpha-MeDA is decreased by glutathione (GSH), but sharply increases in animals pretreated with acivicin, an inhibitor of gamma-glutamyl transpeptidase (gamma-GT) suggesting competition between intact 5-GSyl-alpha-MeDA and GSH for the putative GSH transporter. gamma-GT is enriched in blood-brain barrier endothelial cells and is the only enzyme known to cleave the gamma-glutamyl bond of GSH. We now show that pretreatment of rats with acivicin (18 mg/kg, ip) inhibits brain microvessel endothelial gamma-GT activity by 60%, and potentiates MDA- and MDMA-mediated depletions in serotonin (5-HT) and 5-hydroxylindole acidic acid (5-HIAA) concentrations in brain regions enriched in 5-HT nerve terminal axons (striatum, cortex, hippocampus, and hypothalamus). In addition, glial fibrillary acidic protein (GFAP) expression increases in the striatum of acivicin and MDA (10 mg/kg) treated rats, but remains unchanged in animals treated with just MDA (10 mg/kg). Inhibition of endothelial cell gamma-GT at the blood-brain barrier likely enhances the uptake into brain of thioether metabolites of MDA and MDMA, such as 5-(glutathion-S-yl)-alpha-MeDA and 2,5-bis-(glutathion-S-yl)-alpha-MeDA, by increasing the pool of thioether conjugates available for uptake via the intact GSH transporter. The data indicate that thioether metabolites of MDA and MDMA contribute to the serotonergic neurotoxicity observed following peripheral administration of these drugs." [Abstract]

McCann UD, Ricaurte GA.
Major metabolites of (+/-)3,4-methylenedioxyamphetamine (MDA) do not mediate its toxic effects on brain serotonin neurons.
Brain Res. 1991 Apr 5;545(1-2):279-82.
"The two major metabolites of (+/-)3,4-methylenedioxyamphetamine (MDA), alpha-methyldopamine (alpha-MeDA) and 3-O-methyl-alpha-methyldopamine (3-O-Me-alpha-MeDA), were administered to rats intracerebroventricularly and into brain parenchyma. In addition, their precursors, (alpha-MeDOPA and 3-O-Me-alpha-MeDOPA, respectively) were administered systemically, individually and in combination. None of these treatments produced a lasting depletion of brain serotonin (5-HT). These findings suggest that neither of MDA's major metabolites mediate its toxic effects on 5-HT neurons and that either a minor metabolite is responsible or that alternate mechanisms are involved." [Abstract]

Largeron M, Neudorffer A, Gramond JP, Fleury MB.
[Biomimetic electrochemical synthesis of quinol-thioether conjugates: their implication in the serotonergic neurotoxicity of amphetamine derivatives]
Ann Pharm Fr. 2003 May;61(3):164-72.
"Injection of 3,4-methylenedioxyamphetamine (MDA) or 3,4-methylenedioxymethylamphetamine (MDMA or ecstasy) directly into the brain fails to reproduce the long-term effects observed after peripheral administration, implying an essential role for systemic metabolites in the development of toxicity. However, the precise identity of the metabolites participating in MDA and MDMA-mediated serotonergic neurotoxicity remains unclear: neither 3,4-alpha-methyldopamine, nor N-methyl-alpha-methyldopamine, major metabolites, produce neurotoxicity following peripheral administration. In vivo, these metabolites are oxidized to the corresponding orthoquinones, that readily react with protein and nonprotein sulphydryls including glutathione (GSH). The resulting quinol-thioether conjugates exhibit a variety of toxicological activities, which can be regulated by intramolecular cyclisation reactions that occur subsequent to oxidation. The ability of quinol-thioether conjugates to redox cycle and produce reactive oxygen species provides a rationale for the potential role of these metabolites in MDA and MDMA neurotoxicity. A biomimetic one-pot synthesis of 5-(GSH-S-yl)-N-Me-alpha-Me-DA involving addition of GSH to the electrogenerated orthoquinone species, is reported to evaluate its in vivo potential neurotoxicity." [Abstract]

Miller RT, Lau SS, Monks TJ.
Effects of intracerebroventricular administration of 5-(glutathion-S-yl)-alpha-methyldopamine on brain dopamine, serotonin, and norepinephrine concentrations in male Sprague-Dawley rats.
Chem Res Toxicol. 1996 Mar;9(2):457-65.
"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." [Abstract]

Easton N, Fry J, O'Shea E, Watkins A, Kingston S, Marsden CA.
Synthesis, in vitro formation, and behavioural effects of glutathione regioisomers of alpha-methyldopamine with relevance to MDA and MDMA (ecstasy).
Brain Res. 2003 Oct 17;987(2):144-54.
"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." [Abstract]

Carvalho M, Milhazes N, Remiao F, Borges F, Fernandes E, Amado F, Monks TJ, Carvalho F, Bastos ML.
Hepatotoxicity of 3,4-methylenedioxyamphetamine and alpha-methyldopamine in isolated rat hepatocytes: formation of glutathione conjugates.
Arch Toxicol. 2004 Jan;78(1):16-24. Epub 2003 Oct 28.
"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." [Abstract]

Carvalho M, Hawksworth G, Milhazes N, Borges F, Monks TJ, Fernandes E, Carvalho F, Bastos ML.
Role of metabolites in MDMA (ecstasy)-induced nephrotoxicity: an in vitro study using rat and human renal proximal tubular cells.
Arch Toxicol. 2002 Oct;76(10):581-8. Epub 2002 Aug 01.
"The metabolism of 3,4-methylenedioxymethamphetamine (MDMA, ecstasy) has recently been implicated in the mechanisms underlying ecstasy-induced neurotoxicity and hepatotoxicity. However, its potential role in ecstasy-induced kidney toxicity has yet to be investigated. Thus, primary cultures of rat and human renal proximal tubular cells (PTCs) were used to investigate the cytotoxicity induced by MDMA and its metabolites methylenedioxyamphetamine (MDA), alpha-methyldopamine (alpha-MeDA), and the glutathione (GSH) conjugates 5-(glutathion- S-yl)-alpha-MeDA and 2,5- bis(glutathion- S-yl)-alpha-MeDA. Cell viability was evaluated using the mitochondrial MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. MDMA and MDA were not found to be toxic to either rat or human PTCs at any concentration tested (100-800 micro M). In contrast, 800 micro M alpha-MeDA caused 60% and 40% cell death in rat and human PTCs, respectively. Conjugation of alpha-MeDA with GSH resulted in the formation of even more potent nephrotoxicants. Thus, exposure of rat and human PTC monolayers to 400 micro M 5-(glutathion- S-yl)-alpha-MeDA caused approximately 80% and 70% cell death, respectively. 5-(Glutathion- S-yl)-alpha-MeDA (400 micro M) was more toxic than 2,5- bis(glutathion- S-yl)-alpha-MeDA to rat renal PTCs but equally potent in human renal PTCs. Pre-incubation of rat PTCs with either acivicin, an inhibitor of gamma-glutamyl transpeptidase (gamma-GT), or bestatin, an inhibitor of aminopeptidase M, resulted in increased toxicity of 5-(glutathion- S-yl)-alpha-MeDA but had no effect on 2,5- bis(glutathion- S-yl)-alpha-MeDA-mediated cytotoxicity. The present data provide evidence that metabolism is required for the expression of MDMA-induced renal toxicity in vitro. In addition, metabolism of 5-(glutathion- S-yl)-alpha-MeDA by gamma-GT and aminopeptidase M to the corresponding cystein- S-yl-glycine and/or cystein- S-yl conjugates is likely to be associated with detoxication of this compound. Thus, it appears that toxicity induced by thioether metabolites of ecstasy at the apical membrane of renal proximal tubular cells is the result of extracellular events, presumably redox cycling." [Abstract]