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TOXICOLOGY
Rede de Química e Tecnologia (REQUIMTE), Toxicology Department, Faculty of Pharmacy, University of Porto, Porto, Portugal (J.P.C., F.R., M.L.B., F.C.); Neurology Department, Charité Hospital, Humboldt University, Berlin, Germany (A.M.); REQUIMTE/Centro de Química Fina e Biotecnologia (CQFB), Chemistry Department, Faculty of Science and Technology, University of Nova de Lisboa, Monte de Caparica, Portugal (A.R.A., P.S.B., L.M.F., A.M.L.)
Received July 14, 2005; accepted September 22, 2005.
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Abstract |
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-methyldopamine (N-Me--MeDA) and -methyldopamine (-MeDA), respectively, both of which are catechols that can undergo oxidation to the corresponding ortho-quinones. In the presence of glutathione (GSH), ortho-quinones may be conjugated with GSH to form glutathionyl adducts. In this study, we evaluated the neurotoxicity of MDMA and three of its metabolites obtained by synthesis, N-Me--MeDA, -MeDA, and 5-(GSH)--MeDA [5-(glutathion-S-yl)--methyldopamine] in rat cortical neuronal serum-free cultures under normal (36.5°C) and hyperthermic (40°C) conditions. Cell viability was assessed, and the mechanism of cell death was also evaluated. Our study shows that these metabolites are more neurotoxic [5-(GSH)--MeDA being the most toxic] than the parent compound MDMA. The neurotoxicity of MDMA metabolites was partially prevented by the antioxidants N-acetylcystein and also, in a minor extent, by -phenyl-N-tert-butyl nitrone. All the tested compounds induced apoptotic cell death in cortical neurons, and their neurotoxic effect was potentiated under hyperthermic conditions. These data suggest that MDMA metabolites, especially under hyperthermic conditions, contribute to MDMA-induced neurotoxicity.
). It is widely acknowledged that MDMA produces neurotoxic damage of 5-HT (serotonin) nerve endings, although the serotonergic cell bodies do not appear to be destroyed by the drug (Green et al., 2003). Moreover, studies also report MDMA-induced neuronal degeneration in the parietal cortex, insular/perirhinal cortex, ventromedial/ventrolateral thalamus, and in the tenia tecta (Commins et al., 1987; Schmued, 2003). Evidence for the occurrence of MDMA-induced neurotoxic damage in human users remains a matter of debate, especially due to polydrug abuse among consumers (Green et al., 2003).
Several studies failed to prove serotonergic neurotoxicity when MDMA and MDA were injected directly into the brain (Paris and Cunningham, 1992; Esteban et al., 2001). Since they could not reproduce the serotonergic neurotoxicity seen after the peripheral administration of the drugs, it was postulated that systemic metabolism is needed for the occurrence of neurotoxic events (Monks et al., 2004).
Metabolism of MDMA involves N-demethylation to MDA. MDMA and MDA are O-demethylenated to N-methyl--methyldopamine (N-Me--MeDA) and -methyldopamine (-MeDA), respectively (Lim and Foltz, 1988; Kumagai et al., 1991), both of which are catechols that can undergo oxidation to the corresponding o-quinones. These quinones are highly redox-active molecules that can undergo redox cycling, which originates semiquinone radicals and leads to the generation of ROS and RNS (Bolton et al., 2000; Remião et al., 2002). The catecholamine oxidation process can be catalyzed under physiological conditions by oxidative enzymes, such as xanthine oxidase, peroxidases, lipoxygenase, several copper-containing catechol oxidases, or in the presence of metal ions such as Cu2+, Mn2+, Fe3+, and several copper and ferric chelates (Bindoli et al., 1992). Alternatively, since the reactive o-quinone intermediates are Michael acceptors, cellular damage can occur through alkylation of crucial cellular proteins and/or DNA. In the presence of GSH, o-quinone may be conjugated with GSH to form a glutathionyl adduct, 5-(glutathion-S-yl)--methyldopamine [5-(GSH)--MeDA] (Hiramatsu et al., 1990; Carvalho et al., 2004c). This GSH conjugate remains redox-active, being readily oxidized to the quinone thioether, which, after the reductive addition of a second molecule of GSH, yields a 2,5-bis-glutathionyl conjugate (for detailed insights on MDMA metabolism, see Fig. 1). Taken together, MDMA metabolism leading to the formation of reactive intermediates, ROS, and/or toxic oxidation products may represent the triggering factors responsible for the toxicity exerted by this amphetamine (Carvalho et al., 2004b,c).
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). Misuse of MDMA in crowded conditions with a high ambient temperature, physical activity, and dehydration, i.e., under the conditions that MDMA is used at "rave" parties, may all contribute to increase the hyperthermic response induced by MDMA (Green et al., 2003).
The underlying mechanisms of MDMA-induced neurotoxicity, namely the possible role of its metabolite(s), remain to be completely elucidated. Therefore, it was the aim of this study to evaluate and characterize the neurotoxicity in rat cortical neuronal serum-free cultures of MDMA and three of its main metabolites: N-Me--MeDA, -MeDA, and 5-(GSH)--MeDA. We have previously shown that exposition to hyperthermia (40°C) leads to a potentiation of the MDMA-induced neurotoxic effect (J. P. Capela, K. Ruscher, M. Lautenschlager, D. Freyer, U. Dirnagl, A. R. Gaio, M. L. Bastos, A. Meisel, and F. Carvalho, manuscript submitted for publication). In this study, experiments were conducted to confirm the higher neuronal susceptibility toward MDMA metabolites at hyperthermic temperatures.
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Materials and Methods |
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-phenyl-N-tert-butyl nitrone), NAC (N-acetylcystein), reduced GSH, and mushroom tyrosinase (4.400 units/mg) were obtained from Sigma-Aldrich (St. Louis, MO). The metabolites N-Me--MeDA, -MeDA, and 5-(GSH)--MeDA were synthesized by REQUIMTE/CQFB, Chemistry Department, Faculty of Science and Technology, Universidade Nova de Lisboa. All other chemicals were purchased from Sigma-Aldrich of the highest grade commercially available.
Synthesis of N-Me--MeDA, -MeDA, and 5-(GSH)--MeDA. Solvents were dried by standard methods and distilled before use. Analytical thin-layer chromatography was conducted on Merck Kieselgel 60 F254 silica gel 0.2-mm-thick plates (Merck, Darmstadt, Germany); column chromatography was performed on Merck Kieselgel 60 (240–400 mm) silica gel or reverse-phase RP-18 modified silica. Melting points were recorded on a Reichert-Thermovar hot-stage apparatus (Leica, Wetzlar, Germany) and are reported uncorrected. Infrared spectra were recorded on PerkinElmer Spectrum 1000 (PerkinElmer Life and Analytical Sciences, Boston, MA) as potassium bromide pellets or as film over sodium chloride (NaCl) windows. Proton and carbon nuclear magnetic resonance spectra (1H and 13C NMR) were recorded on Bruker ARX 400 spectrometer (Bruker, Newark, DE) at 400 and 100.62 MHz, respectively. Chemical shifts are expressed in parts per million downfield from tetramethylsilane (
= 0) as an internal standard; J values are given in Hertz. The exact attribution of NMR signals was performed using two-dimensional NMR experiments. Mass spectra were acquired with a Micromass GC-TOF mass spectrometer (Waters, Milford, MA). High-performance liquid chromatography was conducted on a Merck Hitachi system consisting of an L-7100 pump, a Rheodynetype injector, a D-7000 interface, and an L7450A diode array spectrometric detector (Hitachi Software Engineering, Yokohama, Japan).
N-Me--MeDA and -MeDA were prepared following the procedure of Borgman et al. (1974), starting from the corresponding benzaldehyde and nitroethane. 5-(GSH)--MeDA was prepared according to previously published methods (Hiramatsu et al., 1990; Miller et al., 1995) with modifications as reported below.
5-(GSH)--MeDA. To a solution of -methyldopamine (0.020 g, 8.06 x 10–5 mol) in sodium phosphate buffer (80 ml, pH 7.4, 50 mM) at 25°C, mushroom tyrosinase (8000 units) was added. The solution became red, indicating the formation of o-quinone. GSH (124 mg, 4.03 x 10–4 mol) was added, and the solution's red color changed with time to yellow (1 h). The solution was carefully concentrated by rotary evaporation without heating and dissolved in 1 ml of water. The product purification was performed by reverse-phase RP-18 modified silica column chromatography first with water (150 ml), and the product separated using 10 x 7.5 ml of formic acid/water/methanol (1:49:50). Each fraction was checked for the presence of adduct using a UV-visible detector. Fractions containing maxima at 232, 264, and 294 nm were separated and lyophilized to dryness. The product 5-(GSH)--MeDA (27 mg) was obtained as oil in 60% yield. The compound purity was checked by high-performance liquid chromatography using a LiChrospher 100 RP-18 column (Merck), with two mobile phase solvents. Solvent A was prepared by adding concentrated trifluoroacetic acid to deionized water until pH was 2.15. Solvent B was prepared by adding trifluoroacetic acid to a 1:1 mixture of methyl cyanide and deionized water until pH was 2.15. The following mobile phase gradient was used: 0 to 10 min, 0 to 2% of solvent B; 10 to 15 min, 2 to 100% of solvent B; and 15 to 20 min, 100 to 0% of solvent B. The compound eluted in 3 min. The peaks were monitored at 290 nm. 1H NMR (400 MHz, D2O, ): 6.72 (1H, s, H2/6), 6.64 (1H, s, H2/6), 4.27 (1H, m, Cys-), 3.66 (1H, m, Glu-), 3.58 (2H, m, Gly-), 3.41 (1H, m, CH), 3.24 (1H, m, Cys-
), 3.06 (1H, m, Cys-), 2.64 (d, J = 6.6 Hz, 2H, CH2), 2.35 (t, J = 7.1 Hz, 2H, Glu-
), 1.98 (2H, m, Glu-), 1.15 (d, J = 6.2 Hz, 3H, CH3); 13C NMR (100.62 Hz, D2O, ): 174.8 (COGly), 174.6(COGlu-
), 173.9 (COGlu), 172.1(COCys), 144.6 (CAr3), 144.2 (CAr4), 128.8 (CAr1), 126.3 (CAr2/6), 119.4 (CAr5), 117.4 (CAr2/6), 54.1 (CGlu-), 53.2 (CCys-), 49.1 (CH), 42.3 (CGly-), 39.3 (CH2), 34.9 (CCys-), 31.4 (CGlu-), 26.2 (CGlu-), 17.5 (CH3); mass spectrometry (field desorption) m/z 473 (MH+).
Cell Culture. Primary neuronal cultures of cerebral cortex were obtained from embryos (E-18) of Wistar rats. Cultures were prepared according to Lautenschlager et al. (2000). The cerebral cortex was dissected, meninges were removed, and tissue was incubated for 15 min in trypsin/EDTA [0.05:0.02% (w/v) in PBS] at 37°C; the cultures were rinsed twice with PBS and once with dissociation medium (modified Eagle's medium with 10% fetal calf serum, 10 mM HEPES, 44 mM glucose, 100 U penicillin plus streptomycin/ml, 2 mM L-glutamine, and 100 insulin units/l), dissociated by Pasteur pipette in dissociation medium, pelleted by centrifugation (210g for 2 min), redissociated in starter medium (neurobasal medium with supplemental B27, 100 U penicillin + streptomycin/ml, 0.5 mM L-glutamine, and 25 µM glutamate), and plated in 48-well plates in a density of 1.5 x 105 cells/well. Wells were pretreated by incubation with poly-L-lysine [0.25% (w/v) in PBS] overnight at 4°C and then rinsed with PBS, followed by incubation with coating medium [dissociation medium with 0.03 (w/v) collagen G] for 1 h at 37°C; then they were rinsed twice with PBS before the cells were seeded in starter medium. Cultures were kept at 36.5°C and 5% CO2 and fed at the 4th day in vitro (DIV) with cultivating medium (starter medium without glutamate) by replacing half the medium. The cultures were used for experiments after the 8th DIV, containing 
10% astroglial cells. Since these neuronal cultures are serum-free, microglia are virtually absent in the cultures at the day of the experiments. In fact, our cultures only reach about 1% microglia in respect to the total population at the 28th DIV, so only as a late-stage event in old primary serum-free cultures as described before (Lautenschlager et al., 2000). Therefore, microglia in this culture model are not likely to interfere with the mechanism of neurotoxicity.
Experimental Protocol. Previous experiments established 40°C as the temperature for the MDMA/hyperthermia experiments (J. P. Capela, K. Ruscher, M. Lautenschlager, D. Freyer, U. Dirnagl, A. R. Gaio, M. L. Bastos, A. Meisel, and F. Carvalho, manuscript submitted for publication). Cultures were treated after the 8th DIV with MDMA and metabolites (concentration range 100–800 µM; single application without feeding for the following 24 or 48 h) and were incubated under normal temperature (36.5°C). For hyperthermic experiments, cells were placed under hyperthermic temperature (40°C) for 24 h and incubated with MDMA and the MDMA metabolites N-Me--MeDA and -MeDA. In experiments using protective agents, the free radical scavenger PBN (100 µM) and NAC (1 mM) were applied to the culture 1 h before the metabolites. The concentrations of NAC and PBN were chosen after screening experiments. Although higher concentrations of PBN were tested, the degree of protection was similar until 1 mM (data not shown). Since the protection was similar, we used the 100 µM concentration for PBN. For NAC, the highest degree of protection was attained with 1 mM. Protection experiments for 5-(GSH)--MeDA were performed at the 24-h time period. For N-Me--MeDA and -MeDA, the 48-h time point for the protection experiments was used. Drugs were diluted in medium or purified water. Controls received an equivalent amount of vehicle. Cultured cells were assessed morphologically by phase-contrast microscopy and viability by life-death assay at two different time points, 24 and 48 h.
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Ethidium Bromide and Acridine Orange Staining. The fluorescent DNA-binding dyes ethidium bromide and acridine orange were used to distinguish necrotic from apoptotic cells. The ethidium homodimer cannot penetrate intact cellular membranes and, therefore, stains the nucleus of cells red only when the membranes are disrupted, whereas acridine orange is membrane-permeable and stains living cells green. Primary cortical neurons, after 24-h treatment with MDMA and its metabolites, were incubated with 2 µg/ml acridine orange and 2 µg/ml ethidium bromide for 5 min before imaging, using a fluorescence microscope with a standard fluorescein excitation filter (Leica Geosystems AG, Heerburg, Switzerland).
Statistical Analysis. Results are presented as mean ± S.E.M. To avoid possible variations of the cell cultures depending on the quality of dissection and seeding procedures, data were pooled from three representative experiments. The means for different treatment groups were compared using the Kruskal-Wallis test (one-way analysis of variance on ranks), since normality conditions were not always satisfied, followed by the Student-Newman-Keuls post hoc test once a significant p value had been obtained. Details of the statistic analyses are described in each figure legend. Significance was accepted at a p value of less than 0.05.
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Results |
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-MeDA-, and -MeDA-induced toxicity both at normothermic and hyperthermic conditions, as shown in Fig. 2. At this time point, there was a concentration-dependent-induced toxicity at both normothermia and hyperthermia for these compounds. Meanwhile, the hyperthermic condition did not change cell viability in control cells comparatively to the normothermic condition the observed toxicity was potentiated at 40°C for all the studied compounds. Figure 2 shows the marked increase of the MDMA- and metabolite-induced neuronal toxicity under hyperthermia. Under normothermic conditions, after the 24-h incubation period, the toxicity elicited by the MDMA metabolites N-Me--MeDA (Fig. 2B), -MeDA (Fig. 2C), and 5-(GSH)--MeDA (Fig. 3) were shown to be more potent neurotoxins than MDMA itself. 5-(GSH)--MeDA proved to be the most toxic.
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-MeDA, and -MeDA for a longer time period (48 h) showed an increase in the neurotoxic effect (Fig. 4). Once more, N-Me--MeDA and -MeDA showed to be more toxic than MDMA for the 48-h time period. As a result of its high toxicity after 24-h incubation, 5-(GSH)--MeDA was not tested at the 48-h time period.
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-MeDA, and -MeDA were shown to be neurotoxic in a concentration-, temperature-, and time-dependent manner. These metabolites are more neurotoxic than the parent compound MDMA, with 5-GSH--MeDA being the most toxic.
Protective Effect of NAC (1 mM) and PBN (100 µM) on the Neurotoxicity Induced by MDMA Metabolites. The addition of NAC (1 mM), a precursor of GSH, and PBN (100 µM), a free radical scavenger, 1 h prior to culture stimulation with 5-(GSH)--MeDA provided protection against the metabolite-induced neurotoxicity in cortical neurons for the 24 h, as revealed by the MTT test (Fig. 5). The stronger degree of protection afforded by NAC against the neurotoxicity of 5-(GSH)--MeDA was obtained for the 200 µM concentration, where about 60% of cell viability was preserved. The same protective effects of NAC and PBN could also be observed against the N-Me--MeDA (Fig. 6) and -MeDA (Fig. 7) neurotoxic effects after 48-h incubation. The stronger degree of protection afforded by NAC against the neurotoxicity of -MeDA and N-Me--MeDA was obtained for the 400 µM concentration, where about 35% of cell viability was preserved.
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Cellular morphology was preserved when 1 mM NAC was coapplied with 800 µM N-Me--MeDA or with 800 µM -MeDA after 24-h incubation, as observed in the microphotographs included in Fig. 8. The metabolite-induced apoptosis was also reduced with NAC, as the morphology of cells depict.
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MDMA Metabolites Induced Programmed Neuronal Cell Death. Phase-contrast microscopy showed that the mechanism of cell death induced by MDMA metabolites was typical of apoptotic cell death. Microphotographs included in Fig. 8 are an example for the apoptotical-type of cell death induced by N-Me--MeDA and -MeDA. Neurons exposed to increasing MDMA metabolites concentrations during 24 h show progressive signs of neurite disintegration, chromatin condensation, membrane blebbing, cytoplasmic shrinkage, nuclear fragmentation, membrane integrity loss, and neuritic processes. In the ethidium bromide/acridine orange staining (Fig. 9), living cells appear as cells with a regularsized green fluorescent nucleus, whereas early apoptotic cells have a green fluorescent condensed, shrunken, or fragmented nucleus, and late apoptotic cells have a red fluorescent condensed, shrunken, or fragmented nucleus. Necrotic cells exhibit a red fluorescent regular-sized or increased nucleus. The pictures in Fig. 9 suggest a higher degree of apoptosis at higher concentrations in a concentration-dependent manner for N-Me--MeDA and -MeDA.
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Discussion |
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-MeDA, -MeDA, and 5-(GSH)--MeDA induced cortical neurotoxicity in serum-free cultures of cortical neurons; 2) neuronal death followed an apoptotic pattern; 3) the observed neurotoxicity was temperature-, time-, and concentration-dependent; 4) the tested metabolites were more neurotoxic than MDMA; 5) 5-(GSH)--MeDA proved to be the most neurotoxic; and 6) NAC and PBN partially protected against MDMA metabolite-induced neurotoxicity.
In vivo studies performed in rats demonstrated that the deleterious effects of MDMA are diverse, including cell death in several brain regions such as the cortex, striatum, and thalamus (Commins et al., 1987; Schmued, 2003). Recent studies showed that MDMA-induced cell death in cultures of rat cortical neurons is accompanied by activation of neuronal apoptotic pathways (Stumm et al., 1999; J. P. Capela, K. Ruscher, M. Lautenschlager, D. Freyer, U. Dirnagl, A. R. Gaio, M. L. Bastos, A. Meisel, and F. Carvalho, manuscript submitted for publication). In the present study, we showed that N-Me--MeDA-, -MeDA-, and 5-(GSH)--MeDA-induced neurotoxicity is typical of apoptotical cell death. Herein, we also showed that MDMA itself is neurotoxic. To our knowledge, this is the first study using neuronal cultures to evaluate the neurotoxicity elicited by MDMA metabolites. In previous experiments, we have proven that MDMA-induced neurotoxicity in these cultures is dependent on direct MDMA 5-HT2A-receptor activation (J. P. Capela, K. Ruscher, M. Lautenschlager, D. Freyer, U. Dirnagl, A. R. Gaio, M. L. Bastos, A. Meisel, and F. Carvalho, manuscript submitted for publication).
MDA, N-Me--MeDA, and -MeDA are major hepatic metabolites of MDMA (Monks et al., 2004). Carvalho et al. (2004a,c) have proven in vitro that the concentrations of N-Me--MeDA and -MeDA decrease over time in biological media due to their oxidation to the correspondent aminochromes and conjugation with GSH. The reactive intermediates produced during the oxidation of these catecholamines into reactive ortho-quinones and/or aminochromes can be conjugated with GSH to form the corresponding glutathionyl adducts. Aminochromes can also undergo further oxidation, leading to the formation of a melanin-type polymer formation (Zhang and Dryhurst, 1994). In fact, a dark brown/black turbidity, characteristic of these polymers, appeared in the neuronal medium, not only after incubation with N-Me--MeDA and -MeDA but also after incubation with 5-(GSH)--MeDA as a late-stage event (hours later). In accordance, it was previously shown that GSH depletion is one of the early toxic events observed in rat cardiomyocytes exposed to N-Me--MeDA and -MeDA (Carvalho et al., 2004c). The importance of the redox status in the conjugated or nonconjugated MDMA metabolite neurotoxicity was underlined in the present study, since protection was observed when 1 mM NAC, a GSH synthesis precursor in the cells, was added to the neuronal cultures. NAC also attenuated the apoptosis induced by MDMA metabolites. Similar results were reported using freshly isolated rat hepatocytes, with 1 mM NAC providing protection against N-Me--MeDA-induced toxicity (Carvalho et al., 2004b).
Conjugation of electrophiles with GSH usually results in detoxification and their subsequent elimination as mercapturic acids (Monks et al., 2004). However, several examples exist where conjugation of GSH with electrophiles results in preservation or enhancement of biologic (re)activity. Ortho-Quinones, aminochromes, and GSH conjugates are known to cause irreversible inhibition of enzymes that possess either a GSH binding site and/or cysteine residues critical for enzyme function (Monks et al., 2004). Likewise, inhibition of glutathione reductase and glutathione S-transferase by quinones, as well as glutathione reductase, selenium-dependent glutathione peroxidase, and glutathione S-transferase by aminochromes has been reported (Remião et al., 2002). Quinone thioethers have the ability to interfere with redox cycle, produce ROS, and arylate tissue macromolecules (Kleiner et al., 1998). Therefore, all accounts for the potential role of MDMA thioether metabolites in MDMA-induced neurotoxicity (Monks et al., 2004). In this study, the GSH-conjugated derivative 5-(GSH)--MeDA showed a potent neurotoxic effect in rat cortical neurons.
5-(GSH)--MeDA is metabolized via the mercapturic acid pathway within the central nervous system, forming 5-(cystein-S-yl)--MeDA and 5-(N-acetylcystein-S-yl)--MeDA (Miller et al., 1995). 5-(GSH)--MeDA is also readily oxidized to the corresponding quinone-GSH conjugate and undergoes the addition of a second GSH molecule to form 2,5-bis-(glutathion-S-yl)--MeDA. Intracerebroventricular (i.c.v.) injections of 5-(N-acetylcystein-S-yl)--MeDA and 5-(GSH)--MeDA into rats produced neurobehavioral changes characteristic of peripheral administration of MDMA/MDA as well as acute increases in brain 5-HT and dopamine concentrations (Miller et al., 1996). 2,5-Bis-(glutathion-S-yl)--MeDA also proved to be a serotonergic neurotoxicant (Miller et al., 1997). In addition to the effects observed after i.c.v. administration of 5-(N-acetylcystein-S-yl)--MeDA and 5-(GSH)--MeDA, their direct injections into the striatum, cortex, and hippocampus produced prolonged depletions in 5-HT and neurobehavioral changes similar to those obtained after in vivo administration of MDA and MDMA (Bai et al., 1999). 5-(N-Acetylcystein-S-yl)--MeDA was also shown to be an extremely potent serotonergic toxicant (Bai et al., 1999). The existence of transporters capable of transferring GSH and systemic-formed GSH conjugates into the brain across the blood-brain barrier was previously suggested (Kannan et al., 1990). Moreover, recent experiments using in vivo microdialysis have provided direct evidence for the presence of GSH and N-acetylcysteine conjugates of MDMA metabolites in the brain, the latter metabolite being toxic to serotonergic neurons, after in vivo s.c. administration of MDMA (Jones et al., 2005). Therefore, the present study corroborates the important role of the GSH-conjugated metabolites in MDMA-induced toxicity.
Colado et al. (1997) provided direct evidence that MDMA administration increased free radical formation in the rat brain. Sprague and Nichols (1995) also showed that MDMA administration to rats increased brain lipid peroxidation, a marker of free radical-induced damage. In accordance, herein we showed that PBN, a free radical scavenger, partially protected against the metabolite-induced neurotoxicity. The protective effect of PBN against MDMA-induced neurotoxicity in rats was previously reported by Yeh (1999). Hyperthermia has also been shown to increase free radical formation, an effect that may lead to neurotoxicity (Halliwell, 1992). Many in vivo studies support that hyperthermia plays a major role in MDMA-induced neuronal death. MDMA was shown to cause acute dose-dependent hyperthermia in rats, and, in humans, MDMA-induced hyperthermia can be fatal (Henry, 1992; Green et al., 2003). Taking into account that MDMA metabolites are pro-oxidant compounds, the hyperthermia-induced oxidative stress will probably potentiate their toxicity. In animal studies, researchers are faced with several factors affecting body temperature. In this study, the high body temperature was simulated by placing the cortical neurons in an incubator at an environment temperature of 40°C after addition of MDMA and MDMA metabolites. This procedure has the advantage of a tight temperature control throughout the whole experiment. Herein, we showed that the neurotoxicity of MDMA and MDMA metabolites was potentiated by hyperthermia.
The neurotoxicological evaluation of MDMA and/or its metabolites needs to address concentrations that simulate those obtained during chronic drug abuse. On the other hand, it is important to evaluate the mechanistic interactions of MDMA and its metabolites with cellular components, which is only possible using worst-case approach concentrations. The MDMA concentrations used in this study were in accordance with other reports (Simantov and Tauber 1997; Stumm et al., 1999) that showed MDMA induced apoptotical cell death in cultured cells. More importantly, Chu et al. (1996) have shown that s.c. administration of MDMA to rats at doses of 20 and 40 mg/kg results in brain concentrations of approximately 206 µM (1 h after) and 466 µM (1.5 h after), respectively, falling squarely in the range of those used in this study. Ricaurte et al. (2000), using an adjustment for body mass/surface area and drug clearance, calculated that the equivalent dose in humans of a 20-mg/kg rat dose (an accepted neurotoxic dose to this species) to be 1.28 mg/kg or approximately 96 mg in a 75-kg individual. Human MDMA users typically use MDMA single dosages of 75 to 125 mg. The fact that some individuals report using up to 10 to 25 individual dosages (tablets) of MDMA per occasion (Parrott, 2005) further suggests that there is little or no margin of safety between the recreationally used and neurotoxic dosages of MDMA. Moreover, although all human studies on MDMA brain concentration are post-mortem analyses, it was found that brain concentrations of MDMA and its metabolites are substantially higher (up to 30 times) than blood concentrations (García-Repetto et al., 2003). Most of the reported cases of serious toxicity or fatality have involved MDMA blood levels ranging from 0.5 to 10 mg/l approximately 2 to 44 µM (García-Repetto et al., 2003). Notably, it was already reported that in humans, N-Me--MeDA, a major toxic metabolite in the present study, reaches plasma concentrations similar to those of MDMA (Segura et al., 2001). N-Me--MeDA was previously detected in the brain of male Sprague-Dawley rats after the s.c administration of a single high dose (40 mg/kg) of Ecstasy (Chu et al., 1996). Jones et al. (2005) measured brain concentration of 5-(GSH)--MeDA to be 40 to 50 µM after a single s.c. MDMA dose of 20 mg/kg. In fact, a multiple-dosage regimen is often used in animal studies (Green et al., 2003) to match the pattern of MDMA use by abusers, suggesting that these metabolites may accumulate in the brain following multiple-drug administration. It seems, then, reasonable to believe that the metabolites used in this study may achieve neurotoxic concentrations in vivo.
In conclusion, the present findings provide further support for the contribution of MDMA to metabolism and hyperthermia to the neurotoxicity exerted by Ecstasy. More studies are fundamental to determine the exact mechanisms by which MDMA metabolites access the brain and produce neurotoxicity.
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Footnotes |
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ABBREVIATIONS: MDMA, 3,4-methylenedioxymethamphetamine (Ecstasy); 5-HT, serotonin; MDA, 3,4-methylenedioxyamphetamine; N-Me--MeDA, N-methyl--methyldopamine; -MeDA, -methyldopamine; ROS, reactive oxygen species; RNS, reactive nitrogen species; GSH, glutathione; 5-(GSH)--MeDA, 5-(glutathion-S-yl)--methyldopamine; NAC, N-acetylcysteine; PBS, phosphate-buffered saline; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; PBN, -phenyl-N-tert-butyl nitrone; NAC, N-acetylcystein; DIV, day(s) in vitro.
Address correspondence to: João Paulo Capela, REQUIMTE, Toxicology Department, Faculty of Pharmacy, University of Porto, Rua Aníbal Cunha, 164, 4099-030 Porto, Portugal. E-mail: joaocapela{at}ff.up.pt
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